ZOOLOGICAL RESEARCH · 2014-12-16 · Zoological Research 2011; Pound et al, 2014), to name just a few. The key advantage of using NHPs is that NHPs are phylogenetically the most

ZOOLOGICAL RESEARCH

Volume 35, Issue 6 18 November 2014

CONTENTS

Special Topic for Primates and Animal Models of Human Diseases

Review

Experimental primates and non-human primate (NHP) models of human diseases in China: current status and

progress ·····················································································································································

·················Xiao-Liang ZHANG, Wei PANG, Xin-Tian HU, Jia-Li LI, Yong-Gang YAO, Yong-Tang ZHENG (447)

Articles

Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed

macaques (Macaca leonina)·······························································Hong-Yi ZHENG, Ming-Xu ZHANG,

Lin-Tao ZHANG, Xiao-Liang ZHANG, Wei PANG, Long-Bao LYU, Yong-Tang ZHENG (465)

Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha,

Yunnan··················································································· Jin-Fa LI, Yu-Chao HE, Zhi-Pang HUANG,

Shuang-Jin WANG, Zuo-Fu XIANG, Juan-Jun ZHAO, Wen XIAO, Liang-Wei CUI (474)

Experimental infection of tree shrews (Tupaia belangeri) with Coxsackie virus A16 ··············· Jian-Ping LI,

Yun LIAO, Ying ZHANG, Jing-Jing WANG, Li-Chun WANG, Kai FENG, Qi-Han LI, Long-Ding LIU (485)

Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree

shrews··············Gui LI, Ren LAI, Gang DUAN, Long-Bao LYU, Zhi-Ye ZHANG, Huang LIU, Xun XIANG (492)

Articles

Acoustic signal characteristic detection by neurons in ventral nucleus of the lateral lemniscus in mice

······························································································· Hui-Hua LIU, Cai-Fei HUANG, Xin WANG (500)

Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae)

·············································· Hong-Fa LU, Li-Na DU, Zhi-Qiang LI, Xiao-Yong CHEN, Jun-Xing YANG (510)

Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu

Nature Reserve based on microsatellite variation··············Ya HE, Zheng-Huan WANG, Xiao-Ming WANG (528)

Complete mitochondrial genome of yellow meal worm (Tenebrio molitor) ············Li-Na LIU, Cheng-Ye WANG (537)

Zoological Research again recognized for Excellence and Impact in Science and Technology Publishing in 2014 (484)

广告：Comin 牌检测试剂盒 ·························································································································· (546)

Cover image: Macaca nemestrina. Photo by Xiao-Feng MA

Zoological Research Website: http://www.zoores.ac.cn/

Zoological Research 35 (6): 447−464 DOI:10.13918/j.issn.2095-8137.2014.6.447

Science Press Volume 35 Issue 6

Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress

Xiao-Liang ZHANG1,3, Wei PANG1, Xin-Tian HU1,2, Jia-Li LI1,2, Yong-Gang YAO1,2, Yong-Tang ZHENG1,2,3,*

1. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology,

Chinese Academy of Sciences, Kunming Yunnan 650223, China

2. Kunming Primate Research Center of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan

650223, China

3. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming Yunnan 650500, China

Abstract: Non-human primates (NHPs) are phylogenetically close to humans, with many similarities in terms of physiology,

anatomy, immunology, as well as neurology, all of which make them excellent experimental models for biomedical research.

Compared with developed countries in America and Europe, China has relatively rich primate resources and has continually aimed to

develop NHPs resources. Currently, China is a leading producer and a major supplier of NHPs on the international market. However,

there are some deficiencies in feeding and management that have hampered China’s growth in NHP research and materials.

Nonetheless, China has recently established a number of primate animal models for human diseases and achieved marked scientific

progress on infectious diseases, cardiovascular diseases, endocrine diseases, reproductive diseases, neurological diseases, and

ophthalmic diseases, etc. Advances in these fields via NHP models will undoubtedly further promote the development of China’s life

sciences and pharmaceutical industry, and enhance China’s position as a leader in NHP research. This review covers the current

status of NHPs in China and other areas, highlighting the latest developments in disease models using NHPs, as well as outlining

basic problems and proposing effective countermeasures to better utilize NHP resources and further foster NHP research in China.

Keywords: Non-human primates; Experimental primates; Animal models; Current status

Employing animal models for biomedical research has been done to speed along research by bypassing ethical restrictions on using humans as experimental subjects, which averts risks associated with direct research on humans, especially in clinical trials. There are likewise numerous advantages to using animal models such as strong controllability of experimental conditions, high repeatability, ease of scale, comparabi-lity of results. However, the effectiveness of animal mod-els is hampered by intrinsic species differences between animal models and humans. For example, experimental data obtained from rodents, who are phylogenetically distant from humans, does not always appropriately predict the efficacy of a drug treatment or its potential toxicity in humans (Van Der Worp et al 2010; Xu, 2011). To bridge the gaps, non-human primates (NHPs) have

been widely used in lab settings since the 1950s. 1There has been a corresponding increase in the prominence and importance of using NHP models that have been widely used as subjects in infectious diseases (Kaushal et al, 2012; Liu & Zhang, 2010), mental and neurological disorders (Perretta, 2009), cardio-cerebrovascular diseases (Sy et al, 2014), and endocrine diseases (Kamath et al,

Received: 11 June 2014; Accepted: 15 August 2014

Foundation items: This work was supported by the National Natural

Science Foundation of China (81172876, 81273251, U1202228, 81471620); the National Special Science Research Program of China

(2012CBA01305); the National Science and Technology Major

Project (2013ZX10001-002, 2012ZX10001-007) and the Knowledge

Innovation Program of CAS (KSCX2-EW-R-13, KJZD-EW-L10-02) *Corresponding author, E-mail: [email protected]

448 ZHANG, et al.

Zoological Research www.zoores.ac.cn

2011; Pound et al, 2014), to name just a few. The key advantage of using NHPs is that NHPs are

phylogenetically the most proximate models to humans, bearing many distinctive similarities not found in other animal models. Previous reports found that DNA sequence similarities between NHPs and humans can reach up to 98.77% (Fujiyama et al, 2002) and the concordances rates of gene transcription levels of spleen, peripheral blood monocyte cells (PBMCs) and liver between NHPs and humans can reach as high 91.41%, 84.36%, and 74.29%, respectively (Lu et al, 2008). As a consequence, NHPs remain the first or the only choice for investigating major human diseases including HIV/AIDS, measles, malaria, hepatitis, but also in the study of human cognition and brain diseases (Xu et al, 2013). In addition, NHPs are also used for human cancer research (Xia & Chen, 2011). The data garnered from such NHP experiments is invaluable as a starting point, because it ensures greater efficiency and surety of future clinical application, making NHPs the “gold standard” for preclinical studies (He et al, 2013).

It was half a century ago that the NHP model was gained traction in Europe and America where most of the scientific research were conducted at the time. They began to develop the NHP resources by importing NHPs from other countries, developing husbandry programs and exploring the nature of NHPs. As more recent advances in medical research and biotechnology invol-ving gene modification, gene knockout, epigenetics, precision gene editing and fine regulation of local gene expression in experimental primates become more cost-effective and refined, we expect that there will be greater need for NHP resources to meet the demands of many new applications, which developed countries with advanced knowledge of NHPs may struggle to meet. At this point, countries with indigenous NHPs that can also advance effective protection, sustainable exploitation and utilization of their NHP resources may stand to benefit. Indeed, as its growing life sciences and pharmaceutical industries, China has a great opportunity to become a leader in NHP resources and even research (Cyranoski, 2004; Hao, 2007).

This review evaluates the ongoing situation of NHPs in China and other areas as well as the latest development of human disease models using NHPs in China. We analyze the existing problems for further developing NHP models and propose targeted counte-measures that may allow for better exploitation and

utilization of NHP resources in China.

Status of experimental primates abroad There is a disagreement on the total number of

primate species in existence. According to the 2005 Mammal Species of the World (http: //vertebrates.si.-edu/msw/mswCFApp/msw/index.cfm), there are 376 pri-mate species. However, the latest statistics from IUCN Red List of Threatened Species (http: //www.iucnredlist.org) declare 420 primate species. Occasionally, new primate species are described, such as Rhinopithecus strykeri that was discovered recently (Geissmann et al, 2011; Long et al, 2012), make the numbers subject to change. More important to the total number of primates are threats from humans, whose development often interferes with primate habitat. As a result, most primate species are considered to be endangered. To overcome the threats to NHPs, numerous countries have adopted strict protective measures on NHP resources, and some have began to pay greater detail to utilizing these resources more rationally.

While America has no wild NHPs, it is home to primate research institutes including 8 national primate research centers (NPRCs) funded by the National Institutes of Health (NIH) that develop NHP models for basic and applied studies of human health: California National Primate Research Center, New England Primate Research Center, Southwest National Primate Research Center, Tulane National Primate Research Center, Washington National Primate Research Center, Wisconsin National Primate Research Center, Yerkes National Primate Research Center, and Oregon National Primate Research Center (Kaiser, 2013). These 8 NPRCs have a total number of 26, 000 NHPs (Hayden, 2008), representing more than 20 species of NHPs, mostly macaques (largely for practical purposes and ease of research). Annually the US federal government invests about $100 million each year into these NPRCs to financially support ongoing studies of NHPs (http:// www.humanesociety.org/animals_in_research/general_information_on_animal_research/an_introduction_to_primate_issues.html). As advances in research techniques and methodology using primate models have been created, there has been a consummate increase in investment. From 1999 to 2006, funding through the US federal government for NHPs research increased 7.9-fold, ($15.4 million to $122 million), while the total number NHPs at the eight NPRCs increased from 2 4 182 to 2 7 914 (http: //www.all-creatures.org/saen/res-nprcs-8yr.html). Other

Experimental primates and non-human primate (NHP) models of human diseases in China: current status and progress 449

Kunming Institute of Zoology (CAS), China Zoological Society Volume 35 Issue 6

institutions with strong focuses on NHP research also receive federal and state funding, including the New Iberia Research Center at the University of Louisiana at Lafayette, the University of Texas MD Anderson Cancer Center, Alamogordo Primate Facility and Primate Foundation of Arizona, the Caribbean Primate Research Center, a squirrel monkey colony of the University of South Alabama and a baboon breeding facility of the University of Oklahoma. Even a few private facilities in NHP breeding receive government grants to maintain the US’s overall capacity for conducting NHP research (http: //www.sourcewatch.org/index.php/National_Primate_Research_Center_System). One further aspect of NHP research in the US worth noting is that the US is home to the largest colony of Chimpanzees used for research, with around 1 000 currently in government sponsored labs (Wadman, 2011a). Chimpanzees are the only great apes known to be captured for biomedical research, even though they are also an endangered species, and given that they are the primates most closely related to humans, are used in invasive rese-arch, and defined as “inoculation with an infectious agent and/or drug testing.”

Compared to the US, Europe is the next largest center of NHP research (Carlsson et al, 2004), though

with some different trends. While the number of NHPs used in experiments in America has grown annually from 2001 to 2010 (Figure 1, data from http: //www.aphis.-usda.gov), the total amount of NHPs used for research in the EU remains lower than in America, without stable growth. Within EU, total NHPs used for research in 2003, 2005 and 2008 were 10 360, 10 450 and 9 600, respectively (https: //www.gov.uk). The most popular NHPs used for research were rhesus macaques (RMs) and cynomolgus macaques (CMs), with pig-tailed macaques, squirrel monkeys, African green monkeys, common marmosets, cotton-topped tamarins, baboons, chimpanzee, among others taking a minor share. No apes were used in experiments anywhere in the EU from 2002−2008 according to a survey by the European Com-mission (http: //vertebrates.si.edu/msw/mswCFApp/msw/-index.cfm). There is much debate regarding whether National Institutes of Health (NIH) research chimp-anzees be retired (Wadman, 2011b) and US primate centre faces scrutiny with stricter criteria (http: //www.-nature.com/news/us-primate-centre-faces-scrutiny-1.10317). In June 2013, NIH announced that it will retire to sanctuary nearly all of its research chimpanzees (http: //blogs.nature.com/news/2013/06/nih-retires-most-rese-arch-chimpanzees.html).

Figure 1 Number of NHPs used in experiments in America per year from 2001 to 2010

While the US and Europe are the key centers of

NHP research, Southeast Asian countries that possess indigenous NHPs have recently begun to pay more attention to these resources. Southeast Asia has made rapid advances in its NHP capacity: several NHP research institutions and breeding facilities have been set up in the area, and a few NHP breeding facilities have passed the American Association for Accreditation of

Laboratory Animal Care (AAALAC) certification. For example, Thailand has NHP facilities and primate research centers, among which Primate Research Insti-tute of Thailand (PRIT; http: //www.primate.or.jp/se-mi/Abst_f2nd/suchinda.pdf) possesses 14 species of primates, and focuses on primate breeding and producing animal models suitable for diseases research, especially for tropical diseases research. These advances have made

450 ZHANG, et al.


the PRIT a key biomedical research center on primates and an information center for NHPs among Southeast Asia. The presence of such facilities and their corresponding resources greatly enhances the entire region’s NHP research capacity, and complements the growth in NHP resources and research being conducted in China.

Status of experimental primates in China

Compared to US and Europe, NHP resources are rich in China. More than 24 species were found. It accounts for about 6% of primate species around the world. Yunnan Province in particular possess the most NHP resources, and undertakes a bulk of the earliest and present work on NHPs (Shen & Yang, 2003; Pan & Ben, 1984; Li & Lin, 1983). The anchor institution of this research, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS), was established in the late 1950s. KIZ is among the first institutions in China to start a primate breeding center to conduct basic research and clinical applications on NHPs in taxonomy, ecology, anatomy, genetics, behavior, evolution, reproductive biology, neurobiology, immunology, and with a series of laboratories dedicated to animal microbial detection, animal nutrition, clinic pathology of animal; KIZ has also produced a series of monographs on NHPs including The Anatomy of the Golden Monkey, The Anatomy of Macaca mulatta. The primate centre in KIZ hosts currently about 2 800 NHPs, including M. mulatta, M. leonine, M. fascicularis, M. assamensis, M. arctoides, Rhinopithecus roxellana, and other species. The facility is well-equipped to deal with these NHP resources, as it has received AAALAC certification, and the centre’s NHP resources and research has received a worldwide attention (Cyranoski, 2004; Hao, 2007). Alongside KIZ, Institute of Medical Biology (IMB), Chinese Academy of Medicine Science, which was set up in 1958 and before 1980s hosted the greatest number of RMs in China (Shen & Yang, 2003), focuses on the research and development of medical products using experimental primates, particularly for vaccines. IMB is currently the largest base of the research and production of both attenuated poliovirus oral live vaccine and hepatitis A vaccine in China.

Prior to China’s development of domestic NHP research into exploitation and utilization of NHP reso-urces, China was—and remains—a leading producer and a major supplier country for NHPs to the United States,

Europe, Japan, and now Korea. In 2013, the number of breeding experimental monkeys in China, mainly rhesus macaques and cynomolgus macaques, in China there were about 250 000 breeding cynomolgus macaques and 40 000 breeding rhesus macaques (data from The China Laboratory Primates Breeding and Development Asso-ciation), with approximately 50 000 to 60 000 experi-mental monkeys born in breeding facilities each year. Though many NHPs are used for domestic research projects, a large number are exported abroad. For example more than 65% of the experimental monkey imports in the US from Asia are from China, and in Europe, around 70% of research primate imports are from China. Essentially, though NHP research remains predominately a Euro-American venture, China is the leading supplier of resources necessary to this research.

To support China’s shift from an NHP resource producer to an NHP research driver, the Chinese government has increased investment for experimental primates and new drug development. Over the last decade, there has been marked progress on research and innovation into experimental primates. Specifically, the Ministry of Science and Technology (MOST) of the People's Republic of China has set up an experimental primate germplasm resources center in Hainan and Suzhou, and in 2011 MOST launched a number of large science projects under the platform construction of promoting the development of primate animal models of human major diseases. The General Administration of Quality Supervision, Inspection and Quarantine of China has likewise set up a state key laboratory for experi-mental primate animal quarantine in Guangxi Province, and as we mentioned previously the CAS established the Kunming Primate Research Center of Chinese Academy of Sciences in Yunnan. In addition, the governments are providing more support and financial resources to NHP research. In short, these actions will undoubtedly accelerate the innovation and capacity for NHP research in China. Prior to 2005, most high-quality experimental NHPs were exported to European, America and other “developed” countries, while the domestic consumption was relatively limited, but thanks to the additional support and development of the Chinese scientific community, outsourcing of services related to experimental NHPs have continued to spring up and domestic services have exploded in Beijing, Kunming, Shanghai, Suzhou, Chengdu, Guangzhou, among others, to improve the development of experimental NHPs in



China.

Current status and progress on the NHP models of human diseases in China

Chinese researchers have established numerous NHP models to model a variety of human diseases, which have made large contributions to the study, preve-ntion and treatment of human diseases. In China, most NHP models for human diseases focus on exploring infectious diseases, cardio-cerebrovascular diseases, endocrine diseases, reproductive diseases, mental and neurological disorders, and ophthalmic diseases. Simi-larly, some basic research into NHPs by Chinese researchers have greatly extended the usability of NHPs as animal models by exploring transgenic strategies in experimental primates and in 2010 researchers of Kunming Institute of Zoology, CAS created the first transgenic monkey in China (Niu et al, 2010).

Primate animal models for infectious diseases

Though advances in medicine and medical technology have effectively controlled or eradicated infectious diseases, a variety of new infectious diseases have continuously emerged, largely as a result of human activities’ adverse impact on the surrounding environ-ment and bringing individuals into closer contact with these situations. As a developing country with the worl-d's largest population, China is actually threatened by many infectious diseases that are exacerbated by population density, intense land-usage, and industrial-ization, such as HIV/AIDS tuberculosis (TB), viral hepatitis, foot-and-mouth disease, avian influenza. This problem is only magnified on the global scale, especially among developing countries that experience the bulk of population growth and the increase in population densities as a consequence of urbanization and industrialization. Given the nature of infectious diseases and the larger global trends, developing more ideal and targeted animal models is a crucial step for opening up new strategies against them.

HIV/AIDS

To our knowledge, no animal models for HIV/AIDS research are more suitable than NHPs. HIV-1, for instance, infects only humans and a handful of primate animals such as the chimpanzee, gibbon and pigtailed macaque (Kuang et al, 2009). While HIV-1 can infect these NHPs, for several reasons the disease's progression is quite different from that in humans, though AIDS

models of RMs infected with SIV closely resemble the human disease progression of AIDS. Consequently, NHPs are the most widely used animal models for HIV/ AIDS research (Lei et al, 2013). Though NHP AIDS models were established using simian retrovirus (SRV) in the early years, NHP AIDS models created with SIV better simulate human AIDS. Currently, most NHP AIDS models in China for developing vaccines and therapeutics are established via infection of SIV or SHIV, a simian/human immunodeficiency chimeric virus.

An initial Chinese RM/SIV model established by Li et al (2007) was accomplished by infecting Chinese RMs with SIVmac239. Furthermore, Xia et al (2010, 2011) and Li et al (2012) investigated the dynamic and functional changes of dendritic cell subsets and Treg cells in SIVmac239-infected Chinese RM. The results confirmed a previous study and showed that disease symptoms of SIV-infected Chinese RMs were more similar to human responses to HIV-1. Feng et al (2007) also set up RM models of SIVmac251 infection using several different routes (intravenous, intravaginal and intrarectal routes) and observed differences in disease course after SIVmac251 infection resulting from the different routes; notably, this was the first case of a Chinese NHP AIDS animal model infected with SIV through a mucosal route. Later, Cong et al (2011b) established a Chinese RM model of SHIV-SF162p3, an HIV-1 subtype B, infected through repeated low-dose intravaginal inoculation, which more closely resembles the natural transmission of HIV infection in humans. Though HIV-1 subtype C comprises the predominant strains in China (Wu et al, 2006), Cong et al’s model provides technical reference for establishing further AIDS animal model for evaluating vaccines and treatment strategies suitable for China's predominant epidemic strains, which may change given growing infection rates and variances, especially in Yunnan. Additionally, researchers have also successfully established different HIV domestic Chinese RM models (Wang et al, 2011b; Cong et al, 2011a; Yao et al, 2011). The characteristics of these models remains to be elucidated in future, however, as HIV/AIDS infections continue to increase globally in general and in China (and Yunnan) in particular, these models may become increasingly important research models.

While the existing SIV/SHIV-infected macaque models are useful for application in AIDS research, genetic differences between SIV/SHIV and HIV-1

452 ZHANG, et al.


significantly limit the use of SIV/SHIV (Ambrose et al, 2007; Van Rompay, 2012). Accordingly, NHP AIDS models established with HIV/HSIV infection should be a better model of human HIV/AIDS infections and pathology; fortunately, researchers at the KIZ are currently establishing northern pig-tailed macaque AIDS model with HIV/HSIV. If successful, such a novel model may provide an ideal platform for the study, drug screening and the vaccine evaluation of AIDS.

Tuberculosis (TB)

Recent studies reveal that mouse models are quite questionable, but NHP models avoid many of the pitfalls that make rodent models less popular; in particular, NHP models of TB for immunological research and vaccine test show promising results (Kaushal et al, 2012; Guo & Ho, 2014). The study of NHP models of TB was later than other countries in China, despite the ongoing prevalence of the disease. For TB, there is a certain gap in the types and construction methods of NHP models, for example, the NHP models of TB latency and reactivation have not been done in China.

Currently, there are several reports on the ongoing construction of NHP models for TB study in China. For instance, Wuhan University began to establish of NHP models of TB using fiberoptic bronchoscopes since 2006, and later delivered Mycobacterium tuberculosis (MTB) into the lungs of Chinese RMs (Xian et al, 2010; Zhang et al, 2011a). This laboratory has become the fifth standard laboratory for TB vaccine study in the world (http: //news.sohu.com/20060715/n244270131.shtml), and models have been used in preclinical testing of candidate vaccines and drugs. Wang et al (2012) later established Chinese RM models of TB via bronchoscopic and intratracheal instillation into the lungs and found the clinical manifestations, disease progression and pathological changes of these models closely resembled human primary TB and hematogenous disseminated TB.

NHP models of TB have marked advantages in immunological research and test of drugs as compared with non-primate models. Unfortunately, the lack of currently available inbred strains of NHPs makes pathological changes varied. The obvious solution is to strengthen the system research of pathological features, such as lobe fibro-cavitary lesions, etc. Likewise, studies suggest that there is a high rate of HIV and MTB co-infection, which is thought to be one of factors contributing to the global resurgence of TB (Harrington,

2010; Kaushal et al, 2012). In theory, infection with HIV accelerates the activity of latent MTB and then increases the incidence of TB and, at the same time, TB seems to speed the HIV replication rate (Enserink, 2001), making them a potentially lethal complication. Establishing NHP models of TB then provides a foundation for the constr-uction of NHP models of MTB/HIV co-infection, which may prove more ideal than NHP models of MTB/SIV co-infection (Diedrich et al, 2010) currently in place.

Influenza virus

Of the many infectious disease pandemics, infl-uenza has been one of the worst (Kilbourne, 2006). This is especially poignant for China, as outbreaks of highly pathogenic H5N1 avian influenza viruses occurred in Hong Kong in 1997, killing 6 people (Mounts et al, 1999), which was the first worldwide report of avian influenza virus directly infecting humans. Following the report, avian influenza A infection in humans was also reported in Southeast Asia, Europe and North Africa (Obenauer et al, 2006), and outbreaks of H7N9 in humans in China last year received local and global attention (Gao et al, 2013a, b; Hu et al, 2013; Liu et al, 2013). Since rodent animals are not the natural hosts of influenza viruses and show no symptoms of upper-respiratory-tract infections (Zak & Sande, 1999), rodent models are not good systems for studying human influenza infections. NHP models are, however, excellent at mimicking human infections of influenza viruses. Foreign researchers have established NHP models infected with H1N1, H3N2, H5N1, etc (Baas et al, 2006; Carroll et al, 2008; Cillóniz et al, 2009; Fan et al, 2009; Itoh et al, 2009; Kobasa et al, 2007; Laddy et al, 2009; Rimmelzwaan et al, 2001). Recently, Chen et al (2009) infected Chinese RMs with the highly pathogenic avian influenza virus A/Tiger/Harbin/01/2002 (H5N1) intrana-sally and noted that RMs exhibited symptoms similar to humans, such as fever, loss of appetite, interstitial pneumonia, etc. These results indicated that Chinese RMs could be potentially successful infection models of the H5N1 viruses as they exhibit similar symptoms. To date, this model has some shortcomings, since H5N1 only reproduced in the respiratory system and that RMs showed low sensitivity to H5N1 infection. In view of these limitations, Lü et al (2011) infected Chinese RM with A/SZ/406H/2006(H5N1) by endotracheal incuba-tion, and found that this method yielded a more sensitive NHP model of H5N1 virus infection, and that the virus



could replicate in many other organs outside the lung and better resembling severe cases seen in humans.

Since the influenza virus is primarily transmitted via air, it is necessary to assess the contributions of small droplet aerosols and larger respiratory droplets to better understand epidemiological vectors for the disease and public responses to influenza virus outbreaks. Technol-ogical hurdles currently make it difficult, such as lack of inoculations for influenza viruses in NHPs and the diffi-culty in isolating and quantifying viable influenza viruses from dilute aerosols make this currently impractical.

Hepatitis virus

Compared to other infectious diseases, China has a long history of research into hepatitis. Over 30 decades ago, Ge et al (1989) performed long-term liver biopsies of M. assamensis, M. Speciosa, Nycticebus Coucang infected with HBV, successfully setting up NHP models of HBV infection. Zhang et al (1998) infected RMs with HGV and established the first RM model of HGV infection, while more recently Yang et al (2006) infected Chinese RMs with HEV gene type-IV via intravenous injection. Following Yang’s injection, Chinese RMs exhibited typical acute hepatitis, indicating Chinese RMs made viable animal models of HEV gene type-IV. Chinese researchers also successfully established Chinese RM models of HEV1 infection, and through these models found E2 IgG can serve as a marker of HEV infection (Zhang et al, 2003; 2004).

Chinese researchers have used HAV, HBV, HEV and HGV to infect NHPs (Ge et al, 1989; Zhang et al, 1998; Zhang et al, 2003; 2004). But there have been no reports of the establishment of domestic NHP models for HCV infection. Chimpanzee models of HCV infection have already been established by foreign researchers, though this is expected since chimpanzees are the only known animal to be susceptible to HCV infection (Bukh et al, 2001). However, ethical and financial restrictions largely limit the use of chimpanzees for such research, especially in the US and Europe (Akari et al, 2009). GBV-B, a close relative to HCV, provides an effective model and has been used to infect New World monkeys and the GBV-B infection models which closely resemble HCV infection in humans (Akari et al, 2009; Bukh et al, 1999). In spite of the value of the GBV-B model, the establishment and development of an HCV/GBV-B chimeric virus model has higher application value for a preclinical study of antiviral vaccines and drugs, and

accordingly foreign researchers have created monkey models infected with an HCV/GBV-B chimera (Li et al, 2014; Rijnbrand et al, 2005). In future, we should focus on developing NHP surrogate models for HCV infection.

Enteroviruses

Enteroviruses (EV) affect millions worldwide each year. The enterovirus is a genus of RNA viruses assoc-iated with several human diseases, and among them Enterovirus 71 (EV71) is highly contagious and causes foot and mouth disease (HFMD) , which has increasingly been a serious threat for Asian children (Lee & Chang, 2010). CMs infected with EV71 via lumbar injection or intravenous route show some symptoms similar to those found in humans, but these CM models do not exhibit certain important infection symptoms, such as myocarditis and neurogenic pulmonary edema. Likewise, most of the EV71 applied to these CM models are host-adapted EV71 strains (Hashimoto & Hagiwara, 1982; 1983; Hashimoto et al, 1978; Nagata et al, 2004; Nagata et al, 2002), and there can be marked differences between host-adapted EV71 strains and the epidemic strains. Unfortunately, NHPs are not susceptible to infection with epidemic strains of EV71 ( Liu & Zhang, 2010).

The study of EV71 infection NHP models began relatively late in China, with the first models appearing in 2010. Zhang et al (2011b) infected adult Chinese RM with EV71 FY-23 strain via intracerebral, intravenous, respiratory and digestive routes and showed similar symptoms to human cases but without herpetic lesions, the reason for which may be old-age of these experimental Chinese RMs. Perhaps neonatal monkeys would prove better animal models for the study of EV71 pathogenesis, and accordingly Liu et al (2011) infected neonatal RMs with EV71 FY-23 strain via the respiratory tract by tracheoscopy and the results indicated these neonatal RM models more closely resembled infection of human neonates during lactation. Wang et al (2011a) likewise infected neonatal RMs with EV71 via nasal spray, which further confirmed that a respiratory infection route makes models more susceptible to EV71 infection than the digestive tract infection route, providing some valuable information for future studies of childhood EV71 transmission.

NHP models of cardio-cerebrovascular disease

Myocardial infarction, a type of cardiovascular

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disease, has high morbidity and mortality, and is com-mon in Europe and America (Yeh et al, 2010). Previous studies indicated that the treatment strategy—proved to be effective in a murine model—applied to NHPs failed to show any efficacy (Norol et al, 2003; Orlic et al, 2001). Yang et al (2011) recently established RM models of myocardial infarction by ligating the left anterior descending artery (LAD) and the changes of histopathology and electrocardiogram demonstrated that this model should be excellent for studying human myocardial infarction, but the main cause of myocardial infarction in human is cardiovascular blockage or spasm caused by the long-term effects of several factors (hypertension, hyperlipidemia, etc.). The pathogenesis of these RM models created by ligation of the coronary artery existed many differences compared with human, which currently limits drug discovery and development. It is necessary that new NHP models should be developed for studing human myocardial infarction in future.

Cerebrovascular disease is the second leading cause of mortality in China. Ischemic cerebrovascular disease accounts for 80% of cerebrovascular diseases (Feigin et al, 2003). Pan et al (2006) injected polyvinyl alcohol into a branch of middle cerebral artery to make thrombed, and CT scanning and related behavioral changes showed significant damage to brain tissue in the area of vascular occlusion. These results indicate that NHP models of ischemic cerebral infarction were successfully establi-shed and they owned good stability and were suitable in the study of treating acute and chronic diseases. Zhu et al (2008) also established CM models of photochemical thrombosis by focusing the cold lamp-house on the precentral gyrus, and the construction of these models is simple and highly reproducible, making them excellent for the basic study and investigation of treatments for cerebral infarction, especially regarding the study of transplantation of stem cells. Xu et al (2012) recently generated Chinese RM models of middle cerebral artery occlusion by injecting auto-blood clots into middle cerebral artery through the femoral artery with a micro-catheter, a method that is easily repeatable, with high stability and low mortality. Likewise, the extent of embolization of the cerebral infarction could be timely detected. The aforementioned advantages have led to this technique becoming more common and ideal for the study of cerebral ischemia. Zhu et al (2009) previously established CM models of intracerebral hemorrhage by

injecting antiautologous blood slowly and gently into the caudate nucleus. This study indicated that the volume, site and speed of injection are key factors for establishing a successful model, which is instructive to development of the NHP models of intracerebral hemorrhage.

While there has been significant progress in establishing NHP models of cardio-cerebrovascular disease, some shortcomings still exist. The NHPs chosen to be animal models for cardio-cerebrovascular disease are usually young and healthy, despite the fact among humans the elderly are at the highest risk for cardio-cerebrovascular disease (Baccini et al, 2008). Similarly, the patients with cardio-cerebrovascular disease usually suffer from other health conditions such as hypertension, hyperlipidemia or diabetes which may significantly affect cardio-cerebrovascular symptoms. Currently avail-able studies of NHP models of cardio-cerebrovascular disease that accompany these additional diseases is quite limited, to create viable NHP models of cardio-cerebro-vascular disease which is close to clinical nature will be our future task.

NHP models of diabetes

Diabetes is already quite prevalent in developed countries, mainly type 1 and type 2, and it is quickly rising in China now, with an estimated 11% of the population currently afflicted to some extent. Although rodents have been used as the primary model in studying diabetes, rodent models do not adequately mimic human diabetes and make them quite limited for clinical application (Anderson & Bluestone, 2005; Delovitch & Singh, 1997). NHPs, due to their similarities with human, are believed to be the most effective models for human diabetes.

NHP diabetic models are relatively new in China. Liu et al (2009) divided the Chinese RM into a 125 mg/kg STZ group, a 75 mg/kg STZ group and a 50 mg/kg STZ group and then different doses of STZ were injected into corresponding subjects intravenously. The models induced by the dose of 125 mg/kg and 75 mg/kg STZ were not stable, and mortality was high due to the toxic effects of STZ. Most subjects in the 50 mg/kg STZ group retained a high level of blood glucose and their pancreas β cells were severely damaged, but their mortality was quite low, suggesting that 50 mg/kg of STZ may be optimal for inducing diabetes in Chinese RM models. Xu et al (2009) likewise injected STZ at the dose of 45mg/kg into the CMs intravenously and found



that drinking volume, food intake, urine output increased and body weight was reduced, while fasting glucose was increased and the pancreas showed atrophy and fibrosis, indicating that the CM diabetic models created with this method were viable. Given the serious toxic effects of STZ on experimental NHPs, Qiao et al (2009) and Jin et al (2010) established RM models of type 1 diabetes by the partial pancreatectomy combined with low-dose STZ. This strategy is generally safer and more reproducible as compared with single high-dose STZ or total pancreatectomy.

Currently, the most commonly used method to make type 2 diabetic animal models is high fat feeding combined with drugs induction. Typical clinical features of type 2 diabetes was found by injection of STZ into CM at a dose of 35 mg/kg after the appearance of hyperlipidemia and obesity based on high-glucose and fat diet feeding, (Lu et al, 2013) and the models showed. Since pathogenesis of type 2 diabetes induced by STZ is unnatural as it significantly differs from the pathogenesis of human type 2 diabetes, theoretically, screening of monkeys for spontaneous diabetes would lead to a more natural pathogenesis in NHP models. It will make this model more valuable for translational medicine. Wang et al (2004) previously screened 3 RMs of spontaneous diabetes from 100 RMs by both the glucose tolerance and glycosuria tests. These 3 RMs were in middle and elderly age, indicating that onset age and the morbidity rate of spontaneous diabetes in RMs is consistent with that found in humans. After one year of observation, the similarity in clinical symptoms (urinating more and consuming more aggravated gradually and fasting blood glucose was significantly higher than normal) to humans suggested this method was feasible for screening RM models of spontaneous diabetes. Unfortunately, the current number of spontaneous diabetic NHP models is limited and cannot adequately meet the growing demand for effective scientific research.

Given that the morbidity rate of human type 2 diabetes increases annually as long as increases in the number of obese people (mainly caused by factors such as eating patterns, among others), monkey models of type 2 diabetes induced by diet can be accomplished more quickly, and compared with models induced by drugs, more closely resemble the pathogenesis of human type 2 diabetes. Zhang et al (2012) recently divided 36 experimental CMs into 4 groups and fed them standard basic diet, high-sugar diet, high-fat diet and high-sugar

combined with high-fat diet respectively for 24 months. The progression of type 2 diabetes among CMs with high-energy diets showed that similar methods were successful in establishing NHP models of type 2 diabetes, and that it effectively modeled different stages of type 2 diabetes. These findings also provided data on further optimizing dietary strategies that could establish even more effective animal models for human type 2 diabetes research. While type 2 diabetic mouse models have been made (Kadowaki, 2000), the successful establishment of transgenic NHP models of type 2 diabetes should vastly improve NHP study of type 2 diabete.

NHP models of mental and neurological disorders

To our knowledge, none of animal models can fully recapture the neurological symptoms for neurodegene-rative and psychological disorders due to their multiple origins. While NHPs are close to human in terms of genetics, there are numerous differences in the evolution and composition in brain. Currently, NHP models of mental and neurological disorders such as heroin addiction, morphine addiction, Parkinson’s disease (PD), depression syndrome, Alzheimer’s disease (AD), spinoc-erebellar ataxia, spinal cord injury, motor cortex lesion, epilepsy, etc (Chen et al, 2013; Chu et al, 2014; Ding et al, 2008; Liu et al, 1992; Lu et al, 1999; Qiao et al, 2007; Tian & Ma , 2014; Zha et al, 2006; Zhang et al, 1999 ) have been established in China. However, there is a gap in the types of NHP models for mental and neurological disorders as compared with similar models established in other countries.

An increased understanding regarding natural and artificial repair of central nervous system injuries as resulted as more researchers have begun focusing on the study of nerve regeneration following spinal cord injuries. To model these injuries in NHPs, the main strategy is to inflict spinal cord injury via selective resection of the spinal cord with a sharp knife (Tian et al, 2010). Ni et al (2005) made NHP models of spinal cord hemisection by T-11 laminectomy and then resection of a 1-mm long hemispinal cord, while Zha et al (2006) made the C3-5 exposed via a dorsal midline incision in the neck of RMs and transected the left hemispinal cord with a knife under the microscope after C4 laminectomy. The results showed this method to establish RM models of spinal cord injury were simple, effective, stable, accurate and repetitive, even if the method was quite different from naturally occurring human spinal cord injuries. For

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epilepsy, models are usually created by the intentional application of penicillin, aluminum hydroxide, metrazol and coriaria lactone. Chen et al (2013) injected kainic acid into the right intra-hippocampus of RMs by the stereotacic technique, and the resulting seizure pattern, imaging observation and histopathologic tests showed this model closely resembled human temporal lobe epilepsy. Though promising, the long-term stability of this RM models induced by kainic acid requires further study.

Modeling PD has likewise been met with several successes in China. Liu et al (2006) injected 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) into right side internal carotid artery and then performed behavioral testing and imaging detection, which showed the method to be both safe and simple for establishing of NHP models of PD. However, this method made it difficult to control the extent of nigrostriatal damage (Herrero et al, 1991). In order to minimize the nigrostriatal damage, Yan et al (2014) established a chronic NHP model of PD by intramuscularly injecting MPTP. The time of appearance and the severity of each symptom of this model could be controlled by the daily dosage and frequency. This model gradually displayed PD symptoms and mimicked the development of human PD well. However, the PD symptoms were reversible in some MPTP-induced model, being quite different from the degenerative nature of PD in humans, and limiting the study of stem cell therapy for nervous system disorders. Interestingly though, substantia nigra neurons damage caused by 6-hydroxydopamine (6-OHDA) is irreversible, and the extent of nigrostriatal damage can be controlled by the injection amount and the injection sites. Accordingly Zhang et al (2006) generated RM models of PD by injecting 6-OHDA stereotaxically into the right substantia nigra. In testing potential treatments of PD, optimal treatment period is a key issue, and one that can be addressed using NHP models of early stage PD. Accordingly, Ding et al (2008) established NHP early stage models of PD by surgery to make the common carotid artery (CCA) of RMs exposed and then injecting low-dose MPTP into the exposed CCA. Compared with late-stage PD NHPs, NHP early stage models may better mimic the gradual development of human PD. One of the purposes of model development is to find new treatments. Yan et al (2014) reported that morphine could alleviate tremors in a MPTP-induced NHP model, suggesting the therapeutic effects of morphine complement those of L-

Dopa, a classic therapy, on PD symptoms. But due to MPTP toxicity to the digestive system, digestive system disorders of the NHP models induced by MPTP gradually appeared and would affect immune function and nutritional metabolism. Such models are difficult to maintain over long durations, making the future development of a long-term stable NHP PD models an urgent issue.

Recently, studies have established a link between increased endogenous formaldehyde (FA), a methanol metabolite, and AD pathology (He et al, 2010; Li et al, 2008; Tong et al, 2011, 2012). Studies conducted by Hu and He’s Labs in China on RM showed chronic feeding 3% methanol ad libitum led to specific damages to the brain similar to those of Alzheimer’s disease. These were the first studies to show that methanol toxicity inflicted all the major hallmarks of AD on rhesus monkey: cognitive decline, tau phosphorylation, and amyloid plaques formation (Yang et al, 2014).

Recent advances in optogenetic technology of have been proved as a revolutionary tool for studying neural systems. Optogenetics can be used for precisely targeting a specific area of neural systems as well as even in freely moving mammals. Researchers can then turn different neurons on or off, rapidly and safely, using this technology (Deisseroth, 2010; Liu & Tonegawa, 2010). Currently, research of optogenetic approaches has been done on NHPs by foreign researchers and some progress of this research has been made (Dai et al, 2013), but there is lack of such research being done domestically in China.

NHP models of ophthalmic diseases

Ophthalmic diseases present interesting complic-ations for animal models. Compared with other animals, the NHP visual system is closer to that of humans than any other animal, making experimental data from NHPs safer and more effective when translated into clinic settings. However, both cost restrictions and availability of experimental NHPs hamper the development of NHP models of ophthalmic diseases. Chinese researchers have successfully established several NHP models of ophthalmic diseases, such as corneal endothelial injury, amblyopia, glaucoma and so on (Dai et al, 2005; Zhu et al, 2013), but there are some gaps in the types and construction methods of NHP models of ophthalmic diseases between China and other developed countries.

Zhu et al (2013) established NHP models of corneal



endothelial injury by phacoemulsification damaging corneal endothelial cells. This method proved easy to manipulate and is easily replicable, and it causes negli-gible damage to other tissues, making it a useful technique for constructing models that can accurately evaluate the effect of drugs and other factors on the endothelial healing. Similarly, Dai et al (2005) established NHP models of chronic hypertensive glauco-ma by semiconductor frequency-doubled 532 laser and argon laser, respectively, aiming at 360° functional trab-ecular meshwork. The light coagulation times of the successful induction of high intraocular pressure in RMs fluctuate greatly, so refining the existing methods of photocoagulation should improve modeling efficiency, though this requires greater study to verify.

NHP models of reproductive diseases

Reproductive diseases seriously affect human health and even psychological well-being. Infertility caused by reproductive diseases affect an average 8-12% of couples worldwide (Sellandi et al, 2012), and is quite pronounced in developed countries. Reproductive physiology of NHPs is quite similar to human, and the data gathered from studies on reproductive diseases in human using NHPs cannot be replaced using any other experimental animals. The progress in the establishment of NHP models of reproductive diseases, such as polycystic ovary syndrome (PCOS), oligozoospermia, metrorrhagia and postpartum hemorrhage (PPH), has been made by Chinese researchers.

Tang et al (2012) gave RMs two cycles of subcut-aneous injections of propionic acid testosterone (PAT), and then muscle injections of human chorionic gon-adotropin (HCG). The results of this study indicated PAT combined with HCG could successfully induce RM PCOS, which was similar to human PCOS. Sun et al (2006) earlier set up an RM model of oligozoospermia induced by orally administered gossypol acetic acid and provided a suitable NHP model for the study of infertility in men. Until recently, most studies on PPH still were built on the retrospective analysis, with few reports evaluating or using animal models of PPH. Notably, Huang (2010) established CM models of PPH with uterine atony induced by medication such as oxytocin, which provided a useable platform for further research. Likewise, You et al (2003) had earlier set up RM models of metrorrhagia induced by endometritis and the symptoms of this model could closely resemble those of

human metrorrhagia caused by endometritis.

Problems, Challenges and prospects While China is a key producer of NHP resources, it

still lags behind other countries in its capacity for innovative NHP model research. As medical research and biotechnology advances—genetic modification, gene knockout and cloning, etc.—it is likely that a remarkable increase of NHP resources will be developed to meet the needs of more intensive and diverse application. It makes for some interesting possibilities in NHP research. Due to the economic crises and the subsequent “austerity” in budgets across developed countries in America and Europe, funding support for primate research is reduced or kept stagnant. Concurrently, growing criticism from animal-rights activists in these countries has brought a great deal of pressure and research institutions have to limit or even abolish primate research (Cyranoski, 2003; Wadman, 2011b). Given the advances and advantages provided by using NHP primates, researchers on NHP models from developed countries are increasingly seeking collaborations with primate research institutes in China and other developing countries in Southeastern Asia (Cyranoski, 2004). It is possible to bring unprecedented opportunities and challenges to life sciences and biomedical research in China. The key issue is how to cope with these opportunities and challenges to further develop NHP resources effectively.

Development of domestic Chinese NHP resources has made significant progress since its inception, however, in contrast to countries in America, Europe and East Asia (notably Japan and Korea), NHP resources in China are not widely or effectiely being used. In other words, while China has a rich diversity of species, many factors such as subspecies and geographical distribution of NHP resources, differences among subspecies in genetics and physiology and the occurrence of subspecies hybrid populations in breeding and over the course of experiments make the reproducability of experimental results poor in working towards etablishing of animal models, evaluating drugs, etc. Nontheless, some common experimental primate resources (espec-ially exotic species such as Cercopithecus aethiops) are rare or even non-existent in China, which restricting human diseases research and the development of corresponding drugs, but other more general factors such as technical skill, experience, and existing resources are also critical. We must realize that Chinese NHP research

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can only be accomplished by bringing important experimental primates lacking in China—and researchers familiar with them—while simultanesouly strengthening the application existing experimental primates resources in China.

Another factor making Chinese NHPs ineffective is a lack of the principle knowledge about NHPs. For example, compared with some experimental primate species such as Indian RMs, common experimental primate species such as Chinese RMs lack sufficient information regarding the genetic background of these primates, and it is also little known about the normal value of their important biological parameters (Xia et al, 2009). Thankfully, recent genome sequencing and analysis of CMs and Chinese RMs offers a great deal more basic data and useful reference points for relevant NHP model research (Yan et al, 2011). It is necessary to complement basic biological data of experimental primates in China and establish subspecies breeding populations whose basic biological data (such as genetic backgrounds) are definite. Recently, the Kunming Primate Research Center is making efforts to raise a northern pig-tailed macaques (NPMs, M. leonina) colony and Prof. Zheng’s lab has collected many basic biological data of this colony of NPMs (Pang et al, 2013; Zhang et al, 2014), in order to promote the standar-dization and reliability of experimental primate data.

Another key issue for NHP research is the demand for specific pathogen-free (SPF) experimental animals. SPF animals are in great demand right now. While it is available for many traditional models, experimental primates often carry zoonotic pathogens which not only pose health risks to experimenters but also partly interfere with experimental results. In the US, all 8 NPRCs funded by grants through NIH can offer SPF experimental animals (Zheng, 2012), but to date China has not established SPF core groups of experimental primates. Presuming that research into drug development and microbe research on diseases such as HIV continues to grow, if China wants to be a leading player in NHP models, it must establish SPF core groups of experi-mental primates and all breeding efforts should be done only with careful caution and planning.

In recent years, experimental primate husbandry in China has experienced rapid development and the product has been increased, but not always in tandem with the demand. As we mentioned earlier, the influence of the global economic crisis in developed countries in

2008 as well as interference from animal-rights activists have led to a plateau in primate imports from China. On the Chinese side, this unexpected lack of growth has led to rampant overproduction. Likely one reason for Chinese overproduction is that many NHP breeding facilities in China lack effective communication with their international counterparts, making it difficult to project and then match demand. To remedy the situation and ensure adequate supply of relevant and in-demand NHP resources, it is vital to establish an information and communication platform that can effectively offer NHP breeding information services and simultaneously carry out strategic plans for cooperation between NHP breeding facilities and relevant governing bodies and institutions.

Two other factors could affect China’s position on NHP research and supply. First, animal welfare activists have had some effects on NHP research. While it is true that advocacy of animal welfare helps promote harmony between experimental animals and humans (and conse-quently decreases the danger of injuries to experimenters and often offers higher-quality experimental results), animal welfare has begun to attract extensive attention which will result in increased legislation on animal welfare. China has just recently begun to legislate animal welfare. On a cultural level, Chinese citizens’ awareness of animal welfare is weak. Awareness of animal welfare has placed economic pressure on NHP breeding facilities and research institutions. The dual-faced nature of this dilemma means that effective implementation of experimental primate welfare in China is difficult. To bring China more in line with international standards while continuing to develop China’s domestic research capacity, relevant policies should be made for strengthening propaganda, supervision, regulation and support of experimental primate welfare, in such a way to support the healthy development of the life sciences and medical industry in China. Second, it is a very crucial step to develop transgenic NHP resources. Since the birth of the first transgenic mouse, researchers have established a series of transgenic mice for modeling many types of human diseases. Transgenic mice have made greater contributions to the study and the treatment of human diseases, but their phylogenetic distance to humans hampers the usefulness of their results. Establishing primate transgenic models should be able to overcome these limitations and better predict the efficacy and toxicity of novel treatments in humans. The



development of these transgenic primates is still in its infancy; the first transgenic primate was born in 2001(Chan et al, 2001), and over the past decade, few transgenic NHPs have been born (Chen et al, 2012). The major obstacles in producing transgenic NHPs are technical in nature. However, thanks to more efficient gene-editing techniques involving ZFN, TALEN and CRISPR/Cas9-based methods, researchers can avoid some shortcomings—notably low efficiency and high toxicity—of previous transgenic methods using retroviral or lentiviral vectors (Shen, 2013). Chinese researchers first applied TALENs and CRISPR/Cas9 to establish site-specific gene mutant RMs and CMs (Liu et al, 2014b; Niu et al, 2014). The myriad studies have demonstrated that using technologies involving TALENs and CRISPR/Cas9, multiple and single genetic mutations can be effectively performed in monkeys (Liu et al, 2014a; Niu et al, 2014).

In addition to the above-mentioned problems, there are still many other key problems with NHP models to resolve, for example, high cost, difficulties regarding how to have a well-defined phenotype characterization, and a genotype-phenotype correlation for NHPs and how to use cutting-edge molecular imaging system to characterize the NHP brain structure and activity for better understanding mental disease.

In summary, NHPs are ideal experimental models for biomedical research. Compared with developed countries in America and Europe, China has advantages in developing NHPs resources. China is a leading producer and a major supplier of NHPs. Although deficiencies in feeding and management may delay China’s growth in NHP research and materials, a number of primate animal models for human diseases on infectious diseases, cardiovascular diseases, endocrine diseases, reproductive diseases, neurological diseases, and ophthalmic diseases have been established. Particularly, the site-specific gene mutant RMs and CMs have successfully been born in China. We believe that advances in these studies by using NHP models will undoubtedly further promote the development of China's life sciences and pharmaceutical industry, and enhance China’s position as a leader in NHP research.

Acknowledgments: The opinions expressed in this

review paper reflect personal views of the authors. Due to the limited space of this paper and/or our ignorance and negligence, we are sorry that we did not include all these related works regarding primate studies in China although we aimed to have a comprehensive review of NHP models of human diseases. We sincerely apologize to all those colleagues whose important work is not cited.

References

Akari H, Iwasaki Y, Yoshida T, Iijima S. 2009. Non-human primate surrogate model of hepatitis C virus infection. Microbiology and Immunology, 53(1): 53-57.

Ambrose Z, Kewalramani VN, Bieniasz PD, Hatziioannou T. 2007. HIV/AIDS: in search of an animal model. Trends in Biotechnology, 25(8): 333-337.

Anderson MS, Bluestone JA. 2005. The NOD mouse: a model of immune dysregulation. Annual Review of Immunology, 23(1): 447-485.

Baas T, Baskin CR, Diamond DL, García-Sastre A, Bielefeldt-Ohmann H, Tumpey TM, Thomas MJ, Carter VS, Teal TH, Van Hoeven N, Proll S, Jacobs JM, Caldwell ZR, Gritsenko MA, Hukkanen RR, Camp DG 2nd, Smith RD, Katze MG. 2006. Integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics. Journal of Virology, 80(21): 10813-10828.

Baccini M, Biggeri A, Accetta G, Kosatsky T, Katsouyanni K, Analitis A, Anderson HR, Bisanti L, D'Ippoliti D, Danova J, Forsberg B, Medina S, Paldy A, Rabczenko D, Schindler C, Michelozzi P. 2008. Heat effects on mortality in 15 European cities. Epidemiology, 19(5): 711-719.

Bukh J, Apgar CL, Yanagi M. 1999. Toward a surrogate model for

hepatitis C virus: an infectious molecular clone of the GB virus-B

hepatitis agent. Virology, 262(2): 470-478.

Bukh J, Apgar CL, Govindarajan S, Emerson SU, Purcell RH. 2001.

Failure to infect rhesus monkeys with hepatitis C virus strains of

genotypes 1a, 2a or 3a. Journal of Viral Hepatitis, 8(3): 228-231.

Carlsson HE, Schapiro SJ, Farah I, Hau J. 2004. Use of primates in

research: a global overview. American Journal of Primatology, 63(4):

225-237.

Carroll TD, Matzinger SR, Genescà M, Fritts L, Colòn R, Mcchesney

MB, Miller CJ. 2008. Interferon-induced expression of MxA in the

respiratory tract of rhesus macaques is suppressed by influenza virus

replication. The Journal of Immunology, 180(4): 2385-2395.

Chan A, Chong K-Y, Martinovich C, Simerly C, Schatten G. 2001.

Transgenic monkeys produced by retroviral gene transfer into mature

oocytes. Science, 291(5502): 309-312.

Chen N, Meng FG, Zhang JG, Zhang K, Liu HG, Meng DW, Liu C, Ge

Y. 2013. The establishment of macaque models of temporal lobe

epilepsy. Chinese Journal of Neurosurgery, 29(5): 533-537. (in Chinese)

Chen YX, Deng W, Jia CS, Dai XW, Zhu H, Kong Q, Huang L, Liu Y,

Ma CM, Li JM, Xiao C, Liu Y, Wei Q, Qin C. 2009. Pathological

460 ZHANG, et al.


lesions and viral localization of influenza A (H5N1) virus in

experimentally infected Chinese rhesus macaques: implications for

pathogenesis and viral transmission. Archives of Virology, 154(2): 227-

233.

Chen YC, Niu YY, Ji WZ. 2012. Transgenic nonhuman primate models

for human diseases: approaches and contributing factors. Journal of

Genetics and Genomics, 39(6): 247-251.

Chu XX, Rizak JD, Yang SC, Wang JH, Ma YY, Hu XT. 2014. A

natural model of behavioral depression in postpartum adult female

cynomolgus monkeys (Macaca fascicularis). Zoological Research,

35(3): 174-181.

Cillóniz C, Shinya K, Peng X, Korth MJ, Proll SC, Aicher LD, Carter

VS, Chang JH, Kobasa D, Feldmann F, Strong JE, Feldmann H,

Kawaoka Y, Katze MG. 2009. Lethal influenza virus infection in

macaques is associated with early dysregulation of inflammatory

related genes. PLoS Pathogens, 5(10): e1000604.

Cong Z, Jiang H, Jin G, Chen T, Wang W, Tao Z, Yao N, Xiong J, Wu

FX, Chen ZW, Wei Q. 2011a. Titration of a SHIV1157ipd3N4 stock by

intravenous inoculation of Chinese-origin rhesus macaques. Chinese

Journal of Comparative Medicine, 21(4): 12-15. (in Chinese)

Cong Z, Liu H, Duo JY, Wang W, Jiang H, Gao H, Yang ZW, Fleury S,

Wei Q, Qin C. 2011b. Efficient repeated low-dose intravaginal

infection with SHIV (SF162p3) in Chinese-origin rhesus macaques.

Chinese Journal of Comparative Medicine, 21(2): 44-48. (in Chinese)

Cyranoski D. 2003. China launches primate centre to broaden medical

use of monkeys. Nature, 424(6946): 239-240.

Cyranoski D. 2004. China takes steps to secure pole position in primate

research. Nature, 432(7013): 3.

Dai J, Brooks DI, Sheinberg DL. 2013. Optogenetic and electrical microstimulation systematically bias visuospatial choice in primates. Current Biology, 24(1): 63-69.

Dai Y, Sun XH, Guo WY, Yang YM, Yu XB, Qian SH, Shen Y, Jin XH. 2005. Establishment of chronic glaucoma model in rhesus monkeys and evaluation of their related biological characteristics. Acta Laboratorium Animalis Scientifica Sinica, 13(2): 68-71. (in Chinese)

Deisseroth K. 2010. Optogenetics. Nature Methods, 8(1): 26-29.

Delovitch TL, Singh B. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity, 7(6): 727-738.

Diedrich CR, Mattila JT, Klein E, Janssen C, Phuah J, Sturgeon TJ, Montelaro RC, Lin PL, Flynn JL. 2010. Reactivation of latent tuberculosis in cynomolgus macaques infected with SIV is associated with early peripheral T cell depletion and not virus load. PLoS One, 5(3): e9611.

Ding F, Luan L, Ai Y, Walton A, Gerhardt GA, Gash DM, Grondin R, Zhang Z. 2008. Development of a stable, early stage unilateral model of Parkinson's disease in middle-aged rhesus monkeys. Experimental Neurology, 212(2): 431-439.

Enserink M. 2001. Driving a stake into resurgent TB. Science,

293(5528): 234-235.

Fan SF, Gao YW, Shinya K, Li CK, Li Y, Shi JZ, Jiang YP, Suo YB,

Tong TG, Zhong GX, Song JS, Zhang Y, Tian GB, Guan YT, Xu XN,

Bu Z, Kawaoka Y, Chen H. 2009. Immunogenicity and protective

efficacy of a live attenuated H5N1 vaccine in nonhuman primates.

PLoS Pathogens, 5(5): e1000409.

Feigin VL, Lawes CM, Bennett DA, Anderson CS. 2003. Stroke

epidemiology: a review of population-based studies of incidence,

prevalence, and case-fatality in the late 20th century. The Lancet

Neurology, 2(1): 43-53.

Feng YF, Wang W, Xu Y, Cong Z, Jiang H, Tong W, Wu XX, Lu YZ,

Wei Q. 2007. Comparative study on acute manifestation of rhesus

monkeys infected with SIVmac251 by different routes. Chinese


Fujiyama A, Watanabe H, Toyoda A, Taylor TD, Itoh T, Tsai SF, Park

HS, Yaspo ML, Lehrach H, Chen Z, Fu G, Saitou N, Osoegawa K, de

Jong PJ, Suto Y, Hattori M, Sakaki Y. 2002. Construction and analysis

of a human-chimpanzee comparative clone map. Science, 295(5552):

131-134.

Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, Lu SH, Yang YD,

Fang Q, Shen YZ, Xi XM, Gu Q, Zhou XM, Qu HP, Yan Z, Li FM,

Zhao W, Gao ZC, Wang GF, Ruan LX, Wang WH, Ye J, Cao HF, Li

XW, Zhang WH, Fang XC, He J, Liang WF, Xie J, Zeng M, Wu XZ, Li

J, Xia Q, Jin ZC, Chen Q, Tang C, Zhang ZY, Hou BM, Feng ZX,

Sheng JF, Zhong NS, Li LJ. 2013a. Clinical findings in 111 cases of

Influenza A (H7N9) virus infection. New England Journal of Medicine,

368(24): 2277-2285.

Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu

K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z,

Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J,

Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y,

Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y. 2013b.

Human infection with a novel avian-origin influenza A (H7N9) virus.

New England Journal of Medicine, 368(20): 1888-1897.

Ge XM, Huang GY, Liu W, Chen J, Jiang YL, Huang DC, Li RJ, Wang

SS. 1989. A study of developmental pathology of experimental

infection by human hepatitis(HHBV) in Macaca Assamensis. Chinese

Journal of Zoonoses, 5(5): 24-26. (in Chinese)

Geissmann T, Lwin N, Aung SS, Aung TN, Aung ZM, Hla TH,

Grindley M, Momberg F. 2011. A new species of snub-nosed monkey,

genus Rhinopithecus Milne-Edwards, 1872 (Primates, Colobinae),

from northern Kachin state, northeastern Myanmar. American Journal

of Primatology, 73(1): 96-107.

Guo M, Ho WZ. 2014. Animal models to study Mycobacterium

tuberculosis and HIV co-infection. Zoological Research, 35(3): 163-

169.

Hao X. 2007. Monkey research in China: developing a natural resource.

Cell, 129(6): 1033-1036.

Harrington M. 2010. From HIV to tuberculosis and back again: a tale

of activism in 2 pandemics. Clinical Infectiouse Diseases, 50(S3):

S260-S266.

Hashimoto I, Hagiwara A. 1982. Studies on the pathogenesis of and

propagation of enterovirus 71 in poliomyelitis-like disease in monkeys.

Acta Neuropathologica, 58(2): 125-132.

Hashimoto I, Hagiwara A. 1983. Comparative studies on the

neurovirulence of temperature-sensitive and temperature-resistant



viruses of enterovirus 71 in monkeys. Acta Neuropathologica, 60(3-4):

266-270.

Hashimoto I, Hagiwara A, Kodama H. 1978. Neurovirulence in

cynomolgus monkeys of enterovirus 71 isolated from a patient with

hand, foot and mouth disease. Archives of Virology, 56(3): 257-261.

Hayden EC. 2008. US plans more primate research. Nature, 453(7194):

439.

He RQ, Lu J, Miao JY. 2010. Formaldehyde stress. Science China Life

Sciences, 53(12): 1399-1404.

He S, Wang D, Wei L. 2013. Practical and critical instruction for

nonhuman primate diabetic models. Transplantation Proceedings,

45(5): 1856-1865.

Hu YW, Lu SH, Song ZG, Wang W, Hao P, Li JH, Zhang XN, Yen HL,

Shi BS, Li T, Guan WC, Xu L, Liu Y, Wang S, Zhang X, Tian D, Zhu Z,

He J, Huang K, Chen H, Zheng L, Li X, Ping J, Kang B, Xi X, Zha L,

Li Y, Zhang Z, Peiris M, Yuan Z. 2013. Association between adverse

clinical outcome in human disease caused by novel influenza A H7N9

virus and sustained viral shedding and emergence of antiviral resistance.

The Lancet, 381(9885): 2273-2279.

Huang J. 2010. Establishment of the Postpartum Hemorrhage Model of

Cesarean Section in Macaca fascicularis with Uterine Atony Induced

by Medicine. Master Thesis, Guangzhou Medicine College. (in Chinese)

Itoh Y, Shinya K, Kiso M, Watanabe T, Sakoda Y, Hatta M, Muramoto

Y, Tamura D, Sakai-Tagawa Y, Noda T, Sakabe S, Imai M, Hatta Y,

Watanabe S, Li C, Yamada S, Fujii K, Murakami S, Imai H, Kakugawa

S, Ito M, Takano R, Iwatsuki-Horimoto K, Shimojima M, Horimoto T,

Goto H, Takahashi K, Makino A, Ishigaki H, Nakayama M, Okamatsu

M, Takahashi K, Warshauer D, Shult PA, Saito R, Suzuki H, Furuta Y,

Yamashita M, Mitamura K, Nakano K, Nakamura M, Brockman-

Schneider R, Mitamura H, Yamazaki M, Sugaya N, Suresh M, Ozawa

M, Neumann G, Gern J, Kida H, Ogasawara K, Kawaoka Y. 2009. In

vitro and in vivo characterization of new swine-origin H1N1 influenza

viruses. Nature, 460(7258): 1021-1025.

Jin X, Zeng L, He S, Chen Y, Tian B, Mai G, Yang G, Wei L, Zhang Y,

Li H, Wang L, Qiao C, Cheng J, Lu Y. 2010. Comparison of single

high-dose streptozotocin with partial pancreatectomy combined with

low-dose streptozotocin for diabetes induction in rhesus monkeys.

Experimental Biology and Medicine, 235(7): 877-885.

Kadowaki T. 2000. Insights into insulin resistance and type 2 diabetes

from knockout mouse models. Journal of Clinical Investigation, 106(4):

459-465.

Kaiser J. 2013. Research in limbo as harvard moves to close center.

Science, 340(6132): 535-535.

Kamath S, Chavez AO, Gastaldelli A, Casiraghi F, Halff GA,

Abrahamian GA, Davalli AM, Bastarrachea RA, Comuzzie AG,

Guardado-Mendoza R, Jimenez-Ceja LM, Mattern V, Paez AM, Ricotti

A, Tejero ME, Higgins PB, Rodriguez-Sanchez IP, Tripathy D,

DeFronzo RA, Dick EJ Jr, Cline GW, Folli F. 2011. Coordinated

defects in hepatic long chain fatty acid metabolism and triglyceride

accumulation contribute to insulin resistance in non-human primates.

PLoS One, 6(11): e27617.

Kaushal D, Mehra S, Didier PJ, Lackner AA. 2012. The non-human

primate model of tuberculosis. Journalof Medical Primatology, 41(3):

191-201.

Kilbourne ED. 2006. Influenza pandemics of the 20th century.

Emerging Infectious Diseases, 12(1): 9-14.

Kobasa D, Jones SM, Shinya K, Kash JC, Copps J, Ebihara H, Hatta Y,

Kim JH, Halfmann P, Hatta M, Feldmann F, Alimonti JB, Fernando L,

Li Y, Katze MG, Feldmann H, Kawaoka Y. 2007. Aberrant innate

immune response in lethal infection of macaques with the 1918

influenza virus. Nature, 445(7125): 319-323.

Kuang YQ, Tang X, Liu FL, Jiang XL, Zhang YP, Gao GX, Zheng YT.

2009. Genotyping of TRIM5 locus in Northern pig-tailed macaques

(Macaca leonina), a primate species susceptible to human

immunodeficiency virus type 1 infection. Retrovirology, 6(1): 58.

Laddy DJ, Yan J, Khan AS, Andersen H, Cohn A, Greenhouse J, Lewis

M, Manischewitz J, King LR, Golding H, Draghia-Akli R, Weiner DB.

2009. Electroporation of synthetic DNA antigens offers protection in

nonhuman primates challenged with highly pathogenic avian influenza

virus. Journal of Virology, 83(9): 4624-4630.

Lee MS, Chang LY. 2010. Development of enterovirus 71 vaccines.

Expert Review of Vaccines, 9(2): 149-156.

Lei AH, Pang W, Zhang GH, Zheng YT. 2013. Use and research of

pigtailed macaques in nonhuman primate HIV/AIDS models.

Zoological Research, 34(2): 77-88. (in Chinese)

Li MH, Li SY, Xia HJ, Wang L, Wang YY, Zhang GH, Zheng YT. 2007.

Establishment of AIDS animal model with SIVmac239 infected

Chinese rhesus monkey. Virologica Sinica, 22(6): 509-516.

Li FX, Lu J, Xu YJ, Tong ZQ, Nie CL, He RQ. 2008. Formaldehyde-

mediated chronic damage may be related to sporadic neurodegeneration.

Progress in Biochemistry and Biophysics, 35(4): 393-400.

Li SY, Xia HJ, Dai ZX, Zhang GH, Fan B, Li MH, Wang RR, Zheng

YT. 2012. Dynamics and functions of CD4+ CD25high regulatory T

lymphocytes in Chinese rhesus macaques during the early stage of

infection with SIVmac239. Archives of Virology, 157(5): 961-967.

Li T, Zhu S, Shuai L, Xu Y, Yin S, Bian Y, Wang Y, Zuo B, Wang W,

Zhao S, Zhang L, Zhang J, Gao GF, Allain JP, Li CY. 2014. Infection of

common marmosets with hepatitis C virus/GB virus-B chimeras.

Hepatology, 59(3): 789-802.

Li ZX, Lin ZY. 1983. Classification and distribution of living primates

in Yunnan, China. Zoological Research, 4(2): 111-120. (in Chinese)

Liu D, Shi W, Shi Y, Wang D, Xiao H, Li W, Bi Y, Wu Y, Li X, Yan J,

Liu W, Zhao G, Yang W, Wang Y, Ma J, Shu Y, Lei F, Gao GF. 2013.

Origin and diversity of novel avian influenza A H7N9 viruses causing

human infection: phylogenetic, structural, and coalescent analyses. The

Lancet, 381(9881): 1926-1932.

Liu JN, Zhang LF. 2010. Human enterovirus 71 infection in

experimental animals. Acta Laboratorium Animalis Scientia Sinica,

18(3): S5-S9. (in Chinese)

Liu H, Chen Y, Niu Y, Zhang K, Kang Y, Ge W, Liu X, Zhao E, Wang

C, Lin S, Jing B, Si C, Lin Q, Chen X, Lin H, Pu X, Wang Y, Qin B,

Wang F, Wang H, Si W, Zhou J, Tan T, Li T, Ji S, Xue Z, Luo Y, Cheng

L, Zhou Q, Li S, Sun YE, Ji W. 2014a. TALEN-mediated gene

Mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell, 14(3):

323-328.

462 ZHANG, et al.


Liu L, Zhao H, Zhang Y, Wang J, Che Y, Dong C, Zhang X, Na R, Shi

H, Jiang L, Wang L, Xie Z, Cui P, Xiong X, Liao Y, Zhao S, Gao J,

Tang D, Li Q. 2011. Neonatal rhesus monkey is a potential animal

model for studying pathogenesis of EV71 infection. Virology, 412(1):

91-100.

Liu S, Wang W, Yu XP, Mao J, Rong PF. 2006. Establishment of hemi-

Parkinsonism model of rhesus induced by MPTP. Chinese Journal of

Medical Imaging Technology, 22(3): 353-356. (in Chinese)

Liu S, Su ZH, Ai ZD, Li W, Wang W. 2009. Primate models of diabetes

induced by streptozotocin. Journal of Clinical Rehabilitative Tissue

Engineering Research, 13(50): 9917-9923. (in Chinese)

Liu X, Tonegawa S. 2010. Optogenetics 3.0. Cell, 141(1): 22-24.

Liu YJ, Zhang GF, Lu Q. 1992. A monkey paralysis model of unilateral

motor cortex lesion. Journal of Capital Institute of Medicine, 13(4):

264-268. (in Chinese)

Liu Z, Zhou X, Zhu Y, Chen ZF, Yu B, Wang Y, Zhang CC, Nie YH,

Sang X, Cai YJ, Zhang YF, Zhang C, Zhou WH, Sun Q, Qiu Z. 2014b.

Generation of a monkey with MECP2 mutations by TALEN-based

gene targeting. Neuroscience Bulletin, 30(3): 381-386.

Long YC, Momberg F, Ma J, Wang Y, Luo YM, Li HS, Yang GL, Li M.

2012. Rhinopithecus strykeri Found in China. American Journal of

Primatology, 74(10): 871-873.

Lu SN, Wang GL, Zhang YC, Zhang HD, Zheng JW. 1999. Treatment

effect of lingyi capsule for the withdrawal syndromes in morphine-

dependent monkeys. Chinese Journal of Drug Dependence, 8(1): 61-63.

(in Chinese)

Lu SY, He ZL, Yang FM, Yu WH, Zhao Y, Li YY, Wang JB, Chen LX,

Qi SD. 2013. Establishment of a rhesus monkey model of type 2

diabetes mellitus and analysis of some of its clinical features. Chinese


Lu YR, Wang LN, Jin X, Chen YN, Cong C, Yuan Y, Li YC, Tang WD,

Li HX, Wu XT, Li YP, Wang L, Cheng JQ. 2008. A preliminary study

on the feasibility of gene expression profile of rhesus monkey detected

with human microarray. Transplantation Proceedings, 40(2): 598-602.

Lü Q, Deng W, Bao LL, Xu LL, Li FD, Chen T, Zhan LJ, Qin C. 2011. Endotracheal intubation in oculation of H5N1 virus and detection of virus in organ tissues in the rhesus macaques. Chinese Journal of Comparative Medicine, 21(12): 56-60. (in Chinese)

Mounts AW, Kwong H, Izurieta HS, Ho Y, Au T, Lee M, Buxton Bridges C, Williams SW, Mak KH, Katz JM, Thompson WW, Cox NJ, Fukuda K. 1999. Case-control study of risk factors for avian influenza A (H5N1) disease, Hong Kong, 1997. Journal of Infectious Diseases, 180(2): 505-508.

Nagata N, Shimizu H, Ami Y, Tano Y, Harashima A, Suzaki Y, Sato Y, Miyamura T, Sata T, Iwasaki T. 2002. Pyramidal and extrapyramidal involvement in experimental infection of cynomolgus monkeys with enterovirus 71. Journal of Medical Virology, 67(2): 207-216.

Nagata N, Iwasaki T, Ami Y, Tano Y, Harashima A, Suzaki Y, Sato Y, Hasegawa H, Sata T, Miyamura T, Shimizu H. 2004. Differential localization of neurons susceptible to enterovirus 71 and poliovirus type 1 in the central nervous system of cynomolgus monkeys after intravenous inoculation. Journal of General Virology, 85(10): 2981-2989.

Ni W, Li YM, Guan YG, Zhu XB, Wang TH, Feng ZT. 2005.

Evaluation of neurological function following establishment of spinal

cord hemisection. Journal Sichuan University, 36(3): 328-330. (in

Chinese)

Niu YY, Shen B, Cui YQ, Chen YC, Wang JY, Wang L, Kang Y, Zhao

XY, Si W, Li W, Xiang AP, Zhou JK, Guo XJ, Bi Y, Si CY, Hu B, Dong

GY, Wang H, Zhou ZM, Li TQ, Tan T, Pu XQ, Wang F, Ji SH, Zhou Q,

Huang XX, Ji WZ, Sha JH. 2014. Generation of gene-modified

cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-

cell embryos. Cell, 156(4): 836-843.

Niu YY, Yu Y, Bernat A, Yang SH, He XC, Guo XY, Chen DL, Chen

YC, Ji SH, Si W, Lü YQ, Tan T, Wei QA, Wang H, Shi L, Guan JA,

Zhu XM, Afanassieff M, Savatier P, Zhang K, Zhou Q, Ji WZ. 2010.

Transgenic rhesus monkeys produced by gene transfer into early-

cleavage–stage embryos using a simian immunodeficiency virus-based

vector. Proceedings of the National Academy of Sciences of the United

States of America, 107(41): 17663-17667.

Norol F, Merlet P, Isnard R, Sebillon P, Bonnet N, Cailliot C, Carrion C,

Ribeiro M, Charlotte F, Pradeau P. 2003. Influence of mobilized stem

cells on myocardial infarct repair in a nonhuman primate model. Blood,

102(13): 4361-4368.

Obenauer JC, Denson J, Mehta PK, Su X, Mukatira S, Finkelstein DB,

Xu X, Wang J, Ma J, Fan Y, Rakestraw KM, Webster RG, Hoffmann E,

Krauss S, Zheng J, Zhang Z, Naeve CW. 2006. Large-scale sequence

analysis of avian influenza isolates. Science, 311(5767): 1576-1580.

Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-

Ginard B, Bodine DM, Leri A, Anversa P. 2001. Mobilized bone

marrow cells repair the infarcted heart, improving function and survival.

Proceedings of the National Academy of Sciences of the United States

of America, 98(18): 10344-10349.

Pan QH, Ben KL.1984. The review and prospect of primatology in

China. Zoological Research, 5(4S): 1-6. (in Chinese)

Pan XH, He B, Pang RQ, Tang LW, Li JL, Chen XG. 2006. Establishment and evaluation of the ischemic cerebral infarction model in monkey. Journal of Tropical Medicine, 6(4): 400-402. (in Chinese)

Pang W, Lü LB, Wang Y, Li G, Huang DT, Lei AH, Zhang GH, Zheng YT. 2013. Measurement and analysis of hematology and blood chemistry parameters in northern pig-tailed macaques (Macaca leonina). Zoological Research, 34(2): 89-96. (in Chinese)

Pérez-Otaño I, Herrero MT, Oset C, De Ceballos ML, Luquin MR, Obeso JA, Del Río J. 1991. Extensive loss of brain dopamine and serotonin induced by chronic administration of MPTP in the marmoset. Brain Research, 567(1): 127-132.

Perretta G. 2009. Non-human primate models in neuroscience research. Scandinavian Journal of Laboratory Animal Science, 36(1): 77-85.

Pound LD, Kievit P, Grove KL. 2014. The nonhuman primate as a

model for type 2 diabetes. Current Opinion in Endocrinology, Diabetes

and Obesity, 21(2): 89-94.

Qiao CF, Tian BL, Mai G, Wei LL, Jin X, Ren Y, Chen YN, Li HX, Li

YP, Wang L, Cheng JQ, Lu YR. 2009. Induction of diabetes in rhesus

monkeys and establishment of insulin administration strategy.

Transplantation Proceedings, 41(1): 413-417.

Qiao M, Zhao Q, Zhang H, Wang H, Xue L, Wei S. 2007. Isolating



with physical restraint low status female monkeys during luteal phase

might make an appropriate premenstrual depression syndrome model.

Journal of Affective Disorders, 102(1-3): 81-91.

Rijnbrand R, Yang Y, Beales L, Bodola F, Goettge K, Cohen L, Lanford

RE, Lemon SM, Martin A. 2005. A chimeric GB virus B with

5′nontranslated RNA sequence from hepatitis C virus causes hepatitis

in tamarins. Hepatology, 41(5): 986-994.

Rimmelzwaan GF, Baars M, van Amerongen G, van Beek R,

Osterhaus AD. 2001. A single dose of an ISCOM influenza vaccine

induces long-lasting protective immunity against homologous

challenge infection but fails to protect Cynomolgus macaques

against distant drift variants of influenza A (H3N2) viruses.

Vaccine, 20(1-2): 158-163.

Sellandi TM, Thakar AB, Baghel MS. 2012. Clinical study of tribulus

terrestris Linn. in oligozoospermia: a double blind study. Ayurveda,

33(3): 356-364.

Shen H. 2013. Precision gene editing paves way for transgenic

monkeys. Nature, 503(7474): 14-15.

Shen PQ, Yang SF. 2003. The current status and the future of laboratory

primates in China. Laboratory Animal Science and Management,

20(S1): 41-45. (in Chinese)

Sun XM, Chen Y, Li CH, Peng H, Lin SL, Dai JJ. 2006. Study on

establishment of oligozoospermia animal model in cynomolgus monkey.

Laboratory Animal Science and Management, 23(2): 22-25.

Sy C, Liu L, Ding Y. 2014. Ongoing progress and new developments in

the clinical approach to stroke and cerebrovascular disease-memos

from the 2014 Tiantan International Stroke Conference. Neurological

Research, 36(5): 389-390.

Tang XH, Cao YL, Yang ZX, Zhao FX. 2012. Reproductive traits of

polycystic ovary syndrome in female rhesus monkeys. Zoological

Research, 33(1): 37-42.

Tian CY, Ma YY. 2014. Involve both genetic and environmental factors

to build monkey models of mental disorders. Zoological Research,

35(3): 170-171.

Tian W, Zhang Y, Sun L, Wang ZM. 2010. Establishment and

evaluations of the spinal cord injury model. Chinese Journal of

Rehabilitation Theory and Practice, 16(3): 221-223. (in Chinese)

Tong ZQ, Han CS, Luo WH, Wang XH, Li H, Luo HJ, Zhou JN, Qi JS,

He RQ. 2012. Accumulated hippocampal formaldehyde induces age-

dependent memory decline. AGE, 35(3): 583-596.

Tong ZQ, Zhang JL, Luo WH, Wang WS, Li FX, Li H, Luo HJ, Lu J,

Zhou JN, Wan Y, He RQ. 2011. Urine formaldehyde level is inversely

correlated to mini mental state examination scores in senile dementia.

Neurobiology of Aging, 32(1): 31-41.

Van Der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S,

O'collins V, Macleod MR. 2010. Can animal models of disease reliably

inform human studies? PLoS Medicine, 7(3): e1000245.

Van Rompay KK. 2012. The use of nonhuman primate models of HIV

infection for the evaluation of antiviral strategies. AIDS Research and

Human Retroviruses, 28(1): 16-35.

Wadman M. 2011a. Animal rights: chimpanzee research on trial. Nature,

474(7351): 268-271.

Wadman M. 2011b. Chimp research under scrutiny. Nature, 480(7378):

424-425.

Wang JJ, Li W, Diao HL, Liu LZ, Tang DH, Yang LX, Dong ZH, Na

RX, Yu LC, Zhang Y. 2011a. Biological characteristics related to the

transmission of enterovirus 71 in neonatal rhesus monkeys. Journal of

Microbies and Infections, 6(3): 133-138. (in Chinese)

Wang W, Liu Q, Xu Y, Feng YF, Cong Z, Tong W, Jiang H, Yang GB,

Wei Q, Qin C. 2011b. Establishment of a Chinese-origin rhesus

macaque model of SHIV-KB9 virus infection. Chinese Journal of

Comparative Medicine, 21(2): 1-6. (in Chinese)

Wang YJ, Ye HH, Shao JS. 2004. Discussion of rhesus monkey model

of the spontaneous diabetes. Chinese Journal of Laboratory Animal

Science, 14(1): 13-15. (in Chinese)

Wang Y, Mo PZ, Tang ZJ, Xian QY, Huang ZX, Rao Y, Bao R, Guo M,

Wang X, Li XD, Huo WZ. 2012. Establishment and evaluation of a

Chinese rhesus model of tuberculosis. Chinese Journal of Tuberculosis

and Respiratory Diseases, 35(11): 843-848. (in Chinese)

Wu Z, Sun X, Sullivan SG, Detels R. 2006. HIV testing in China.

Science, 312(5779): 1475-1476.

Xia HJ, Chen CS. 2011. Progress of non-human primate animal models

of cancers. Zoological Research, 32(1): 70-80.

Xia HJ, Ma JP, Zhang GH, Han JB, Wang JH, Zheng YT. 2011. Effect

of plasma viremia on apoptosis and immunophenotype of dendritic

cells subsets in acute SIVmac239 infection of Chinese rhesus macaques.

PLoS One, 6(12): e29036.

Xia HJ, Zhang GH, Ma JP, Dai ZX, Li SY, Han J-B, Zheng YT. 2010.

Dendritic cell subsets dynamics and cytokine production in

SIVmac239-infected Chinese rhesus macaques. Retrovirology, 7: 102.

Xia HJ, Zhang GH, Wang RR, Zheng YT. 2009. The influence of age

and sex on the cell counts of peripheral blood leukocyte subpopulations

in Chinese rhesus macaques. Cellular and Molecular Immunology, 6(6):

433-440.

Xian QY, Tang ZJ, Li XD, Wang Y, Bao R, Guo M, Liu JY. 2010.

Application of fiberoptic bronchoscope in establishment of nonhuman

primate model for mycobacterium tuberculosis infection. Medical

Journal of Wuhan University, 31(6): 713-716. (in Chinese)

Xu CL, Chen YM, Xu ZY, Lu Q, Xu Q, Shen YP, Su JF, Long Y. 2009.

Establishment of cynomolgus monkey diabetic models. Journal of

Guangzhou University of Traditional Chinese Medicine, 26(1): 91-94.

(in Chinese)

Xu JH, Deng YX, Qui WJ, Zhou ZP, Zhang HY, Zeng YD. 2012. The

establishment and evaluation of middle cerebral artery occlusion model

in monkeys by interventional management. Journal of Interventional

Radiology, 21(7): 578-581. (in Chinese)

Xu L. 2011. Animal models of human diseases. Zoological Research,

32(1): 2-3. (in Chinese)

Xu L, Zhang Y, Liang B, Lü LB, Chen CS, Chen YB, Zhou JM, Yao

YG. 2013. Tree shrews under the spot light: emerging model of human

diseases. Zoological Research, 34(2): 59-69. (in Chinese)

Yan G, Zhang G, Fang X, Zhang Y, Li C, Ling F, Cooper DN, Li Q, Li Y,

van Gool AJ, Du H, Chen J, Chen R, Zhang P, Huang Z, Thompson JR,

Meng Y, Bai Y, Wang J, Zhuo M, Wang T, Huang Y, Wei L, Li J, Wang

464 ZHANG, et al.


Z, Hu H, Yang P, Le L, Stenson PD, Li B, Liu X, Ball EV, An N, Huang

Q, Zhang Y, Fan W, Zhang X, Li Y, Wang W, Katze MG, Su B, Nielsen

R, Yang H, Wang J, Wang X, Wang J. 2011. Genome sequencing and

comparison of two nonhuman primate animal models, the cynomolgus

and Chinese rhesus macaques. Nature Biotechnology, 29(11): 1019-

1023.

Yan T, Rizak JD, Yang SC, Li H, Huang BH, Ma YY, Hu XT. 2014.

Acute morphine treatments alleviate tremor in 1-methyl-4-phenyl-1, 2,

3, 6-tetrahydropyridine-treated monkeys. PLoS One, 9(2): e88404.

Yang MF, Miao JY, Rizak JD, Zhai RW, Wang ZB, Anwar TH, Li T,

Zheng N, Wu SH, Zheng YW, Fan XN, Yang JZ, Wang JH, Ma YY, Lü

LB, He RQ, Hu XT. 2014. Alzheimer's disease and methanol toxicity

(part 2): lessons from four rhesus macaques (Macaca mulatta)

chronically fed methanol. Journal of Alzheimers Disease, 41(4): 1131-

1147.

Yang P, Han P, Hou J, Zhang L, Song H, Xie Y, Chen Y, Xie H, Gao F,

Kang YJ. 2011. Electrocardiographic Characterization of rhesus

monkey model of ischemic myocardial infarction induced by left

anterior descending artery ligation. Cardiovascular Toxicology, 11(4):

365-372.

Yang ZG, Xu JZ, Ju JL, Xu YH, Qiu H, Yue MY, Meng JH. 2006.

China rhesus model of HEV gene type- infection. Ⅳ Jiangsu Medical

Journal, 32(2): 147-148. (in Chinese)

Yao N, Wang W, Cong Z, Chen T, Jin G, Tao Z, Chen ZW, Wei Q. 2011.

RT-SHIV infected and passaged in Chinese-origin rhesus monkeys.

Chinese Journal of Comparative Medicine, 21(4): 16-20. (in Chinese)

Yeh RW, Sidney S, Chandra M, Sorel M, Selby JV, Go AS. 2010.

Population trends in the incidence and outcomes of acute myocardial

infarction. New England Journal of Medicine, 362(23): 2155-2165.

You ZL, Ma HX, Chen JM, Wang RG, Liang XY, Liu WE. 2003.

Establishment of animal model with metrorrhagia induced by

endometritis in experimental rhesus monkey. Chinese Journal of


Zak O, Sande MA. 1999. Handbook of Animal Models of Infection.

New York: Academic Press.

Zha WG, Guo XM, Wang XX. 2006. The lateral hemisection of cervical

spinal cord injury model in rhesus monkey. Chinese Journal of

Neurosurgery, 22(5): 309-311. (in Chinese)

Zhang DM, Guo XH, Guo L, Zhang ZM, Zhang ZX, Hou YC, Zheng

CL, Zeng QY, Jiang JX. 1999. Establishment of animal model of

heroin-dependent macaca mulatta. Chinese Journal of Modern Applied

Pharmacy, 16(6): 12-14. (in Chinese)

Zhang L, Jiang CC, Xu B, Xu HJ, Li XJ, Huang KM. 2006.

Establishment of the parkinson’s disease rhesus monkey models under

the stereotactic unilateral substantia nigra lesion by 6-OHDA. Chinese

Journal of Experimental Surgery, 23(12): 1463-1465. (in Chinese)

Zhang J, Ge SX, Huang GY, Li SW, He ZQ, Wang YB, Zheng YJ, Gu Y,

Ng MH, Xia NS. 2004. Significance of serological markers and

virological marker for hepatitis E in rhesus monkey model. Chinese

Journal of Hepatology, 12(1): 7-10. (in Chinese)

Zhang J, Ge SX, Huang GY, Li SW, He ZQ, Wang YB, Zheng YJ,

Gu Y, Ng MH, Xia NS. 2003. Evaluation of antibody-based and

nucleic acid-based assays for diagnosis of hepatitis E virus

infection in a rhesus monkey model. Journal of Medical Virology,

71(4): 518-526.

Zhang J, Ye YQ, Wang Y, Mo PZ, Xian QY, Rao Y, Bao R, Dai M, Liu

JY, Guo M, Wang X, Huang ZX, Sun LH, Tang ZJ, Ho WZ. 2011a. M.

tuberculosis H37Rv infection of chinese rhesus macaques. Journal of

Neuroimmune Pharmacology, 6(3): 362-370.

Zhang WZ, Zhang ZJ, Liu XL, Rong JQ, Mao PY, Zhao JM, Mao YL.

1998. Study on the experimental model of hepatitis G of macaques.

Chinese Journal of Laboratory Animal Science, 8(3): 152-156. (in

Chinese)

Zhang XL, Pang W, Deng DY, Lü LB, Feng Y, Zheng YT. 2014.

Analysis of immunoglobulin, complements and CRP levels in serum of

captive northern pig-tailed macaques (Macaca leonina). Zoological

Research, 35(3): 196-203.

Zhang Y, Cui W, Liu L, Wang J, Zhao H, Liao Y, Na R, Dong C, Wang

L, Xie Z, Gao J, Cui P, Zhang X, Li Q. 2011b. Pathogenesis study of

enterovirus 71 infection in rhesus monkeys. Laboratory Investigation,

91(9): 1337-1350.

Zhang YC, Jin LS, Gao LH, Peng BL, He H, Ji F, Hao XF, Zhang XJ,

Tang XB, Rao JH, Liu XM. 2012. Study on the model of T2DM of

cynomolgus iduced by high-energy diet. Progress in Veterinary

Medicine, 33(8): 47-52. (in Chinese)

Zheng X. 2012. Monitoring of the Virus Antibodies During the Process

of SPF Non-human Primates Populations Establishment. Master Thesis,

Soochow University. (in Chinese)

Zhu H, Li Q, Feng M, Chen YX, Li H, Sun JJ, Zhao CH, Wang RZ,

Qin C. 2009. Establishment and evaluation on the intracerebral

hemorrhage model of cynomolgus macaques. Chinese Journal of


Zhu H, Li Q, Xu YF, Feng M, Li H, Sun JJ, Zhao CH, Wang RZ, Qin C.

2008. The Establishment of photochemical thrombosis animal model in

cynomolgus macaques. Chinese Journal of Comparative Medicine,

18(9): 32-34. (in Chinese)

Zhu Q, Xiao ZN, Sun XM, Hu M, Liu H, Hu ZL. 2013. Model

establishment of corneal endothelial injury by phacoemulsification in

rhesus monkeys. Recent Advances in Ophthalmology, 33(2): 110-112.

(in Chinese)



Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina)

Hong-Yi ZHENG1,2, Ming-Xu ZHANG1,3, Lin-Tao ZHANG1,4, Xiao-Liang ZHANG1,4, Wei PANG1, Long-Bao LYU1,5, Yong-Tang ZHENG1,2,3,5,*



2. School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230026, China

3. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming Yunnan 650204, China

4. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming Yunnan 650500, China

5. Kunming Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan 650223, China

Abstract: Pig-tailed macaques (Macaca nemistrina group) have been extensively used as non-human primate animal models for various human diseases in recent years, notably for AIDS research due to their sensitivity to HIV-1. Northern pig-tailed macaques (M. leonina) are distributed in China and other surrounding Southeast Asia countries. Although northern pig-tailed macaques have been bred on a large scale as experimental animals since 2012, the reference value of normal levels of leukocytes is not available. To obtain such information, 62 blood samples from male and female healthy northern pig-tailed macaques at different ages were collected. The normal range of major leukocyte subpopulations, such as T lymphocytes, B lymphocytes, natural killer (NK) cells, monocytes, and the expression levels of activation or differentiation related molecules (CD38, HLA-DR, CCR5, CD21, IgD, CD80 and CD86) on lymphocytes were analyzed by flow cytometry. The counts of B cells decreased with age, but those of CD8+ T cells and NK cells and the frequency of CD38+HLA-DR+CD4+ T cells were positively correlated with age. The counts of leukocyte subpopulations were higher in males than those in females except for CD4+ T cells. Males also showed higher expression levels of IgD and CD21 within B cells. This study provides basic data about the leukocyte subpopulations of northern pig-tailed macaques and compares this species with commonly used Chinese rhesus macaques (M. mulatta), which is meaningful for the biomedical application of northern pig-tailed macaques.

Keywords: Northern pig-tailed macaque; Flow cytometry; Leukocyte subpopulation; Age; Sex

Pig-tailed macaques (Macaca nemistrina group, PTM), rhesus macaques (M. mulatta, RM) and cynom-olgus macaques (M. fascicularis, CM), all belonging to the Cercopithecidae family of Old World monkeys, have become more widely used models for AIDS pathogenesis in recent years (Lei et al, 2013; Zhu et al, 2010). As their name suggests, PTM are characterized by the short, semi-erect ‘pig-like’ tail and the flat vertex. They diverged from RM and CM approximately 5 million years ago, while the divergence between RM and CM was generated at one another 2.4 million years later (Baroncelli et al, 2008).

PTM have been widely employed as animal models of sexually transmitted diseases due to the similarities

between the anatomic structure of the genital tract and the menstrual cycle with humans, the feature that they breed in all1seasons and have a relatively large body size. By contrast, it is difficult to perform colposcopic examinations and multiple biopsies on CM as they have much smaller vaginal canals and cervix diameters than Received: 08 May 2014; Accepted: 10 August 2014

Foundation items: This work was supported by the National Special

Science Research Program of China (2012CBA01305); the National

Natural Science Foundation of China (81172876, U0832601,

81273251, U1202228); the Knowledge Innovation Program of CAS

(KSCX2-EW-R-13) and the National Science and Technology Major

Project (2013ZX10001-002, 2012ZX10001-007) *Corresponding author, E-mail: [email protected]

466 ZHENG, et al.


PTM. The reproductive physiology of RM is also quite different from humans as their constant breeding season (Patton et al, 2009). More importantly, PTM have the overriding advantages on their abilities in acute human immunodeficiency virus-1 (HIV-1) infection and exceptional susceptibility to simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). In fact, PTM are the only reported species of Old World monkeys that could be infected by HIV-1 (Agy et al, 1992; Batten et al, 2006; Bosch et al, 2000).

Although they have been extensively applied in biomedical areas, such as cognitive neuroscience, pharm-acology and infectious etiology, PTM did not obtain a clear taxonomic status until Gippoliti et al (2001) sepa-rated the initial taxonomic three subspecies (nemestrina, leonina, pagensis) of the M. nemistrina group at the species level. Sunda pig-tailed macaques (M. nemestrina) are found in the southern half of the Malay Peninsula (only just extending into southernmost Thailand), Borneo, Sumatra and Bangka Island. Mentawai macaques (M. pagensis) are located in the Mentawai islands. Northern pig-tailed macaques (M. leonina) range from about N8° in Peninsular Thailand through Burma and Indochina into Bangladesh, India extending north as far as to the Brahmaputra, and the southernmost of Yunnan, China (Malaivijitnond et al, 2012).

Due to the lack of primate models that can be productively infected by HIV-1, especially given that HIV-1 infection is more efficiently restricted by tripartite motif protein 5α (TRIM5α), apolipoprotein B mRNA-editing enzyme and catalytic polypeptide-like 3 (APOBEC3) proteins of macaques than those of humans, the pathogenic mechanisms of HIV-1 have not been fully elucidated. The early block to HIV-1 infection in monkey cells was relieved by interference with TRIM5α expression (Stremlau et al, 2004). In our previous study, we for the first time reported that northern PTM do not express TRIM5α but rather TRIM5-CypA fusion gene, in which the B30.2/ SPRY domain of TRIM5α is substituted by retrotransposed CypA in the 3'-UTR, contributing to the invalidation of restriction to HIV-1 replication in these animals (Kuang et al, 2009). Recent studies showed that PTM rather than RM or CM are susceptive to siman-tropic HIV-1 (stHIV-1), in which the HIV-1NL4-3 vif gene was replaced by the vif genes from SIVmac or HIV-2ROD to suppress the antiretroviral activity of several APOBEC3 proteins (Hatziioannou et al, 2009; Thippeshappa et al, 2011). Thus northern PTM may is a

desirable animal model for AIDS studies. Wild northern PTM mainly distribute in southwe-

stern Yunnan, China and southeastern Tibet. Because of the scarcity of this primate species, they are under First Grade State Protection of China. In 2012, Kunming Primate Research Center, China, first introduced nort-hern PTM from Vietnam for large-scaled breeding for laboratory use. However, the limited knowledge of their immune systems has restricted their applications. In this study, we determined the phenotypic characteristics of leukocytes in northern PTM ranging from 2 to 11 years of age to provide detailed immunological data.

MATERIALS AND METHODS

Animals and sample collection Cage-bred northern PTM were maintained according

to the regulations approved by the American Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) at the Kunming Primate Research Center, Kunming Institute of Zoology, CAS. Ethyle-nediamine tetraacetic acid (EDTA) stabilized blood sam-ples were collected from male (n=15) and female (n=14) juveniles (mean age=2.4 years (2−3 years)), as well as male (n=17) and female (n=16) adults (mean age=6.1 years (5−11 years)) without apparent external symptoms (acute infections, trauma and severe diarrhea) for flow cytometric analysis. For comparison purposes, data from age-appropriate male (n=9) and female (n=8) juvenile (mean age=2.4 years (2−3 years)), as well as male (n=14) and female (n=12) adult (mean age=7.5 years (5−10 years)) Chinese rhesus macaques (ChRM) were also collected. Animals’ age and sex information is shown in Table 1. All procedures were performed under the guidance of the Ethics Committee of Kunming Institute of Zoology.

Antibodies

Mouse anti-human CD molecule monoclonal and polyclonal antibodies that cross-reacted with PTM were used according to standard procedures and appropriate concentrations in this study. Anti-CD159a PE (clone Z199) mAb was obtained from Beckman Coulter. Anti-CD38 FITC (clone AT-1) mAb was obtained from STE-MCELL. Streptomycin-PE-Cy7 was obtained from Biol-egend. Anti-IgD biotin pAb used by conjunction with streptomycin-PE-Cy7 was obtained from Southern Biot-ech. Anti-CD14 APC (clone M5E2), anti-CD20 PerCP-Cy5. 5(clone 2H7), anti-CD21 APC (clone B-ly4), anti-CD3

Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina) 467


Table 1 Study group of northern pig-tailed macaques and Chinese rhesus macaques

Northern pig-tailed macaques Chinese rhesus macaques

Males Females Males Females Age groups (years)

n Age (years) n Age (years) n Age (years) n Age (years)

Juvenile (2−3) 15 2.3±0.5 14 2.5±0.5 9 2.3±0.5 8 2.5±0.5

Adult (4−11) 17 6.6±1.6 16 5.4±1.7 14 7.0±1.6 12 8.1±1.5

Total 32 4.6±2.5 30 4.0±2.0 23 5.2±2.6 20 5.9±3.0

APC-Cy7 (clone SP34-2), anti-CD4-PerCP-Cy5.5/ FITC (clone L200), anti-CD8α PE-Cy7 (clone RPA-T8), anti-CD80 PE (clone HB15e), anti-CD86 PE (clone FUN-1), anti-CCR5 PE (clone 3A9) and anti-HLA-DR APC (clone L243) mAbs were all obtained from BD Pharmigen.

Absolute quantification of major leukocyte subpopu-lations

The methods of direct cell surface staining for whole blood and absolute number analysis were as described previously (Xia et al, 2009). Briefly, 50 μL of whole blood samples were added in TruCount tube (BD Bio-sciences) and incubated with fluorochrome-conjugated antibodies of CD3, CD4, CD8α, CD20, CD14 and CD159a mAbs for 30 min on ice. Erythrocytes were lysed with FACS lysing solution (BD Biosciences), and the samples were analyzed with BD FACSVerse cytometer directly. The absolute numbers of T cells (CD3+CD20-), CD4+ T cells (CD4+CD8a-CD3+), CD8+ T cells (CD4-CD8a+CD3+), B cells (CD3-CD20+), NK cells (CD3-CD8a+CD159a+) and monocytes (CD3-CD20-

CD14+) were calculated using the following formula: Cell concentration (cell numbers/μL)=(events in gated region (n)×total number of TruCount beads (n))/(number of acquired beads (n)×sample volume (μL)) (Figure 1).

Phenotype analysis by flow cytometry

As shown in Figure 1, to determine CCR5+ T cells, activated (CD38+HLA-DR+) T cells, activated (CD80/ CD86+) B cells, resting (CD21+) B cells and IgD/IgM secreting (IgD+) B cells, 100 μL of fresh whole blood used for each analysis were lysed with FACS lysing solution for 10 min at room temperature, followed by washing and resuspension with Dulbecco’s phosphate-buffered saline (DPBS) with 2% of new-born calf serum and 0.09% sodium azide (staining buffer). The suspe-nding leukocytes were then stained with the relevant directly conjugated mAbs for 30 min on ice and fixed using PBS containing 4% paraformaldehyde.

Flow cytometric acquisition was performed on the BD FACSVerse cytometer driven by the FACSuite software (version 1.0.3; BD). The acquired cell number was at least 100 000 CD3+ T cells or 50 000 CD20+ B cells. Analysis of the acquired data was performed using FlowJo software (version 7.6.1; TreeStar).

Statistical analysis

Statistical differences between two groups were determined by the nonparametric Mann-Whitney U rank sum test. The Spearman’s test was performed for corre-lation analysis. Two-tailed P<0.05 was considered statis-tically significant. Data were presented as mean±SD. All statistical analyses were performed via GraphPad Prism software (version 5.01; GraphPad Software).

RESULTS

The absolute number of leukocyte subpopulations in peripheral blood of northern PTM

The absolute number of T cells, CD4+ T cells, CD8+ T cells, B cells, monocytes and NK cells in different groups are listed in Table 2. Numbers of B cells were negatively correlated with age (r=−0.426, P<0.001). CD8+ T cell counts (r=0.336, P=0.008) and NK cell cou-nts (r=0.329, P=0.01) were slightly increased with the increasing of age. Adult group (1 117±548/μL) had dramatically less B cells than juvenile group (1 782± 741/μL, P<0.001), however, no significant cell count differences of CD8+ T cells and NK cells were found between adult and juvenile groups.

In adult groups, compared with females, males showed higher counts of T cells (male 3 544±1 341/μL, female 2 875±1 192/μL; P=0.032), CD8+ T cells (male 1310±56/μL, female 1 046±533/μL; P=0.026), B cells (male 1 594±621/μL, female 1 251±789/μL; P=0.016;) and NK cells (male 701±617/μL, female 378±303/μL; P=0.004). However, no significant differences of age or sex were found in their counts of CD4+ T cells and monocytes.

468 ZHENG, et al.


Figure 1 Gating strategies for flow cytometric analysis A: Gating strategies for T cells, B cells, NK cells and monocytes. CD14/side-scatter scattergram used for gating monocytes, TruCount beads and remaining

lymphocytes. CD3/CD20 dot polt gated on lymphocytes used to count T and B cells. T cells were further divided into CD4+ and CD8+ T cells. NK cells were

defined as CD3-CD8a+CD159a+ phenotype gating on lymphocytes. B: The expression levels of CCR5 were analyzed in each T cell subset. C: CD38+HLA-DR+

cells in each T cell subset were defined as activated cells. D: The expression levels of CD21, CD80/CD86 and IgD were analyzed in B cells.

Table 2 Cell counts (cell numbers/µL) of leukocyte subpopulations in different age and sex groups

Age (years) Sex T cells CD4+ T cells CD8+ T cells B cells NK cells Monocytes

Juvenile M 3 433±1 469 1 886±874 1 209±533 1 751±698 548±411 442±319

(2−3) F 3 524±1 322 1 933±701 1 295±633 1 815±809 421±368 577±330

Adult M 3 642±1 254 1 941±662 1 399±597 1 455±526 836±741 636±412

(4−11) F 2 306±701 1 303±491 828±304 757±281 340±240 414±235

Total M 3 544±1 341 1 915±756 1 310±567 1 594±621 701±617 545±378

F 2 875±1 192 1 597±668 1 046±533 1 251±789 378±303 490±290

All 3 220±1 305 1 761±727 1 182±562 1 428±722 563±510 518±337

Range 1 141−6 635 564−3 794 321−3 001 378−3 282 66−3178 154−1 844

Expressions of activation or differentiation related molecules on lymphocytes of northern PTM

The expression values of activation or differentiat-ion related markers on each lymphocyte subset in diffe-rent groups were shown in Table 3. CD38+HLA-DR+ subset

increased progressively with the increaseing of age in CD4+ T cells (r=0.256, P=0.045), and more CD38+HLA-DR+ sub-set within CD4+ T cells (juvenile 1.3±0.4%, adult 2.0±0.9%; P=0.006) as well as CD8+ T cells (juvenile 4.1±1.8%, adult 5.0±1.9%; P=0.02) were observed in adult groups.



Figure 2 Comparisons between northern pig-tailed macaques and Chinese rhesus macaques

Table 3 Expression levels (%) of activation or differentiation associated markers in different groups

Age (years)

Sex CD38+DR+

CD4+ T cells CD38+DR+

CD8+ T cells CCR5+

CD4+ T cellsCCR5+

CD8+ T cells CD21+ B cells

IgD+ B cells

CD80/CD86+ B cells

Juvenile M 1.25±0.31 3.85±1.46 3.53±2.15 11.14±6.36 67.99±12.76 72.75±12.93 44.79±12.51

(2−3) F 1.32±0.53 4.32±2.21 3.48±1.98 11.85±4.77 64.67±11.53 65.71±13.97 48.19±9.48

Adult M 2.27±0.92 5.49±2.18 4.07±1.45 12.02±3.73 66.81±12.05 69.97±11.94 41.78±11.11

(4−11) F 1.63±0.81 4.58±1.50 4.01±2.20 14.35±7.19 60.03±14.13 56.12±13.89 60.14±17.42

Total M 1.79±0.86 4.72±2.03 3.82±1.81 11.61±5.07 67.37±12.20 71.27±12.29 43.19±11.69

F 1.49±0.70 4.46±1.84 3.76±2.08 13.19±6.21 62.19±12.98 60.60±14.53 54.57±15.30

All 1.64±0.79 4.59±1.93 3.79±1.93 12.37±5.66 64.86±12.75 66.11±14.35 48.70±14.61

Range 0.49−3.84 1.48−10.70 1.00−8.79 3.42−28.80 33.80−90.50 35.80−93.10 17.00−80.10

Males showed higher frequency of IgD+ B cells

(male 71.3±12.3%, female 60.6±14.5%; P=0.006) but lower frequency of CD80/86+ B cells (male 43.2±11.7%, female 54.6±15.3%; P=0.004) than females. However, no statistically significant age and gender differences in the frequencies of CCR5+CD4+ T cells, CCR5+CD8+ T cells and CD21+B cells were found.

Comparisons of lymphocyte subpopulations between northern PTM and ChRM

As shown in Figure 2, a flow cytometric analysis was performed to study the differences in lymphocyte subpopulations between PTM and ChRM. Compared with ChRM, northern PTM exhibited less T cells (north-

ern PTM 3 220±1 305/μL, ChRM 3 998±1 561/μL; P=0.006), less CD8+ T cells (northern PTM 1 182±562/μL, ChRM 1 634±921/μL; P=0.009), whereas higher freque-ncies of both CD4+ T cells (northern PTM 55.1±7.3%, ChRM 47.3±10.1%; P=0.0001) and CD21+ B cells (northern PTM 69.4±12.8% compared to 54.2±18.8%; P=0.001) as well as increased CD4/CD8 ratio (northern PTM 1.62±0.61, ChRM 1.38±0.72; P=0.016). Further analysis demonstrated that male northern PTM had less T cells (northern PTM 3 544±1 341/μL, ChRM 4498±1458/μL; P=0.03), less CD8+ T cells (northern PTM 1 310±567/μL, ChRM 1 985±991/μL; P=0.01; northern PTM 36.8±6.9%, ChRM 44.0±13.8%; P=0.013), but higher frequency of CD4+ T cells (northern PTM

470 ZHENG, et al.


54.3±7.2%, ChRM 45.9±11.0%; P=0.003) and CD4/CD8 ratio (northern PTM 1.58±0.65, ChRM 1.25±0.75; P=0.01) than male ChRM. Whereas, females showed lower frequency of CD21- B cells (northern PTM 37.8±13.0%, ChRM 54.6±15.4%; P<0.0001) compared to female ChRM.

DISCUSSION

PTM group (M. nemestrina, M. leonine and M. pagensis) have served an important role in the field of biomedicine, notably in HIV/AIDS research, in recent years (Lei et al, 2013). However, no detailed taxonomic description of these animals was found in most studies that use the collectively called “pig-tailed macaques” (Agy et al, 1992; Batten et al, 2006; Bosch et al, 2000). We previously demonstrated that northern PTM express TRIM5-CypA fusion gene, resulting in its sensitivity to HIV-1 and improving the application value of these new laboratory animals in HIV/AIDS research (Kuang et al, 2009). We also provided the reference values of immu-noglobulins, complements, C reactive protein (CRP), hematological and biochemical indexes of north PTM, and elucidated that gender, age and weight can influence these indexes (Pang et al, 2013; Zhang et al, 2014). However, the lack of the immune system characteristics of northern PTM may have restricted its application as AIDS animal models. Here we evaluated the absolute number and the expression of activation or differen-tiation related markers of major lymphocyte subpop-ulations in 62 male and female northern PTMs ranging from 2 to 11 years of age, and compared the lymphocyte subpopulations of PTM with those of the most widely used ChRM.

Serving as a major risk factor of morbidity and mortality for many infections caused by various patho-gens, age influences the immune system in both human and non-human primates (NHP) (High, 2004). Wiener et al (1990) found that the absolute number of both CD19+ B cells and CD3+ T cells decreased while the absolute number of CD4+ and CD8+ T cells and the CD4/CD8 ratio were well maintained with increasing age. The following Swedish OCTO immune longitudinal study showed decreased CD4 subset, increased CD8 subset, and a lower CD19+ B cell percentages in aged population (Ferguson et al, 1995). Fagnoni et al (1996) reported a decrease in the absolute number of both CD4+ and CD8+ T cells in people older than 60 years. However, no age

correlations of the absolute or relative cell numbers of lymphocyte subpopulations were found in recent study (Klose et al, 2007). Although the results were contr-adictory in these studies, a widely accepted theory suggests that aging is characterized by the decline in B cell numbers and an accumulation of CD8+ T cells rather than a loss of CD4+ T cells in peripheral blood (Frasca et al, 2008; Koch et al, 2007). On the other hand, Studies have suggested that aging is associated with numerous alterations in innate immunity which is the first line of defense against pathogens and plays a key role in regulating the responses of adaptive immunity (Shaw et al, 2010). Positive correlations were observed between age and CD16+CD56+ NK cell numbers, and it is generally accepted that the CD56dim peripheral NK cell population expands with age (Jiao et al, 2009; Mahbub et al, 2011). Tollerud et al (1989) evaluated the influence of age, race, and gender on the immune system, and did not observe age related effects for CD14+ cells. However, other recent research reported that the number of monocytes was significantly higher in the elderly than in the young group (Della Bella et al, 2007).

Because they share greater genetic and physiological similarities with humans than rodent models, NHP has been used for biomedical research for several decades. Most of our understanding of the immune system in Old World monkeys comes from studies utilizing rhesus macaques (Messaoudi et al, 2011). However, only few reports focused on the age-related changes in the immune system of PTM, especially northern PTM. Asquith et al (2012) measured age-related changes in T cell homeostasis in Indian rhesus macaques (InRM) ranging from 1 to 30 years old, and indicated that the frequency of CD8+ T cells expands with age while the frequency of CD4+ T cells remains stable, whereas age-related changes of the absolute number of both CD4+ and CD8+ T cells were not found in InRM (Didier et al, 2012). The CD4/CD8 ratio in juvenile InRM is approximately 2, and starts to decline after the first 2 months of life, whereas no further decline is detected between 5 and 7 years of age (Dykhuizen et al, 2000). Similarly, B cells in RM are identified based on the expression of CD20 and HLA-DR, and show a sharp decrease during aging (Didier et al, 2012). Early studies indicated that the number of circulating CD56+CD16+ NK cells in RM reduces with age (Coe & Ershler, 2001). However, highly different from humans, NK cells in peripheral blood of macaques express high levels of



CD8α and almost all of them express NKG2A, but do not express CD56, which make them a CD3-CD8α+

NKG2A+ phenotype (Hong et al, 2013). A recent study of rhesus macaques ranging from 2 to 24 years old did not find any correlation between the circulating NK cells and age (Didier et al, 2012). Though the frequency of circulating monocytes increases in InRM older than 5 years, the numbers of these cells reduces with aging (Asquith et al, 2012).

It has been reported that CD38+HLA-DR+ subset within CD8+ T cells has a strong positive correlation with HIV-1 disease progression and CD38+HLA-DR+ CD4+ T cells are highly susceptible to infection with R5-tropic virus because of elevated CCR5 expression (Kestens et al, 1992; Meditz et al, 2011). CCR5 plays an essential role in HIV pathogenesis as a main co-receptor responsible for viral transmission. CCR5+CD4+ T cells are the target of R5-tropic virus and peripheral blood CCR5+CD8+ T cells accumulate during HIV infection (Brenchley et al, 2004). In addition, a loss in the expression of CD21 and IgD, and increased levels of activation markers, including CD80 and CD86, on circul-ating B cells are suggested be linked to HIV viremia (Moir & Fauci, 2008). Klatt et al (2012) demonstrated that higher pre-infection levels of immune activation than RM may contribute to rapid disease progression in PTM. Thus it is also necessary to examine these in both healthy humans and laboratory animals. The frequency of CCR5+ cells within CD4+ T cells in northern PTM (3.79±1.93%) was the lowest compared to Sunda PTM (14.54±6.3%) and RM (7.76±4.8%) (Klatt et al, 2012). The frequency of CD38+HLA-DR+ cells was 4.4±2.5% for CD4+ T cells and 14.5±7.0% for CD8+ T cells in healthy human as compared with 1.64±0.79% and 4.59±1.93% for CD4+ and CD8+ T cells, respectively, from northern PTM and 3.03±2.84% for CD8+ T cells from rhesus macaques (Brignolo et al, 2004; Onlamoon et al, 2005). Elevated levels of CD21- B cells are believed to account for the poor proliferative responses of B cells. This population is significantly expanded in most patients with systemic lupus erythematosus (SLE) and HIV-1 and its normal level is approximately 15% in healthy people, 45% in CM, 46% in RM and 36% in northern PTM (Das et al, 2011; Kling et al, 2011; Zandieh et al, 2013). The B cells in the peripheral blood of healthy people have an average 75% IgD+ subset, while northern PTM have lower frequency (66%) (Klein et al, 1998).

ChRM infected with SIV commonly have lower plasma viral loads and develop AIDS more slowly than InRM, whereas PTM are susceptible to many lentiviral infections, including infection with SIV, HIV-1, HIV-2, SHIV and stHIV-1, and their progress to AIDS after SIV infection are also more rapid than RM (Ling et al, 2002). A comparison of lymphocytes between ChRM and northern PTM may provide clues to explaining this phenomenon. Previous studies demonstrated that ChRM experience significant age-related changes in leukocyte subpopulations, including decreased lymphocyte numbers, CD4+ T cell frequency and CD4/CD8 ratio, increased CD8+ T cell frequency, but stable monocytes (Xia et al, 2009; Zheng et al, 2014). Similarly, northern PTM showed increased numbers of NK cells and CD8+ T cells, reduced B cells, but stable CD4+ T cells and monocytes in our study. In addition, northern PTM also showed higher CD4+ T cell frequency but fewer CD8+ T cells and CD21- B cells than ChRM.

The sex effects on circulating lymphocytes have been reported in both humans and NHP. Uppal et al (2003) demonstrated that women have a higher number and frequency of CD4+ T cells, whereas men have a higher frequency of CD8+ T cells. However, no differences in circulating B cells, NK cells and CD14+ cells were found between men and women (Choong et al, 1995; Tollerud et al, 1989). However, the influence of sex on the leukocytes in macaques is less clear. Tryphonas et al (1996) revealed significant gender differences in the CD4+ T cell percentage (females> males) and CD4/CD8 ratio (females>males) of infant CM. Recent studies on circulating lymphocytes in InRM did not find differences between males and females (Asquith et al, 2012; Didier et al, 2012). However, our previous study revealed that female ChRM have higher counts of CD4+ T cells, CD8+ T cells, B cells and NK cells than males (Xia et al, 2009). In this study, northern PTM also showed numerous gender differences in leukocytes and activation markers, and males may have stronger immune response due to their higher levels of CD8+ T cells, B cells and NK cells than females. Howe-ver, further work is required to determine the character-istics of immune function between males and females.

In summary, the present study not only provided data on the immunological characteristics of the leukocyte subpopulation, but also demonstrated age and sex effects on those in northern PTM. Moreover, the comparison of northern PTM with ChRM provided clues

472 ZHENG, et al.


to understanding the big differences between these two species when infected with HIV-1 or SIV.

Acknowledgments: We thank Mr. Zhen-Fei Hu,

Gui Li and Dong-Ti Huang of Kunming Primate Research Center for their assistance with the exp-eriments.

References

Agy MB, Frumkin LR, Corey L, Coombs RW, Wolinsky SM, Koehler J, Morton WR, Katze MG. 1992. Infection of Macaca nemestrina by human immunodeficiency virus type-1. Science, 257(5066): 103-106.

Asquith M, Haberthur K, Brown M, Engelmann F, Murphy A, Al-Mahdi Z, Messaoudi I. 2012. Age-dependent changes in innate immune phenotype and function in rhesus macaques (Macaca mulatta). Pathobiology of Aging & Age Related Diseases, 2.

Baroncelli S, Negri D R, Michelini Z, Cara A. 2008. Macaca mulatta, fascicularis and nemestrina in AIDS vaccine development. Expert Review of Vaccines, 7(9): 1419-1434.

Batten CJ, De Rose R, Wilson KM, Agy MB, Chea S, Stratov I, Montefiori DC, Kent SJ. 2006. Comparative evaluation of simian, simian-human, and human immunodeficiency virus infections in the pigtail macaque (Macaca nemestrina) model. AIDS Research and Human Retroviruses, 22(6): 580-588.

Bosch ML, Schmidt A, Chen J, Florey MJ, Agy M, Morton WR. 2000. Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina). Journal of Medical Primatology, 29(3-4): 107-113.

Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, Nguyen PL, Khoruts A, Larson M, Haase AT, Douek DC. 2004. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. Journal of Experimental Medicine, 200(6): 749-759.

Brignolo L, Spinner A, Yee JL, Lerche NW. 2004. Subsets of T cells in healthy rhesus macaques (Macaca mulatta) infected with simian T-lymphotropic virus type 1. Comparative Medicine, 54(3): 271-274.

Choong ML, Ton SH, Cheong SK. 1995. Influence of race, age and sex on the lymphocyte subsets in peripheral blood of healthy Malaysian adults. Annals of Clinical Biochemistry, 32(Pt 6): 532-539.

Coe CL, Ershler WB. 2001. Intrinsic and environmental influences on immune senescence in the aged monkey. Physiology & Behavior, 73(3): 379-384.

Das A, Veazey RS, Wang XL, Lackner AA, Xu HB, Pahar B. 2011. Simian immunodeficiency virus infection in rhesus macaques induces selective tissue specific B cell defects in double positive CD21+CD27+ memory B cells. Clinical Immunology, 140(3): 223-228.

Della Bella S, Bierti L, Presicce P, Arienti R, Valenti M, Saresella M, Vergani C, Villa ML. 2007. Peripheral blood dendritic cells and monocytes are differently regulated in the elderly. Clinical Immunology, 122(2): 220-228.

Didier ES, Sugimoto C, Bowers LC, Khan IA, Kuroda MJ. 2012. Immune correlates of aging in outdoor-housed captive rhesus macaques (Macaca mulatta). Immunity & Ageing, 9(1): 25.

Dykhuizen M, Ceman J, Mitchen J, Zayas M, MacDougall A, Helgeland J, Rakasz E, Pauza CD. 2000. Importance of the CD3

marker for evaluating changes in rhesus macaque CD4/CD8 T-cell ratios. Cytometry, 40(1): 69-75.

Fagnoni FF, Vescovini R, Mazzola M, Bologna G, Nigro E, Lavagetto G, Franceschi C, Passeri M, Sansoni P. 1996. Expansion of cytotoxic CD8+ CD28- T cells in healthy ageing people, including centenarians. Immunology, 88(4): 501-507.

Ferguson FG, Wikby A, Maxson P, Olsson J, Johansson B. 1995.

Immune parameters in a longitudinal study of a very old population of

Swedish people: a comparison between survivors and nonsurvivors.

The Journals of Gerontology. Series A: Biological Sciences and

Medical Sciences, 50(6): B378-B382.

Frasca D, Landin AM, Riley RL, Blomberg BB. 2008. Mechanisms for

decreased function of B cells in aged mice and humans. The Journal of

Immunology, 180(5): 2741-2746.

Gippoliti S. 2001. Notes on the taxonomy of Macaca nemestrina

leonina Blyth, 1863 (Primates: Cercopithecidae). Hystrix - Italian

Journal of Mammalogy, 12(1): 51-54.

Hatziioannou T, Ambrose Z, Chung NPY, Piatak M Jr, Yuan F, Trubey

CM, Coalter V, Kiser R, Schneider D, Smedley J, Pung R, Gathuka M,

Estes JD, Veazey RS, KewalRamani VN, Lifson JD, Bieniasz PD. 2009.

A macaque model of HIV-1 infection. Proceedings of the National

Academy of Sciences of the United States of America, 106(11): 4425-

4429.

High KP. 2004. Infection as a cause of age-related morbidity and

mortality. Ageing Research Reviews, 3(1): 1-14.

Hong HS, Rajakumar PA, Billingsley JM, Reeves RK, Johnson RP.

2013. No monkey business: why studying NK cells in non-human

primates pays off. Frontiers in Immunology, 4: 32.

Jiao Y, Qiu ZF, Xie J, Li DJ, Li TS. 2009. Reference ranges and age-

related changes of peripheral blood lymphocyte subsets in Chinese

healthy adults. Science in China. Series C: Life Sciences, 52(7): 643-

650.

Kestens L, Vanham G, Gigase P, Young G, Hannet I, Vanlangendonck F,

Hulstaert F, Bach BA. 1992. Expression of activation antigens, HLA-

DR and CD38, on CD8 lymphocytes during HIV-1 infection. AIDS,

6(8): 793-797.

Klatt NR, Canary LA, Vanderford TH, Vinton CL, Engram JC,

Dunham RM, Cronise HE, Swerczek JM, Lafont BAP, Picker LJ,

Silvestri G, Brenchley JM. 2012. Dynamics of simian immuno-

deficiency virus SIVmac239 infection in pigtail macaques. Journal of

Virology, 86(2): 1203-1213.

Klein U, Rajewsky K, Kuppers R. 1998. Human immunoglobulin (Ig)

M+IgD+ peripheral blood B cells expressing the CD27 cell surface

antigen carry somatically mutated variable region genes: CD27 as a

general marker for somatically mutated (memory) B cells. Journal of

Experimental Medicine, 188(9): 1679-1689.



Kling HM, Shipley TW, Norris KA. 2011. Alterations in peripheral

blood B-cell populations in SHIV89.6P-infected macaques (Macacca

fascicularis). Comparative Medicine, 61(3): 269-277.

Klose N, Coulibaly B, Tebit DM, Nauwelaers F, Spengler HP, Kynast-Wolf G, Kouyaté B, Kräusslich HG, Böhler T. 2007. Immunohematological reference values for healthy adults in Burkina Faso. Clinical and Vaccine Immunology, 14(6): 782-784.

Koch S, Larbi A, Ozcelik D, Solana R, Gouttefangeas C, Attig S, Wikby A, Strindhall J, Franceschi C, Pawelec G. 2007. Cytomegalovirus infection: a driving force in human T cell immunosenescence. Annals of the New York Academy of Sciences, 1114: 23-35.

Kuang YQ, Tang X, Liu FL, Jiang XL, Zhang YP, Gao GX, Zheng YT. 2009. Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina), a primate species susceptible to Human Immunodeficiency Virus type 1 infection. Retrovirology, 6: 58.

Lei AH, Pang W, Zhang GH, Zheng YT. 2013. Use and research of pigtailed macaques in nonhuman primate HIV/AIDS models. Zoological Research, 34(2): 77-88. (in Chinese)

Ling BH, Veazey RS, Luckay A, Penedo C, Xu KY, Lifson JD, Marx PA. 2002. SIV(mac) pathogenesis in rhesus macaques of Chinese and Indian origin compared with primary HIV infections in humans. AIDS, 16(11): 1489-1496.

Mahbub S, Brubaker AL, Kovacs EJ. 2011. Aging of the innate immune system: an update. Current Immunology Reviews, 7(1): 104-115.

Malaivijitnond S, Arsaithamkul V, Tanaka H, Pomchote P, Jaroenporn S, Suryobroto B, Hamada Y. 2012. Boundary zone between northern and southern pig-tailed macaques and their morphological differences. Primates, 53(4): 377-389.

Meditz AL, Haas MK, Folkvord JM, Melander K, Young R, McCarter M, MaWhinney S, Campbell TB, Lie Y, Coakley E, Levy DN, Connick E. 2011. HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo. Journal of Virology, 85(19): 10189-10200.

Messaoudi I, Estep R, Robinson B, Wong SW. 2011. Nonhuman primate models of human immunology. Antioxidants and Redox Signaling, 14(2): 261-273.

Moir S, Fauci AS. 2008. Pathogenic mechanisms of B-lymphocyte dysfunction in HIV disease. Journal of Allergy and Clinical Immunology, 122(1): 12-19.

Onlamoon N, Tabprasit S, Suwanagool S, Louisirirotchanakul S, Ansari AA, Pattanapanyasat K. 2005. Studies on the potential use of CD38 expression as a marker for the efficacy of anti-retroviral therapy in HIV-1-infected patients in Thailand. Virology, 341(2): 238-247.

Pang W, Lü LB, Wang Y, Li G, Huang DT, Lei AH, Zhang GH, Zheng YT. 2013. Measurement and analysis of hematology and blood chemistry parameters in northern pig-tailed macaques (Macaca leonina). Zoological Research, 34(2): 89-96. (in Chinese)

Patton DL, Sweeney YT, Paul KJ. 2009. A summary of preclinical topical microbicide rectal safety and efficacy evaluations in a pigtailed macaque model. Sexually Transmitted Diseases, 36(6): 350-356.

Shaw AC, Joshi S, Greenwood H, Panda A, Lord JM. 2010. Aging of the innate immune system. Current Opinion in Immunology, 22(4): 507-513.

Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. 2004. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature, 427(6977): 848-853.

Thippeshappa R, Polacino P, Yu Kimata MT, Siwak EB, Anderson D, Wang WM, Sherwood L, Arora R, Wen M, Zhou P, Hu SL, Kimata JT. 2011. Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1. Journal of Virology, 85(8): 3767-3779.

Tollerud DJ, Clark JW, Brown LM, Neuland CY, Pankiw-Trost LK, Blattner WA, Hoover RN. 1989. The influence of age, race, and gender on peripheral blood mononuclear-cell subsets in healthy nonsmokers. Journal of Clinical Immunology, 9(3): 214-222.

Tryphonas H, Lacroix F, Hayward S, Izaguirre C, Parenteau M, Fournier J. 1996. Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis. Journal of Medical Primatology, 25(2): 89-105.

Uppal SS, Verma S, Dhot PS. 2003. Normal values of CD4 and CD8 lymphocyte subsets in healthy indian adults and the effects of sex, age, ethnicity, and smoking. Cytometry. Part B: Clinical Cytometry, 52(1): 32-36.

Wiener D, Shah S, Malone J, Lowell N, Lowitt S, Rowlands DT Jr. 1990. Multiparametric analysis of peripheral blood in the normal pediatric population by flow cytometry. Journal of Clinical Laboratory Analysis, 4(3): 175-179.

Xia HJ, Zhang GH, Wang RR, Zheng YT. 2009. The influence of age and sex on the cell counts of peripheral blood leukocyte subpopulations in Chinese rhesus macaques. Cellular & Molecular Immunology, 6(6): 433-440.

Zandieh A, Izad M, Fakhri M, Amirifard H, Khazaeipour Z, Harirchian MH. 2013. Cytometric profiling in various clinical forms of multiple sclerosis with respect to CD21+, CD32+, and CD35+ B and T cells. Translational Neurodegeneration, 2(1): 14.

Zhang XL, Pang W, Deng DY, Lü LB, Feng Y, Wang Y, Zheng YT. 2014. Analysis of immunoglobulin, complements and CRP levels in serum of captive northern pig-tailed macaques (Macaca leonina). Zoological Research, 35(3): 196-203.

Zheng HY, Zhang MX, Pang W, Zheng YT. 2014. Aged Chinese rhesus macaques suffer severe phenotypic T- and B-cell aging accompanied with sex differences. Experimental Gerontology, 55: 113-119.

Zhu L, Zhang GH, Zheng YT. 2010. Application studies of animal models in evaluating safety and efficacy of HIV-1 microbicides. Zoological Research, 31(1): 66-76. (in Chinese)



Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan

Jin-Fa LI1,#, Yu-Chao HE2,#, Zhi-Pang HUANG1, Shuang-Jin WANG1, Zuo-Fu XIANG3, Juan-Jun ZHAO4, Wen XIAO4,*, Liang-Wei CUI1,*

1. Key Laboratory of Forest Disaster Warning and Control in Yunnan Province, Southwest Forestry University, Kunming, Yunnan 650224, China

2. Bureau of Yunling Provincial Nature Reserve, Lanping, Yunnan 671400, China

3. College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, Hunan 410004, China

4. Institute of Eastern-Himalaya Biodiversity Research, Dali University, Dali, Yunnan 671003, China

Abstract: Seasonal variation in environmental factors is vital to the regulation of seasonal reproduction in primates. Consequently,

long-term systematic data is necessary to clarify the birth seasonality and pattern of primates in highly seasonal environments. This

study indicated that black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha exhibited strict birth seasonality with a

pulse model. Infants were born with a certain degree of synchronization. Birth distribution showed three birth peaks, and the birth

pattern showed a “V” style in even-numbered years and a gradual increase in odd-numbered years. The beginning date, end date and

median birth date were earlier in even-numbered years than those in odd-numbered years. The higher latitude of their habitats, earlier

birth date, shorter birth period, fewer birth peaks and stronger birth synchrony might be adaptations for strongly seasonal variation in

climate and food resources. After the summer solstice when daylight length began to gradually shorten, R. bieti at Mt. Lasha started

to breed during the period with the highest environmental temperature and food availability, which implied that photoperiod may be

the proximate factor triggering the onset of estrus and mating. It appears that R. bieti coincided conception and mid-lactation with the

peak in staple foods, and weaning with the peak in high quality of foods. Thus, food availability was the ultimate factor regulating

reproductive seasonality, and photoperiod was the proximate factor fine-turning the coordination between seasonal breeding and food

availability.

Keywords: Rhinopithecus bieti; Birth seasonality; Birth pattern; Birth synchrony

In wild environments, birth seasonality exists in many animals. Primates show reproduction continuum from seasonality to un-seasonality like other mammals (Brockman & van Schaik, 2005). The typical reproduc-tion patterns of primates include seasonality (births within a specific period), peaks (birth peaks in certain months) and irregularity (births within the entire bree-ding cycle without regularity) (Struhsaker & Leland, 1987; Brockman & van Schaik, 2005). In high and moderate birth seasonality, ≥67% and 33%−67% of infa-nts were found to be born within three months, respecti-vely (van Schaik et al, 1999). External environmental factors (e.g. temperature, precipitation, food resources, photoperiod, seasonal climate, and food availability) and biological rhythms affect reproductive seasonality (Bron-

son & Heideman, 1994; Negus & Berger,11972; Andelm-an, 1986; Bronson & Heideman, 1994; Brockman & van Schaik, 2005; Tecot, 2010; van Schaik & Pfannes, 2005). In natural environments, seasonal food resources are the most important factor that determine birth pattern, while

Received: 4 March 2014; Accepted: 17April 2014

Foundation items: This study was supported by the National Natural

Science Foundation of China (31160422, 30960084), China

Postdoctoral Science Foundation (2013M542379), Program for New

Century Excellent Talents in University (NCET-12-1079), and Key

Subject of Wildlife Conservation and Utilization in Yunnan Province

*Corresponding authors, E-mails: [email protected];

[email protected]

#Authors contributed equally to this work

Birth seasonality and pattern in black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan 475


precipitation, temperature and daylight cycle function as proximate factors (Wikleski et al, 2000). Animals synchronize their critical reproductive period with the optimal phase of environments to maximize survival (Negus & Berger, 1972). Photoperiod and temperature in temperate regions, as well as precipitation in tropical areas, affect seasonal food resources, and eventually induce reproductive seasonality (Vivien-Roels & Pevet, 1983). Under some circumstances, however, photoperiod has no influence on conception, and food availability is the only determining factor of reproductive seasonality (Huang et al, 2012; Carnegie et al, 2011; Kowalewski & Zunino, 2004). With increases in both latitude and altitude, climate and food seasonality change signific-antly. In certain primate species (e.g. Macaca thibetana), the median birth date occurs earlier with the increase in altitude (M. fuscata: Fooden & Aimi, 2003; P. entellus: Newton, 1987). During the breeding season, an individual’s weight gain indicates the accumulation of nutrients, which not only guarantees the nutritional req-uirements of mating individuals, infants and nursing activities, but also ensures that the critical period of infant survival falls into the next autumn with enriched food resources (Zhao & Deng, 1988). Reproductive seasonality is more obvious among species living in areas with high altitude and latitude (Janson & Verdolin, 2005; R. brelichi: Yang et al, 2009; R. roxellana: Qi et al, 2008; Ren et al, 2003; Zhang et al, 2000; P. entellus: Newton, 1987; C. polykomos: Dasilva, 1989; P. pileatus: Stanford, 1990). In addition, reproductive peaks can be significantly correlated with climate and food seasonality (e.g. P. entellus: Newton, 1987), while relatively stable food acquisition (e.g. provision or crop stealing) can induce reproductive irregularity (Bishop, 1979).

Black-and-white snub-nosed monkeys (Rhinopi-thecus bieti) are endemic to the Trans-Himalayas, which are bound by the upper rivers of the Yangtze to the east and the Mekong to the west (N26°14′−N29°20′) (Long et al, 1994). From north to south, the altitudes of groups of this species decrease, habitat transits from dark coni-ferous into dark coniferous-broadleaf mixed forest, and gradient trends manifest in photoperiod, temperature, precipitation and food availability (Xiang & Sayers, 2009; Ding & Zhao, 2004; Huang et al, 2012). The birth period of the Xiaochangdu group (N29°15′) was found to occur from February 4 to March 14, with the median birthdate on February 24 and standard deviation of 6.5 days (Xiang & Sayers, 2009). The birth period of the Mt.

Lasha group (N26°20′) was found to occur from February 15 to April 7 in 2009−2010, with the median birth date on March 27 (Huang et al, 2012), but was from February 19 to April 12 in 2011, with the median birth date on March 17 (Wang et al, 2012). It appears, therefore, that reproductive patterns are influenced by differences in habitat altitude and latitude, with monkeys in high altitude and latitude areas showing earlier birth dates but shorter breeding periods, as well as by differences year to year. Mating behaviors are triggered by various factors, such as photoperiod (Sadlier, 1972), so for some species living in highly seasonal environments, maintaining high energy levels may not always be necessary for conception (Brockman & van Schaik, 2005; Drent & Daan, 1980).

Reproductive seasonality in black-and-white snub-nosed monkey groups (especially the Xiaochangdu group) is not induced by photoperiod, but is determined by the enriched food resources during the conception period (Huang et al, 2012; Xiang & Sayers, 2009). Reproduction of R. beiti is also characterized by birth synchrony, with spatial-temporal differences (Huang et al, 2012; Wang et al, 2012). Reproductive synchrony refers to the way species maximize food quality and availability with high energy consumption periods (e.g. nursing and weaning) (van Schaik & van Noordwijk, 1985). Black-and-white snub-nosed monkeys, also called snow monkeys (Elliot, 1912), inhabit areas with high altitude (Long et al, 1994), as well as significant seasonal climate changes and food resources (Kirkpatrick et al, 1998; Xiang & Sayer, 2009; Huang et al, 2012). However, limited short-term data have been unable to provide a strong interpretation of the reproductive seasonality and corresponding environmental factors in the breeding of black-and-white snub-nosed monkeys. In this study, reproductive data of the Mt. Lasha black-and-white snub-nosed monkey group were collected to determine the: (1) reproductive seasonality; (2) birth pattern (peaks and variations); and (3) mechanisms of regulating reproductive seasonality.


Study areas and animal subjects Our study site is located at Mt. Lasha (N26°20′,

E99°15′) in the Yunling National Reserve, Lanping County, Nujiang Prefecture, Yunnan Province, China. The highest peak is 3 854 m in elevation. Only one black-and-white snub-nosed monkey population with

476 LI, et al.


130 individuals has been observed at Mt. Lasha, and is comprised of eleven one-male multi-female units (OMUs) and two all-male-units (AMUs) (Huang et al, 2012; Wang et al, 2012). Home range of this population covers an area of 14 km2 and is surrounded by villages and farmland at low altitude (<2 800 m), as well as alpine pastures at high altitude (>3 600 m). Its habitat is patchy with sporadic pastures, fire grasslands, and charcoal burning lands (from oak). The vegetation from low to high altitude forms zonal distributions of coniferous forest, mixed broadleaf-conifer forest, broad-leaved deciduous forest and dark coniferous forest (Huang et al, 2012; Wang et al, 2012).

Annual average temperature at Mt. Lasha is 12.42±6.81 °C, with lowest and highest monthly average temperatures of −1.88±7.15 °C (−4.15−1.00 °C, January) and 25.84±5.97 °C (15.86−34.06 °C, October), respect-ively. An extreme temperature fluctuation occurred from August 2012 to July 2013 (–4.15–34.16 °C). Annual precipitation is 767.60 mm, with the highest proportion falling from March to September (695.60 mm), and can be subdivided into precipitation peak (July to September) and sub-peak (March to May). The snow season occurs from November to the following March.

Data collection and statistical analyses

Data collection was scheduled ahead of the monkey breeding season (from February to April) (Huang et al, 2012; Wang et al, 2012). At panoramic view points (e.g. ridges and rocks) opposite monkey activation areas, we recorded numbers of infants and female adults when they are crossing an open area and foraging in the wide-field forests using a monocular telescope (Leica, T77). Newborns were characterized by overall white hair (expect a hint of dark hair on the head), black tip of the tails (one third), as well as red and hairless auricles and extremities. Newborns less than 1.5 months old are carried by adult females. To ensure the accuracy of the data, only observations with more than half of adult females of the group were used for further analysis since its interbirth interval is two years (Huang et al, 2012; Wang et al, 2012). When multiple observations were carried out in one day, only the most reliable (e.g. best view, most individuals included) was used in statistical analysis. Reproductive seasonality was analyzed by considering the numbers of infants and the ratio of infants to female adults (I/F).

We used each 7-day period to calculate all births, to infer the median birth date (Md), mean birth date (Mn)

and standard deviation (SD) (Caughley, 1977, but see Eisenberg et al, 1981). We then transformed each day into degrees of a circle (360°) for testing birth seasonality by circular statistics, the mean vector length r was calculated to measure birth season dispersion, ranging from 0 (uniform) to 1 (clustered) (Batschelet, 1981). The Rayleigh test (Z=nr2) was used to determine whether the birth data were unevenly distributed throughout the year (Batschelet, 1981; Zar, 1999; Huang et al, 2012).

The I/F data of the Xiaochangdu group obtained from either close tracking recording or field scope scanning were used to determine birth pattern. When an I/F was obtained by both methods at the same time, the former had priority (Xiang & Sayers, 2009).

RESULTS

Birth timing The first R. bieti infant at Mt. Lasha was born on

February 20, 2013. The I/F value indicated that the breeding season ended on April 14. The numbers of new-born infants peaked (13) on April 8, and thereafter, stabilized on 13. Because the I/F value was rather unstable than the I-value, and the F-value also included adult females that had given birth in the last year, the I-value was the more reliable index reflecting the birth timing. The end of the breeding season inferred from the I-value was April 8 (Figure 1A). Therefore, in 2013, there were 13 new-born infants at Mt. Lasha and the breeding season was from February 20 to April 8. By referring to the I-values, the birth data of R. bieti at Mt. Lasha from 2009 to 2012 was analyzed (Figure 1). Although the beginning of the breeding season was consistent in the two estimations based on the I-value and I/F, the latter was overall postponed with deviations (−1 to 8 days) (Table 1).

Table 1 Breeding seasons of Mt. Lasha monkey group inferred from different indexes

End of breeding season Year

Beginning of

breeding season I/F I Deviations (day)

2009 2-21 4.02 4-03 −1

2010 2-15 4.04 4-04 0

2011 2-19 4.20 4-12 8

2012 2-17 4.07 4-06 1

2013 2-20 4.14 4-08 6

I: Infants; F: Female adults; I/F: Ratio of infants to female adults.

Birth seasonality

Strict birth seasonality with a pulse type (SD<30 days) was observed in R. bieti at Mt. Lasha. The



breeding season lasted 57 days. Reproductive variables had certain similarities every two years (e.g. beginning phase, ending phase, and Md). The breeding season began and ended earlier in even-numbered years than those in odd-numbered years, and Md was postponed by ten days (Table 2).

Birth pattern

The birth of new-born infants at Mt. Lasha

exhibited certain synchrony, and showed three peaks on February 15−26, March 11−19 and March 20−April 12, respectively (Figure 2). The data on infant births using 3-day intervals indicated that the third peak occupied 50% of the entire breeding season, whereas the first and the second peaks accounted for 25% and 23.5%, respectively. The highest birth peak in both odd- and even-numbered years fell in the third peak period, whereas in odd-numbered years, the sub-peak fell in the second peak

478 LI, et al.


Figure 1 Values of I/F and I of Rhinopithecus bieti at Mt. Lasha from 2009 to 2013 A: 2013; B: 2012; C: 2011; D: 2010; E: 2009.

Table 2 Indexes of birth seasonality of Rhinopithecus bieti at Mt. Lasha

Year Breeding period (day) F I I/F Mn Md SD (day) R Z

2009 2-21−4-03 (42) 24 11 0.46 3-26 3-21 11.58 0.974 10.44**

2010 2-15−4-04 (49) 27 16 0.59 3-23 3-31 19.48 0.938 14.08**

2011 2-19−4-12 (53) 29 13 0.45 3-24 3-17 17.23 0.960 11.98**

2012 2-17−4-06 (50) 33 15 0.45 3-28 3-28 17.27 0.966 14.00**

2013 2-20−4-08 (48) 34 13 0.38 3-24 3-19 16.90 0.961 12.00**

2009-10 2-15−4-04 (49) − 27 − 3-24 3-27 16.95 0.953 24.51**

2009-11 2-15−4-12 (57) − 40 − 3-25 3-22 16.72 0.955 36.47**

2009-12 2-15−4-12 (57) − 55 − 3-25 3-24 16.73 0.958 50.46**

2009-13 2-15−4-12 (57) − 68 − 3-25 3-22 16.86 0.958 62.43**

Odd-numbered year 2-19−4-12 (53) − 37 − 3-25 3-19 15.81 0.964 34.37**

Even-numbered year 2-15−4-06 (51) − 31 − 3-25 3-29 18.19 0.952 28.07**

I: Infants; F: Female adults; Mn: Mean birthdate; Md: Median birthdate; SD: Standard deviation; **: P<0.001.



Figure 2 Birth patterns of Rhinopithecus bieti at Mt. Lasha from 2009 to 2013 Disconnected points mean no data was available or fewer infants were recorded than three days earlier.

period and in even-numbered years, the sub-peak fell in the first peak period (Figure 2). Birth pattern comparison of the northern and south-ern monkey groups

The Xiaochangdu and Mt. Lasha monkey groups were 3° apart in altitude. With increasing altitude, the beginning phase, ending phase and Md of the monkeys shifted to an earlier date; moreover, the breeding season shortened and birth synchrony was strengthened (SD decreased by ten days) (Table 3). The birth pattern of R. bieti at Mt. Lasha was triple-pulse-type, whereas,

that of the Xiaochangdu group was double-pulse-type (peak: February 27−March 7, sub-peak: February 6−11) (Figure 3). However, no differences were found in the percentage of new-born infants between birth peaks of the two groups (Xiaochangdu vs. Mt. Lasha: 62.2% vs. 50%, proportion test: Z=1.20, P=0.11).

DISCUSSION

Regulation of birth seasonality Reproduction is strongly influenced by biological

rhythms and external stimulation (e.g., photoperiod,

Table 3 Birth indexes of Rhinopithecus bieti at Xiaochangdu and Mt. Lasha

Populations Breeding season (day) Mn Md SD (day) Altitude (m) Latitude Reference

Xiaochangdu 2-04−3-13 (38) 2-28 2-24 6.50 3 500−4 250 N29°15’ Xiang & Sayers, 2009

Mt. Lasha 2-15−4-12 (57) 3-25 3-22 16.86 2 850−3 800 N26°20’ This study

Mn: Mean birthdate; Md: Median birthdate; SD: Standard deviation.

Figure 3 Accumulative numbers of Rhinopithecus bieti infants at Xiaochangdu and Mt. Lasha. Data on Xiaochangdu group was collected in 2005 and data on Mt. Lasha group was from 2009 to 2012

480 LI, et al.


temperature, precipitation and food abundance) (Amroso & Marshall, 1960; Lancaster & Lee, 1965). Energy requirements and allocation have been considered the ultimate regulation factors in reproductive patterns. Other factors may play roles in regulating birth seaso-nality, including temperature and food abundance, to meet the requirements of infant survival, nursing and development (Brockman & van Schaik, 2005; Janson & Verdolin, 2005). Reproduction in Xiaochangdu and Mt. Lasha groups showed strong seasonality. However, because Xiaochangdu group’s habitat was 3° higher in altitude than that of the Mt. Lasha group, its reproduction was more concentrated over a shorter breeding season with an earlier beginning date (Table 3).

More obvious seasonality was found in the climate

and food resources in Xiaochangdu area (Table 4). The mating period of both Xiaochangdu and Mt. Lasha groups was from July to October (Xiang & Sayers, 2009; Huang et al, 2012; Wang et al, 2012; Li, unpublished). The breeding season of black-and-white snub-nosed monkeys began after midsummer with shortening daytime, high temperature, abundant food and they started mating in July. This indicated that photoperiod initiated estrus and mating. Huang et al (2012) found that low temperature, especially the lowest temperature during midsummer, triggered repro-ductive behavior in the Mt. Lasha group. Cozzilino et al (1992) also found that temperature and its variations initiated the beginning of estrus and mating in Macaca fuscata.

Table 4 Seasonality of food resources in the Xiaochangdu and Mt. Lasha monkey groups

Xiaochangdu (Xiang et al, 2007; Xiang & Sayer, 2009) Mt. Lasha (Huang et al, 2012; Li, unpublished)

Climate Food Climate Food

Maximum precipitation from June

to August;

Usnea thrives, other vegetation buds

in mid-May;

Maximum precipitation from June

to October; Vegetation buds in late-February;

Monthly average temperature

(10.2−12.5 °C);

Buds, leaves, flowers and fruits/nuts

are in abundance from June to

August;

Monthly average temperature

(15.0−18.2 °C) from June to

September;

Usnea and leaves are in abundant

from June to September, buds,

flowers and fruits/nuts are in

abundance from April to May;

Monthly average lowest

temperature (3.7−5.4 °C) Leaves fall in mid-October

Monthly average lowest

temperature (12.2−14.7 °C) from

June to September;

Leaves fall in mid-November

Photoperiod, temperature and precipitation affect

food abundance, and, in turn, the reproduction period (Bronson, 1988; Sadleier, 1969). Based on birth date (Xiang & Sayer, 2009) and lactation period (195−204 days) of black-and-white snub-nosed monkeys (Ji et al, 1998), the conception period of Xiaochangdu group was calculated to be from July 24 to September 2 (median August 13), whereas, the conception period of the Mt. Lasha group was from August 6 to September 30 (median September 9). R. bieti at Xiaochangdu began to conceive after one month of food abundance peak, and monkeys at Mt. Lasha did after two months of food peak. Although the conception of Xiaochangdu and Mt. Lasha groups began at different time points, they all terminated before the end of the food summit. Moreover, the median date of conception of the two monkey groups occurred in the middle of the last month of the peak in food availability. The long cold winter with limited food consumed the energy stores of the monkeys. Breeding individuals recovered, restored energy and completed

conception during the peak in food availability. Therefore, complete conception during the peak in food availability played an important role in regulating reproductive seasonality, and food abundance instead of photoperiod initiated ovulation and conception (Huang et al, 2012; this study).

The decrease in both food abundance and tempe-rature during the conception period means that energy requirements increased. Infants were born during a period of low temperature and relatively limited food. R. bieti infants at Mt. Lasha began to wean at 13−14 months old (March−April of the following year) and ended at 18-months old (June of the following year) (Li, unpublished). This “bottle phase” (weaning) fell in the period with increased growth in “soft” and high nutrition foods (buds, leaves and flowers). Compared with the Mt. Lasha area, the peak in food availability at Xiaochangdu was one month shorter, buds formed 2.5 months later, and leaves fell one month earlier. Although we lacked weaning information on R. bieti infants at Xiaochangdu,



according to the relatively short food supply and the fact that infants at Mt. Lasha rarely foraged independently at six-months-old (Li, unpublished), we assumed that the chances of Xiaochangdu infants being weaned during the same year were quite low, and more likely occurred in late-May to early-June of the following year when abun-dant young vegetation was available. If no appropriate food is available during weaning, reproduction will ultimately fail and lead to the demise of an entire popula-tion, or perhaps even species. Thus, the weaning of R. bieti during a period of abundant high nutrition food is not only a prerequisite for successful reproduction, but also an adaptive strategy to environmental seasonality.

Early- and late-lactation period of this species occurred during relatively low temperature and limited food periods, whereas, highly energy-consumptive mid-lactation overlapped with the peak in food availability. Monkeys stored energy during pre-conception and mid- lactation to alleviate energy requirements during early- and mid-to-late-lactation. Compared with the Mt. Lasha monkeys, Xiaochangdu infants were born one month earlier and weaned 2.5 months later. Their lactation period was prolonged with relatively lower temperatures, and higher energy consumption phase (mid-lactation) fell during the second peak of food resources. We concluded that the high energy requirement of lactation may have induced the high infant mortality, which was 54% at Wuyapuya (N28°35’) (Kirkpatrick et al, 1998), whereas that of the Mt. Lasha group was much more lower (6.7% in 2012) (Li, unpublished). To avoid energy overdraft of adult females during lactation (especially during winter) and increase infant survival, high frequency of male-infant-caretaking was found in Xiaochangdu group (Xiang et al, 2010), whereas such behaviors were only observed at low frequency in the Mt. Lasha group (Wang et al, 2012). The short breeding season with early start in the Xiaochangdu group appears to be an adaptive stra-tegy to high environmental seasonality. Research on M. fuscata groups at higher altitude showed that infants born early but with a prolonged developmental period were able to survive the low temperature and lack of food experienced during through winter (Food & Aimi, 2003).

The reproduction of many high altitude species is affected by photoperiod, with the length of daytime and its variation triggering related neuroendocrine mechan-isms (Van Horn, 1980; Bronson & Heideman, 1994; Goldman, 2001). The habitat of Xiaochangdu monkeys was located at a higher altitude than that of Mt. Lasha

monkeys; therefore, more obvious seasonality in both climate and food was shown. Although there were diffe-rences in birth patterns of two monkey groups, their birth peaks stabilized during the following 6 to 7 months after the peak in food availability. Thus, the relative stability of the breeding season seems to be an adaptation to seasonal environments, which was critical for its succe-ssful reproduction (Table 3). Previous research has sho-wn that 100% of wild black-and-white snub-nosed mon-keys and 96.3% of captive individuals start reproduction after mid-summer (Cui et al, 2006), indicating that phot-operiod is the triggering factor of their estrus and mating behaviors. Although the stable food supply decreased the seasonality and synchrony of infants born, a birth peak was still observed in captive Rhinopithecus monkeys (R. bieti: Cui et al, 2006; R. roxellana: Zhang et al, 2000). These findings indicate the existence of boilogical reproductive rhythms that are not influenced by either photoperiod or temperature (Fooden & Aimi, 2003). This study indicated that conception and mid-lactation of R. bieti overlapped with the peak in staple food availability, and weaning overlapped with the abundance of high quality foods (new suitable vegetation). Therefore, food availability was the ultimate factor determining the reproductive seasonality of R. bieti, while other enviro-nmental factors, such as precipitation, temperature and photoperiod, functioned as proximate factors for fine-turning coordination between seasonal breeding and food availability (Wikleski et al, 2000). However, whether conception or weaning has priority in this regulation of reproductive seasonality still needs long-term, systematic observations of monkeys in different habitats.

Birth synchrony

Restrictions in environmental conditions induce seasonal synchrony (Power, 2013). When reproduction of Northern Plains Gray Langur (Semnopithecus entellus) is not tied with seasonality, synchrony is lost. Obvious reproductive signals (such as menstruation) in females enable males to control mating, and thereafter decrease food competition and increase their fitness. However, the more restricted reproduction is by seasonality, the more significant the reproductive synchrony. Females may conceal reproductive signals to confuse paternity and or decrease the chance of adult males initiating infanticide (Power, 2013). In addition, strong reproductive seasonality results in greater synchronicity in female ovulation and birth (Power, 2013). R. bieti births at

482 LI, et al.


Xiaochangdu were more synchronized than those at Mt. Lasha (SD was 6.5 and 16.9 days, respectively). This indicated that higher habitat altitudes resulted in stronger birth synchronicity, and may also be correlated with highly synchronized ovulations in female monkeys.

Reproductive synchrony of females, including estrus, birth and length of breeding season, prevents males from monopolizing mating (Carnegie et al, 2011). The more females share reproductive synchrony, the more difficult it is for males to control the reproduction process (Power, 2013). Breeding patterns, such as polygamy and multi-male-female groups, are determined by reproductive seasonality because even the strongest male is unable to totally monopolize multiple females in synchronized pregnable states (Srivastava & Dunbar, 1996). As a result, reproductive synchrony is prone to induce monkey groups with multiple males and females, whereas polygenous groups are more common with reproductive non-synchrony, which is beneficial in ensuring paternity (Power, 2013). The Xiaochangdu monkeys showed a double peak in births, while the monkey at Mt. Lasha displayed three peaks. These phenomena resulted from the strong reproductive synchrony (brief breeding season) of the high altitude monkey groups, the rather large group size (Xiaochangdu group was 210 in 2003 and the Mt. Lasha group was 130 in 2012), and the birth synchrony among different OMUs.

Annual differences in birth patterns

The birth peak of the Mt. Lasha group was “V” shaped in even-numbered years, but exhibited an increasing trend in odd-numbered years. In odd-numbered years, the Md shifted ten days earlier than that in even-numbered years. Moreover, the commencement and termination of the breeding season occurred earlier. These phenomena may be correlated with annual differences in food resources or strategies adopted by

OMUs to decrease food competition; however, more evidence from long-term food detection and systematic research is needed.

Decrease in reproductive rate

Adult female R. bieti at Mt. Lasha gradually increased; however, no such trend was found in infants. From 2009−2011, all females successfully reproduced. In 2012, there were 33 adult females (including four females that reached maturation in 2012) in the group, but there were only 15 infants. In 2013, the numbers of adult females and infants were 34 and 13, respectively. These results indicate that either some adult females did not reproduce (e.g., in 2012, five females did not reproduce and in 2013, six did not), or some infants died. Further research is required to determine if this low reproductive rate is caused by individual aging, female sexual competition, habitat saturation, or by infant deaths.

Breeding season determination

Black-and-white snub-nosed monkeys inhabit original coniferous forests at high altitude in temperate regions. Due to the steep terrain and harsh climates of their habitats and the nature of monkeys to avoid humans, it is difficult to perform close observation or achieve individual recognition. Under such circumstances, only partial I/F data was acquired with one observation, and only I/F can be used to estimate breeding season (Xiang & Sayers, 2009). However, if all adult females and infants could be recognized in multiple observations, then the I-value alone is sufficient to infer accurate breeding seasonality and birth pattern (this study, Huang et al, 2012).

Acknowledgements: We thank the directors and

staff from the Administrative Bureau of Yunling National Reserve, Lanping County, Nujiang Prefecture, Yunan Province, the residents of Dashanqing village and assistants Qing-Sheng Su and Jin-Fu Zhang.

References

Amroso EC, Marshall FHA. 1960. External factors in sexual periodicity. In: Parkes AS. Marshall’s Physiology of Reproduction. 3rd ed. London: Longmans Green, 707-831.

Andelman SJ. 1986. Ecological and social determinants of cercopithecine mating systems. In: Rubenstein DI, Wrangham RW. Ecological Aspects of Social Evolution. Princeton: Princeton University of Press, 201-216.

Batschelet E. 1981. Circular Statistics in Biology. New York: Academic

Press, 593-663.

Bishop NH. 1979. Himalayan langurs: temperate colobines. Journal of

Human Evolution, 8(2): 251-281.

Brockman DK, van Sckaik CP. 2005. Seasonality in Primates. New

York: Cambridge University Press.

Bronson FH, Heideman PD. 1994. Seasonal regulation of reproduction in mammals. In: Knoil E, Neil JD. The Physiology of Reproduction. 2nd



ed. New York: Raven Press, 541-583.

Carnegie SD, Fedigan, LM, Melin AD. 2011. Reproductive seasonality in female capuchins (Cebus capucinus) in Santa Rosa (Area de Conservacion Guanacaste), Costa Rica. International Journal of Primatology, 32(5), 1076-1090.

Caughley G. 1977. Analysis of Vertebrate Population. New York: John Wiley and Sons, 72-74.

Cui LW, Sheng AH, He SC, Xiao W. 2006. Birth seasonality and interbirth interval of captive Rhinopithecus bieti. American Journal of Primatology, 68(5): 457-563.

Cozzilino R, Cordischi C, Aureli F, Scucchi S. 1992. Environmental temperature and reproductive seasonality in Japanese macaques (Macaca fuscata). Primates, 33(3): 329-336.

Dasilva GL. 1989. The Ecology of the Western Black and White Colobus (Colobus polykomos polykomos Zimmerman 1780) on a Riverine Island in Southeastern Sierra Leone. Ph. D. thesis, University of Oxford, England.

Ding W, Zhao QK. 2004. Rhinopithecus bieti at Tacheng, Yunnan: Diet and daytime activities. International Journal of Primatology, 25(3): 583-598.

Drent RH, Daan S. 1980. The prudent parent: energetic adjustments in avian breeding. Ardea, 68(1-4): 225-252.

Eisenberg JF, Dittus WPJ, Fleming TH, Green K, Struhsaker T, Trorington RW, JR. 1981. Techniques for the Study of Primate Population Ecology. Washington: National Academy Press, 136-143.

Elliot DG. 1912. A Review of Primates. New York: American Museum of National History.

Fooden J, Aimi M. 2003. Birth-season variation in Japanese macaques, Macaca fuscata. Primates, 44(2): 109-117.

Goldman BD. 2001. Mammalian photoperiodic system: formal properties and neuroendocrine mechanisms of photoperiodic time measurement. Journal of biological rhythms, 16(4): 283-301.

Huang ZP, Cui LW, Scott MB, Wang SJ, Xiao W. 2012. Seasonality of reproduction of wild black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha, Yunnan, China. Primates, 53(3): 237-245.

Janson C, Verdolin J. 2005. Seasonality of primate births in relation to climate. In: Brockman DK, van Schaik CP. Seasonality in Primates: Studies of Living and Extinct Human and Nonhuman Primates. Cambridge: Cambridge University Press, 307-350.

Kirkpatrick RC, Long YC, Zhong T, Xiao L. 1998. Social organization and range use in the Yunnan Snub-nosed Monkey Rhinopithecus bieti. International Journal of Primatology, 19(1): 13-51.

Kowalewski M, Zunino GE. 2004. Birth seasonality in Alouatta caraya in northern Argentina. International Journal of Primatology, 25(2): 383-400.

Lancaster JB, Lee RB. 1965. The annual reproductive cycle in monkeys and apes. In: Devore I. Primate Behavior. New York: Holt, Rinehart and Winston, 486-513.

Long YC, Kirkpatrick RC, Zhong T, Xiao L. 1994. Report on the distribution, population, and ecology of the Yunnan snub-nosed

monkey (Rhinopithecus bieti). Primates, 35(2): 241-250.

Negus NC, Berger PJ. 1972. Environmental factors and reproductive processes in mammalian populations. In: Velardo JT, Kasprow BA. Biology of Reproduction: Basic and Clinical Studies. New Orleans: Pan American Association of Anatomy, 89-98.

Newton PN. 1987. The social organization of forest hanuman langurs (Presbytis entellus). American Journal of Primatology, 8(3): 199-232.

Power C. 2013. The Seasonality Thermostat: Female Reproductive Synchrony and Male Behavior in Monkeys, Neanderthals, and Modern Humans. PaleoAnthropology, 2013: 33-60.

Qi XG, Li BG, Ji WH. 2008. Reproductive parameters of wild female Rhinopithecus roxellana. American Journal of Primatology, 70(4): 311-319.

Ren BP, Zhang SY, Xia SZ, Li QF, Liang B, Lu MQ. 2003. Annual reproductive behavior of Rhinopithecus roxellana. International Journal Primatology, 24(3): 575-589.

Sadlier RMFS. 1972. Environmental effects. In: Austin CR, Short RV. Reproduction in Mammals. Book 4: Reproductive Patterns. Cambridge: Cambridge University Press, 69-94.

Stanford CB. 1990. The capped langur in Bangladesh: behavioral ecology and reproductive tactics. Contributions to Primatology, 21: 1-179.

Struhsaker TT, Leland L. 1987. Colobines: Infanticide by adult male. In: Smuts BB, Cheney DL, Seyfarth RM, Wrangham RW, Struhsaker TT. Primate Society. Chicago: The University Of Chicago Press, 83-97.

Srivastava A, Robin IMD. 1996. The mating system of Hanuman langurs—a problem in optimal foraging. Behavioral Ecology and Sociobiology, 39(4): 219-226.

Tecot SR. 2010. It’s all in the timing: birth seasonality and infant survival in Eulemur rubriventer. International Journal Primatology, 31(5): 715-735.

van Schaik CP, van Noordwijk MA. 1985. Interannual variability in fruit abundance and the reproductive seasonality in Sumatran long-tailed macaques (Macaca fascicularis). Journal of Zoology, 206(4): 533-549.

van Schaik CP, van Noordwijk MA, Nunn CL. 1999. Sex and social evolution in primates. In: Lee P. Comparative Primate Socioecology. Cambridge: Cambridge University Press, 204-231.

van Schaik CP, Pfannes KR. 2005. Tropical climates and phenology: A primate perspective. In: Brockman DK, van Schaik CP. Seasonality in Primates: Studies of Living and Extinct Human and Non-human Primates. Cambridge: Cambridge University Press, 24-54.

Vivien-Roels B, Pevet P. 1983. The pineal gland and the synchronization of reproductive cycles with variations of the environmental climatic conditions, with special reference to temperature. Pineal Research Reviews, 1, 91-143.

Van Horn RN. 1975. Primate breeding season: photoperiodic regulation in captive Lemur catta. Folia Primatologica, 24(2-3): 203-220.

Wang SJ, Huang ZP, He YC, He XD, Li DH, Sun J, Cui LW, Xiao W. 2012. Mating behavior and birth seasonality of black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Mt. Lasha. Zoological

484 LI, et al.


Research, 33(3): 241-248. (in Chinese)

Wikleski M, Hau M, Wingfield JC. 2000. Seasonality of reproduction in a neotropical rain forest bird. Ecology, 81(9), 2458-2472.

Xiang ZF, Sayers K. 2009. Seasonality of mating and birth in wild black-and-white snub-nosed monkeys (Rhinopithecus bieti) at Xiaochangdu, Tibet. Primates, 50(1): 50-55.

Xiang ZF, Huo S, Xiao W. 2010. Male allocare in Rhinopithecus bieti at Xiaochangdu, Tibet: Is it related to energetic stress? Zoological Research, 31(2): 189-197.

Yang MY, Sun DY, Zinner D, Roos C. 2009. Reproductive parameter in Guizhou snub-nosed monkeys (Rhinopithecus brelichi). American Journal of Primatology, 71(3): 266-270.

Zar JH. 1999. Biostatistical Analysis. 4th ed. New Jersey: Prentice Hall.

Zhang SY, Liang B, Wang LX. 2000. Seasonality of matings and births in captive Sichuan golden monkeys (Rhinopithecus roxellana). American Journal of Primatology, 51(4): 265-269.

Zhao QK, Deng ZY. 1988. Macaca thibetana, at Mt. Emei, China. II. Birth seasonality. American Journal of Primatology, 16(3): 261-268.

Zoological Research again recognized for Excellence and Impact in Science and

Technology Publishing in 2014 At the September 2014 conference hosted by the Institute of Scientific and Technical Information of China (ISTIC)

in Beijing, Zoological Research (ZR) was ranked among the top 300 journals classified as “Outstanding S&T Journals of China” for 2014 due to its high academic value and the leading role it plays in advancing the quality of the S&T journals of China. Few journals in China ever receive this ranking, but this is the second time ZR has now been recognized by this award twice since 2008.

ISTIC also launched a new project to promote scientific communications and the growing internationalization of

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All in all, it has been a good year for ZR.



Experimental infection of tree shrews (Tupaia belangeri) with Coxs-ackie virus A16

Jian-Ping LI, Yun LIAO, Ying ZHANG, Jing-Jing WANG, Li-Chun WANG, Kai FENG, Qi-Han LI*, Long-Ding LIU*

Institute of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical Colleg, Kunming 650118, China

Abstract: Coxsackie virus A16 (CA16) is commonly recognized as one of the main human pathogens of hand-foot-mouth disease (HFMD). The clinical manifestations of HFMD include vesicles of hand, foot and mouth in young children and severe inflammatory CNS lesions. In this study, experimentally CA16 infected tree shrews (Tupaia belangeri) were used to investigate CA16 pathogenesis. The results showed that both the body temperature and the percentages of blood neutrophilic granulocytes / monocytes of CA16 infected tree shrews increased at 4−7 days post infection. Dynamic distributions of CA16 in different tissues and stools were found at different infection stages. Moreover, the pathological changes in CNS and other organs were also observed. These findings indicate that tree shrews can be used as a viable animal model to study CA16 infection.

Keywords: Coxsackie virus A16; Infection; Tree shrew

Hand-foot-mouth disease (HFMD) is a pathogenesis that mainly affects infantile populations. Although most infection cases show only mild symptoms (Mao et al, 2013), there are a small number of individuals suffering from neurological symptoms of encephalitis and severe heart and lung failure (Tan et al, 2014). From the view of the pathogen, HFMD is mainly caused by enterovirus type 71 (EV71) and Coxsackie virus group A type16 (CA16) infection (Yang et al, 2014). Currently, EV71 vaccine has completed phase III clinical trial (Chen et al, 2014; Li et al, 2014; Zhu et al, 2014), whereas a CA16 vaccine is still under development. Due to the lack of systematic analysis on the infection and immunization of CA16 virus, an effective animal model for CA16 infection is urgently necessary. The conventional animal models of CA16 infection, such as mice or rats (Liu et al, 2014), can neither fully reflect the etiological or pathological characteristics of infection and immuni-zation, nor meet the comprehensive assessment require-ments of vaccine safety and efficacy. Non-human prim-ate animal models are of special importance in vaccine evaluation, e.g., Zhang et al (2014) used infant rhesus monkeys (Macaca mulatta) to explore the efficacy and safety of EV71 vaccine. However, high cost and limited

animal resources has significantly limited its application. Due to a variety of unique characteristics, e.g., small adult body size, short reproductive1cycle and life span (Wang et al, 2013), and especially a close affinity to primates, tree shrews (Tupaia belangeri) have been widely applied in biomedical research, especially in virology research (Fan et al, 2013). In this study, 2-month-old tree shrews were used to investigate GX18 infection and their potential as an animal model for CA16 infection was assessed.


Virus and cells The CA16 virus used in this study was an isolated

GX18 strain from an infected child with mild clinical symptoms in Guilin, China, 2012. The GX18 strain was

Received: 11 April 2014; Accepted: 20 May 2014

Foundation items: This work was supported by the National High-

Tech R&D Program (2014ZX09102042), the National Natural

Science Foundation of China (81373142) and the Natural Science

Foundation of Yunnan Province (2012ZA009)

*Corresponding authors, E-mails: [email protected]; longdingl

@gmail.com

486 LI, et al.


identified as a CA16 B2 sub-genotype virus by RT-PCR. The virus grown in Vero cells (cultured by Institute of Medical Biology) was harvested for freezing at –20 °C with a concentration of 106.5 cell culture infectious doses (CCID50)/mL. The Vero cells were maintained in 5% MEM containing fetal bovine serum (provided by Institute of Medical Biology) and grown to a confluent monolayer in a T-25 vessel or in 96-well plates for viral isolation and antibody neutralization assays.

Virus titration and neutralization assay

CA16 harvested from cell culture or isolated from different organs and tissues of infected tree shrews were analyzed by a microtitration assay using a standard protocol (Arita et al, 2006). The neutralization test was performed according to the standard protocol (WHO, 1988). A mixture of diluted serum containing anti-CA16 antibodies (provided by Institute of Medical Biology) and the virus at a titer of 500–1 000 CCID50 in 100 µL of PBS (provided by Institute of Medical Biology) was incubated at 37 °C for 1 h. The cellular pathogenic effect (CPE) of the virus was examined by inoculating the mixture onto Vero cells grown in 96-well plates. The plaque assay was performed in 6-well plates containing 90% confluent Vero monolayer cells. The cells were inoculated with samples from blood, throat swabs or feces. After 1 h incubation at 37 °C, the infection media were aspirated off and each well was then covered with 3 mL of agar overlay medium and incubated for 2 days at 37 °C in a CO2 incubator. At the end of the incubation, the cells were fixed with formaldehyde and stained with 0.1% Crystal Violet as a standard protocol (Hung et al, 2010).

Tree shrews

Fifteen healthy female tree shrews (80±10 g, 2-month-old) were divided into a infection group (n=12) and a mock-infection control group (n=3). All animal procedures were approved by the Office of Laboratory Animal Management of Yunnan Province, China. Animals were individually caged and fed according to the guidelines of the Committee of Experimental Anim-als at the Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS). Individuals were conf-irmed free of antibodies against CA16 prior to the experiment via neutralization test.

CA16 infection

Our previous study on EV71 infection in rhesus

monkeys showed that virus distributions in blood and other organs can be observed via intravenous and intratracheal inoculation routes (Zhang et al, 2011). Ong et al (2008) found that infections via intravenous route in cynomolgus monkeys (Macaca fascicularis) can not yield a similar distribution of the virus.

In this study, 12 tree shrews were infected with CA16 (104.5 CCID50) via the respiratory tract by nasal spraying. The other 3 tree shrews were mock sprayed by saline as uninfected negative controls. The animals were monitored daily, and their body temperatures were measured rectally by a digital stick thermometer (MC-BOMR, Omron Co.) from day 2 post infection (p.i.). In parallel, venous blood samples were taken into EDTA coated capillary tubes daily for routine detections of biological indicatorsand viral loads (Veterinary Multi-species Hematology System, Hemavet 950FS, Drew Scientific Co.). Pathogenic and histopathological examinat-ions were performed on all the tissues and organs of tree shrews sacrificed by anesthesia at day 4, 7, 10 and 14 p.i..

Viral RNA extraction and quantitative RT-PCR

Viral RNA was extracted from whole blood (100 µL), fresh tissue homogenate (10%, 100 mg) or fecal homo-genate (5%, 100 mg) by TRNzol-A+, according to manufacturer’s protocols (Tiangen, China). The viral RNA was eluted in a final volume of 20 µL. For quantification, a single-tube, real-time Taqman RT-PCR assay was performed using the Taqman 1-step RT-PCR Master Mix in the 7500 Fast Real-time RT-PCR system (Applied Biosystems, Foster City, CA, U.S.). The reaction mixture (20 µL) contained the Taqman Univ-ersal PCR master mix, primers (each at 20 µmol/L), the FAM/TAMRA labeled probe (10 µmol/L) (Takara Co., Ltd. Dalian, China), and RNA (2 µL). The sequences of the CA16-specific primers and probe were as following: forward primer (5'-ACACTCCATTACCCTGAGGGT GTA-3'); probe (5'-ATGAGAATCAAACACGTCAG GGCATGGAT-3'); and reverse primer (5'-ACACTCC ATTACCCTGAGGGTGTA-3'). The following protocol was used for all PCR assays: 5 min at 42 °C and 10 s at 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The standard reference curve was obtained by the measurement of the serially diluted virus RNA generated by in vitro transcription from a DNA construct that contains the Vp1 region. Viral copies were quantified according to vitro-synthesized RNA by spectrophotometry quantification, and the quantity was

Experimental infection of tree shrews (Tupaia belangeri) with Coxsackie virus A16 487


expressed as a relative copy number, determined by the equation: [(μg of RNA/μL)/(molecular weight)] ×Avog-adro's number= viral copy number/μL.

Histopathological examination and immunohisto-chemical analysis

The tissue samples from different organs were fixed in 10% formalin in PBS, dehydrated in ethanol gradients and embedded in paraffin before obtaining 4 µm sections for further H-E staining. Histopathological analysis of the tissue sections from each organ was performed under a light microscope. For the immunohistochemical analy-sis, tissue samples were embedded in an optimal cutting temperature (OCT) compound (Miles Inc., Elkhart, Ind.) and frozen in liquid nitrogen. The frozen tissues were then cut into 4 µm sections, placed on poly-l-lysine-coated glass slides and fixed in 3.7% paraformaldehyde. The endogenous peroxidase activity of the tissues was inhibited by treating with hydrogen peroxide (2.5%). The CA16 antigen was detected by mouse anti-CA16 sera and horseradish peroxidase (HRP)-conjugated anti-

mouse IgG antibodies (Sigma, Deisenhofen, Germany) followed by color development with diaminobenzidine for the detection of the antigen-antibody reaction.

Statistical Analysis

All the data were expressed as the mean of three samples from all experiments (mean±SE).

RESULTS

Clinical observations of CA16-infected tree shrews No typical papules or vesicles on limbs or mouths

were found in infected tree shrews, but increased body temperatures p.i. (Figure 1A), as well as increased percentages of neutrophilic granulocytes and monocytes in some animals (Figure 1B, C) from day 5 to 7 p.i. with the decrease of lymphocyte (Figure 1D) were observed. These findings suggest that similar clinical CA16 infection symptoms as humans (Lo et al, 2011), such as fever and inflammatory responses (reflected in blood cell analysis), can be found in tree shrews.

Figure 1 Clinical symptoms of CA16 infected tree shrews via respiratory route A: Body temperatures of CA16-infected tree shrews; B: The percentage of neutrophilic granulocytes in leukocytes of CA16-infected tree shrews; C: The

percentage of monocytes in leukocytes of CA16-infected tree shrews; D: The percentage of lymphocytes in leukocytes of CA16-infected tree shrews.

Dynamic profiles of virus in blood and stool of CA16-infected tree shrews

We analyzed viral load dynamic profiles in blood and stool at different stages of infection. The peak levels

of viral load were observed in the blood (500 copies/100 µL) at day 6 p.i. and in stool (2 000 copies/100 mg) at day 7 p.i., respectively. These findings indicate that the viremia of the infected tree shrews develops from day 3

488 LI, et al.


to day 7 p.i., whereas, the virus shedding in feces is detectable from day 3 to day 14 p.i. (Figure 2B). These virus dynamic profiles imply that the clinical symptoms of CA16-infected tree shrews are correlated with the viremia manifestations.

Viral distributions in tissues from CA16-infected tree shrews

The target organs of CA16 infection in humans involve several different tissues (Wang et al, 2004). To understand the CA16 distributions in tree shrews, the viral loads in various tissues were determined at different stages of infection. CA16 virus was detectable in the lymph node, lung, spleen, kidney, thigh muscle and brain from day 7 p.i.. Relatively high viral loads were found in

the spleen (356 copies/100 mg), cerebellum (290 copies/100 mg), lung (294 copies/ 100 mg), lymph node (200 copies/100 mg), parotid gland (155 copies/100 mg) and thigh muscles (290 copies/ 100 mg), whereas no detectable viral loads were observed in the same tissues of non-infected control tree shrews. In general, viral replication peaked on day 6 or 7 p.i. in the lymph nodes and glands (Figure 3B, C), whereas, peaked on day 10 p.i. in the CNS and spleen, lung and muscles (Figure 3A, D), which might be the major target organs of infection in tree shrews.

It was reported that in certain cases, CA16 is able to target CNS and lead to viral encephalitis (Chang et al, 1999; Goto et al, 2009). In this present study, the total

Figure 2 Dynamic distributions of CA16 in the blood and stool of infected tree shrews via respiratory route

A: Viral RNA extracted from blood specimens; B: Viral load in feces.

Figure 3 Viral distributions in tissues of CA16-infected tree shrews A: Viral load in the major organs of infected tree shrews; B: Viral load in glands of infected tree shrews; C: Viral load in the lymph nodes of infected tree

shrews; D: Viral load in CNS of infected rhesus monkeys.



distributions of viral loads increased from day 4 to day 10 p.i. (Figure 3B), and higher loads were found specifically in the cerebellum (291 copies/100 mg) and lobus frontalis (294 copies/100 mg) at day 10 p.i., which were consistent with the observation that viral loads peaked in muscles and lungs on day 10 p.i.. Viral antigen detection in tissues from CA16-infected tree shrews

To investigate CA16 replications, antigen expres-sions of CA16 in the related tissues were examined immunohistochemically. High expressions of viral antigen in the heart (Figure 4A), spleen (Figure 4B) and lung (Figure 4C) were observed. Viral antigens were also observed in the CNS including cerebellum (Figure 4d). These findings are consistent with the patterns of viral load distributions of widespread infections.

Figure 4 Viral antigen expressions in tissues from CA16 infected tree shrews

A: Sample of the heart collected on day 10 p.i.; B: Sample of the spleen

collected on day 10 p.i.; C: Samples of the lung collected on day 10 p.i.;

D: Samples of the cerebellum collected on day 10 p.i.; Arrows indicate the

stained CA16 antigen; Images are shown at 200× magnification.

Pathological changes in tissues from CA16-infected tree shrews

In this study, the pathogenic changes of CA16 infected tree shrews were evaluated by systematic pathologic analysis on various tissues, including the liver, kidney, spleen, heart, lung, muscles, spinal cord and brain. Pathological responses, such as cell damage and inflammatory cell infiltration (Figure 5A−D) were found in the lung, kidney and muscles, correlating with the presence of high viral loads. The observed neuropath-ological lesions in the cerebellum indicate that the

Figure 5 Pathological analyses of CA16-infected tree shrews The typical features of pathological changes as infiltration of inflammatory

cells (black arrow) in the lung (A), liver (B), kidney (C) and muscles (D) of

CA16 infected tree shrews. Images show the proliferations of neuroglial and

neuronal lesions in the thalamus (e) andspinal cord (f) of CA16 infected tree

shrews. Images are shown at 200× magnification.

490 LI, et al.


pathogenic changes in CNS may contribute to the degen-eration of neural cells, infiltration and inflammatory cell aggregation (Figure 5E, F). Moreover, as expected, all the observed pathological changes were correlated with the viral antigen expressions. These results demonstrate that CA16 has a tropism to the CNS and lung and thereafter may induce severe lesions of these tissues.

DISCUSSION The establishment of an viable animal model plays

critical roles in understanding infectious diseases, such as HFMD, and vaccine evaluations. Animal models of EV71 infection include suckling mice and neonatal rhesus monkeys (Wang et al, 2004; Zhang et al, 2011). The conventional animal model in the studies of CA16 infection is still neonatal mice. The mouse model provides evidence of the protective immunity of CA16 vaccine (Dong et al, 2011) but fails to provide convi-ncing data on the infection pathology and the compreh-ensive evaluation of vaccine. According to previous reports, tree shrews can be widely used as a potential animal model for many infectious diseases, such as hepatitis C, measles, atypical pneumonia (SARS), tuberculosis, etc (Han et al, 2011; Xu et al, 2013; Yang et al, 2013).

In the present study, experimentally CA16-infected tree shrews have been used to investigate the etiology and pathology of HFMD. In the CA16-infected tree shrews, although no typical vesicles or papules on limbs

and mouth were observed, certain clinical features, such as fever and lymphocyte-related inflammatory responses could mimic some manifestations in human patients with HFMD (Mou et al, 2014; Wang et al, 2013).

The distributions of CA16 virus in tree shrews were determined by viral loads and pathological changes. The high viral loads found in the lymph nodes, heart, lung, spleen, kidney, thigh muscles and brain imply that the respiratory route is one of potential natural infection routes of viral transmissions. The viral load in blood and the antigen expression in the lymph nodes, lung, muscles and CNS indicate that CA16 can spread throughout the whole body of infected animals. Moreover, in this study, high viral loads were detected in cerebellum from day 4 to day 10 p.i., and neuropathological lesions were obse-rved via histopathological examinations. These findings indicate that CA16 infected tree shrews can eventually display a broad range of viral transmission, including CNS. In addition, the viral shedding found in the feces of the infected tree shrews indicate that the typical sprea-ding route of enterovirus can also be demonstrated in this animal model.

Although this study only demonstrates limited clin-ical manifestations of HFMD, the clinical symptoms and pathological changes found in CA16 infected tree shrews still reveal a complete process of CA16 infection and str-ongly support the application of tree shrews as an animal model in CA16 infection research and vaccine evaluation.

References

Arita M, Nagata N, Sata T, Miyamura T, Shimizu H. 2006. Quantitative analysis of poliomyelitis-like paralysis in mice induced by a poliovirus replicon. The Journal of General Virology, 87(Pt 11): 3317-3327.

Chang LY, Lin TY, Huang YC, Tsao KC, Shih SR, Kuo ML, Ning HC, Chung PW, Kang CM. 1999. Comparison of enterovirus 71 and coxsackievirus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998. The Pediatric Infectious Disease Journal, 18(12): 1092-1096.

Chen YJ, Meng FY, Mao QY, Li JX, Wang H, Liang ZL, Zhang YT, Gao F, Chen QH, Hu YM, Ge ZJ, Yao X, Guo HJ, Zhu FC, Li XL. 2014. Clinical evaluation for batch consistency of an inactivated enterovirus 71 vaccine in a large-scale phase 3 clinical trial. Human Vaccines & Immunotherapeutics, 10(5): 1366-1372.

Dong CH, Liu LD, Zhao HL, Wang JJ, Liao Y, Zhang XM, Na RX, Liang Y, Wang LC, Li QH. 2011. Immunoprotection elicited by an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys. Vaccine, 29(37): 6269-6275.

Fan Y, Huang ZY, Cao CC, Chen CS, Chen YX, Fan DD, He J, Hou HL,

Hu L, Hu XT, Jiang XT, Lai R, Lang YS, Liang B, Liao SG, Mu D, Ma YY, Niu YY, Sun XQ, Xia JQ, Xiao J, Xiong ZQ, Xu L, Yang L, Zhang Y, Zhao W, Zhao XD, Zheng YT, Zhou JM, Zhu YB, Zhang GJ, Wang J, Yao YG. 2013. Genome of the Chinese tree shrew. Nature Communications, 4: 1426.

Goto K, Sanefuji M, Kusuhara K, Nishimura Y, Shimizu H, Kira R, Torisu H, Hara T. 2009. Rhombencephalitis and coxsackievirus A16. Emerging Infectious Diseases, 15(10): 1689-1691.

Han JB, Zhang GH, Duan Y, Ma JP, Zhang XH, Luo RH, Lü LB, Zheng YT. 2011. Sero-epidemiology of six viruses natural infection in Tupaia belangeri chinensis. Zoological Research, 32(1): 11-16. (in Chinese)

Hung HC, Chen TC, Fang MY, Yen KJ, Shih SR, Hsu JT, Tseng CP. 2010. Inhibition of enterovirus 71 replication and the viral 3D polymerase by aurintricarboxylic acid. Journal of Antimicrobial Chemotherapy, 65(4): 676-683.

Li RC, Liu LD, Mo ZJ, Wang XY, Xia JL, Liang ZL, Zhang Y, Li YP, Mao QY, Wang JJ, Jiang L, Dong CH, Che YC, Huang T, Jiang ZW,



Xie ZP, Wang LC, Liao Y, Liang Y, Nong Y, Liu JS, Zhao HL, Na RX, Guo L, Pu J, Yang EX, Sun L, Cui PF, Shi HJ, Wang JZ, Li QH. 2014. An inactivated enterovirus 71 vaccine in healthy children. The New England Journal of Medicine, 370(9): 829-837.

Liu QW, Shi JP, Huang XL, Liu F, Cai YC, Lan K, Huang Z. 2014. A murine model of coxsackievirus A16 infection for anti-viral evaluation. Antiviral Research, 105: 26-31.

Lo SH, Huang YC, Huang CG, Tsao KC, Li WC, Hsieh YC, Chiu CH, Lin TY. 2011. Clinical and epidemiologic features of Coxsackievirus A6 infection in children in northern Taiwan between 2004 and 2009. Journal of Microbiology, Immunology and Infection, 44(4): 252-257.

Mao Q, Wang Y, Yao X, Bian L, Wu X, Xu M, Liang Z. 2013. Coxsackievirus A16: Epidemiology, diagnosis, and vaccine. Human Vaccines & Immunotherapeutics, 10(2): 360-367.

Mou J, Dawes M, Li Y, He Y, Ma H, Xie X, Griffiths S, Cheng J. 2014. Severe hand, foot and mouth disease in Shenzhen, South China: what matters most? Epidemiology and Infection, 142(4): 776-788.

Tan CW, Lai JK, Sam IC, Chan YF. 2014. Recent developments in antiviral agents against enterovirus 71 infection. Journal of Biomedical Science, 21: 14.

Ong KC, Badmanathan M, Devi S, Leong KL, Cardosa MJ, Wong KT. 2008. Pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis. Journal of Neuropathology and Experimental Neurology, 67(6): 532-542.

Wang CY, Li LF, Wu MH, Lee CY, Huang LM. 2004a. Fatal coxsackievirus A16 infection. The Pediatric Infectious Disease Journal, 23(3): 275-276.

Wang YF, Chou CT, Lei HY, Liu CC, Wang SM, Yan JJ, Su IJ, Wang JR, Yeh TM, Chen SH, Yu CK. 2004b. A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection. Journal of Virology, 78(15): 7916-7924.

Wang YR, Sun LL, Xiao WL, Chen LY, Wang XF, Pan DM. 2013b. Epidemiology and clinical characteristics of hand foot, and mouth disease in a Shenzhen sentinel hospital from 2009 to 2011. BMC Infectious Diseases, 13: 539.

WHO. 1988. Procedure for Using the Lyophilized LBM Pools for Typing Enterovirus, Geneva.

Xu L, Zhang Y, Liang B, Lü LB, Chen CS, Chen YB, Zhou JM, Yao YG. 2013. Tree shrews under the spot light: emerging model of human diseases. Zoological Research, 34(2): 59-69.

Yang E, Cheng C, Zhang Y, Wang J, Che Y, Pu J, Dong C, Liu L, He Z, Lu S, Zhao Y, Jiang L, Liao Y, Shao C, Li Q. 2014. Comparative study of the immunogenicity in mice and monkeys of an inactivated CA16 vaccine made from a human diploid cell line. Human Vaccines & Immunotherapeutics, 10(5): 1266-1273.

Yang MB, Li N, Li F, Zhu QQ, Liu X, Han QY, Wang YW, Chen YP, Zeng XY, Lv Y, Zhang PP, Yang CL, Liu ZW. 2013. Xanthohumol, a main prenylated chalcone from hops, reduces liver damage and modulates oxidative reaction and apoptosis in hepatitis C virus infected Tupaia belangeri. International Immunopharmacology, 16(4): 466-474.

Zhang XM, Zhao HL, Wang JJ, Liao Y, Na RX, Wang LC, Liu LD, Gao JH, Tang DH, Wang CZ, Li QH. 2011a. Evaluation of immune responses and related patho-inflammatory reactions of a candidate inactivated EV71 vaccine in neonatal monkeys. National Medical Journal of China, 91(28): 1977-1981.

Zhang Y, Cui W, Liu LD, Wang JJ, Zhao HL, Liao Y, Na RX, Dong CH, Wang LC, Xie ZP, Gao JH, Cui PF, Zhang XM, Li QH. 2011b. Pathogenesis study of enterovirus 71 infection in rhesus monkeys. Laboratory Investigation, 91(9): 1337-1350.

Zhang Y, Yang EX, Pu J, Liu LD, Che YC, Wang JJ, Liao Y, Wang LC, Ding D, Zhao T, Ma N, Song M, Wang X, Shen D, Tang DD, Huang HT, Zhang ZX, Chen D, Feng MF, Li QH. 2014. The gene expression profile of peripheral blood mononuclear cells from EV71-infected rhesus infants and the significance in viral pathogenesis. PloS One, 9(1): e83766.

Zhu FC, Xu WB, Xia JL, Liang ZL, Liu Y, Zhang XF, Tan XJ, Wang L, Mao QY, Wu JY, Hu YM, Ji TJ, Song LF, Liang Q, Zhang BM, Gao Q, Li JX, Wang SY, Hu YS, Gu SR, Zhang JH, Yao GH, Gu JX, Wang XS, Zhou YC, Chen CB, Zhang ML, Cao MQ, Wang JZ, Wang H, Wang N. 2014. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. The New England Journal of Medicine, 370(9): 818-828.



Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews

Gui LI1, Ren LAI1,2,*, Gang DUAN3, Long-Bao LYU1, Zhi-Ye ZHANG1, Huang LIU1, Xun XIANG3

1. Kunming Primate Research Center of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan

650223, China



3.Yunnan Agricultural University, Kunming Yunnan 650201, China

Abstract: Endosymbionts influence many aspects of their hosts’ health conditions, including physiology, development, immunity, metabolism, etc. Tree shrews (Tupaia belangeri chinensis) have attracted increasing attention in modeling human diseases and therapeutic responses due to their close relationship with primates. To clarify the situation of symbiotic bacteria from their body surface, oral cavity, and anus, 12 wild and 12 the third generation of captive tree shrews were examined. Based on morphological and cultural characteristics, physiological and biochemical tests, as well as the 16S rDNA full sequence analysis, 12 bacteria strains were isolated and identified from the wild tree shrews: body surface: Bacillus subtilis (detection rate 42%), Pseudomonas aeruginosa (25%), Staphlococcus aureus (33%), S. Epidermidis (75%), Micrococcus luteus (25%), Kurthia gibsonii (17%); oral cavity: Neisseria mucosa (58%), Streptococcus pneumonia (17%); anus: Enterococcus faecalis (17%), Lactococus lactis (33%), Escherichia coli (92%), Salmonella typhosa (17%); whereas, four were indentified from the third generation captive tree shrews: body surface: S. epidermidis (75%); oral cavity: N.mucosa (67%); anus: L. lactis (33%), E. coli (100%). These results indicate that S. epidermidis, N. mucosa, L. lactis and E. coli were major bacteria in tree shrews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa were species-specific flora. This study facilitates the future use of tree shrews as a standard experimental animal and improves our understanding of the relationship between endosymbionts and their hosts.

Keywords: Tree shrew; Microbial; Separation; Identification

Symbiotic bacteria are bacteria living in symbiosis with their hosts. They influence almost every aspect of the physiological prosess of the host, including the growth and development, physiology and biochemistry, gene expression, metabolism, immunology, etc. Recent years, tree shrews (Tupaia belangeri chinensis) have been rapidly and widely applied in biomedical research as a novel experimental animal model, especially in studies on human diseases (Wang et al, 2010; Huang et al, 2013; Xu et al, 2013). Domestic tree shrews of China include northern tree shrews (Tupaia belangeri) and six suspecies (Simpson, 1945). Currently, laboratory tree shrews are mainly from the field, and therefore their unclarified genetic background, physical condition and situations of symbiotic bacteria have brought many difficulties into their application in research into the development of novel antimicrobial medications, the

bacterial diseaseas, etc. Although scientists have carried out studies on the symbiotic bacteria of healthy wild tress shrews (Gao et al, 2009; Wang et al, 1987; Wang et al, 2011; Xing et al, 2012; Zhang et al, 2009), there is still no report on symbiotic bacteria from the body surface, oral cavity and anus of captive tree shrews, especially the third generation captive tree shrews. The most commonly used method in studying symbiotic bacteria is the meta-genomic methods basing on the second generation sequ-encing tools, which means, the extracted DNA of flora will connect with T-vector before Sanger sequencing. 1

Received: 23 March 2014; Accepted: 25 June 2014

Foundation items: This study was supported by the National 863

Project of China ( 2012AA021801) and the Project of Frontier Study of

Foundation, CAS (KSCX2-EW-R-11, KSCX2-EW-J-23)

*Corresponding author, E-mail: [email protected]

Isolation and identification of symbiotic bacteria from the skin, oral, and rectal of wild and captive tree shrews 493


In this study, the isolated symbiotic bacteria from the body surface, oral cavity and anus of wild and third generation captive tree shrews were identified based on their morphological, cultural, physiological and bioche-mical characteristics, as well as the results of 16S rDNA whole sequence analysis. These findings not only pro-vide basic data in setting microorganism standard of laboratory tree shrews but also are metagenomic refe-rence of studies on the symbiotic bacteria in tree shews.


Experimental animals Adult (12-month-old, body weight=130−150 g),

healthy wild (n=12, six males and six females) and the third generation captive tree shrews (n=12, six males and six females) were provided by the Laboratory Animal Center of Kunming Institute of Zoology, CAS. Wild tree shrews were captured from the western suburb of Kunming, Yunnan, China, and their symbiotic bacteria were sampled on the same day of capture. The rearing environment of captive tree shrews was well ventilated, at 16−25 in temperature, 40%−80% in humidity, 12 h/ 12 h (0800h−2000h lights on) in light/dark cycle. Tree shrews were fed a grain premixture (main ingredients include corn meal, wheat flour, fish meal, milk powder, bone meal, sugar, salt, yeast, dregs of beans and vitamins), fruits (apples and bananas), terzebrio molitors and cooked eggs. The laborotary animal production and utilization numbers are SCXK (Yunnan) K2012-0001 and SYXK (Yunnan) K2012-0003, respectively.

Experimental reagents and facilities

Columbia blood agar bases, Salmonella Shigella agar bases and Mac Conkey agar bases were all from OXIOD (UK); Biochemical identification tubes were from Biome-rieux (France); Luria Bertani (LB) agar bases were homemade.

The instruments and facitities used in this study incl-ude PCR instrument (Biorad Mycycler), CO2 incubator (The-rmo scientific Series 8000DH), thermostatical rocking plate (COS-211C), sterile operation platform (BAKER A2), auto-matic autoclave (THA-3560C), transmission electron micr-oscope (HITACHI, H-7650), spectrophotometer (mode 721).

Experimental methods

Symbiotic bacteria from the body surface, oral cavity and rectum of wild and third generation captive tree shrews were sampled, cultured and isolated. The isolated bacteria stains were identified and systematically

classified based on their morphological, cultural, physiological and biochemical characteristics, combining the results of 16S rDNA whole sequence analysis. The flowchart of experimental procedures (approve number: SYBW20110416-1) is shown in Figure 1.

Figure 1 Flowchart of experimental procedures

Bacteria sampling and pure culture Next to the alcohol burner inside of the sterile

operation platform, symbiotic bacteria from the body surface (sampling a bit of fur by a sterile biceps), (using sterile cotton swab) and rectum (using sterile cotton swab) of 24 tree shrews were sampled. Body surface and oral specimens were inoculated onto the Columbia blood agar bases and LB agar bases, whereas, rectal specimens were inoculated onto the SS agar bases and Mac Conkey agar bases. Bacterial species can be initially and roughly identified according to their growth situations on differ-ent agar bases. Innoculated agar bases were cultured in 37 incubator. The morphology, color, hemolysis and pigments of the colonies were observed 18−72 h later. Single colony (numbered from hs1 to hs12) was picked by a sterile toothpick and placed on a fresh surface for futher indentification.

Morphological identification

Gram-staining used in this study was ammonium oxalate crystal violet staining (Katznelson et al, 1964). Specimens were fixed in 2.5% glutaraldehyde, stained with 0.1mol/L tungstophosphoric acid and then observed under a transmission electron microscope.

Biochemical characteristics identification

Main biochemical indexes of isolated bacteria strain were determined via the biochemical identification tubes. Strains cultured overnight at 30 were inoculated into

the biochemical identification tube via the sterile inoculating loop.Every strain inoculation were triplicated. Negative control tubes were void of bacteria. Results were read within 12−72 h. Positive results went through

494 LI, et al.


a three-generation continuous inoculation. Indentification conclusions were obtained by comparing the data against the characterics of other close bacteria strains.

Physiological characteristics identification

The physiological characters of bacteria strains mainly include growth temperature, pH tolerance range and optimal pH, as well as salt endurance.

Fresh bacteria fluid (5%) was inoculated onto the improved LB liquid agar bases, shaking cultured by thermostatical rocking plates (180 r/min) at 5, 10, 25, 30, 35, 37, 40, 42, 45 and 50 , respectively. OD600 light obsorption values were obtained at 24, 48, 72 h, respectively, to determine the growth range at different temperatures.

To obtain the optimal pH value, fresh bacteria fluid (5%) were inoculated onto the improved LB liquid agar bases with pH at 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and shaking cultured at 30 , 180 p/min. OD600 light absorption values were obtained at 24, 48, 72 h, respectively, to determine the pH tolerance range.

Fresh bacteria fluid were inoculated onto the improved LB liquid agar bases with different NaCl concentrations (0%−10%) and cultured at 30 for 48 h, then their OD600 light absorption values were obtained to determine their salt endurance range and the optimal salt concentration.

16S rDNA sequence analysis

The unindentified bacteria strains were under pure cultures and small scale amplifications. Universal primers were designed based on the high conservativeness of 16S rDNA sequence in prokaryotes and produced by Sangon Biotech (Shanghai, China) (forward: 5'-AGAGTTTGA TCCTGGCTCAG-3'; reverse: 5'-AAG GAGGTGATCC AAGCCGCA-3'). Bateria DNA were templates and the PCR products were larger than 1 400 bp. The reaction solution (50 µL) included 2.5 ng templated DNA, 10 µL forward primer, 10 µL reverse primer, 0.25 µL TaKaRa (5 U/µL), 5.0 µL 10× PCR Buffer (Mg2+ Free), 4.0 µL MgCl2 dNTP mixture and 35.75 µL sterile distilled water. PCR reactions were pre-denaturation at 94 for 4 min, denaturation at 94 for l min, renaturation at 52 for l min, extension at 72 for 2 min, incubate at 72 for 10 min after 30 cycles and then kept at 8 . PCR products were detected by 1% agarose gel electrophoresis and then sequenced by Genscript (Nanjing, China).

16S rRNA sequences were obtained via Chromas and DNASTAR.Lasergene.v7.1, and then went through the phylogenetic analysis via Blast (NCBI) (http:

www.ncbi. nlm.nih.gov/Blast/).

Bacteria identification The isolated bacteria stains were identified and syst-

ematically classified based on their morphological, cultu-ral, physiological and biochemical characteristics, combi-ning the results of 16S rDNA whole sequence analysis.

RESULTS

Identification results of the isolated symbiotic strains The results of staining microscopy, physiological

characteristics and 16S rDNA whole sequence analysis of the isolated symbiotic bacteria were shown in Table 1.

High homologies were found by comparing the 16S rDNA sequences of these isolated strains with the gene sequences of the online registed stains (http: //www.ncbi. nlm.nih.gov). When the results of phylogenetic tree show that an isolated unknown strain has more than 99% homology with a known strain, then according to its morphology, physiological and biochemical characte-ristics, this isolated unknown strain can be identified.

Physiological and biochemical characteristics of the isolated bacterial strains

The physiological and biochemical characteristics of the isolated bacterial strains, from hs1 to hs12, were shown in Table 2 to Table 13, respectively.

Detected bacteria from the wild (Table 14) and third generation captive tree shrews (Table 15)

In this study, 12 bacteria species were identified from the wild tree shrews, body surface: B. subtilis (42%), P. aeruginosa (25%), S. aureus (33%), S. epidermidis (75%), M. luteus (25%) and K. gibsonii (17%); oral cavity: N. mucosa (58%), S. pneumoniae, (17%); anus: E. faecalis (17%), L. lactis (33%), E. coli (92%) and S. typhosa (17%). Four bacteria specises were identified from the third generation captive tree shrews, body surface: S. epidermidis (75%); oral cavity: N. mucosa (67%); rectum: L. lactis (33%) and E. coli (100%). S. epidermidis, N. mucosa, L. lactis and E. coli were the major symbiotic bacteria strains of tree shews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa are species-specific bacteria strains of tree shrews.

DISCUSSION

Symbiotic bacteria affect many aspects of the host’s physiological conditions, including the growth and development, gene expression, metabolism, biochemical



Table 1 Results of staining microscopy, physiological characteristics and electrophoregram of the isolated symbiotic bacteria

Microscopy Temperature ()pH

tolerence rangeNaCl agar base (%)

Bacteria Gram’s

stain Morphology

Brood body

Flagellum CapsuleGrowth range

Optimal Growth range

Optimal

Electrophoregram

B. subtilis + Rhabditiform; paired or in chains; round or squre ends

Y 5−50 25−30 6.5−9.5 0.6−1.2 1.0

E. coli −

Brevibacterium; median sized; blunt ends; scattered or paired

N 5−40 20−30 6.5−9.5 0.1−1.0 0.6

E. faecalis + Globular or oval N Y N 10−45 37 4.2−4.6 0.1−6.5 4.0

P. aeruginosa

−

Thallus slender and in various sizes; shape in rod or linear; paired or in short chains

N Y 25−42 25−30 4.2−4.6 0.1−6.5 4.0

K. gibsonii + Blunt ends; scattered N Y 25−42 25−30 4.2−4.6 0−6.0 4.0

N.mucosa − Small thallua, reniformed; in dual rank

N Y 25−38 30 7.0−7.5 0−6.0 4.0

S.epidermidis + N N N 20−45 37 6.0−9.0 1.0−10.0 5.0

S. epidermidis

+ Globular or slightly oval; aciniformed

N N N 25−40 37 4.5−9.8 1.0−8.5 3.0

L. lactis + Globular or oval N N 5−40 30 6.5−9.8 0.1−7.5 2.0

M. luteus + Cellular spherical; paired, in quadruplet or clustered

N 5−40 25−37 5.5−9.0 0.1−7.5 2.5

S. pneumoniae

+ Globular or oval; paired or in chains

N N N 5−40 37 6.5−8.5 0.1−8.5 4.0

S. typhosa − Brevibacterium; blunt ends; scattered

N N 5−40 35 6.5−9.5 0.1−1.0 0.6

+: Positive in Gram’s stain; −: Negative in Gram’s stain; Y: Presence; N: Absence.

496 LI, et al.


Table 2 Physiological and biochemical characteristics of hs1

Characteristics Results Characteristics Results

Catalase + Nitrate-reducing +

Carbamide − Amylohydrolysis +

Gelatin liquefaction + Citrate +

Sucrose + D-Xylose +

D-mannitol + D-Mannose +⁄−

Sorbierite − Maltose −

D-raffinose − L-pectinose +

+: Positive or capable of utilizing; −: negative or incapable of utilizing; +/−:

Subtle differences between strains of one genus.



Mannite + V-P test −

Lactose + Phaseomannite −

Esculiw + D+ (−) glycogen −

Maltose + Salicin −

Glucose + Methyl red +

Raffinose + Mushroom sugar +

Arabinose + Peroxidase +

Citrate − Tyrosine −

Xylose + Indole +

Carbamide − Melibiose +

Phenylalanine − Oxidase −

H2S − Casein protein breakdown

−

Fructose + Amylohydrolysis +

Sorbierite + Gelatin liquefaction −

+: Positive or capable of utilizing; −: Negative or incapable of utilizing.



Lactose + Hemolytic +

Maltose + Catalase −

Mannite + Sorbierite +

Glucose + 10% gail +

Raffinose − 40% gail +

Xylose − Salicin +

Arabinose − B-Galactose glucoside enzyme +

Rhamnose − Oxidase −

Amylase − Arginine hydrolysis +

Esculin hydrolysis + Sucrose +

Nitrate-reducing +




Maltose − Glutin +

Mannite − Mushroom sugar −

Sucrose − Xylose −

Galactose − Acetamide +

Carbamide + H2S −

Arabinose − Nitrate-reducing +

Fructose − Oxidase +

Indole − Arginine hydrolysis +

Glucose; acid-producingGlucose; aerogenesis

+ −

Citrate +

+: Positive or capable of utilizing; − : Negative or incapable of utilizing.



Arabinose − Mannite −

V-P test − Seminose −

Esculin hydrolysis − Indole −

Rhamnose − Fucose −

Lactose − A-methyl-D-glucoside −

Methyl red − Nitrate-reducing −

Fructose − Acetate −

Sucrose − Sorbose −

Raffinose − Phaseomannite −

Maltose − Β-galactosidase −

Galactose − Melibiose −

Sorbierite − Glycerol −

Glucose; acid-producing Glucose; aerogenesis

− Citrate −

Urease − Gelatin liquefaction −

Amylase − Oxidase −

Melampyrite − Catalase +




Sucrose + Nitrate +

Glucose + Nitrite +

Peroxidase + Oxidase +

Maltose: acid-producing Aerogenesis

+

Indole −

Lactose −






Robiocina + Sucrose +

Plasma-coagulase + lactose +

Oxidase + maltose +

Carbamide + glucose +

Methyl red + arginine +

Mannite + V-P test Weak +

Gelatin Liquefaction

+ nitrate +




Gelatin liquefaction + Glucose +

V-P test + Mannite −

Urease − Sucrose +

Plasma-coagulase − Lactose +

H2S − Maltose +

M．R + Fructose +




Arginine hydrolase + Maltose +

Glucose − Melibiose −

Amylase − Galactose +

Indole − Lactose +

Methyl red + Ribose +

Oxidase − Fructose +

Glutin − Melizitose −

Catalase − Raffinose −




Mannite − Lactose −

Gelatin hydrolysate + Inorganic nitrogen agar +

Glycerol − Glucose −

Oxidase + Arginine −

Catalase + Nitrate −

Esculin hydrolysis + Citrate −




Starch + Synanthrin −

Mannite − Lactose −

50% gall + Maltose +

Sucrose + Arabinose +

Glucose − Sorbierite +

Raffinose −




Arabinose − Sucrose −

Ornithine + Mannite: acid-producing Aerogenesis

+ +

Maltose + Carbamide −

D-tartrate −

Lactose − A-methyl-D-glucoside −

V-P test − Melampyrite −

Sorbierite − Methyl red +

Glucose: acid-producing Glucose: aerogenesis

+

+ Phenylalanine −

Fructose − Glutin −

Indole − Citrate +

+: Positive or capable of utilizing; −: Negative or incapable of utilizing. changes and immunity. Therefore, it is important to understand the classifications of symbiotic bacteria.

The symbiotic bacteria hosted in tree shrews are affected by the living environment of microorganisms

The symbiotic bacteria hosted in tree shrews are diversified and complicated, and are greatly affected by the living environment of microorganisms. In this study, 12 bacteria strains were isolated and identified from the wild tree shrews. B. subtilis widely exsits in earth and soiled organisms, and multiplies quickly in hay infusion. In this study, B. subtilis was identified only from the body surface of wild tree shrews, with detectable rate at 50%, indicating it is a commonly hosted bacterium in wild tree shrews. P. aeruginosa, which was indentified from wild tree shrews with detectable rate at 25% in this study, is one of the most commonly found bacteria in earth. S. aureus is highly pathogenic and exsits in air, water, dirt and excretions, whereas, S. epidermidis breeds on body surfaces and is a normal flora. The detectable rates of these two Staphlococcus bacteris, which were 33% and 75%, respectively, are probably due to their

498 LI, et al.


Table 14 Detected bacteria of 12 wild tree shrews

Sampling area Numbers of the detected bacteria (n) Genus Species

Body surface Oral Rectal

Detectable rate (%)

Bacillus B. subtilis 3 2 42

Escherichia E. coli 6 5 92

Enterococcus E. faecalis 1 1 17

Pseudomonas P. aeruginosa 1 2 25

Kurthia K. gibsonii 1 1 17

Kurthia N. mucosa 3 4 58

Staphlococcus S. aureus 2 2 33

Staphlococcus S. epidermidis 5 4 75

Lactococus L. lactis 2 2 33

Micrococcus M. luteus 1 2 25

Streptococcus S. pneumoniae 1 1 17

Salmonella S. typhosa 1 1 17

: Positive; : Negative.

Table 15 Detected bacteria of 12 third generation captive tree shrews

Sampling area Numbers of the detected bacteria (n) Genus Species

Body surface Oral Rectal Detectable rate (%)

Escherichia E. coli 6 6 100

Neisseria N. mucosa 4 4 67

Staphlococcus S. epidermidis 5 4 75

Lactococus L. lactis 2 2 33

: Positive; : Negative.

wide distrbutions in natural environments. M. luteus can be found in air, earth, water, as well as on the body surface of animals and plants. This conditioned pathogen may cause local or severe infections in tissues. K. gibsonii aften grows in animal excretions or meat products and so far, there are no reports of K. gibsonii as a pathogen. In this study, 17% K. gibsonii were detected from the body surface of wild tree shrews. The detectable rate of oral N. mucosa was 67%. It is mainly hosted in the oral mucosa of mammals and is a non-pathogenic bacterium. S. pneumoniae exists in natural environments, animal excretions and the pharynx nasalis of healthy human beings and was detected in two wild tree shrews. E. faecalis was only found in wild tree shrew rectums. L. lactis can be found in diary and plantation products. Our results show that its detectable rates in both wild and third generation tree shrew were both 33%, indicating its probiotic advantages in tree shrews. E. coli are widely distributed in natural environments and mostly are non-panthogenic. Although the detectable rates of Escherichia coli were extremely high in this study (92%−100%), we consider them as intestinal tract normal flora, because all the experimental tree shrews were healthy and did not show any clinical disorders. S. typhosa is one of the bacteria that can easily cause infections and exists in almost every natural environment, including the air,

water, food, vegetable, dirt and animal excretions. We only found Salmonella typhosa in two wild tree shrews, indicating it is a rare strain in tree shrews.

The influences of artificial environments to the sym-biotic bacteria hosted in tree shrews

The succeful artifical reproductions of tree shrews require several aspects the artifical environments, including the facilities, nutritions, as well as the proper managing and reproducing methods (Li et al, 2009; Jiang et al, 2011; Zhao et al, 2013). The long-term and frequent application of sterilization measures determined that less bacteria exist in artifical environments than in wild.

In this study, four bacteria strains were isolated and identified from the third generation of captive tree shre-ws, suggesting that the living environments are signif-icantly correlated with the species of microorganisms.

As a novel animal to study human diseases and from the angle of evolution, to understand the similarities and differences of symbiotic bacteria from the same boby part of human and tree shrewsis necessary. Now, Human Microbiome Project (HMP) has carried out detailed investigations on the bacteria flora of human skin, oral cavities and intestinal tracts to determine the factors influencing the growth of symbiotic bacteria, such as gender, race, geography distribution, diet and body weight.



CONCLUSIONS

The tree shrews used in this study were all in good healthy condition at time of sampling and 30 days after sampling. Therefore, we assume that the indentified bacteria are major parasitic bacteria of tree shrews. The high detectable rate indicates that E.coli is the major normal parasitic bacteria, which is consistant with the study of Gao et al (2009). In other conventional laboratory animals, Staphlococcus bacteria is rare; however, in both wild and third generation captive tree shrews, its detectable rates were high (75%). Our results also show a high occurrence of pathogens in wild tree shrews (17%−33%), whereas no pathogens were found in the third generation captive tree shrews.

As a novel laboratory animal, the applications of tree shrews are still lack of national standards (Shen et al, 2011). For example, for the conventional animal model rhesus monkeys (Macaca mulatta), it is clearly pointed out that laboratory rhesus monkeys have to be clean of Salmonella spp. Pathogenic dermal fungi and Cam-pylobaceter jejuni. Wang et al (1987) reported that C.

jejuni and Shigella exsit in the intestinel tract of healthy adult tree shrews. So far, no Brucella spp., Leptospira spp., Mycobacterium tuberculosis or Yersinia enteroc-olitica have been found in tree shrews, suggesting that bacteria strains may function species-specifically in different parts of different animals.

In recent years, the advantages of tree shrews as a novel animal model have become apparent, because they are close to primates, they have been widely applied in studies on virus (Li et al, 2011; Wang et al, 2012b), neuronal peptide Y (Dong et al, 2011), disbetes (Wu et al, 2013), depression (Wang et al, 2012a), cerebral ischemia (He et al, 2011), etc. Therefore, it is nessassary and to set standards to normalize tree shrews as a laboratory animal model.

Based on the enriched tree shrew resources of Yunnan Province, our lab for the first time has successfully isolated and identified the symbiotic bacteria of the third generation captive tree shrews. These findings not only provide basic data in setting the microorganism standard of laboratory tree shrews but also are metagenomic reference of studies on the symbiotic bacteria in tree shews.

References

Dong L, Lü LB, Lai R. 2011. Molecular cloning of Tupaia belangeri chinensis neuropeptide Y and homology comparison with other analogues from primates. Zoological Research, 33(1): 75-78.

Gao JH, Jang QF, Luo ZW, Sun XM, Dai JJ. 2009. Culture, isolation and identification of normal intestinal bacterial flora and their antibiotic susceptibility in tree shrew. Chinese Journal of Comparative Medicine, 19(12): 24-26.

He B, Han D, He L, Zhang FY, Li SQ. 2012. The research of magnetic resonance imaging of thrombotic cerebral ischemia in tree shrews. Journal of Clinical Radiology, 31(10): 1492-1496.

Huang XY, Xu J, Sun XM, Dai JJ. 2013. Development of application of tree shrew in human disease animal models research. Laboratory Animal Science, 30(2): 59-64.

Jiang QF, Kuang DX, Tong PF, Sun XM, Dai JJ. 2011. Scale breeding of tree shrews and the establishment of breeding population. Laboratory Animal Science, 28(6): 35-38.

Katznelson H, Gillespie DC, Cook FD. 1964. Studies on the relationships between nematodes and other soil microorganisms. 3. Lytic action of soil myxobacters on certain species of nematodes. Canadian Journal of Microbiology, 10, 699-704.

Li G, Yan Y, Chang YY, Lü LB. 2009. New method of the tree shrews (Tupaia belangeri chinensis) breeding in laboratory. Husbandry and Veterinary of Modern, (3): 18-21.

Li Y, Dai JJ, Sun XM, Xia XS. 2011. Progress in studies on HCV receptor of Tupaia as a potential hepatitis C animal model. Zoological Research, 32(1): 97-103.

Shen PQ, Zheng H, Liu RW, Chen LL, Li B, He BL, Li JT, Ben KL, Cao YM, Jiao JL. 2011. Progress and prospect in research on laboratory tree shrew in China. Zoological Research, 32(1): 109-114.

Simpson GG. 1945. The principles of classification and a classification

of mammals. Bull Amer Mus Nat Hist, 95: 1-22.

Wang J, Zhou QX, Lü LB, Xu L, Yang YX. 2012a. A depression model of social defeat etiology using tree shrews. Zoological Research, 33(1): 92-98.

Wang WG, Huang XY, Xu J, Sun XM, Dai JJ, Li QH. 2012b. Experimental studies on infant Tupaia belangeri chineses with EV71 infection. Zoological Research, 33(1): 7-13.

Wang XJ, Yang C, Su JJ. 2010. Development of application of tree shrew in experimental medical research. Chinese Journal of Comparative Medicine, 20(2): 67-70.

Wang XX, Li JX, Wang WG, Sun XM, He CY, Dai JJ. 2011. Preliminary investigation of viruses to the wild tree shrews (Tupaia belangeri chinese). Zoological Research, 32(1): 66-69.

Wang YC, Jin HX, Tang YM, Liao GY, Xu JY. 1987. Analysis of intestinal pathogen for the healthy tree shrews. Medical Biology Research, 1: l4.

Wu XY, Li YH, Chang Q, Zhang LQ, Liao SS, Liang B. 2013. Streptozotocin induction of type 2 diabetes in tree shrew. Zoological Research, 34(2): 108-115.

Xing J, Feng YF, Fu R, Yue BF, Sun XM, Dai JJ, He ZM. 2012. The survey of culturable bacteria and fungi in wild tree shrews. Laboratory Animal Science, 29(3): 34-38.

Xu L, Zhang Y, Liang B, Lü LB, Chen CS, Chen YB, Zhou JM, Yao YG. 2013. Tree shrews under the spot light: emerging model of human diseases. Zoological Research, 34(2): 59-69.

Zhang CJ, Shi M, Chen F, Chen L, Shen PQ, Bao FK, Li ML. 2009. Isolation and identification of common bacteria in tree shrews. Journal of Pathogen Biology, 4(12): 899-900.

Zhao Y, Wu TT, Li YF, Sun Y, Sun H, Hu LN, Qu HH, Wang QG. 2013. Breeding management method of long-term artificial cultivation of tree shrew in Beijing area. Chinese Journal of Comparative Medicine, 23(2): 64-68.



Acoustic signal characteristic detection by neurons in ventral nucleus of the lateral lemniscus in mice Hui-Hua LIU, Cai-Fei HUANG, Xin WANG*

College of Life Sciences and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, China

Abstract: Under free field conditions, we used single unit extracellular recording to study the detection of acoustic signals by neurons in the ventral nucleus of the lateral lemniscus (VNLL) in Kunming mouse (Mus musculus). The results indicate two types of firing patterns in VNLL neurons: onset and sustained. The first spike latency (FSL) of onset neurons was shorter than that of sustained neurons. With increasing sound intensity, the FSL of onset neurons remained stable and that of sustained neurons was shortened, indicating that onset neurons are characterized by precise timing. By comparing the values of Q10 and Q30 of the frequency tuning curve, no differences between onset and sustained neurons were found, suggesting that firing pattern and frequency tuning are not correlated. Among the three types of rate-intensity function (RIF) found in VNLL neurons, the proportion of monotonic RIF is the largest, followed by saturated RIF, and non-monotonic RIF. The dynamic range (DR) in onset neurons was shorter than in sustained neurons, indicating different capabilities in intensity tuning of different firing patterns and that these differences are correlated with the type of RIF. Our results also show that the best frequency of VNLL neurons was negatively correlated with depth, supporting the view point that the VNLL has frequency topologic organization.

Keywords: Ventral nucleus of the lateral lemniscus; Firing pattern; Frequency tuning; Intensity tuning; Mouse

The ventral nucleus of the lateral lemniscus (VNLL) is the largest nucleus of the lateral lemniscus (LL) (Batra & Fitzpatrick, 2002). As a station in the auditory ascending pathway, the VNLL plays a vital role in acoustic signal processing by receiving afferent projections from the contralateral ventral cochlear nucleus (VCN), ipsilateral trapezoid body (TB), superior olivary complex (SOC) and contralateral dorsal cochlear nucleus (DCN), and casts efferent projections into the inferior colliculus (IC) (Benson & Cant, 2008; Schofield & Cant, 1997; Kelly et al, 2009; Huffman & Covey, 1995; Prather, 2013).

Electrophysiological studies on the VNLL indicate that VNLL neurons can detect, process and encode the characteristics of acoustic signals and have various responding reactions. The firing patterns of VNLL neurons include onset firing (instantaneous firing to the onset of acoustic signals) and sustained firing (sustained firing during the whole interval of acoustic signals) (Zhang & Kelly, 2006). Recently, it was reported that the firing pattern and ability of VNLL neurons to detect

acoustic signal characteristics are closely correlated. For example, the first spike latency (FSL) of onset neurons is short and with increasing sound intensity,1 the FSL shor-tens insignificantly, whereas those of the sustained neurons are opposite (Zhang & Kelly, 2006). The minimal threshold (MT) of onset neurons is also higher than that of sustained neurons (Aitkin et al, 1970). Regarding firing tuning in VNLL neurons, the value of Q10 (Q10=best frequency/the band width at 10 dB above threshold) is used to assess the sharpening of the frequency tuning cures (FTC). Studies on different animals show that the value of Q10 in onset neurons is smaller than in sustained neurons (Aitkin et al, 1970; Covey & Casseday, 1991). Regarding intensity tuning in VNLL neurons, the dynamic range (DR, according to the Received: 15 March 2014; Accepted: 01 June 2014

Foundation items: This study was supported by the National Natural

Science Foundation of China (31000493) and the Central China

Normal University Independent Scientific Research Project Fund for

Youth Scholars (11A01025)

*Corresponding author. E-mail: [email protected]

Acoustic signal characteristic detection by ventral nucleus neurons in the lateral lemniscus in mice 501


rate-intensity function, RIF, of neurons, DR is usually set at 10% higher than the smallest firing spikes and 10% lower than the highest firing spikes) is used to assess intensity tuning abilities. The study on Dutch-belted rabbits (Oryctolagus cuniculus) indicates that the DR of onset neurons is smaller than that of sustained neurons (Batra & Fitzpatrick, 1999).

We assume that the firing pattern, intensity and frequency of VNLL neurons have some sort of conne-ction. Despite being an animal model for acoustic studies, detection characteristics and frequency topologic organ-ization of VNLL neurons to acoustic signals in Kunming mice (Mus musculus) remain unclear. Here, using single unit extracellular recording, we observed the firing patterns of VNLL neurons and compared FSL, frequency and intensity tuning characteristics under different firing patterns. Our results are helpful in more fully understanding correlations between VNLL neuron firing patterns and acoustic signal detection characteristics, and exploring the physiological functions of VNLL neurons and the effects of ascending projections on the IC.

MATERIALS AND ANIMALS

Animal surgery and electrode preparation Kunming mice (n=54, 18−28 g) with normal

hearing were obtained from the Laboratory Animal Center, Institute of Hubei Preventive Medicine, China. Animal surgical procedures are referenced in previous studies (Wang et al 2007; Tang et al, 2007). All experi-ment procedures were performed strictly in accordance with the guidelines published in the NIH Guide for the care and use of Laboratory Animals. Anesthesia was induced by injection of mebumal sodium (45−50 mg/kg, bw). After the mouse was immobilized in a stereotaxic apparatus, the VNLL was exposed (4.16−4.48 mm posterior to the bregma, 1.10−1.95 mm lateral to the midline, 2.85−4.10 mm in dorsal ventral depth) by removing skull and dura over the VNLL (Paxinos & Franklin, 2001). An electrode was inserted into the small hole (200−500 μm in diameter) over the VNLL. After surgery, the mouse was transferred onto a shake proof bench in the anechoic shielded chamber and its head was immobilized on a homemade bracket with the aid of a tack (1.8 cm in length) stuck on the skull. The animal’s eyes, ears and the center of the speaker were at the same level. Background noise inside the anechoic shielded chamber was 29 dB SPL and the temperature was set at 25−28 .

During the experiment, 0.6% mebumal sodium solution was used to keep the animal anesthetized and paralyzed.

By referencing previous studies (Cheng et al, 2013; Mei et al, 2012), a homemade bipolar tungsten electrode made of two insulated tungsten wire electrodes (FHC lnc, Bowdoin, ME, USA) was used to record neuronal responses of the VNLL and to confirm the recording site after experimentation. The electrode was <10 μm in diameter, 1−5 MΩ in impedance and the two poles were 30−80 μm apart.

Acoustic signal system and neuronal response recor-ding

Under free field conditions, the speaker was placed at 0° in the pitching position, and 60° in the contralateral meridional direction (opposite to the animal’s external auditory canal opening). Acoustic signals were produced by the Tucker-Davis Technology System 3 (Alachua, USA). Parameters, such as frequency, intensity and duration were controlled by computer and calibrated by a sound meter (2610, B&K, Denmark) and 1/4-inch microphone (4936, B&K, Denmark). Acoustic signals were tone bursts with a duration of 40 ms, rise and fall of 5.0 ms, and a frequency of 2 stimuli/s. The bipolar tungsten electrode was vertically inserted into the VNLL via a hydraulic drive (Kopf 640, USA). Tone burst was adopted to locate acoustic signal sensitive neurons. The responding characteristics of the neurons of the dorsal nucleus of the lateral lemniscus (DNLL) (unpublished data) were used as parallel comparisons to improve accuracy when locating the nucleus. Neuronal responses were amplified by bioelectric amplifier (ISO-DAM, WPI, USA) via the bipolar tungsten electrode, displayed on the oscilloscope (PM3084, FLUKE, USA), monitored by the monitoring apparatus (AM9, GRASS, USA) and then stored in a computer. The depth, best frequency (BF) and MT of the acoustic signal sensitive neuron were recorded. Each tone burst stimulus was repeated 32 times and the induced neuronal firings were profiled into a peri-stimulus time histogram (PSTH). Under the BF, starting from the MT, firing times with the intensity of acoustic signal increasing every 10 dB segment were recorded to infer the RIF. Meanwhile, the highest and lowest frequencies of the neuron were found and their FTCs were inferred.

Electrical burning and histological examination

At the end of the experiment, the recording site in

502 LIU, et al.


the anesthetic mouse was burned for 10 s by a 0.1 mA direct current for precise location later. Current stimula-tion was generated by a stimulator and introduced to the mouse via a direct current stimulation isolator (Model SEN-7203, Nihon Kohden Co, Shinjuku, Tokyo, Japan) and the bipolar tungsten electrode. After a 2 h perfusion with 200 mL saline water and 4% paraformaldehyde, the brain was isolated and fixed in 4% paraformaldehyde for 24 h. Regular paraffin sections (40 μm) were prepared with a vibration slicing machine (VT 1000S, Leica CO, USA) and under Nissl’s staining (toluidine blue) for immunohistochemistry examination. The burned location on the section was observed under a light microscope (Leica, DM4000B, Germany) and photographed.

Statistical analysis

All statistical analyses were completed using SPSS 13.0 and all figures were generated with Sigmaplot 10.0. Data are expressed as mean±SD. One-way ANOVA and t-tests were used to compare differences.

RESULTS

Characteristics of VNLL neurons For the 98 VNLL neurons we recorded in this study,

the dorsal ventral depth was 3 368±156.2 μm (2 844−4 090 μm); BF was 19.89±11.12 kHz (2.6−39 kHz); MT was 52.66±13.11 dB SPL (13.83−71.61 dB SPL); and

FSL was 5.78±2.79 ms (2−18.5 ms). Linear regression analysis showed linear relationships between BF and dorsal ventral depth (R=−0.36, P<0.001) (Figure 1A). We found that neurons sensitive to high frequencies are located in the dorsal VNLL. From dorsal to ventral, the BF of VNLL neurons decreased, indicating frequency topological organization along the dorsal-ventral-axis of the VNLL. No linear relationships were found among FSL, MT and dorsal ventral depth (R=0.1, P>0.05; R=0.06, P>0.05) (Figure 1B, C).

Temporal firing pattern of VNLL neurons

As one of the basic features of central auditory neurons, the temporal firing pattern reflects firing changes in neurons before and after stimulation. With acoustic signals at BF and MT+20 dB, the firing patterns of 98 VNLL neurons were recorded. Following the research of Zhang & Kelly (2006), our observed firing patterns were categorized into two types: onset and sustained. The onset type can be sub-categorized into: (1) phasic: only one or two spikes to the onset of the aco-ustic signals; (2) phasic burst: several spikes to the onset of the acoustic signals; and (3) double burst: fixed intervals can be found between the spikes to the onset of the acoustic signal. Likewise, the sustained type can also be sub-categorized into: (1) onset plus sustained: significant response can be found to the onset of the acoustic signals,

Figure 1 Correlations between recording depth and BF (A), FSL (B) and MT (C) in VNLL neurons and Nissl-stained coronal section of the VNLL and surrounding structures (D)

n: Numbers of neurons; R: correlation coefficient.



then decreases and lasts through the entire duration; (2) tonic: a fixed firing rate lasts through the entire duration; and (3) primary-like: the firing rate to the onset of the acoustic signals quickly reaches maximum, then decreases to a certain level. The representative post-stimulus time histograms (PSTH) of these six firing patterns and neuron numbers are shown in Figure 2.

Among them, phasic burst had the highest percentage, followed by tonic, phasic and onset plus sustained, respectively; primary-like and double burst had the smallest percentages. Although differences were found among the percentages of various firing patterns, the quantities of onset (52/98, 53.06%) and sustained (46/98, 46.94%) patterns were comparable.

Figure 2 Types of onset (A-1, A-2, A-3) and sustained (B-1, B-2, B-3) patterns of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3118, 53.83, 16.2 (A-1); 3739, 64.28, 35 (A-2); 3970, 66.79,

10.6 (A-3); 2908, 38.14, 13.7 (B-1); 4090, 54.22, 3.4 (B-2); 3042, 3.14, 17.8 (B-3), respectively.

Under intensity at MT+20 dB, the FSL of onset

VNLL neurons was 5.47±1.86 ms (2−11 ms), with 67.31% of the FSL within the range of 2.5−5 ms; the FSL of sustained VNLL neurons was 7.1±3.13 ms (2−19.5 ms) with a relatively wide distribution (Figure 3A, C). The FSL of onset VNLL neurons was shorter than that of sustained neurons (P<0.001, unpaired t-test). When the intensity of the acoustic signal increased every 10 dB segment to MT+50 dB, no significant changes in the FSL of onset VNLL neurons were observed (one-way ANOVA, P>0.05), whereas, the FSL of sustained neurons decreased (one-way ANOVA, P<0.001) (Figure 3B, D).

Frequency tuning of VNLL neurons

Frequency is a basic parameter of acoustic signals.

Auditory neurons analyze and encode external acoustic

stimulation, which play an important role in the behavior

of humans and other animals. FTC are commonly used in

assessing the frequency tuning of auditory neurons.

According to Suga (1997) and our previous data, the

FTC of the 71 VNLL neurons recorded here were

divided into six types: (1) V-shape: with increasing

intensity, the verges of high and low frequency are

broadening; (2) U-shape: the changes in FTC are

independent of intensities [the V- and U-shape can be

differentiated by bandwidth level intolerance (BLI,

BLI=BWmax/BW10), if BLI<3, it is U-shape, if BLI≥3,

it is V-shape]; (3) Lower-tail-upper-sharp (LTUS): the

low frequency verge is characterized with a long tail and

the high frequency verge is very sharp; (4) Upper-tail-

lower-sharp (UTLS): the high frequency verge is charac-

terized with a long tail and the low frequency verge is

very sharp [in LTUS and UTLS, the SlopeMT+30−MT+10≥30%,

otherwise, if SlopeMT+30−MT+10<30%, the FTC is V- or U-

shape]; (5) W-shape: two peaks can be found in FTC and

the two frequency tuning segments are partitioned by an

auditory completely insensitive area only under low

intensity, but not under high intensity; and (6) Double V-

shape: two peaks can be found in FTC and the two

frequency tuning segments are partitioned by an auditory

completely insensitive area under both low and high

intensities (Figure 4).

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Figure 3 Distribution of FSL in onset neurons (A) and sustained neurons (C) and FSL-intensity function in onset neurons (B) and sustained neurons (D)

Abscissa of B and D were normalized by each neuron’s MT as the control of tone intensity; numbers above the vertical bars in B and D are the numbers of

neurons calculated statistically.

Figure 4 Types of frequency tuning curves of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3114, 32.22, 16.8 (A); 3925, 49.63, 11.2 (B); 3001, 61.66, 25.5

(C); 3513, 55.81, 11.5 (D); 3009, 58.03, 40.3 (E); 3179, 67.09, 51.8 (F), respectively.

Among the 71 neurons, 38 were onset and 33 were sustained. The MT of the onset neurons was 55.33±10.83 dB SPL (27.09−69.34 dB SPL) and that of the sustained neurons was 48.83±12.85 dB SPL (13.83−73.54 dB SPL) (P<0.05, unpaired t-test) (Figure 5 A). The value of Q10 of the FTC of onset neurons was 2.49±1.67 (0.65−7.01) and that of the

sustained neurons was 3.43±3.83 (0.8−19.26) (P>0.05, unpaired t-test), whereas, the value of Q30 of the FTC of onset neurons was 0.94±0.52 (0.26−2.45) and that of the sustained neurons was 0.96±0.26 (0.13−1.99) (P>0.05, unpaired t-test) (Figure 5 B, C), indicating that the firing patterns and frequency tuning of VNLL neurons are irrelevant.



Figure 5 Differences in the MT (A), Q10 value (B) and Q30 value (C) between onset neurons and sustained neurons

*: P<0.05.

Intensity tuning of VNLL neurons

Intensity reflects the amplitude of acoustic vibration. Within certain range, most of the auditory neurons increase spiking with increasing intensity. Under relatively high intensity, different neurons show different changes in firing rate function. With an acoustic signal at BF, 40 ms, and intensity increases every 10 dB segment, the firing numbers of VNLL neurons changed and showed various characteristics of intensity tuning. Accor-ding to previous work and our own data (Tang et al, 2007; Zhou & Jen, 2000), under increasing intensity, if the firing numbers keep increasing, neurons are recognized as monotonic RIF; if the firing numbers do not change or decrease to <25%, neurons are recognized as saturated RIF; and if the firing numbers decrease to≥25%, neurons are recognized as non-monotonic RIF. The representative curves of these three types of RIF and numbers of neurons are shown in Figure 6. The monotonic RIF had the highest percentages, followed by saturated and then non-monotonic RIF.

Of the 91 VNLL neurons we recorded in this study, 49 were onset and 42 were sustained. The percentages of monotonic, saturated and non-monotonic RIF of onset neurons were 48.98% (24/49), 42.86% (21/49) and

8.16% (4/49), respectively; of sustained neurons the percentages were 61.9% (26/42), 21.43% (9/42) and 16.67% (7/42), respectively (Figure 7A). Therefore, in onset and sustained neurons, the distribution patterns of RIF from high to low were monotonic, saturated and non-monotonic, respectively. The DR of the onset neurons was smaller than that of sustained neurons (P<0.01, unpaired t-test). In neurons with monotonic and saturated RIF, the DR of the onset neurons was smaller than sustained neurons (P<0.01; P<0.05, unpaired t-test); in neurons with non-monotonic RIF, no differences were found between onset and sustained neurons (P>0.05, unpaired t-test) (Figure 7B).

DISCUSSION Firing patterns and temporal features of VNLL neurons

The percentages of onset and sustained patterns were comparable, 53.06% (52/98) and 46.94% (46/98), respectively, consistent with previous findings on big brown bats (Eptesicus serotinus) (Covey & Casseday, 1991), rats (Zhang & Kelly, 2006) and rabbits (Batral & Fitzpatrick, 1999). The 2.04% (2/98) double-burst pattern we found has only been reported in rats (Zhang &

Figure 6 Types of rate-intensity function of recorded VNLL neurons n: Numbers of neurons; Recording depth (μm), MT (dB SPL) and BF (kHz) of these neuron are 3403, 50.52, 33 (A); 3259, 27.09, 24.8 (B); 3211, 38.89, 19.8

(C), respectively.

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Figure 7 Differences in percentage of RIF (A) and DR (B) between onset neurons and sustained neurons

M: monotonic; Sat: saturated; NM: non-monotonic. *: P<0.05; **: P<0.01.

Kelly, 2006) and its biological meaning in acoustic signal processing remains unclear.

The FSL of onset neurons was shorter than sustained neurons. With the increase in intensity, the FSL of onset neurons remained stable (Figure 3B), indicating a feature of precise timing of onset neurons. This specific feature of onset VNLL neurons may be attributable to projections of octopus cells in the cochlear nucleus (CN) (Adams et al, 1997; Pollak et al, 2011). The octopus cell have large, irregularly shaped cell bodies with three to five thick primary dendrites emerging from only one side of the cell body. The sizable low voltage activated potassium channels on its membranes endow the cell with a low impedance and fast time constant, which bestow its projections to the VNLL with temporal advantages (Johnston et al, 2010; Bal & Ocrtel, 2007). The precise timing of onset VNLL neurons is not only beneficial in the encoding of acoustic signal onset, but also affects the process of IC neurons to temporal information via its ascending projections. For example, studies on rats (Nayagam et al, 2005), Mexican free-tailed bats (Tadarida brasilensis mexicana) (Xie et al, 2007) and big brown bats (Voytenko & Galazyuk, 2008) show that an inhibitory postsynaptic potential (IPSP) can be observed ahead of the excitations of some IC neurons, which means an IPSP occurs before depolarization. It is assumed that because of the temporal advantage of VNLL neurons possess, the inhibitory projection from VNLL neurons will reach the IC ahead of the excitatory projection, thereafter, an early IPSP is initiated.

The FSL of sustained VNLL neurons decreased with increasing intensity (Figure 3D). This phenomenon is not specific to sustained VNLL neurons (Zhang & Kelly, 2006) and has also been observed in the CN (Goldberg & Brownell, 1973), SOC (Sanes & Rubel, 1988), IC (Tan et al, 2008) and auditory cortical (AC) (Heil, 1997). Therefore, it is assumed that FSL encodes the intensity of the onset of the acoustic signal. The larger

the intensity, the shorter the FSL, and the quicker the signal can be recognized. However, correlations between FSL and intensity are unclear. Pharmacological studies show that neitherγ-aminobutyric acid (GABA) nor glyc-ine (Gly) affects correlations between FSL and intensity in AC and IC neurons (Zhang et al, 2005), indicating that these correlations may be encoded by auditory neurons below the IC, but this requires further exploration.

Frequency tuning and frequency topologic organiza-tion of VNLL neurons

Although the percentages of six different types of FTC are consistent with previous studies on rats (Zhang & Kelly, 2006) and rabbits (Batra & Fitzpatrick, 1999), the degrees of sharpening are quite different among different species. For example, in this study, the average value of Q10 of the FTC of VNLL neurons in Kunming mice was 2.9, but was 7.1 (Zhang & Kelly, 2006) in rats and 9.1 (Covey & Casseday, 1991) in bats, respectively, indicating that different species are characterized with different capabilities of frequency tuning. The sharp-ening of FTC and GABAergic inhibitory projection are correlated with the formation of the inhibitory area of frequency tuning (Chen & Jen, 2000). The FTC of VNLL neurons in mice have smaller Q10 and low degrees of sharpening. Because the VNLL in mice receives less inhibitory projections from the ipsilateral trapezoid body, but has a wide frequency receptive range to excitatory projections from the contralateral cochlear nucleus, perhaps the FTC of VNLL neurons is not characterized by significant sharpening.

No differences were found in the Q10 of the FTC in onset and sustained neurons (Figure 5). However, studies on big brown bats and cats show that the Q10 in onset neurons is smaller than sustained neurons (Covey & Casseday, 1991; Aitkin et al, 1970). Based on these discrepancies, it is assumed that different species have different neural circuits corresponding to specific behaviors.



For example, because the frequency tuning of the onset VNLL neurons in Mexican free-tailed bats affects the reor-ganization of IC neurons to some special communication signals, they show certain corresponding firing patterns and features of frequency tuning (Pollak et al, 2011).

Frequency topological organization of VNLL neu-rons remains controversial. When the IC of rats was infected with retrograde tracing agents, low frequency neurons in the VNLL distributed along the band, whereas, high frequency neurons were evenly distributed (Kelly et al, 1998). Electrophysiological experiments on bats (Covey & Casseday, 1991) and cats (Aitkin et al, 1970) show that low frequency neurons are distributed dorsally of the VNLL, whereas, high frequency neurons are distributed ventrally of the VNLL. Although differences exist in the distribution patterns of different species, the logical distribution of low and high frequency neurons supports the view of frequency topologic organization in the VNLL. However, an electrophysiological experiment on rats (Nayagam et al, 2006) and the study on infecting retrograde tracing agents in the IC of cats (Glendenning & Hutson, 1998) and the primary auditory field (AI) of Mongolian gerbils (Budinger et al, 2013) indicate a lack of topologic organization in the VNLL. Possible reasons of these discrepancies are differences in animal models and experimental methods. In this study, the BF and dorsal ventral depth of VNLL neurons were negatively correlated (Figure 1A). Neurons with high frequencies were located dorsally of the VNLL and from dorsal to ventral, the BF of neurons decreased, indicating frequency topologic organization along the dorsal-ventral-axis of the VNLL.

Intensity tuning of VNLL neurons

In the 91 VNLL neurons recorded here, the percentage of non-monotonic RIF (12.09%) was lower than that in the advanced auditory nuclei of mice, such as the IC (Tang et al, 2007) and AC (Qi et al, 2013). The occurrence of non-monotonic type is due to inhibitory

input tuning (Chen & Jen, 2000; Andrew & Ellen, 2009 Wu et al, 2006; Tang et al, 2008). The low level of non-monotonic RIF in VNLL neurons may be consistent with less inhibitory inputs in the VNLL. Previous histoc-hemical studies show that excitatory projections in the VNLL are mainly from the CN (Suneja et al, 1995), whereas low levels of inhibitory projections are mainly from the TB (Yavuzoglu et al, 2010).

The intensity tuning of onset and sustained neurons were compared and values of DR were used as an index to assess the sensitivity of neurons to acoustic signal intensity. The smaller the DR, the narrower the intensity tuning and the higher the sensitivity, meaning that variation in intensity can induce significant changes in the neuronal spiking of humans and other animals. The smaller DR in onset neurons compared to sustained neurons indicates different sensitivity in these two types of neurons and implies that the intensity encoded by neuronal impulses is affected by neuronal firing patterns. Although previous studies have reported similar phenomena in the VNLL (Batra & Fitzpatrick, 1999), DNLL (unpublished data) and IC (Bal et al, 2002), if differences in DR can be observed among neurons with different firing patterns then correlations with RIF remain unclear. The DR of two neuron types with three different RIF was compared. In monotonic and saturated RIF, the DR of onset neurons was smaller than sustained neurons, but in non-monotonic RIF, no differences were found (Figure 7B), indicating that DR is correlated with RIF type and in general, onset neurons are more sensitive than sustained neurons in intensity tuning.

The firing patterns of VNLL neurons are affected by multiple factors and truthfully reflect their ability in inte-nsity tuning, The firing patterns of certain VNLL neurons change with changes in acoustic signals (Zhang & Kelly, 2006) indicating that their firing pattern and intensity are regulated by different signal pathways and integrations of excitatory and inhibitory inputs within the time window.

References

Adams JC. 1997. Projections from octopus cells of the posteroventral cochlear nucleus to the ventral nucleus of the lateral lemniscus in cat and human. Auditory Neuroscience, 3(4): 335-350.

Aitkin LM, Anderson DJ, Brugge JF. 1970. Tonotopic organization and discharge characteristics of single neurons in nuclei of the lateral lemniscus of the cat. Journal of Neurophysiology, 33(3): 421-440.

Andrew K, Ellen C. 2009. Functional role of GABAergic and glycinergic inhibition in the intermediate nucleus of the lateral lemniscus of the big brown bat. Journal of Neurophysiology, 101(6): 3135-3146.

Bal R, Oertel D. 2007. Voltage–activated calcium currents in octopus cells of the mouse cochlear nucleus. Bal R, Oertel D. 2007. Voltage–

508 LIU, et al.


activated calcium currents in octopus cells of the mouse cochlear nucleus. Journal of the Association for Research in Otolaryngologyl, 8(4): 509-521.

Bal R, Green GG, Rees A, Sanders DJ. 2002. Firing patterns of inferior colliculus neurons-histology and mechanism to change firing patterns in rat brain slices. Neuroscience Letters, 317(1): 42-46.

Batra R, Fitzpatrick DC. 1999. Discharge patterns of neurons in the ventral nucleus of the lateral lemniscus of the unanesthetized rabbit. Journal of Neurophysiology, 82(3): 1097-1113.

Batra R, Fitzpatrick DC. 2002. Monaural and binaural processing in the ventral nucleus of the lateral lemniscus: a major source of inhibition to the inferior colliculus. Hearing Research, 168(1-2): 90-97.

Benson CG, Cant NB. 2008. The ventral nucleus of the lateral lemniscus of the gerbil (Meriones unguiculatus): organization of connections with the cochlear nucleus and the inferior colliculus. The Journal of Comparative Neurology, 510(6): 673-690.

Budinger E, Brosch M, Scheich H, Mylius J. 2013. The subcortical auditory structures in the Mongolian Gerbil: II. frequency–related topography of the connections with cortical field AI. The Journal of Comparative Neurology, 521(12): 2772-2797.

Chen QC, Jen PH. 2000. Bicuculline application affects discharge patterns, rate-intensity functions, and frequency tuning characteristics of bat auditory cortical neurons. Hearing Research, 150(1-2): 161-174.

Cheng L, Mei HX, Tang J, Fu ZY, Jen PHS, Chen QC. 2013. Bilateral collicular interaction: modulation of auditory signal processing in frequency domain. Neuroscience, 235: 27-39.

Covey E, Casseday JH. 1991. The monaural nuclei of the lateral lemniscus in an echolocating bat: parallel pathways for analyzing temporal features of sound. The Journal of Neuroscience, 11(11): 3456-3470.

Glendenning KK, Hutson KA. 1998. Lack of topography in the ventral nucleus of the lateral lemniscus. Microscopy Research and Technique, 41(4): 298-312.

Goldberg JM, Brownell WE. 1973. Discharge characteristics ofneurons in anteroventral and dorsal cochlear nuclei of cat. Brain Research, 64: 35-54.

Heil P. 1997. Auditory cortical onset responses revisited. I. First spike timing. Journal of Neurophysiology, 77(5): 2616-2642.

Huffman RF, Covey E.1995. Origin of ascending projections to the nuclei of the lateral lemniscus in the big brown bat, Eptesicus fuscus. The Journal of Comparative Neurology, 357(4): 532-545.

Johnston J, Forsythe LD, Kopp–Scheinpflug C. 2010. Going native: voltage–gated potassium channels controlling neuronal excitability [J]. The Journal of Physiology, 588(17): 3187-3200.

Kelly JB, van Adel BA, Ito M. 2009. Anatomical projections of the nuclei of the lateral lemniscus in the albino rat (Rattus norvegicus). The Journal of Comparative Neurology, 512(4): 573-593.

Kelly JB, Liscum A, van Adel B, Ito M. 1998. Projections from the superior olive and lateral lemniscus to tonotopic regions of the rat's inferior colliculus. Hearing Research, 116(1-2): 43-54.

Mei HX, Cheng L, Tang J, Fu ZY, Wang X, Jen PHS, Chen QC. 2012.

Bilateral collicular interaction: modulation of auditory signal processing in amplitude domain. Plos One, 7(7): e41311.

Nayagam DA, Clarey JC, Paolini AG. 2005. Powerful, onset inhibition in the ventral nucleus of the lateral lemniscus. Journal of Neurophysiology, 94(2): 1651-1654.

Nayagam DA, Clarey JC, Paolini AG. 2006. Intracellular responses and morphology of rat ventral complex of the lateral lemniscus neurons in vivo. The Journal of Comparative Neurology, 498(2): 295-315.

Paxinos G, Franklin KBJ. 2001. The Mouse Brain in Stereotaxic Coordinates. 2nd ed. San Diego: Academic Press.

Pollak GD, Burger RM, Klug A. 2003. Dissecting the circuitry of the auditory system. Trends in Neurosciences, 26(1): 33-39.

Pollak GD, Gittelman JX, Li N, Xie R. 2011. Inhibitory projections from the ventral nucleus of the lateral lemniscus and superior paraolivary nucleus create directional selectivity of frequency modulations in the inferior colliculus: a comparison of bats with other mammals. Hearing Research, 273(1-2): 134-144.

Prather JF. 2013. Auditory signal processing in communication: perception and performance of vocal sounds. Hearing Research, 305: 144-155.

Qi QZ, Si WJ, Luo F, Wang X. 2013. Intensity tuning of neurons in the primary auditory cortex of albino mouse. Progress in Biochemistry and Biophysics, 40(4): 365-373(in Chinese)

Sanes DH, Rubel EW. 1988. The ontogeny of inhibition and excitation in the gerbil lateral superior olive. The Journal of Neuroscience, 8(2): 682-700.

Schofield BR, Cant NB. 1997. Ventral nucleus of the lateral lemniscus in guinea pigs: cytoarchitecture and inputs from the cochlear nucleus. The Journal of Comparative Neurology, 379(3): 363-385.

Suga N.1997. Tribute to yasuji katsuki's major findings: sharpening of frequency tuning in the central auditory system. Acta Otolaryngologica Supplement, 532: 9-12.

Suneja SK, Benson CG, Gross J, Potashner SJ. 1995. Evidence for glutamatergic projections from the cochlear nucleus to the superior olive and the ventral nucleus of the lateral lemniscus. Journal of Neurochemistry, 64(1): 161-171.

Tan AY Y, Atencio CA, Polley DB, Merzenich MM, Schreiner CE. 2007. Unbalanced synaptic inhibition can create intensity-tuning auditory cortex neurons. Neuroscience, 146(1): 449-462.

Tan X, Wang X, Yang W, Xiao Z. 2008. First spike latency and spike count as functions of tone amplitude and frequency in the inferior colliculus of mice. Hearing Research, 235(1-2): 90-104.

Tang J, Xiao ZJ, Shen JX. 2008. Delayed inhibition creates amplitude tuning of mouse inferior collicular neurons. Neuroreport, 19(15): 1445-1449.

Tang J, Wu FJ, Wang D, Jen PHS, Chen QC. 2007. The amplitude sensitivity of mouse inferior collicular neurons in the presence of weak noise. Chinese Journal of Physiology, 50: 187-198.

Voytenko SV, Galazyuk AV. 2008. Timing of sound–evoked potentials and spike responses in the inferior colliculus of awake bats.



Neuroscience, 155(3): 923-936.

Wang X, Jen PHS, Wu FJ, Chen QC. 2007. Preceding weak noise sharpens the frequency tuning and elevates the response threshold of the mouse inferior collicular neurons through GABAergic inhibition. Brain Research, 1167: 80-91.

Wu GK, Li P, Tao HW, Zhang L. 2006. Nonmonotonic synaptic excitation and imbalanced inhibition underlying cortical intensity tuning. Neuron, 52(4): 705-715.

Xie R, Gittelman JX, Pollak GD. 2007. Rethinking tuning: in vivo whole–cell recordings of the inferior colliculus in awake bats. The Journal of Neuroscience, 27(35): 9469-9481.

Yavuzoglu A, Schofield BR, Wenstrup JJ. 2010. Substrates of auditory

frequency integration in a nucleus of the lateral lemniscus.

Neuroscience, 169(2): 906-919.

Zhang H, Kelly JB. 2006. Responses of neurons in the rat's ventral

nucleus of the lateral lemniscus to monaural and binaural tone bursts.

Journal of Neurophysiology, 95(4): 2501-2512.

Zhang J, Qiu Q, Tang J, Xiao ZJ, Shen JX. 2005. The effect of

bicuculline and strychnine on the latency of neuron inferior colliculus

and auditory cortex of BALB/c mouse. Progress in Biochemistry and

Biophysics, 32(11): 1055-1060.

Zhou XM, Jen PHS. 2000. Corticofugal inhibition compresses all types

of rate-intensity functions of inferior collicular neurons in the big

brown bat. Brain Research, 881: 62- 68.



Morphological analysis of the Chinese Cipangopaludina species (Gast-ropoda; Caenogastropoda: Viviparidae)

Hong-Fa LU1,†, Li-Na DU2,†, Zhi-Qiang LI1,*, Xiao-Yong CHEN3,*, Jun-Xing YANG3,*

1. Zhejiang Normal University, Xiaoshan Campus, Hangzhou 311231, China

2. Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

3. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

Abstract: Viviparidae are widely distributed around the globe, but there are considerable gaps in the taxonomic record. To date, 18 species of the viviparid genus Cipangopaludina have been recorded in China, but there is substantial disagreement on the validity of this taxonomy. In this study, we described the shell and internal traits of these species to better discuss the validity of related species. We found that C. ampulliformis is synonym of C. lecythis, and C. wingatei is synonym of C. chinensis, while C. ampullacea and C. fluminalis are subspecies of C. lecythis and C. chinensis, respectively. C. dianchiensis should be paled in the genus Margarya, while C. menglaensis and C. yunnanensis belong to genus Mekongia. Totally, this leaves 11 species and 2 subspecies recorded in China. Based on whether these specimens’ spiral whorl depth was longer than aperture depth, these species or subspecies can be further divided into two groups, viz. chinensis group and cathayensis group, which can be determined from one another via the ratio of spiral depth and aperture depth, vas deferens and number of secondary branches of vas deferens. Additionally, Principal Component Analysis indicated that body whorl depth, shell width, aperture width and aperture length were main variables during species of Cipangopaludina. A key to all valid Chinese Cipangopaludina species were given.

Keywords: Cipangopaludina; Gastropoda; Bellamyinae; Anatomy; Taxonomy; Chinese

The freshwater gastropod Viviparidae is currently divided into three subfamilies (Bouchet & Rocroi, 2005): Viviparinae Gray, 1847, Bellamyinae Rohrbach, 1837 and Lioplacinae Gill, 1863, which together comprise approximately 150 species and 31 genera recognized to date (Franke et al, 2007). Typically, viviparid species inhabit lakes, ponds, and lentic rivers of temperate to tropical region (Strong et al, 2008; Ying et al, 2013), explaining their wide global distribution across every continent save for South America and Antartica (Brown, 1994). In China, the existing taxonomy for Viviparidae includes approximately 61 recognized species in 9 genera (Du et al, 2011; Liu, 1991; Wang & Xie, 2005; Yen, 1943): Viviparus Montfort, 1810, Bellamya Jousse-aume, 1886, Filopaudina Habe, 1964, Mekongia Crosse et Fischer, 1876, Angulyagra Benson, 1836, Margarya Nevill, 1877, Rivularia Heude, 1890 and Trochotaia Brandt, 1974, Cipangopaludina Hannibal, 1912. The last genus, Cipangopaludina is particularly interesting as a

diverse genus mainly distributed in China, Japan, Korea, Thailand, Vietnam, Laos, India, Burma and Malaya (Brandt, 1974; 1Liu et al, 1993) that is comprising of 35 species and subspecies (Global Names Index; http:// gni.Globalnames.org), there has been substantial debate on the taxonomy of Cipangopaludina. Hannibal (1912) had previously proposed Cipangopaludina as a subgenus of Idiopoma, based on invasive specimens collected in California which were identified as Paludina malleata

Received: 02 April 2014; Accepted: 20 July 2014

Foundation items: This research was funded by the National Natural

Science Foundation of China (31301865); the Natural Science Foun-

dation of Zhejiang Province of China (LY12C19006) and the Collec-

tion and Preparation of Display Specimens at Kunming Natural

History Museum of Zoology (KSZD–EW–TZ–005) *Corresponding authors, E-mail: [email protected]; chenxy@mail.

kiz.ac.cn; [email protected] †Authors contributed equally to this work

Morphological analysis of the Chinese Cipangopaludina species (Gastropoda; Caenogastropoda: Viviparidae) 511


Reeve. Meanwhile, Annandale (1920) proposed Lecyth-oconcha as a distinct genus for Paludina lecythis Benson while Prashad (1928) argued that Lecythoconcha was invalid and adopted the name Cipangopaludina.

Even within China, there is no clear verdict on the validity of each species or how many are even distributed in the area. Heude (1890) listed 9 species from China, viz. C. chinensis (Benson (non Gray)), C. lecythoides (Heude), C. diminuta (Heude), C. longispira (Heude), C. fluminalis (Heude), C. leucostoma (Heude), C. cath-ayensis (Heude), C. ventricosa (Heude) and C. aubryana (Heude). Later, comprehensive work by Kobelt (1909) revised Viviparidae, including practically all the forms of the family known at the time, which was one species and 12 subspecies in China, with most of species being designated varieties of the species C. chinensis (Gray): cathayensis (Heude), compacta (Nevill), ventricosa (Heude), longispira (Heude), fluminalis (Heude), hainanensis (Kobelt), diminuta (Heude), leucostoma (Heude), aubryana (Heude), patris (Kobelt), wingatei (Smith), lecythoides (Benson) and its form latissima (Dautzenberg et H. Fischer). Several decades later, Yen (1943) revised species of Chinese Viviparidae, and pointed out that leucostoma and diminuta were synon-yms of chinensis and that lecythoides (Heude, non Benson) distributed in Zhoushan Island, compacta (Ko-belt, non Nevill) distributed in Hainan, and lecythis crassior (Annandale) distributed in western Yunnan were all synonyms of patris (Kobelt), and longispira (Heude) was argued to be synonymous with haasi. Liu (1991) later clarified the distribution and taxonomy of Cipangopaludina and raised 15 of the subspecies to the species level, viz. C. chinensis, C. cathayensis, C. fluminalis, C. aubryana, C. latissima, C. lecythoides, C. ussuriensis (Gerstfeldt), C. yunnanensis, C. dianchiensis, C. ampullacea, C. haasi, C. lecythis, C. longispira, C. ventricosa and C. menglaensis. Finally, Wang & Xie (2005) evaluated the extinction risk of Cipangopaludina species, listing 14 distinct species: Cipangopaludina hainanensis, C. leucostoma, C. menglaensis, C. latissima, C. dianchiensis, C. lecythis C. lecythoides, C. haasi, C. ampullacea, C. ventricosa, C. ampulliformis, C. aubryana, C. longispira and C. ussuriensis. Curiously, these latest works by Liu (1991) and Wang & Xie (2005) made no mention of two species, C. patris and C. wingatei, and ignored some of the earlier work done by Yen (1943).

Despite discrepancies and arguments as well as the

elevation and demotion of species to subspecies and vice versa, the existing literature names 18 potential species of Cipangopaludina distributed in China: C. ampullacea, C. ampulliformis, C. aubryana, C. cathayensis, C. chinensis (synopsis C. leucostoma and C. diminuta), C. dianchiensis, C fluminalis, C. haasi (synopsis C. longispira), C. hainanensis (synopsis C. compacta), C. latissima, C. lecythoides, C. lecythis, C. menglaensis, C. patris, C. ussuriensis, C. ventricosa, C. wingatei and C. yunnanensis. Similarly, the disparate results of the previous studies on the topic also highlight how difficult and complex the identification of Cipangopaludina is, largely due to intraspecific variations in shell shape. This has left the taxonomic position and phylogenetic relationships of these species largely unknown. In this study, we opted to perform a fresh check on the morphology and anatomy of Chinese Cipangopaludina species currently being held at repositories in Beijing and Kunming in order to arrive at a more complete species list. Morphometric analyses of shell variations were conducted on Cipangopaludina species through a Principal Component Analysis.


Sample collection and identification Four hundred and ninety-six, including 11 Cipang-

opaludina species were collected from Jilin, Jiangxi, Zhejiang, Guangxi and Yunnan Provinces. These specim-ens were preserved in Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ). Additionally, 145 Cipangopaludina specimens, including 18 Cipangop-aludina species or its synonyms listed in the existing literature preserved in the Invertebrate Museum of the Institute of Zoology, Chinese Academy of Sciences, Beijing (IOZ) were measured. A morphological analysis was conducted on these specimens and the validity of the currently identified species was determined. Specimens were identified mainly based on original description, type specimens, Liu et al (1979, 1993), Kobelt (1909) and Prashad (1928).

Characters selected for morphometric analysis

To select characters for use in morphometric anal-ysis, we reviewed the available taxonomic descriptions from literature (Liu et al, 1979). Five shell characters were measured with callipers accurate to 0.1 mm: Shell depth (D), the maximum dimension parallel to the axis of coiling; Shell width (W) the maximum dimension

512 LU, et al.


perpendicular to D; Length of the aperture (LA), the maximum dimension from the junction of the outer lip with the penultimate whorl to the anterior edge of the aperture (oblique to coiling axis); width of the aperture (WA), the maximum dimension perpendicular to LA; depth of the body whorl (BW), the dimension from the lower margin of the aperture to the upper suture delimiting the first whorl; and N, the number of whorls.

Embryonic shells were measured to 0.1 mm using an ocular micrometer. Anatomy was studied using a microscope with drawing apparatus. Radulae and embry-onic shells were studied via scanning electron micro-scopy (SEM). The radulae were cleaned enzymatically with proteinase K, as described by Holznagel (1998), then sonicated and mounted on aluminium specimen stubs with adhesive pads. Embryonic shells were clea-ned mechanically, sonicated, and mounted on adhesive carbon-coated pads. Both radulae and embryonic shells were coated with gold-palladium and studied on AMR-AY 1000B scanning electron microscope at 30 kV. The description of shell features and visceral hump follows the general terminology suggested by Simone (2004).

Allometric shell growth

Due to ontogenetic effect of allometric growth have usually caused confusion in morphometric analyses, these individuals within the range of allometric growth were eventually excluded before multivariate analyses (Armbruster, 1995; Chiu et al, 2002; Valovirta & Vaisa-nen, 1986). However, in the present-day study, it is difficult for us to obtain snails of all size classes from each population per species and examine the allometric shell growth. Under this circumstance, the consequence of Chiu et al (2002) was followed that excluded individuals with aperture lengths of less than 1.0 cm. Morphometric data was explored through a Principal Component Analysis by SPSS 10.0 (SPSS for Windows, Chicago, IL, USA).

RESULTS

Systematics Cipangopaludina Hannibal, 1912 Idiopoma (Cipangopaludina) Hannibal 1912, Proc-

eedings of the Malacological Society of London 10: 194 (type species, Paludina malleata Reeve, original desig-nation)

Lecythoconcha Annandale 1920, Records of the Indian Museum. 19: 111 (type species, Paludina lecythis Benson, monotypic); Annandale 1921, Records of the

Indian Museum, 6: 401. Viviparus (Cipangopaludina), Clench & Fuller,

1965, Occasional papers on Mollusks, Museum of Com-parative Zoology, 2: 385–412.

Bellamya (Cipangopaludina), Smith, 2000, Nautilus, 114(2): 31–37.

Diagnosis: Shell medium to large (up to 70 mm), oval, brown or greenish in colour. Apex pointed or obtused. Suture narrow. Umbilicus, big or narrow or closed. Sculpture lacking except growth lines and axial undulations. Aperture subcircular, inner lip whitish blue and outer lip black in colour, easily broken. Young specimens (from brood pouch) with up to 4 whorls; protoconch smooth, three primary rows of chaetae on last whorl and other whorls have two rows of chaetae. Male’s testis with two or three main branches.

Cipangopaludina chinensis was the only species given a detailed description, while diagnosis charac-terstics are listed for all other species. All morphometric and meristic characters of Cipangopaludina species are given in Table 1.

Cipangopaludina ampullacea (Charpentier, 1863) (Figure 1A)

Paludina ampullacea Charpentier: Küster, 1852, Syste-matisches Conchylien–Cabinet. 2 ed.p. 23, pl. 4, fig 2–3.

Material: 25 ex. (2 female and 3 male were anat-omy). KIZ 000110–000117, collected from Qiubei Cou-nty, Yunnan in August 2005 by Cui GH and Du LN; KIZ 000453–000470, collected from Beihai, Tengchong County, Yunnan in April 2006 by Chen XY.

Diagnosis: shell larger, up to 53 mm, brown–greenish, with 5 whorls. Apex obtused. Spiral whorl depth is less than aperture length. Body whorl inflated, shell width about 80.1%–90.6% shell depth. Body whorl depth is less than shell width. Spiral whorls inflated and round, form a flat shoulder. Osphradium ridge-like, shorter or equal with length from anterior tip of osphradium to mantle border. The base of gill filaments wide and apex narrow and round, curve to left. Kidney arch-shape, the dorsal length of kidney right plane about 1.5 times than ventral left plane and posterior plane, and left plane and posterior plane very curve (Figure 2C-II). Vas deferens with 2 main branches and the first anterior branch with 4 parallel secondary branches. Vas deferens opens in last 1/4 of testis. Pouch filled with less than 44–48 embryos in various stages of development. Embryos with 4 whorls, body whorl with 3 chaetae and other whorls have 2 rows of chaetae.



514 LU, et al.


Figure 1 Shell of Cipangopaludina A: C. lecythis ampullacea, KIZ 000453; B: C. lecythis, KIZ000284; C: C. leucostoma, KIZ 000270; D: C. chinensis, IOZ–FG166277; E: C. chinensis fluminalis,

KIZ 000443; F: C. haasi, KIZ 000101; G: C. dianchiensis, KIZ 000536; H: C. hainanensis, IOZ–FG168161; I: C. latissima, IOZ–FG84656; J: C. lecythoides,

IOZ–FG166538; K, L: C. menglaensis, IOZ–FG0142 (Holotype); M: C. yunnanensis, IOZ–FG0152 (Holotype); N: C. aubryana, IOZ–FG 170556; O: C.

ussuriensis, IOZ–FG84819; P: C. ventricosa, KIZ000227. Scale bars=1 cm.

Figure 2 Anatomy and genital system of Cipangopaludina

A: Shell removed showing external features of animal; B: Male with mantle cavity opened mid–dorsally; C: Two type of kidney, I triple shape and II arch shape.

Abbreviations: aa — anterior aorta; ag — albumen gland; an — anal papilla; au —auricle; bp — brood pouch; cm—columellar muscle; ct — ctendium; df —

dorsal fold of buccal mass; eg — egg-shell gland; es — esophagus; ey — eye; ft — foot; int — intestine; jw — jaw; kd — kidney; l — liver; me — mantle edge; mo

— mouth; ne — nephrostome; op — operculum; pa — posterior aorta; pe—penis; pg—prostate gland; re—rectum; sn—snout; te—tentacle; ur—ureter; ve—

ventricle; vd—vas deferens.

Distribution: Southwest of China, Yunnan, Sichuan

provinces and Macao (Liu, 1991). Remarks: Cipangopaludina ampullacea can be

distinguished from C. chinensis, C. fluminalis, C. haasi, C. menglaensis, C. dianchiensis and C. ussuriensis by spiral depth shorter than aperture depth. Cipangop-aludina ampullacea can be distinguished from other species by the following characters: osphradium length

shorter or equal with length from anterior tip of osphradium to mantle border, vs. longer in C. ventricosa, C. patris and C. aubryana; shell larger, apex eroded vs. shell medium and apex pointed in C. yunnanensis; umbilicus small or closed vs. big and round in C. latissima; apex obtused vs. pointed in C. cathayensis and C. lecythoides. Cipangopaludina ampullacea can be distinguished from C. lecythis by apex eroded vs. pointed;



the upper angle of aperture acute vs. straight. However, the anatomy of C. ampullacea and C. lecythis are not obviously different, so it is suitable to treat C. ampullacea as a subspecies of C. lecythis.

Cipangopaludina aubryana (Heude, 1890) (Figure 1N)

Paludina aubryana Heude, 1890, Memoires conce-mant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 175, pl. 39.

Vivipara (chinensis) aubryana Heude, Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 118, pl. 20, fig 6–7.

Viviparus chinensis aubryana (Heude), Yen, 1939, Diese Arbeit erscheint gleichzeitig in den Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft Abhandlung, 444, p. 35, taf. 3.

Viviparus chinensis aubryana (Heude), Yen, 1942, Proceedings of the Malacological Society of London, 190, 200, pl. 13, fig 32.

Cipangopaludina chinensis aubrayana (Heude), Pradshad, 1928, Memoirs of the Indian Museum, Calc-utta, 8: 168.

Cipangopaludina lecythoides aubryana (Heude), Yen, 1943, Nautilus, 56(4): 124–130.

Cipangopaludina aubryana (Heude), Liu, 1991, Proceeding of Tenth International Malacological Con-gress, 590.

Materials: 20 ex. (4 females and 4 males were anat-omy). IOZ–FG166735, IOZ–FG166737, from Wang Co-unty, Sichuan Province in June 1964; IOZ–FG170556, IOZ–FG170553, IOZ–FG170551, IOZ–FG 170549, from Hanshou County, Hunan province in March 1963; KIZ 000087–100, from Pu’er City, Yunnan in April 2011 by Zheng LP.

Diagnosis: shell medium, brown green, with 5 who-rls. Body whorl inflated, but other whorls not, without shoulders. Apex pointed. Spiral whorl depth less than aperture depth. Osphradium ridge-like, longer than length from anterior tip of osphradium to mantle border. The base of gill filaments wide and apex narrow and pointed, does not curve to right. Kidney triple-shape, the dorsal length of kidney right plane about 1.2 times than ventral left plane, 2 times than posterior plane, the left plane and posterior plane do not curve much (Figure 2C-I). Vas deferens with 3 main branches and the first anterior branch with 4 parallel secondary branches. Vas deferens opens in last 1/3 of testis. Pouch filled with less than 10 embryos in various stages of development.

Embryos with 4 whorls, body whorl with 3 chaeta and other whorls have 2 rows of chaetae.

Distribution: Sichuan, Hunan, Guangdong, Guizhou and Yunnan Provinces, China (Liu, 1991).

Remarks: Cipangopaludina aubryana can be disti-nguished from C. chinensis, C. haasi, C. fluminalis, C. dianchiensis, C. ussuriensis and C. menglaensis by spiral depth less than aperture length and shell medium. Cipangopaludin aubryana can be distinguished from other species by the following characteristics: kidney triple-shape vs. arch-shape in C. ampullacea, C. patris and C. ventricosa; acute spiral whorl and without shoulder (vs. spiral whorl inflated and form shoulder in C. lecythoides and C. yunnanensis); shell medium and body whorl not very inflated vs. shell larger and body whorl very inflated in C. latissima; apex pointed, not eroded vs. apex eroded, with only 4 whorls in C. hainanensis; vas deferens opens in last 1/3 of testis vs. 1/4 in C. lecythis and C. l. ampullacea.

Cipangopaludina cathayensis (Heude, 1890) (Figure 1C)

Paludina catayensis Heude, 1890, Memoires conce-mant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 175, pl. 39.

Vivipara chinensis cathayensis Heude, Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 112, pl. 18, fig 5–6.

Cipangopaludina chinensis cathayensis (Heude): Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168; Yen, 1943, Nautilus, 56(4): 124–130.

Cipangopaludina cathayensis (Heude), Liu, 1991, Proceeding of Tenth International Malacological Cong-ress, 590.

Materials: 34 ex. (2 males and 2 females were anatomy) KIZ000016–17, collected from Lake Xingyun, Yunnan in July 2008 by Du LN; KIZ000422–434, collected from Lake Dianchi, Yunnan in September 2006 by Du LN, Jiang YE and Cui GH; KIZ 000076 collected from Debao County, Guangxi in August 2009 by Yu GH and Li Y; KIZ000190–196, collected from Liaoyuan City, Jilin in October 2012 by Du LN.

Diagnosis: shell shin, medium, up to 60 mm, with 6 inflated whorls, spiral whorl with shoulder. Apex pointed. Spiral whorl depth less than aperture depth. Osphradium length similar with length from anterior tip of osphr-adium to mantle border, anterior 2/3 part of osphradium ridge-like and posterior 1/3 part inflated. Gill filaments same with C. chinensis. Kidney triple-shape. Vas defer-

516 LU, et al.


ens with 3 main branches and the first anterior branch with 3–4 parallel secondary branches. Vas deferens opens in last 1/3 of testis. Female pouch filled with more than 60 embryos in various stages of development. Embryos with 3 whorls, body whorl with 3 chaeta and other whorls have 2 rows of chaetae.

Distribution: Jilin, Shanxi, Hebei, Shandong, Anhui, Jiangsu, Zhejiang, Hunan, Sichuan, Guizhou and Yunnan provinces (Liu, 1991).

Remarks: In the original description, this species was spelt catayensis, but later reviewers all spelt it cathayensis, according to the International Code of Zoological Nomenclature (1999). Catayensis is then treated as an inadvertent error, and thus this spelling is incorrect. Cipangopaludina cathayensis can be distinguished from C. chinensis, C. haasi, C. fluminalis, C. dianchiensis and C. menglaensis by spiral depth less than aperture depth. Cipangopaludina cathayensis can be distinguished from other species by the following characteristics: anterior 2/3 of osphradium ridge-like and posterior 1/3 inflated; female’s pouch with more than 60 embryos, and embryo’s have 3 whorls.

Cipangopaludina chinensis (Gray, 1834) (Figure 1D)

Paludina chinensis Gray: in Griffith & Pidgeon, 1834, Animal Kingdom, pl. 1, fig 5.

Paludina chinensis Gray: Gray in Griffith & Pidgeon, 1834, Animal Kingdom, 599.

Vivipara chinensis (Gray), Kobelt, 1909, Abbildun-gen Nach de Natur mit Beschreibngen, p111, pl 23, fig 1.

Cipangopaludina chinensis (Gray): Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168.

Viviparus chinensis chinensis (Gray): Yen, 1939, Diese Arbeit erscheint gleichzeitig in den Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft Abhandlung, 444, 35. Taf. 3.

Viviparus chinensis (Gray): Yen, 1942, Proceedings of the Malacological Society of London, 190, 200, pl. 13, fig 32.

Cipangopaludina chinensis leucostoma (Heude): Yen, 1943, Nautilus, 56(4): 124–130.

Cipangopaludina chinensis diminuta (Heude): Yen, 1943, Nautilus, 56(4): 124–130.

Bellamya (Cipangopaludina) chinensis (Gray): Smith, 2000, Nautilus, 114(2): 31–37.

Cipangopaludina chinensis (Heude): Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168; Liu, 1991, Proceeding of Tenth International Malacological

Congress, 590.

Material: 40 ex. (1 males and 2 females were anatomy). IOZ–FG166277, Shangsi Co. Guangxi Prov-ince in 19th April 1974; IOZ84521, Zhaotong City, Yun-nan Province in May 1932; IOZ166773, IOZ166777, Qionghai Lake, Xichang County, Sichuan Province in 4th July 1964; IOZ166733, Luofang village, Guangdong Province in March 1982; KIZ000164–165, Lake Erhai, Yunnan, collected by Du LN, Jiang YE and David Aldridge in 14 April 2006; KIZ 000294–310, Lake north mountain, Jilin City, Jilin Province, collected by Du LN on 13 October 2012; KIZ 000132–133, KIZ 000149–150, West Lake, Dali City, Yunnan, collected by Du LN in 13 April 2006, Jiang YE and Aldridge D; KIZ 000157–158, Lake Xinyun, Jiangchuan County, Yunnan, collected by Du LN, Jiang YE and Cui GH in September 2009; KIZ 000013015, KIZ 000038040, KIZ 0000363–366, KIZ 000421, Lake Dianchi, Kunming City, Yunnan, collected by Du LN and Aldridge D. in March 2006.

Description

Shell: large (up to 70 mm), oval, brown or greenish in colour, with 6–7 whorls. Apex pointed. Suture narrow. Umbilicus narrow or closed. Sculpture lacking except growth lines and axial undulations. Aperture subcircular, inner lip whitish blue and outer lip black in colour, easily brown. Young specimens (from brood pouch) with up to 4 whorls; protoconch smooth, three primary rows of chaetae on last whorl and other whorls have two rows of chaetae.

Operculum: Occupying entire shell aperture. Centre is brown and outer parts greenish. Nucleus sub-central, located closer to inner margin. Sculptured with conce-ntric growth lines. Inner surface glossy; scar sub-circular, close to inner margin, occupying about 2/3 of opercula area.

External morphology (Figure 2B): head and foot black. Snout cylindrical, anterior margin flat. Length of tentacles about 1.3 times longer than snout length, base at side of snout base. Ommatophore short, located between basal and middle third of outer surface of each tentacle. Foot large, sole simple. Opercular pad larger than its base in dorsal foot surface. Columellar muscle thick.

Radula: Central tooth with wide rounded major denticles and 4 smaller triangular denticles on each side. Lateral tooth with tongue-shaped major denticles and 3 minor denticles on each side. Inner marginal tooth with



tongue-shape major denticles and 3 minor denticles and marginal teeth with 10 almost equal-sized denticles.

Mantle organs (Figure 2A-B): Mantle border simple and thick. Osphradium ridge-like, very close to ctenid-ium, about same length from anterior tip of osphradium to mantle border. Ctenidium long and narrow, about same length as pallial cavity, anterior end at mantle border. Gill filaments very tall and narrow, arched towards right and apex slightly pointed, close to food groove.

Hypobranchial gland lies on left of gill margin. Right margin of mantle cavity filled by oviduct in females. Ureter runs along mantle cavity right margin in males or edging oviduct in females. Rectum on dorsal and left sides of ureter. At right end of mantle border, in females, presenting three openings, most posterior and smaller is ureter pore, anus most anterior, and female pore larger, located between anus and ureter pore.

Alimentary canal: Oval mouth, bounded by fleshy lips, ventral at anterior end of snout. Jaw plates ridge-like (Figure 2A). Esophagus with pair of dorsal, longitudinal folds, about 1.5 times of buccal mass length. Esophagus runs along the hypobranchial gland to posterior of columellar muscle, and then turns right and passes upwards in floor of pericardial chamber to reach digestive gland in upper part of visceral hump where it curves round to open into stomach. Stomach located halfway posterior to pallial cavity, posterior half immersed in digestive gland. Esophagus is inserted in posterior gastric extremity, and as gradually enlarges curves towards left. After this curve, stomach abruptly expands becoming almost as wide as whorl, posterior narrowing, without clear separation with intestine. Intestine forms loop overlying pericardial cavity and when it reaches digestive gland turns sharply forwards to continue as rectum. Rectum passes forwards on right of mantle cavity to open at anus lying just behind mantle edge.

Circulatory and excretory systems: Heart located at anterior ventral of kidney. Auricle connected with ctenidial vein just posterior to gill. Ventricle posteriorally connected to auricle by a thin tube. The size of ventricle is same as the auricle. Kidney triangular (Figure 2 C-I), with four almost plane surfaces: 1) posterior surface towards pericardium; 2) left with pallial cavity; 3) ventral right with ureter; 4) dorsal with mantle. The dorsal length of the kidney’s left plane is similar to the ventral right plane, and about two times longer than

posterior plane. Nephrostome is a round, small pore, located in centre basally inferior region of ventral surface of kidney. Ureter runs between rectum and oviduct (in females) or right pallial cavity edge (in males). Ureter pore just posterior to anus (or just posterior to female pore).

Genital system. Male (Figure 2A): Testis compact, semi-lunar, on

right of mantle cavity. Extends to upper end of mantle cavity, where apex close to pericardial cavity also connected by thin fold of membrane with lower surface of digestive gland. Testis flattened laterally, right surface abuts columellar mussel while left surface abuts right wall of ureter. Vas deferens very narrow, running on columellar and inferior ventral margin of testis, with 2 main branches, and in the anterior branch with 5–6 parallel secondary branches, the branches with many very narrow branches from different portions of testis. Vas deferens opens in last 1/3 of testis and runs to prostate gland in mantle cavity along mantle cavity floor for about 3/4 of its length; left 1/4 length of vas deferens abruptly narrows and is surrounded by very thick, muscular walls. Vas deferens runs all along the right cephalic tentacle, its inner surface relatively broad, with some inner, longitudinal folds. Vas deferens opens in tentacle tip as broad papilla with rounded tip. Right tentacle possesses deep concavity located at right from genital papilla. Papilla can retract into its concavity.

Female. Ovary grey, located in same position as testis. Ovary in close contact with posterior wall of cardiac region of stomach and along course of hepatic artery. Oviduct very narrow; from albumen gland it runs to posterior end of columella, and then turns left, making a loop. Oviduct increases in size and runs to capsule gland along brood pouch. Brood pouch located in dorsal of oviduct. Albumen gland tongue-shaped, slightly curved, capsule gland on dorsal and posterior surface and opens to brood pouch. Walls of brood pouch thin, semi-transparent, smooth. Pouch filled with about 45–84 embryos in various stages of development.

Ecology and habitat: it lives in slow-moving water such as lakes, pond, irrigation canals, ditches and slow moving stream (Pace, 1973). It is a benthic grazer and filter feeder, feeding on benthic and epiphytic diatom (Plinski et al, 1978), often found on sandy to muddy substrates.

Distribution: original range in China, Taiwan, Korea, and Japan, but has also been introduced in freshwater

518 LU, et al.


ponds and lakes in Canada, the north-eastern USA, and Europe (Soes et al, 2011).

Remarks: Although Yen (1943) mentioned that C. leucostoma (Heude) and C. diminuta (Heude) are synonymous with C. chinensis (Gray), the authors thought these two species are synonymous with C. patris (Kobelt) according to apex obtuse and less than 6 whorls. Additionally, Cipangopaludina wingatei (Smith) could be synonymous with C. chinensis according to shell size, spiral depth longer than length of aperture, and number of whorls. Cipangopaludina chinensis, C. dianchiensis, C. fluminalis, C. haasi, C. ussuriensis and C. menglaensis share the characteristic that spiral depth is longer than length of the aperture. However, C. chinensis can be distinguished from C. dianchiensis and C. ussuriensis by its shell smooth rather than having a shell with a ridge; female’s pouch has more than 80 embryos vs. less than 10 embryos in C. dianchiensis; embryo with three primary rows of chaetae on last whorl vs. 3 ridges in C. dianchiensis. Cipangopaludina chinensis can be distinguished from C. fluminalis by the following characters: shell thin vs. thick, umbilicus close vs. big and deeply.

Cipangopaludina dianchiensis Zhang, 1990 (Figure 1F)

Cipangopaludina dianchiensis Zhang, 1990, Acta Zootaxonomica Sinica, 15(1): 25–27.

Materials: 34 ex. (2 males and 3 females were anatomy). KIZ 000233, collected in November 2002 by Cui GH; KIZ 000001–KIZ 000004, collected in June 2008 by Du LN, Jiang YE and Barclay H; KIZ 000005–KIZ 000012, collected in September 2005 by Du LN and Aldridge D; KIZ 000212–KIZ 000217, KIZ 000287–KIZ 000293; KIZ 000311–KIZ 000316, collected in January 2006 by Du LN and Yuan C; KIZ 000373, KIZ 000231 collected in June 2006 by Du LN. All specimens were from Lake Dianchi.

Diagnosis: shell thick. Body whorl with 7–12 ridges (3–4 ridges clearly). Apex pointed. Osphradium ridge–like, shorter than length from anterior tip of osphradium to mantle border. Gill filaments and jaw plate are the same as C. chinensis. The size of ventricle is the same as the auricle. Vas deferens has 3 main branches and the first anterior branch has 4 “V” shaped secondary branches. Vas deferens opens in last 1/3 of testis. Pouch filled with about 6–8 embryos in various stages of development. Embryos with 5 whorls, body whorl with 3 ridges and other whorls with 1 ridge.

Distribution: only known to live in Lake Dianchi, Yunnan.

Remark: Morphology characteristics, such as shell thickness, body whorl with clear ridges, female’s pouch with few (6–8) embryos and embryo shell with ridges can be distinguished clearly from other species of genus Cipangopaludina. Additionally, phylogenetic analysis indicated that C. dianchiensis clustered with species of Margarya in Lake Dianchi (Du et al, 2013). The morp-hology and molecular results indicated that C. dianch-iensis should be placed into the genus Margarya.

Cipangopaludina fluminalis (Heude, 1890) (Figure 1E)

Paludina fluminalis Heude, 1890, Memoires concemant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 175, pl. 39.

Vivipara chinensis fluminalis Heude, Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 116, pl. 19, fig. 4–5.

Viviparus chinensis fluminalis (Heude), Yen, 1939, Diese Arbeit erscheint gleichzeitig in den Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft Abhandlung, 444, p. 35, taf. 3; Yen, 1942, Proceedings of the Malacological Society of London, 190, 200, pl. 13, fig 32.

Cipangopaludina chinensis fluminalis (Heude), Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168.

Cipangopaludina lecythoides fluminalis (Heude): Yen, 1943, Nautilus, 56(4): 124–130.

Cipangopaludina fluminalis (Heude), Liu, 1991, Proceeding of Tenth International Malacological Congress, 590.

Materials: 21 ex (1 male and 1 female were anatomy). KIZ 000181 collected from Jinping County, Honghe prefecture, Yunnan in April, 2011 by Jiang WS; KIZ 000204–000211 collected from West Lake, Dali prefecture, Yunnan in April 2006 by Du LN, Jiang YE and Aldridge D; KIZ 000384 collected from Songming County, Kunming prefecture, Yunnan in December 2005 by Du LN; KIZ 000442–000451 collected from Songming County, Kunming prefecture, Yunnan in December 2004 by Du LN.

Diagnosis: shell is thick, larger, up to 60 mm, with 7 slightly inflated whorls. Apex pointed. Spiral whorl depth longer than aperture depth. Umbilicus big and deep. Osphradium length similar with length from anterior tip of osphradium to mantle border. Gill filaments same as C.



chinensis. Kidney triple shaped, the dorsal length of kidney left plane about same length as ventral right plane, 1.5 times longer than posterior plane. Vas deferens with 3 main branches and the first anterior branch with 5–6 parallel main secondary branches. Vas deferens opens in last 1/3of testis. Pouch filled with 12 embryos in various stages of development. Embryos with 4 whorls, body wh-orl with 3 chaeta and other whorls have 2 rows of chaetae.

Distribution: Yangtze River (Yen, 1943; Liu, 1991). Remarks: Cipangopaludina fluminalis is very

similar with C. chinensis except the shell thick rather than thin; umbilicus big and deep rather than small or closed. The anatomy characteristics were relatively similar with little difference, making it is possible to treat C. fluminalis as subspecies of C. chinensis.

Cipangopaludina latissima (Dautzenberg et H. Fischer, 1905) (Figure 1I)

Paludina lecythoides latissima Dautzenberg et H. Fischer, 1905, Journal de Conchylologie, p418, pl. 10, fig 17; Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 144, pl. 29, fig 1.

Cipangopaludina lecythoides latissima (Dautzen-berg et H. Fischer), Pradshad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168.

Cipangopaludina latissima (Dautzenber et H. Fis-cher): Yen, 1943, Nautilus, 56(4): 124–130; Liu et al, 1991, Proceeding of Tenth International Malacological Congress, 590.

Materials: 15 shell specimens. IOZ–FG84653–84667, collected from Mengzi County, Yunnan in March 1927.

Diagnosis: shell is large, round, up to 53 mm, with 6 whorls. Apex pointed. Body whorl very inflated, shell width is about 85%–95% of shell depth. Due to the body whorl inflated, the upper of body whorl has a flat shoulder. Aperture ellipse. Umbilicus very big, round. Anatomy characteristics unknown.

Distribution: only known to be from Mengzi County, Yunnan.

Remarks: Cipangopaludina latissima can be distin-guished from other species of Cipangopaludina by the body whorl special inflated, and the upper of body whorl with a flat shoulder.

Cipangopaludina lecythoides (Benson, 1842) (Figure 1J)

Paludina lecythoides Benson, 1842, The Annals and Magazine of Natural History, Zoology, Botany and Geo-logy 9, 488; Philippi, 1846, Abbildungen und Beschrei-

bungen neuer oder wenig gekannter Conchylien heraus-gegeben, II. p.133, pl 2, fig 1; Küster, 1852, In Abbildu-ngen nach der Natur mit Beschreibungen. Systematisches Conchylien-Cabinet 2ed, p. 23, pl. 5, fig 1, 2; Reeve, 1862, Conchologia iconica, or, illustrations of the shell of molluscous animals, pl. 4, fig 21; Dautzenberg et H. Fischer, 1905, Journal de Conchylologie, p417–418.

Paludina (Vivipara) lecythoides Benson: Mabille et Le Mesle, 1866, Journal de Conchyliologie, p. 134; Fischer, 1891, Catalogue et Distribution géographique des Mollusques terrestres, fluviatiles et marins d’une partie de l’Indo-Chine (Siam, Laos, Cambodge, Conchi-nchine, Annam, Tonkin), p. 177.

Vivipara (chinensis var.) lecythoides (Benson), Kobelt, 1909, Abbildungen Nach de Natur mit Beschrei-bngen, p 119, pl. 23, fig 8.

Cipangopaludina chinensis lecythoides (Benson), Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 8: 168.

Cipangopaludina lecythoides (Benson), Yen, 1943, Nautilus, 56(4): 124–130; Liu et al, 1991, Proceeding of Tenth International Malacological Congress, 590.

Materials: 1 ex. KIZ 000507, collected from Hang-zhou City, Zhejiang Province in April 2013 by Lu HF;

Diagnosis: shell depth 55 mm, with 6 inflated whorls. Shell dark brown, with clearly growth lines. Apex pointed. Spiral whorl depth shorter than aperture depth. Umbilicus small, nearly closed. Vas deferens has 2 main branches and the first anterior branch with 3 parallel secondary branches. Vas deferens opens in last 1/3 of testis.

Distribution: Zhejiang Province. Remarks: Although Wang & Xie (2005) mentioned

that this species is distributed in Yunnan, it was not collected during field excursions undertaken between 2003–2008 by authors Du LN and Chen XY. Generally, the validity of this species is unclear, as it is difficult to distinguish C. lecythoides and C. cathayensis by shell morphology alone. Further anatomic characteristics are required to determine the validity of this species.

Cipangopaludina lecythis (Benson, 1836) (Figure 1B)

Paludina lecythis Benson, 1836, Journal of Asiatic Society of Bengal, 5: 745.

Paludina chinensis lecythis Benson: Nevill, 1885, Hand list of Mollusca in the Indian Museum: Gastropoda. Prosobranchia–Neurobranchia, p. 20.

Paludina ampullacea Reeve (not Charpentier), 1862, Conchologia iconica, or, illustrations of the shell of

520 LU, et al.


molluscous animals, 12. Vivipara lecythis Benson, Kobelt, 1909, Abbildungen

Nach de Natur mit Beschreibngen, p 148, pl. 30, fig 1–2. Cipangopaludina chinensis lecythis (Benson): Prashad,

1928, Memoirs of the Indian Museum, Calcutta, 8: 168. Cipangopaludina lecythis (Benson): Yen, 1943,

Nautilus, 56(4): 124–130; Liu et al, 1991, Proceeding of Tenth International Malacological Congress, 590.

Materials: 25 ex. (1 female and 2 males). KIZ 000018–000030 collected from Songming County, Kun-ming City, Yunnan in April 2006 by Du LN and Yuan C; KIZ 000044 collected from Lake Dianchi, Yunnan in April 2006 by Du LN and Yang J; KIZ 000144 collected from Lake Erhai in April 2006 by Du LN, Yang YE and Aldridge D; KIZ 000151–156 collected from Lake Xingyun, Yunnan in September by Du LN, Jiang YE and Cui GH; KIZ 000224 collected from Lake Xingyun, Yunnan in April by Du LN; KIZ 000284–286 collected from Lake Dianchi, Yunnan in March 2006 by Du LN.

Diagnosis: shell larger, up to 60 mm, brown gree-nish, with 7 whorls. Apex pointed. Spiral whorl depth sh-orter than aperture depth. Whorls inflated, with a shoul-der. Aperture ellipse, the upper of aperture form a strai-ght shoulder. Umbilicus small or closed. Osphradium ridge-like, shorter than length from anterior tip of osphradium to mantle border. Gill filaments same with C. chinensis. Vas deferens with 2 main branches and the first anterior branch with 4 parallel secondary branches. Vas deferens opens in last 1/4 of testis. Pouch filled with about 37 embryos in various stages of development. Embryos with 3–4 whorls, body whorl with 2 chaetae and other whorls with 1 chaetae.

Distribution: Yunnan, China. Burma (Kobelt, 1909). Remarks: Cipangopaludina lecythis can be distin-

guished from other species of this genus by its special aperture shape, the upper of aperture straight and form a flat shoulder. Kobelt (1909) list C. lecythis ampulliformis (Souleyet) as a subspecies of C. lecythis, which are distributed in both Burma and Yunnan, China. According to Kobelt (1909) it is difficult to distinguish between these two species, so we opted to treat C. lecythis ampu-lliformis as synonymous with C. lecythis.

Cipangopaludina patris (Kobelt, 1906) (Figure 1C)

Paludina patris Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 118, pl. 23, fig 4–5.

Cipangopaludina chinensis patris (Kobelt), Pras-had, 1928, Memoirs of the Indian Museum, Calcutta, 8:

168. Cipangopaludina lecythoides (Heude, non Benson):

Yen, 1943, Nautilus, 56(4): 124–130. Cipangopaludina compacta (Kobelt, non Nevill):

Yen, 1943, Nautilus, 56(4): 124–130. Cipangopaludina lecythis crassior Annandale: Yen,

1943, Nautilus, 56(4): 124–130. Materials: 28 ex (2 females and 2 males were

anatomy). KIZ 000268–000279, collected from Yuanji-ang County, Yunnan in January 2010 by Du LN; KIZ 000369–000372, collected from Jinning County, Yunnan in August 2006 by Du LN; KIZ 000391–000402, collected from Yuanjiang County, Yunnan in January 2013 by Du LN.

Diagnosis: shell medium, up to 50 mm, brownish, with 6 whorls. Apex obtuse. Whorls inflated, especially last two whorls, upper three whorls slightly inflated and always eroded. Spiral whorl depth shorter or equal with aperture depth. Aperture ellipse. Umbilicus closed. The shape of the kidney is similar with C. ampullacea. Osph-radium ridge-like, longer than length from anterior tip of osphradium to mantle border. Gill filaments same with C. chinensis. Vas deferens with 3 main branches and the first anterior branch with 1 “V” shape and 3 parallel sec-ondary branches. Vas deferens opens in last 1/3 of testis. The loop of oviduct exceeds the posterior margin of col-umellar muscle. Pouch filled with 96–109 embryos in various stages of development. Embryos with 3 whorls, body whorl with 3 chaetae and other whorls with 2 chaetae.

Distribution: Yunnan, Guangdong and Zhejiang pro-vinces, China.

Remarks: Cipangopaludina patris can be disting-uished from C. chinensis, C. haasi, C. dianchiensis, C. ussuriensis and C. menglaensis by a spiral depth shorter than aperture length and a medium shell. Cipang-opaludina patris can be distinguished from other species by the following characteristics: larger shell, up to 50 mm (vs. medium, up to 26 mm in C. yunnanensis); the kidney arch-shape is similar with C. ampullacea, (vs. triple-shape in C. cathayensis, C. aubryana and C. lecythoides); medium shell and body whorl are not very inflated (vs. shell larger and body whorl very inflated in C. latissima); shell with 6 whorls (vs. only with 4 whorls in C. hainanensis); the upper of aperture without straight shoulder (vs. with straight shoulder in C. lecythis).

Cipangopaludina haasi (Prashad, 1928) (Figure 1F)

Cipangopaludina chinensis haasi Prashad, 1928,



Memoirs of the Indian Museum, Calcutta, 168. Paludina longispira Heude, 1890, Memoires

concemant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 175, pl. 39.

Vivipara chinensis longispira Heude, Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 115, pl. 19, fig 3.

Viviparus chinensis longispira (Heude), Yen, 1939, Diese Arbeit erscheint gleichzeitig in den Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft Abhandlung, 444, p. 35, taf. 3.

Viviparus chinensis longispira (Heude), Yen, 1942, Proceedings of the Malacological Society of London, 190, 200, pl. 13, fig 32.

Cipangopaludina haasi (Prashad): Yen, 1943, Naut-ilus, 56(4): 124–130.

Cipangopaludina longispira (Heude), Liu, 1991, Proc-eeding Tenth International Malacological Congress, 590.

Materials: 44 ex (2 females and 2 males were anatomy). KIZ 000197–000198, KIZ 000280–000282, collected from Black dragon spring, Songming Town, Kunming, Yunnan in December 2004 by Cui GH and Chen XY; KIZ 000122–131, collected from Xiayu Village, Zhangshu City, Jiangxi Province in February 2006 by Yu GH; KIZ 00101–00108, collected from Lake Yilong, Yunnan in February 2008 by Du LN and Jiang YE; KIZ 000477–000497, collected from Hangzhou City, Zhejiang Province in April 2013 by Lu HF.

Diagnosis: shell larger, up to 65 mm, greenish, with 7 whorls. Apex pointed. Suture shallow. Spiral whorl depth higher than aperture depth. Shell width less than body whorl depth. Whorls slightly inflated, do not form shoulder. Umbilicus small. Aperture ellipse. Osphradium ridge-like, longer than the length from anterior tip of osphradium to mantle border. Gill filaments same as C. chinensis. Esophagus has pair of dorsal, longitudinal folds, about 2 times of buccal mass length. Stomach elliptical, spoon-shape. Heart located anterior ventral of kidney. Ventricle is about 2 or 3 times larger than auricle. Kidney triple-shape. Nephrostome round, small pore, located in posterior 1/5 of ventral surface of kidney. Vas deferens with 2 main branches and the first anterior branch has 8 to 9 parallel secondary branches. Vas deferens opens in last 1/4 of testis. Female’s pouch length is about 4 times longer than egg gland. Pouch filled with 128–188 embryos in various stages of development. Embryos with 4 whorls, body whorl with 3 chaetae and other whorls with 2 chaetae.

Distribution: Hunan, Jiangxi, Sichuan, Zhejiang and Yunnan Provinces, China.

Remarks: Cipangopaludina haasi, C. dianchiensis, C. chinensis, C. ussuriensis and C. menglaensis share the characteristic that spiral depth is longer than length of the aperture. However, C. haasi could be distinguished from C. dianchiensis and C. ussuriensis by shell smooth vs. shell with ridge; female’s pouch with more than 128–188 embryos vs. less than 10 embryos in C. dianchiensis; embryo with three primary rows of chaetae on last whorl vs. 3 ridges in C. dianchiensis. Cipangopaludina haasi could be distinguished from C. chinensis by the following characteristics: body whorl not inflated vs. inflated; body whorl depth same or shorter than body width vs. obviously longer. Cipangopaludina haasi could be distinguished from C. menglaensis by a larger shell, up to 65 mm vs. smaller, up to 30 mm; body whorl not inflated vs. inflated.

Cipangopaludina hainanensis (Möllendorff, 1909) (Figure 1H)

Vivipara (chinensis var.) hainanensis Möllendorff, in Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 117, pl. 19, fig. 6–7.

Cipangopaludina chinensis hainanensis (Möllend-orff): Prashad, 1928, Memoirs of the Indian Museum, Calcutta, 168.

Cipangopaludina hainanensis (Möllendorff): Yen, 1943, Nautilus, 56(4): 124–130.

Materials: 5 ex. (without specimens were anatomy). IOZ–FG 168660 collected from Ledong County, Hainan Island in December 1989; IOZ–FG 168160–168163 collected from Chang Jiang County, Hainan Island in December 1989.

Diagnosis: shell medium, up to 40mm. Apex eroded, left 4–5 inflated whorls. Suture deeply. Umbilicus big and deep.

Distribution: only known from Hainan Island. Remarks: this species was evaluated as being

extinct by Wang and Xie (2005). Existing specimens can be distinguished from other species by medium shell, apex eroded, with 4–5 inflated whorls and umbilicus big and deep.

Cipangopaludina menglaensis Zhang, Liu et Wang, 1981 (Figure 1L)

Cipangopaludina menglaensis Zhang, Liu et Wang, 1981, Acta Zootaxonomica Sinica, 6(1): 40.

Materials: 10 ex. IOZ–FG 0142 (holotype), IOZ–FG 00143–151 (Paratypes) from Manzhazai Village,

522 LU, et al.


Pu’er City, Yunnan in March 1965 by Zhang WZ and Liu YY.

Diagnosis: shell dark brown, small, up to 30 mm, with 6 whorls. Apex pointed. Suture deeply. Central tooth with wide rounded major denticles and 3 or 4 smaller triangular denticles on each side. Lateral tooth with tongue-shaped major denticles and 4 or 5 minor denticles on each side. Inner marginal tooth with tongue-shape major denticles and 3 or 4 minor denticles and marginal teeth with 20 almost equal sized denticles.

Distribution: only known from type locality. Remarks: Without anatomized specimens, the anat-

omy characteristics remain unknown. However, this species can be distinguished from other species of Cipangopaludina by a smaller shell, up to 30 mm and marginal teeth with 20 almost equal sized denticles.

Cipangopaludina ussuriensis (Gerstfeldt, 1859) (Figure 1O)

Paludina ussuriensis Gerstfeldt, 1859, Memoires des savants Strangers, 9: 507, fig 1–4; Reeve, 1862, Conchologia iconica, or, illustrations of the shell of molluscous animals, pl. 8, fig 1–4;

Vivipara ussuriensis (Gerstfeldt), Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p108, pl. 18, fig 1–4.

Cipangopaludina ussuriensis (Gerstfeldt), Yen, 1943, Nautilus, 56(4): 124–130; Liu et al, 1991, Proce-eding Tenth International Malacological Congress, 590.

Material: 1 ex. IOZ–FG84819, collected from Khanka Lake, Heilongjiang Province in 1960.

Diagnosis: shell larger, up to 54 mm, brown–gree-nish, with 6 whorls and two or three brown band. Apex obtuse. Spiral depth longer than aperture depth. Shell width is about 78.5% shell depth. Body whorl with 4 clear ridges, other whorls with 2 ridges. Umbilious small.

Distribution: Lower Amur River. Remarks: Cipangopaludina ussuriensis can be

distinguished from other species of this genus by the following character: apex obtuse, body whorl with 4 ridges and other whorl with 2 ridges.

Cipangopaludina ventricosa (Heude, 1890) (Figure 1P)

Paludina ventricosa Heude, 1890, Memoires conc-emant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 175, pl. 39.

Vivpara chinensis ventricosa (Heude): Kobelt, 1909, Abbildungen Nach de Natur mit Beschreibngen, p 114, pl. 19, fig 1–2.

Cipangopaludina chinensis ventricosa (Heude), Pradshad, 1928, Memoirs of the Indian Museum, Calc-utta, 8: 168.

Cipangopaludina ventricosa (Heude), Yen, 1943, Nautilus, 56(4): 124–130; Liu, 1991, Proceeding Tenth International Malacological Congress, 590.

Materials: 24 ex. (1 female and 2 males were anatomy). KIZ 000031 collected from Yangguang Cou-nty, Yunnan in February 2006 by Du LN; KIZ 000036–38, KIZ 000174, KIZ 000227–228, KIZ 000230 collec-ted from Lake Dianchi, Yunnan in March 2006 by Du LN; KIZ 000143 collected from Lake Erhai, Yunnan in April 2006 by Du LN, Jing YE and Aldridge A; KIZ 000175–180 collected from Jiuzai dragon spring, Yunnan in March 2006 by Du LN; KIZ 000226 collected from Lake Xingyun, Yunnan in April 2006 by Du LN; KIZ 000283 collected from Black dragon spring, Songming County, Yunnan in December 2004 by Cui GH and Chen XY; KIZ 000361–362 collected from Black dragon spring, Songming County, Yunnan in December 2005 by Du LN; KIZ 000437–441 collected from Black dragon spring, Songming County, Yunnan in April 2006 by Du LN.

Diagnosis: shell larger, medium thick, up to 65 mm, with 7 whorls. Body whorl inflated. Aperture inflated and round, but the upper angle does not forms a straight shoulder. Apex pointed. Umbilicus big and deep. Osphradium ridge-like, longer than length from anterior tip of osphradium to mantle border. Gill filaments same with C. chinensis. The dorsal length of kidney left plane about same length with ventral right plane, 1.5 times than posterior plane. Vas deferens with 3 main branches and the first anterior branch with 1 “V” shape and 3 parallel secondary branches. Vas deferens opens in last 1/4 of testis. Pouch filled with 33 embryos in various stages of development. Embryos with 3 whorls, body whorl with 3 chaetae and other whorls with 2 chaetae.

Distribution: Yunnan, Sichuan and Guizhou Provi-nces, China (Liu, 1991).

Remarks: Cipangopaludina ventricosa is similar to C. cathayensis and C. ampullacea with a slightly larger shell, apex pointed and whorls inflated. However, C. ventricosa can be distinguished from C. cathayensis by following characteristics: shell with 7 whorls vs. 6; aperture round vs. ellipse; Osphradium ridge-like, longer than length from anterior tip of osphradium to mantle border vs, anterior 2/3 part of osphradium ridge-like and posterior 1/3 part inflated, osphradium length similar



with length from anterior tip of osphradium to mantle border; Vas deferens opens in last 1/4of testis vs. 1/3. Cipangopaludina ventricosa can be distinguished from C. ampullacea by the following characteristics: upper aperture does not form straight shoulder vs. having a straight shoulder; umbilicus big and deep vs. closed; Osphradium longer than length from anterior tip of osphradium to mantle border vs. shorter; Vas deferens opens in last 1/4 of testis vs. 1/3.

Cipangopaludina yunnanensis Zhang, Liu et Wang, 1981 (Figure 1M)

Cipangopaludina menglaensis Zhang, Liu et Wang, 1981, Acta Zootaxonomica Sinica, 6(1): 40–41.

Materials: 48 ex. IOZ–FG 0152 (holotype), IOZ–FG164354–164400 (Paratypes) from Maliping village, Pu’er City, Yunnan in May 1957 by Zhang WZ and Liu YY.

Diagnosis: shell greenish, small, up to 26 mm, with 5 whorls. Apex obtuse. Shell surface with conspicuous growth lines and irregular malleation. Central tooth with wide rounded major denticles and 3 or 4 smaller trian-gular denticles on each side. Lateral tooth with tongue-shaped major denticles and 3 or 5 minor denticles on each side. Inner marginal tooth with tongue-shape major denticles and 3 or 4 minor denticles and marginal teeth with 13 almost equal-sized denticles.

Distribution: only known from type locality. Remarks: Without anatomized specimens, the

anatomy characters remain unknown. However, it can be distinguished from other species of Cipangopaludina by a smaller shell, up to 30 mm and marginal teeth with 13 almost equal-sized denticles.

Morphometric analysis by PCA

The five shell characteristics are shown in Table 1. The first two factors PCA accounted for 85.4% of the total variance (Table 2). The first factor (55.9% of the variation of the selected variables) separated the ratios W/D, BW/D, BW/LA and BW/WA (factor loadings >80). The second factor (29.5% of the variation of the selected variables) separated the WA/D and WA/LA. A scatter map was made by averaging the first and second PCA factors per species (Figure 3). In PCA 1 axis (X-axis), W and BW are positively, D and LA negatively related to X-axis, respectively. In PCA 2 axis (Y-axis), WA is positive, D and LA negatively related to Y-axis, respectively. These variances are related to the shape of

aperture and body whorl depth. The pot of C. latissima located in the right of region II, it indicated that this species has a taller body whorl and shell width, but has a shorter spiral whorl. To the contrary, C. haasi looks slimmer due to taller spiral whorl and smaller shell width.

Figure 3 Scatterplots of scores on 1st and 2nd priniciple components of Chinese Cipangopaludina

Table 2 Loadings of the first two principal components for 5 shell characteristics of Chinese Cipangopaludina species

PCA 1 PCA 2

W/D 0.832* 0.322

LA/D 0.571 0.513

WA/D 0.397 0.914*

BW/D 0.949* 0.288

WA/LA 0.078 0.858*

BW/LA 0.856* 0.001

BW/WA 0.727* –0.682

Prp. Total 55.9 29.5

* loadings> 0.80.

Additionally, C. haasi, located in left bottom of

region III, has an oval aperture. Cipangopaludina hainanensis and C. cathayensis located in the top of region I and II, they have a bigger WA and relatively smaller LA, indicating these two species have a round aperture.

Key to species of Chinese Cipangopaludina 1 Shell with brown color band and ridge ..................................................................C. ussuriensis – Shell without brown color band or ridge ................... 2 2 Apex pointed.............................................................. 3 – Apex obtuse or eroded .............................................. 11 3 The upper of body whorl form a flat shoulder ................................................................... C. latissima

524 LU, et al.


– The upper of body whorl without a flat shoulder .......4 4 Shell with 5 whorls, spiral whorl not inflated .................................................................. C. aubryana – Shell with 6–7 whorls, and spiral whorl inflated ........5 5 Vas deferens opens in last 1/3 of testis .......................6 – Vas deferens opens in last 1/4 of testis .....................10 6 The first anterior branch of vas deferens with more

than 5 secondary branches..........................................7 – The first anterior branch of vas deferens with less than

3 secondary branches..................................................9 7 Shell width shorter than body whorl depth; the first

anterior branch of vas deferens with 8 secondary branches...........................................................C. haasi

– Shell width longer than body whorl depth; the first ant-erior branch of vas deferens with 5–6 secondary bra-nches ...........................................................................8

8 Shell thin; umbilicus small, nearly closed .................................................................. C. chinensis – Shell thick; umbilicus big and deeply .....C. c. fluminalis 9 Vas deferens with 3 main branches........ C. cathayensis – Vas deferens with 2 main branches ............C. lecythoides 10 Vas deferens with 2 main branches; kidney triple

shape......................................................... .C. lecythis – Vas deferens with 3 main branches; kidney arch shape

.............................................................. C. ventricosa 11 Shell medium, with 4–5 whorls.......... C. hainanensis – Shell larger, with 6 whorls .....................................12 12 Vas deferens with 2 main branches, opens in last 1/4 of

testis.................................................. C. l. ampullacea – Vas deferens with 3 main branches, opens in last 1/3

of testis ................................................ C. leucostoma

DISCUSSION

Alongside numerous difficulties in determining the validity of numerous Cipangopaludina—lack of anatomy samples, discrepancies in the literature, or ambiguous morphological data—the genus itself is also problematic. Smith (2000) treated Cipangopaludina as subgenus of Bellamya, while Sengupta et al (2009) indicated that species of Bellamya from Asia could not belong to this genus. In China, there are currently seven recognized genera of Bellamyinae recorded, viz. Margarya Nevill, 1877, Cipangopaludina Hannibal, 1912, Bellamya Jousseaume, 1886, Mekongia Crosse et Fischer, 1876, Filopaludina Habe, 1964, Angulyagra Benson, 1836 and Trochotaia Brandt, 1974 (Liu et al, 1979, 1993; Du et al, 2011). Generally speaking, it is possible to distinguish

these, as the shell of Cipangopaludina, Margarya, and Trochotaia tend to be larger, while other four genera species are more mid-sized. Similarly, species of Filopaludina and Mekongia have a color band, and Angulyagra has ridges. For more detailed markers, Cipangopaludina can be distinguished from Trochotaia by shell round or oval vs. depressed pyramidally, last whorl with a sharp keel, and from Margarya by shell smooth vs. shell with ridges, nodules or spines; embryo with chaetae vs. ridges or nodules. Based on these criteria, we opted to treat Cipangopaludina as a valid genus.

Generally, the diagnostic characteristics of Cipangopaludina are shell median to large, sculpture lacking except growth lines and axial undulations. Aperture subcircular. Young specimens (from brood pouch) with up to 3–4 whorls; protoconch smooth, three primary rows of chaetae on last whorl and other whorls have two rows of chaetae. However, C. menglaensis and C. yunnanensis are small, up to 30 mm, smooth and solid, suggesting that C. menglaensis and C. yunnanensis belong to the genus Mekongia. It is also possible that C. dianchiensis could belong to the genus Margarya due to several characteristics: shell thick with ridges, female pouch with few big embryos and embryos shell with ridges (not chaetae). Similarly, Cipangopaludina ampullacea and C. lecythis, C. fluminalis and C. chinensis can be distinguished by shell characteristics, but they do not appear to possess any obviously distinctive anatomy, placing C. ampullacea and C. fluminalis as a subspecies of C. lecythis and C. chinensis, respectively. Our analysis also placed C. leucostoma (Heude) and C. diminuta (Heude) as synonymous with C. patris (Kobelt) according to apex obtuse and less than 6 whorls. However, according to the International Code of Zoological Nomenclature (ICZN, 1999), C. leucostoma should be valid due to its publication earlier than C. patris. Finally, Cipangopaludina wingatei and C. ampulliformis were found to be synonyms of C. chinensis and C. lecythis, respectively.

Totally, our analysis of 18 Cipangopaludina species resulted in a revised taxonomy that includes 11 species and 2 subspecies recorded in China, viz. C. aubryana, C. cathayensis, C. chinensis, C. haasi, C. hainanensis, C. latissima, C. lecythis, C. lecythoides, C. leucostoma, C. ussuriensis, C. ventricosa and C. chinensis fluminalis, C. lecythis ampullacea. Based on whether or not the spiral whorl depth was longer than aperture depth, Chinese



Cipangopaludina species could be divided to two groups, the chinensis group with spiral whorl depth longer than aperture depth that includes C. chinensis, C. haasi, C. ussuriensis and C. c. fluminalis, and the other the cathayensis group with spiral whorl depth being shorter than aperture depth, including C. aubryana, C. catha-yensis, C. hainanensis, C. latissima, C. lecythis, C. lecyt-hoides, C. leucostoma, C. ventricosa and C. l. ampul-lacea. Several anatomical characteristics can also help distinguish between the two groups. For example, the chinensis group can be distinguished from cathayensis group by vas deferens: chinensis group species with more than 5 (C. haasi with 8) parallel secondary bran-ches in anterior branch, while cathayensis group species always with 4 secondary branches. The number of secondary branches of vas deferens are in connection with size of body whorl. The shell of chinensis group species was taller, with body whorl depth being the same or longer than body width, making the vas deferens longer and with more secondary branches, especially in C. haasi. However, the body whorl of cathayensis group species is inflated, with body whorl depth shorter than body width, resulting in vas deferens being short and fat, and the secondary branch with many tiny branches, looking rather similar to a lush tree.

Within each group, there are further defining characteristics of individual species. In chinensis group, C. ussuriensis can be easily distinguished from other species by body whorl with 4 ridges and colour band. Cipangopaludina haasi can be distinguished from other species by body whorl not inflated, vas deferens with 8 secondary branches in anterior branch and vas deferens opens in last 1/4 of testis. Cipangopaludina chinensis fluminalis, however, is quite similar with C. chinensis, except for the shell thick vs. thin; umbilicus big and deeply vs. small or closed. In cathayensis group, C. latissima could be distinguished from other species by its markedly enlarged aperture while C. hainanensis can be distinguished from other species by apex eroded, left 4 whorls. According to kidney shape, the other species could be divided to two subgroups. Triple shape, including C. cathayensis, C. aubryana and C. lecythis, and arch shape, including C. l. ampullacea, C. leucostoma and C. ventricosa. Cipangopaludina lecythis can be distinguished from C. cathayensis and C. aubryana by vas deferens with 2 branches and opens in last 1/4 of testis vs. 3 branches and 1/3, and C. catha-yensis can be distinguished from C. aubryana by the

anterior 2/3 part of osphradium ridge-like and posterior 1/3 part inflated. Cipangopaludina leucostoma can be distinguished from C. l. ampullacea and C. ventricosa by vas deferens opening in last 1/3 vs. 1/4, and C. ventricosa can be distinguished from C. l. ampullacea by vas deferens with 3 branches vs. 2 branches.

Similar to our study, most viviparids are primarily delineated by their shells, though a few observations on the anatomy have also been made. Vail (1977) for example mentioned that the reproductive system was a useful characteristics to delineate viviparid subfamilies and genera by anatomical studies of mature male and female Campeloma geniculum (Conrad, 1834), Lioplax pilsbryi Walker, 1905 and Viviparus georgianus (Lea, 1834). Simone (2004) and Rao (1925) also reported intergeneric differences in the central nervous system, stomach lining and form of the ctenidial filaments in the five Asian genera. Du et al (2011) later reported a difference in kidney shape and the number of embryos present in the uterus of four Chinese viviparids, Angulygra, Cipangopaludina, Margarya and Trochotaia. In this study, the size of osphradium, location of vas deferens opens and the number of secondary branches, kidney shape were useful anatomical characteristics to identify interspecies of Cipangopaludina. The allometric growth or environmental effects could result in intraspecific variations in shell morphology and operculum (Chiu et al, 2002), but these anatomical characteristics could be used to identify young specimen.

Given the difficulties in identifying species solely by shell characters, further description of such species anatomical characteristics of both young and mature individuals may prove useful in further refining the taxonomy and identifying species and determining their validity. Unfortunately, until further studies are undertaken that more accurately characterize the anatomy or even underlying genetic differences, there will remain a variety of unanswered or unanswerable questions. To date, the monophyly of Cipangopaludina is still unresolved. A recent study by Du et al (2013) on the phylogeny of Margarya based on combined COⅠ and 16S rRNA sequences indicated that Cipangopaludina is not a monophyly, since species cluster with Margarya; however, the specimens of Cipangopaludina and Margarya are from Lake Dianchi. Likely further studies into the phylogeny and ecology of the Cipangopaludina in different environments and distributions will further enhance our understanding of these organisms.

526 LU, et al.


Acknowledgments: We thank Jiang YE, Cui GH, Zheng LP, Jiang WS, Yuan C, Li Y and Yu GH of Kunming Institute of Zoology, CAS and Aldridge D of Cambridge University for collecting these specimens. We greatly thank Meng K and Jia LJ of Institute of Zoology,

CAS for generous helping when first author check specimens at IOZ. We specially thank Liu YY for showing how to identify these species. We thank the anonymous reviewers for their helpful comments on the manuscript.

References

Annandale N. 1920. The Indian general of Viviparidae. Records of the Indian Museum, 19: 114-115.

Annandale N, Sewell RBS. 1921. The banded pond-snail of India (Vivipara bengalensis). Records of the Indian Museum, 22: , 215-292.

Armbruster G. 1995. Univariate and multivariate analyses of shell variables within the genus Cochlicopa (Gastropoda: Pulmonata: Cochlicopidae). Journal of Mollusca Studies, 61(2): 225-235.

Benson WH. 1836. Descriptive catalogue of a collection of land and fresh-water shells, chiefly contained in the museum of the Asiatic Society. Journal of Asiatic Society of Bengal, 5: 741-750.

Benson WH. 1842. Mollusca, in Theodor Cantor’s general features of Chusan, with remarks on the flora and fauna of that island. The Annals and Magazine of Natural History, including Zoology, Botany and Geology, 9: 481-493.

Bouchet P, Rocroi JP. 2005. Classification and nomenclator of

gastropod families. Malacologia, 47: 85-397.

Brandt RAM. 1974. The non-marine aquatic Mollusca of Thailand. In:

Archiv für Molluskenkunde der Senckenbergischen Naturforschenden

Gesellschaft, vol. 105. Senckenbergische Naturforschende Gesell-

schaft: , 33.

Brown DS. 1994. Freshwater Snails of Africa and Their Medical

Importance, . 2nd ed. London, : Taylor & Francis, .

Chiu YW, Chen HC, Lee SC, Chen CA. 2002. Morphometric analysis

of shell and operculum variations in the viviparid snail, Cipango-

paludina chinensis (Mollusca: Gastropoda), in Taiwan. Zoological

Studies, 41(3): 321-331.

Clench W, Fuller S. 1965. The genus Viviparus in North America.

Occasional Papers on Mollusks, Museum of Comparative Zoology, 2:

385-412.

Dautzenberg P, Fischer H. 1905. Liste des mollusques récoltés par M.

H. Mansuy en Indo-Chine et au Yunnan et description d’espèces

nouvelles. Journal de Conchylologie, 53: 343-417.

Du LN, Yang JX, Chen XY. 2011. A new species of Trochotaia

(Caenogastropoda: Viviparidae) from Yunnan, China. Molluscan

Research, 31, (2): 85-89.

Du LN, Yang JX, von Rintelen T, Chen XY, Aldridge D. 2013.

Molecular phylogenetic evidence that the Chinese viviparid genus

Margarya (Gastropoda: Viviparidae) is polyphyletic. Chinese Science

Bulletin, 58(18): 2154-2162.

Fischer P. 1891. Catalogue et Distribution géographique des Mollusq-

ues terrestres, fluviatiles & marins d’une partie de l’Indo-Chine (Siam,

Laos, Cambodge, Conchinchine, Annam, Tonkin). Dejussieu père et fils.

Franke H, Riedel F, Glaubrecht M, Köhler F, von Rintelen T. 2007. Evolution and biogeography of Southeast Asian viviparids (Gastropoda: Caenogastropoda). In: Jordaens K, Van Houtte N, Van Goethem J, Backeljau T. , World Congress of Malacology, . Antwerp, Belgium, .

Gerstfeldt G. 1859. Ueber Land– und Süsswasser-Mollusken Siberiens und des Amur-ge-bietes. Mémoires des Savants étrangers, 9: 507-548.

Griffith E, Pidgeon E. [1833]−1834. The animal kingdom arranged in conformity with its organization, by the Baron Cuvier, additional descriptions of all the species hitherto named, and of many not before noticed. In: Griffith E. The Mollusca and Radiata. Vol. 12. London: Whittaker and Co.,

Hannibal H. 1912. A synopsis of the Recent and Tertiary freshwater Molluscs of the Californian province, based upon an ontogenetic classification. Proceedings of the Malacological Society of London, 10: 194.

Heude PM. 1882-1890. Notes sur les mollusques terrestres et d’eau douce de la vallée du Fleuve Bleu. Memoires concemant L’Histoire naturelle e L’empire chinois par des peres de la compagnie de Jesus, 173-175, pl. 39.

Holznagel WE. 1998. Research note: A nondestructive method for cleaning gastropod radulae from frozen, alcohol-fixed, or dried material. American Malacological Bulletin, 14(2): 181-183.

ICZN. 1999. International Code of Zoological nomenclature. 4th ed. London: International Trust for Zoological Nomenclature.

Kobelt WS. 1909. Die Gattung Paludina Lam. (Vivipara Montfort): Neue Folge. In: Abbildungen Nach de Natur mit Beschreibngen. Systematisches Conchylien–Cabinet von Martini und Chemnitz, 1 (21).: 97-380.

Küster HC. 1852. Die Gattungen Paludina, Hydrocaena und Valvata. In: Abbildungen nach der Natur mit Beschreibungen. Systematisches Conchylien–Cabinet von Martini und Chemnitz, 1(21): 1-96. Pls. 1-14.

Liu YY. 1991. Studies on the Family Viviparidae in China (Mollusca, Gastropoda). In: Proceedings of the 10th International Malacological Congress (Tiibingen 1989), 587-590.

Liu YY, Zhang WZ, Wang YX. 1993. Medical Malacology. Beijing: China Ocean Press.

Liu YY, Zhang WZ, Wang YX, Wang EY. 1979. Freshwater molluscs. In: Economic Fauna of China. Beijing: Science Press.

Mabille J, Le Mesle G. 1866. Observations sur la faune malacologique de la Cochinchine et du Cambodje, comprenant la description des espéces nouvelles (1). Journal de Conchyliologie, 14: 117-138.

Nevill G. 1885. Hand list of Mollusca in the Indian Museum: Gastropoda. Prosobranchia–Neurobranchia. 1-306.



Pace GL. 1973. The freshwater snails of Taiwan (Formosa). In: Malacological Review Supplement, Vol. 1. University of California: , 1-117.

Philippi RA. 1846. Paludina Tab. II. Abbildungen und Beschreibungen neuer oder wenig gekannter Conchylien herausgegeben, 133.

Plinski M, Ławacz W, Stanczykowska A, Magnin E. 1978. Etude quantitative et qualitative de la nourriture des Viviparus malleatus (Reeve) (Gastropoda, Prosobranchia) dans deux lacs de la région de Montréal. Canadian Journal of Zoology, 56(2): 272-279.

Prashad P. 1928. Recent and fossil Viviparidae. A study in distribution, evolution and palaeogeography. Memoirs of the Indian Museum, , 8: 153-251.

Rao HS. 1925. On the comparative anatomy of Oriental Viviparidae. Records of the Indian Museum, 28: 129-135.

Reeve LA. 1862-1864. Conchologia iconica, or, illustrations of the shells of molluscous animals, Vol. 13-15. London: Monograph of the genus Paludina.

Sengupta ME, Kristensen TK, Madsen H, Jørgensen A. 2009. Molecular phylogenetic investigations of the Viviparidae (Gastropoda: Caenogastropoda) in the lakes of the Rift Valley area of Africa. Molecular Phylogenetics and Evolution, 52, (3): 797-805.

Simone LRL. 2004. Comparative morphology and phylogeny of representatives of the superfamilies of Architaenioglossans and the Annulariidae (Mollusca, Caenogastropoda). Arquivos do Museu Nac-ional, , 62, : 387-504.

Smith DG. 2000. Notes on the taxonomy of introduced Bellamya (Gastropoda: Viviparidae) species in northeastern North America. The Nautilus, 114(2): 31-37.

Soes DM, Majoor GD, Keulen SMA. 2011. Bellamya chinensis (Gray, 1834) (Gastropoda: Viviparidae), a new alien snail species for the

European fauna. Aquatic Invasions, 6(1): 97-102.

Strong EE, Gargominy O, Ponder WF, Bouchet P. 2008. Global diversity of gastropods (Gastropoda: Mollusca) in freshwater. Hydrobiologia, 595(1): 149-166.

Vail VA. 1977. Comparative reproductive anatomy of three viviparid gastropods. Malacologia, 16: 519-540.

Valovirta I, Vaisanen RA. 1986. Multivariate morphological discrimination between Vitrea contracta (Westerlund) and V. crystalline (Muller) (Gastropoda, Zonitidae). Journal of Mollusca Studies, 52: 62-67.

Wang S, Xie Y. 2005. China Species Red List (Volume III). Inverte-brates. Beijing: Higher Education Press.(in Chinese)

Yen TC. 1939. Die chinesischen Land- und Süßwasser-Gastropoden des Natur-Museums Senckenberg. In: Diese Arbeit erscheint gleichzeit-ig in den Abhandlungen der Senckenbergischen Naturforschenden Ges-ellschaft Abhandlung, Vol. 444: Klostermann.

Yen TC. 1942. A review of Chinese gastropods in the British Museum. Proceedings of the Malacological Society of London, 24(5-6): 170-289.

Yen TC. 1943. A preliminary revision of the recent species of Chinese Viviparidae. The Nautilus, 56(4): 124-130.

Ying T, Fürsich FT, Schneider S. 2013. Giant Viviparidae (Gastropoda: Architaenioglossa) from the Early Oligocene of the Nanning Basin (Guangxi, SE China). Neues Jahrbuch für Geologie und Paläontologie, 267(1): 75-87.

Zhang L. 1990. A new species of genus Cipangopaludina from Dianchi Lake in Yunnan Province, China. Acta Zootaxonomica Sinica, 15, (1): 25-27.

Zhang WZ, Liu YY, Wang YX. 1981. Three new species of Viviparidae from Yunnan province, China. Acta Zootaxonomica Sinica, 6(1): 40-43.



Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation

Ya HE1,2, Zheng-Huan WANG1, Xiao-Ming WANG1,2,*

1. School of Life Sciences, East China Normal University, Shanghai 200062, China

2. Shanghai Science & Technology Museum, 2000 Century Avenue, Shanghai 200127, China

Abstract: Cervus sichuanicus is a species of sika deer (Cervus nippon Group). To date, research has mainly focused on quantity surveying and behavior studies, with genetic information on this species currently deficient. To provide scientific evidence to assist in the protection of this species, we collected Sichuan sika deer fecal samples from the Sichuan Tiebu Nature Reserve (TNR) and extracted DNA from those samples. Microsatellite loci of bovine were used for PCR amplification. After GeneScan, the genotype data were used to analyze the genetic diversity and population structure of the Sichuan sika deer in TNR. Results showed that the average expected heterozygosity of the Sichuan sika deer population in TNR was 0.562, equivalent to the average expected heterozygosity of endangered animals, such as Procapra przewalskii. Furthermore, 8 of 9 microsatellite loci significantly deviated from the Hardy-Weinberg equilibrium and two groups existed within the Sichuan sika deer TNR population. This genetic structure may be caused by a group of Manchurian sika deer (Cervus hortulorum) released in TNR.

Keywords: Sichuan sika deer; Microsatellite; Genetic diversity; Population structure

The Sichuan sika deer (Cervus sichuanicus) (Guo et al, 1978) is a species of the Cervus nippon group (Groves and Grubb, 2011). It is an endemic species in China and is listed as a classⅠ endangered species, as well as an endangered species by the International Union for Conservation of Nature and Natural Resources (IUCN). Currently, the Sichuan sika deer is distributed in three unconnected areas located in northwest Sichuan Province and southwest Gansu Province. Compared to other spec-ies in China, Sichuan has the largest sika deer population in the wild, consisting of about 850 individuals (Guo, 2000).

In recent years, most research has focused on macroscopic aspects such as social behavior (Guo et al, 1991), distribution and quantity surveying (Guo, 2000), activity rhythms (Liu et al, 2004), and food habits (Guo, 2001). At the genetic level, Wu et al (2008) chose 16 microsatellite loci to analyze the genetic diversity of Sika deer in China, including 24 samples of Sichuan sika deer. Lü et al (2006) and Liu et al (2003) studied the genetic

relationship of sika deer using mitochondrial DNA, which also had a small number (16) of Sichuan sika deer samples. These studies focused on the differences among species and genetic structure of sika deer in China. Due to the limited samples, however, the above data do not reflect genetic information in one species or population. Moreover, using traditional capture methods makes it difficult to obtain abundant samples to study genetic diversity and population structure within Sichuan sika deer populations. 1

The Tiebu Nature Reserve (TNR) in the Zoige administrative district of northwest Sichuan has the largest sika deer population. According to monitoring data from TNR, there were about 1 000 Sichuan sika deer

Received: 25 March 2014; Accepted: 01 August 2014

Foundation items: The work was supported by the National Natural

Science Foundation of China (30870308 and 31071944) and the

Shanghai Rising-Star Program (10QA1402200)


Genetic diversity and population structure of a Sichuan sika deer (Cervus sichuanicus) population in Tiebu Nature Reserve based on microsatellite variation 529


in the reserve in 2012. TNR has been chosen as the main area to study Sichuan sika deer due to large population size and better protection situation (Li, 2010; Ning et al, 2008; Qi et al, 2010). To date, however, no genetic information studies on TNR sika deer have been conducted and the genetic diversity and possible population substructures remain unknown. Previous studies have shown that the geographical distribution pattern of the population is related to geographical isolation (Boyce et al, 1999; Gavin et al, 1999; Scandura et al, 1998). However, it is unclear whether the rivers, roads, villages, and crop protection fences across the reserve affect gene flow or generate genetic structures within the sika deer population. Therefore, we hypothesized that geographic and artificial factors may affect the gene flow of sika deer, and thus established seven sampling areas in TNR to obtain sika deer fecal samples. Overall, we collected five hides, one tissue sample, and 149 fecal samples. After extracting DNA

from those samples, nine Bovinae microsatellite loci were amplified and the products were used for gene scanning. We then used the genotype data to analyze genetic diversity and population structure. The results of this study provide a scientific basis for the protection of the Sichuan sika deer.


Study area and sampling collection Fecal samples were collected in seven areas in TNR

(E102°46′–103°14′, N33°58′–34°16′, about 206 922 km2), as shown in Figure 1. To avoid cross-contamination when sampling, we only collected one pellet from each fecal heap. We collected five hides, one tissue sample, and 149 fecal samples. The hide samples were collected from dead individuals in the wild. The only tissue sample came from an individual that had been killed by poachers. All samples were stored in 95% ethanol.

Figure 1 Distribution of sampling areas in Tiebu Nature Reserve

DNA extraction and amplification

DNA was extracted using an E.Z.N.A HP Tissue DNA Maxi Kit and a Stool DNA kit (OMEGA) according to the manufacturer’s protocols. The former primers of nine Bovinae microsatellite loci (IDVGA29, BM4107, RT1, TGLA53, RM188, RT13, BM6506, BL42, CSSM019, ETH225, and CERVID14) were mo-

dified fluorescent groups. The PCR mixture contained 1.5 μL genomic DNA (100 ng/μL), 1 μL of each pri-mer (10 pmol/μL), 0.5 μL BSA (20 mg/μL), and 7.5 μL of Premix Ex Taq (Takara), with deionized water used to make the sample volume up to 15 μL. The PCR amp-lification was carried out using an initial denaturation at

94 C for 3 min, followed by 35 cycles at 94 C for 30 s,

530 HE, et al.


annealing for 30 s, primer extension at 72 C for 45 s, and a final extension at 72 C for 10 min. The products were preserved at 4 C. One tissue sample was used as positive control and water as negative

control to ensure that PCR was performed correctly and to avoid contamination. The PCR products were sent to Shanghai MAP Biotechnology Co., Ltd (Shanghai, China) for GeneScan.

Table 1 Information on the nine microsatellite bovine loci used in this research

Locus Repeat motif Primer sequence Size range (bp)Annealing

temperature () Sources of

primers

CCC ACA AGG TTA TCT ATC TCC AG IDVGA29 (AC)n

CCA AGA AGG TCC AAA GCA TCC AC 137−157 60

Konfortov et al (1996)

AGC CCC TGC TAT TGT GTG AG BM4107 (AC)n (TC)n (TG)n

ATA GGC TTT GCA TTG TTC AGG 162−172 60

Bishop et al (1994)

TGC CTT CTT TCA TCC AAC AA RT1 (GT)n

CAT CTT CCC ATC CTC TTT AC 222−240 55

Wilson & White (1998)

CAG CAG ACA GCT GCA AGA GTT AGC TGLA53 (AC)n

CTT TCA GAA ATA GTT TGC ATT CAT GCA G 176−216 50

Hoffmann et al (2004)

GGG TTC ACA AAG AGC TGG AC RM188 (AC)n

GCA CTA TTG GGC TGG TGA TT 141−151 50-52

Barendse et al (1994)

GCC CAG TGT TAG GAA AGA AG RT13 (GT)n

CAT CCC AGA ACA GGA GTG AG 293−324 60

Bishop et al (1994)

GCA CGT GGT AAA GAG ATG GC BM6506 (AC)n

AGC AAC TTG AGC ATG GCA C 196−212 59−63

Moore et al (1994)

CAA GGT CAA GTC CAA ATG CC BL42 (AC)n

GCA TTT TTG TGT TAA TTT CAT GC 246−258 58−60

Bishop et al (1994)

TTG TCA GCA ACT TCT TGT ATC TTT CSSM019 (AC)n

TGT TTT AAG CCA CCC AAT TAT TTG 138−156 52−58

Wilson & White (1998)

GAT CAC CTT GCC ACT ATT TCC T ETH225 (AC)n

ACA TGA CAG CCA GCT GCT ACT 140−193 60

Steffen et al (1993)

TCT CTT GCG TCT CCT GCA TTG AC CERVID14 (AC)n

AAT GGC ACC CAC TCC AGT ATT CTT C 214−231 61−65

De Woody et al (1995)

Species identification and population assessment

We amplified all samples using two pairs of primers of the mitochondrial control region and compared the sequences in GenBank to ensure the samples in our research were from sika deer. We used GeneMapper v. 4 (Applied Biosystems) to obtain the microsatellite fragment sizes and identified the heterozygotes and homozygotes. We did two repeats if the genotypes of the two repetitive PCRs were different; for example, if there was a heterozygote in one repeat but a homozygote in the other repeat. If there was a heterozygote in three of four repeats, we considered this site to be a heterozygous locus; the same for a homozygote locus. The probability of identity (PI) and the PI between siblings were calculated in Gimlet 1.3.3 (Valière, 2002; Liu & Yao, 2013) using genotype data of the nine microsatellite loci. We used the following principle when using Mstools

(Park, 2001) to assess the population number: we allowed one genetic mismatch at one allele for one locus to be considered as two samples belonging to the same individual (Bellemain et al, 2005). To detect the validity of the above principle, we randomly selected 50 fecal samples (accounting for a third of the total number) and amplified the nine loci twice. At the same time, we amplified another two loci (ETH225 and CERVID14) of the selected 50 samples. We tested whether the influence of population number may be caused by the repeat amplification and increasing loci.

Genetic diversity and population structure

We computed the expected heterozygosity (HE), observed heterozygosity (HO), and polymorphism infor-mation content (PIC) using Mstools. Arlequin 3.1 (Exc-offier et al, 2005) and Fstat 2.9.3.2 (Goudet, 2001) were



used to test the Hardy-Weinberg equilibrium and Fis separately. The fixation index (FST) (Weir & Cockerham, 1984) among sampling areas was tested in Arlequin 3.1, using 1 000 random iterations to calculate confidence intervals (CI). Software structure 2.3 (Pritchard et al, 2000) was used to estimate the potential populations (K). The admixture model was used and run three times using 10 replications of K ranging from 1 to 7. For those runs the following conditions were adopted: a burn-in period at 10 000 following 10 000 000 replicates of the MCMC. The ΔK calculated as per Evanno et al (2005) was used to estimate optimal K. The formulas were: ΔK=m|L(K+1)−2L(K)−L(K−1)|/s[L(K)]. We referred to the structure output lnP(D) as L(K). m was the mean value and s was the standard deviation. Software CLUMPP Windows.1.1.2 (Jakobsson & Rosenberg, 2007) and destruct (Rosenberg, 2004) were used to visualize the individual coefficients of membership in the subpopulation calculated by structure 2.3.

RESULTS

Species identification and population assessment Comparing the obtained sequences of mitochondrial

hypervariable region I from our research to the corresponding sequence in GenBank, all samples were obtained from sika deer. Thereinto, 143 of 155 samples belonged to the haplotypes of Sichuan sika deer and the remainder belonged to the haplotypes of Manchurian sika deer (Cervus hortulorum).

Most of the Sichuan sika deer samples could be amplified at all the nine microsatellite loci, few samples (11) had a loss at one locus. Analyzing the united PI values for nine loci, we found that the discrimination power of nine loci was close to 100% (Figure 2). According to the rule of Bellemain, we assessed 76 sika

deer individuals from 143 face samples. After the second amplification of the randomly selected samples, the population number did not change. The additional two loci (ETH225 and CERVID14) increased the population by one individual, accounting for 3% of the total number. The distribution of the 76 individuals in the samplings areas is shown in Table 3.

Figure 2 Multi-loci PI (biased and unbiased) and PI (sibs) for sika deer in increasing order of single-locus values

Genetic diversity and population structure

In the Sichuan sika deer population in TNR, the mean number of alleles / locus was 6.56 and the HE was 0.562. The PIC values were higher than or close to 0.5, except for RT13 (0.129). All detected loci deviated from the Hardy-Weinberg equilibrium significantly (Figure 2) and the P value was 0. In addition to the locus RT13, the Fis values of the remaining eight loci were negative. The values of HE ranged from 0.4292 to 0.6865 in the seven sampling areas and all HO values were higher than those of HE (Table 3). The FST among the seven sampling areas ranged from –0.04781 to 0.05243. The highest FST (0.05243) appeared between Area Ⅳ and Ⅵ but the lowest (–0.04781) appeared between Area Ⅰ and Ⅲ (Table 4).

Table 2 Microsatellite diversity indices of Sichuan sika deer in Tiebu Nature Reserve

Locus No. of alleles HE HO PIC Fis

IDVGA29 5 0.587 0.722 0.516 –0.230

BM4107 7 0.560 0.654 0.495 –0.169

RT1 6 0.633 0.951 0.558 –0.506

CSSM019 6 0.635 0.926 0.560 –0.462

TGLA53 8 0.643 0.899 0.571 –0.401

BL42 9 0.607 0.913 0.523 –0.509

RM188 6 0.656 0.738 0.607 –0.125

BM6506 8 0.604 0.974 0.519 –0.620

RT13 4 0.134 0.026 0.129 0.810

Mean 6.56 0.562 0.756 0.496 –0.347

532 HE, et al.


Table 3 Indices of genetic diversity of Sichuan sika deer in different sampling areas

Sample area Sample size Loci typed HE HO

Ⅰ 10 9 0.4292 0.7160

Ⅱ 13 9 0.5499 0.8034

Ⅲ 7 9 0.4831 0.7778

Ⅳ 8 9 0.6804 0.7284

Ⅴ 20 9 0.6865 0.7444

Ⅵ 11 9 0.4350 0.7063

Ⅶ 7 9 0.5368 0.8571

Table 4 F-statistics (FST, below diagonal) and significant differences among different areas

Sample area Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Ⅶ

Ⅰ + – + + – +

Ⅱ –0.00493 – – – + –

Ⅲ –0.04781 –0.03753 – – – –

Ⅳ 0.03553 –0.0052 –0.00225 – + –

Ⅴ 0.05177 0.00789 0.01646 –0.01778 + –

Ⅵ –0.02823 –0.00142 –0.0334 0.03287 0.05243 +

Ⅶ 0.01389 –0.02825 –0.03837 –0.01869 0.01487 –0.00278

+ denotes significant difference, – in contrast (95% confidence interval).

Population structure

Structure analysis showed that when K=2, the ΔK exhibited an obvious apex (Figure 3). The FST between subpopulations was 0.535. Individual coefficients of membership in subpopulations (Figure 4) showed that when K=2, the genetic amount of cluster 1 (Yellow) was larger and distributed in all sampling areas. In contrast, the genetic amount of cluster 2 (Blue) was less than cluster 1, and had a relatively smaller distribution area. Most of the blue genotype was distributed in Areas Ⅳ and Ⅴ.

Figure 3 Value of ΔK as a function of K based on 10 runs

Figure 4 Individual coefficients of membership in each sampling area

DISCUSSION

Population assessment In recent years, non-invasive sampling methods

have been widely used in genetic studies of endangered animals (He et al, 2010; Wang et al, 2012). Because of the endangered status and fugacious feature of sika deer, non-invasive sampling methods (obtaining fecal samples) have become an important means of population assessment and individual identification. In this study, the stability of repeat amplification of nine microsatellite loci had no effect on assessing the population number, that is, our genotype data were reliable. After adding two loci, there was a 3% error between previous and post assessment results, which is much higher than 2% (Bellemain et al, 2005). Bellemain et al (2005) attributed such errors in population assessment to pseudogenes, pollution and missing data.

Genetic diversity and differentiation

Genetic diversity helps populations adapt to changing environments. With more variation, it is more likely that some individuals in a population will possess variations of alleles that are suited for the environment. The population will continue for more generations because of the success of these individuals (Khater et al, 2011). In our research, the HE of Sichuan sika deer



population was 0.562, close to the mean HE (0.559) of sika deer in China, but much higher than the mean HE for Sichuan sika deer (0.477) and slightly lower than the mean HE for Manchurian sika deer (0.584) and south China sika deer (Jiangxi, 0.585; Zhejiang, 0.589) (Wu et al, 2008). Compared to other Ungulata, such as red deer (Cervus elaphus) (HE=0.78, Hajji et al, 2007; HE=0.804, Pérez-Espona et al, 2008; HE=0.62−0.85, Zachos et al, 2007), forest musk deer (Moschus berezovskii) (HE=0.8−0.9, Zhou et al, 2005), and Mongolian wild ass (Equus hemionus) (HE=0.83, Kaczensky et al, 2011), Sichuan sika deer had a relatively lower HE and was equivalent to Przewalski's Gazelle (Procapra przewalskii) (HE=0.552, Yang & Jiang, 2011) and milu (Elphurus davidianus) (HE=0.46−0.54, Zeng et al, 2007). The HEs values were different in each sampling area. Area Ⅰ had

the lowest HE (0.4292), while Area V had the highest (0.6865). Polymorphism information content (PIC) is a measure of variation between microsatellite loci. It is generally considered that a PIC value greater than 0.5 is for highly polymorphic loci, a PIC value of less than 0.5 and greater than 0.25 is for moderately polymorphic loci, and a PIC of less than 0.25 is for low polymorphic loci (Niu et al, 2001). In this study, the PIC values of six of the nine microsatellite loci were higher than 0.5, indicating highly polymorphic loci, while two were moderately polymorphic loci and one was a low polymorphic locus. Heterozygosity and PIC information indicated Sichuan sika deer in TNR had abundant genetic diversity.

Hartl & Clark (1997) considered that population deviation from the Hardy Weinberg equilibrium was mainly due to small populations, not random mating, gene mutation, migration or other factors. The present study showed that nine microsatellite loci of the Sichuan sika deer population in TNR deviated significantly from the Hardy Weinberg equilibrium. The Sichuan sika deer population numbers in TNR grew steadily from 650 in 2000 (Guo, 2000) to 1 000 in 2012 (monitoring data from TNR). As a result, the deviation from the Hardy Weinberg equilibrium is unlikely attributed to a reduction in the population. The coefficient of genetic differentiation (FST), on the other hand, is an important indicator of genetic differentiation among subpopul-ations. According to Wright (1978), FSTs values bet-ween 0−0.5 suggest no differentiation between sub-groups. The FSTs values in our seven sampling areas were all less than 0.5, ranging from 0.04781 to 0.05243, and

showing negative value in some regions. This indicated that the genetic variation among the seven sampling areas mainly came from inside the sampling area, and the geographical barriers and human factors do not affect gene flow in Sichuan sika deer in TNR. In addition, due to the relatively closed geographical position of TNR, very few little sika deer were distributed in the periphery of the protected areas (Guo, 2000). Gene flow through immigration and emigration could not significantly affect the Sichuan sika deer populations to deviate from the Hardy Weinberg equilibrium.

Microsatellite polymorphism studies of sika deer in China found that 16 microsatellite loci significantly deviated from the Hardy Weinberg equilibrium, but HEs were higher than HOs (Wu et al, 2008). In the present study, HOs were significantly higher than the HEs. The Fis values of eight microsatellite loci were negative, showing heterozygosis excess. When a population experiences a reduction in its effective size, it generally develops heterozygosity excess at selectively neutral loci (Cornuet & Luikart, 1996). Xu et al (2009) used microsatellites to detect the population structure of Himalayan marmots and found heterozygosity excess due to historic population decline. Wu et al (2008) tested bottlenecks in four Chinese sika deer species and found that Sichuan sika deer had not experienced a population bottleneck in recent history. Therefore, the heterozygosity excess of Sichuan sika deer in TNR could not be caused by population reduction.

Population structure

A population composed of individuals from two different source populations will tend to have an excess of heterozygotes (Milkman, 1975). In testing population structure in the present study, we found that when K=2, ΔK exhibited a significant apex, indicating that two subpopulations existed within the Sichuan sika deer population. Considering that the Sichuan sika deer population had heterozygote excess and two subpo-pulations, we speculated that the sika deer in TNR might have two different sources populations. According to Wu (2004) and TNR staff, a captive-bred Manchurian sika deer group was bred and released in TNR in the late 1960s. The mitochondrial data showed that haplotypes of Manchurian sika deer (unpublished data) do exist. Previous research has shown that introduced species often hybridize with local sibling species (Avise et al, 1974; Gutiérrez, 1993; Perry & Goudet, 2001) and

534 HE, et al.


panmictic admixtures can develop rapidly over a short period of time (Echelle & Connor, 1989; Echelle, 1987; 1997). Hybridization abounds in Cervidae, and introduced sika deer have been shown to hybridize with native red deer (Senn & Pemberton, 2009). Therefore, there is a strong possibility that hybridization occurred between the two sika deer species in our research. The released Manchurian sika deer explained the existence of subpopulations in the Sichuan sika deer population in TNR. We speculate that the Manchurian sika deer genotype is the minority – the blue parts in the individual coefficients of membership, which were mainly distributed in Areas Ⅳ and Ⅴ– because the number of

introduced Manchurian sika deer is not clear and of the 155 sika deer fecal samples examined, only 12 belonged to the haplotypes of Manchurian sika deer. The Sichuan sika deer population were in larger quantities than that of Manchurian sika deer. So, the yellow parts in Figure 4

indicate the genotypes of Sichuan sika deer. In the past, researchers have defined the Sichuan

sika deer and Manchurian sika deer as two subspecies of sika deer. Groves & Grubb (2011) compared antecedent research on the morphology and molecular data of sika deer and suggested the Sichuan and Manchurian sika deer were two independent species in the Cervus nippon group. If hybridization and introgression occurred, it may lead to a local genetic extinction of a population (Avise et al, 1997; Templeton, 1986). From the perspective of cons-ervation biology, how genes flow between two species mixed by human influence is a focus for future research.

Acknowledgments: We wish to express our thanks to the staff at Tiebu Nature Reserve and Mr L. Zuo at Zoige Forestry Administration for providing support during our sampling period. Special thanks to Miss J.F. Yang for our sampling guide.

References

Avise JC, Smith MH, Selander RK, Lawlor TE, Ramsey PR. 1974. Biochemical polymorphism and systematics in the genus Peromyscus. V. Insular and mainland species of the subgenus Haplomylomys. Systematic Zoology, 23(2): 226-228.

Avise JC, Pierce PC, Van den Avyle MJ, Smith MH, Nelson WS, Asmussen MA. 1997. Cytonuclear introgressive swamping and species turnover of bass after an introduction. Journal of Heredity, 88(1): 14-20.

Barendse W, Armitage SM, Kossarek LM, Shalom A, Kirkpatrick BW,

Ryan AM, Clayton D, Li L, Neibergs HL, Zhang N, Grosse WM, Weiss

J, Creighton P, McCarthy F, Ron M, Teale AJ, Fries R, McGraw RA,

Moore SS, Georges M, Soller M, Womack JE, Hetzel DJS. 1994. A

genetic linkage map of the bovine genome. Nature Genetics, 6(3): 227-

235.

Bellemain E, Swenson JE, Tallmon D, Brunberg S, Taberlet P. 2005. Estimating population size of elusive animals with DNA from hunter-collected feces: Four methods for brown bears. Conservation Biology, 19(1): 150-161.

Bishop MD, Kappes SM, Keele JW, Stone RT, Sunden SL, Hawkins GA, Toldo SS, Fries R, Grosz MD, Yoo J, Beattie CW. 1994. A genetic linkage map for cattle. Genetics, 136(2): 619-639.

Boyce WM, Ramey RR II, Rodell TC, Rubin ES, Singer RS. 1999. Population subdivision among desert bighorn sheep (Oviscanadensis) ewes revealed by mitochondrial DNA analysis. Molecular Ecology, 8(1): 99-106.

Cornuet JM, Luikart G. 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics, 144(4): 2001-2014.

DeWoody JA, Honeycutt RL, Skow LC. 1995. Microsatellite markers in white-tailed deer. Journal of Heredity, 86(4): 317-319.

Echelle AA, Connor PJ. 1989. Rapid, geographically extensive genetic introgression after secondary contact between two pupfish species (Cyprinodon, Cyprinodontidae). Evolution, 43(4): 717-727.

Echelle AA, Echelle AF, Edds DR. 1987. Population structure of four pupfish species (Cyprinodontidae: Cyprinodon) from the Chihuahuan desert region of New Mexico and Texas: Allozymic variation. Copeia, 3: 668-681.

Echelle AA, Hosgstrom CW, Echelle AF, Brooks JE. 1997. Expanded occurrence of genetically introgressed pupfish (Cyprinodontidae: Cyprinodon pecosensis × variegatus) in New Mexico. Southwestern Naturalist, 42: 336-339.

Evanno G, Regnaut S, Goudet J. 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14(8): 2611-2620.

Excoffier L, Laval G, Schneider S. 2005. Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1: 47-50.

Gavin TA, Sherman PW, Yensen E, May B. 1999. Population genetic structure of the northern Idaho ground squirrel (Spermophilus brunneus brunneus). Journal of Mammalogy, 80(1): 156-168.

Goudet J. 2001. FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Available from http: //www 2.unil.ch/popgen/softwares/fstat.htm. Updated from Goudet (1995)

Groves C, Grubb P. 2011. Ungulate Taxonomy. Baltimore: The Johns Hopkins University Press, 101-105.

Guo YS. 2000. Distribution, numbers and habitat of Sichuan sika deer (Cervus nippon sichuanicus). Acta Theriologica Sinica, 20(2): 81-87. (in Chinese).



Guo YS. 2001. Study on the food habits of Sichuan sika deer (Cervus nippon sichuanicus). Journal of Sichuan Teachers College: Natural Science, 22(2): 112-119. (in Chinese).

Guo YS, Hu JC, Luo DH, Se K, Ren SP, Zou HF. 1991. Studies on the social behavior of Cervus nippon sichuanicus. Acta Theriologica Sinica, 11(3): 165-170. (in Chinese).

Guo ZP, Chen EY, Wang YZ. 1978. A new subspecies of sika deer from Sichuan—Cervus nippon sichuanicus subsp. nov. Acta Zoologica Sinica, 24(2): 187-191. (in Chinese).

Hajji GM, Zachos FE, Charfi-Cheikrouha F, Hartl GB. 2007. Conservation genetics of the imperilled Barbary red deer in Tunisia. Animal Conservation, 10(2): 229-235.

Hartl DL, Clark AG. 1997. Organization of genetic variation. In: Hartl DL, Clark AG. Principles of Population Genetics. 3rd ed. Sunderland, Massachusetts: Sinauer Associates, Inc. Publishers, 74-110.

He L, Zhang YG, Peng HL, Li DQ, Li DQ. 2010. Genetic diversity of Rhinopithecus roxellana in Shennongjia National Nature Reserve as estimated by non-invasive DNA technology. Acta Ecologica Sinica, 30(16): 4340-4350. (in Chinese).

Li M, Zhang G, Ding Y, Yang Y, Gu S, Wang X, Wang Z. Genetic

structure and history of a Sichuan sika deer (Cervus nippon sichuanicus)

population based on mitochondrial DNA. (submit)

Hoffmann I, Marsan PA, Barker JSF, Cothran EG, Hanotte O, Lenstra

JA, Milan D, Weigend S, Simianer H. 2004. New MoDAD marker sets

to be used in diversity studies for the major farm animal species:

Recommendations of a joint ISAG/FAO working group. In:

Proceedings of 29th International Conference Animal Genetic. Tokyo,

Japan, 107.

Jakobsson M, Rosenberg NA. 2007. CLUMPP: a cluster matching and

permutation program for dealing with label switching and

multimodality in analysis of population structure. Bioinformatics,

23(14): 1801-1806.

Kaczensky P, Kuehn R, Lhagvasuren B, Pietsch S, Yang WK, Walzer C.

2011. Connectivity of the Asiatic wild ass population in the Mongolian

Gobi. Biological Conservation, 144(2): 920-929.

Khater M, Salehi E, Gras R. 2011. Correlation between genetic

diversity and fitness in a predator-prey ecosystem simulation. In: Al

2011: Advances in Artificial Intelligence. Berlin Heidelberg: Springer,

422-431.

Konfortov BA, Jørgensen GB, Barendse W, Miller JR. 1996. Five

bovine polymorphic microsatellite markers (AF1-AF5). Animal

Genetics, 27(3): 220-221.

Li M. 2010. Research on Habitat Selection of the Sichuan Sika Deer

during Fetiferous and Antler Growth Period in Tiebu Natural Reserve.

Master Thesis, West China Normal University. (in Chinese)

Liu H, Shi HY, Hu JC. 2004. Daily activity rhythm and time budget of

Sichuan sika deer (Cervus nippon sichuanicus) in spring. Acta

Theriologica Sinica, 24(4): 282-285.(in Chinese)

Liu H, Yang G, Wei FW, Li M, Hu JC. 2003. Sequence variability of the

mitochondrial DNA control region and population genetic structure of

sika deers (Cervus nippon) in China. Acta Zoologica Sinica, 49(1): 53-

60.(in Chinese)

Liu XH, Yao YG. 2013. Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew (Tupaia belangeri chinensis). Zoological Research, 34(E2): E62-E68.

Lü X, Wei FW, Li M, Yang G, Liu H. 2006. Genetic diversity among Chinese sika deer (Cervus nippon) populations and relationships between Chinese and Japanese sika deer. Chinese Science Bulletin, 51(4): 433-440. (in Chinese)

Milkman R. 1975. Heterozygote excess due to population mixing. Annals of Human Genetics, 38(4): 505-506.

Moore SS, Byrne K, Berger KT, Barendse W, McCarthy F, Womack JE, Hetzel DJS. 1994. Characterization of 65 bovine microsatellites. Mammalian Genome, 5(2): 84-90.

Ning JZ, Guo YS, Zheng HZ. 2008. Vocalization behaviour of Sichuan sika deer (Cervus nippon sichuanicus) during rut. Acta Theriologica Sinica, 28(2): 187-193. (in Chinese).

Niu R, Shang HT, Wei H, Huang ZB, Zeng YZ. 2001. Genetic analysis of 35 microsatellite loci in 5 lineages of Xishuangbanna miniature pig inbred line. Acta Genetica Sinica, 28(6): 518-526.

Park SDE. 2001. Trypanotolerance in West African Cattle and the Population Genetic Effects of Selection. Ph. D. thesis, University of Dublin.

Pérez-Espona S, Pérez-Barbería FJ, Mcleod JE, Jiggins CD, Gordon IJ, Pemberton JM. 2008. Landscape features affect gene flow of Scottish Highland red deer (Cervus elaphus). Molecular Ecology, 17(4): 981-996.

Perry N, Goudet J. 2001. Inbreeding, kinship, and the evolution of natal dispersal. In: Clobert J, Danchin E, Dhondt AA, Nichols JD. Dispersal. Oxford: Oxford University Press, 123-142.

Pritchard JK, Stephens M, Donnelly P. 2000. Inference of population structure using multilocus genotype data. Genetics, 155(2): 945-959.

Qi WH, Yue BS, Ning Jz, Jiang XM, Quan QM, Guo YS, Mi J, Zuo L, Xiong YQ. 2010. Behavior ethogram and PAE coding system of Cervus nippon sichuanicus. Chinese Journal of Applied Ecology, 21(2): 442-451. (in Chinese).

Rosenberg NA. 2004. Distruct: a program for the graphical display of population structure. Molecular Ecology Notes, 4(1): 137-138.

Scandura M, Tiedemann R, Apollonio M, Hartl GB. 1998. Genetic variation in an isolated Italian population of fallow deer Dama dama as revealed by RAPD-PCR. Acta Therioloica, (Suppl. 5): 163-169.

Senn HV, Pemberton JM. 2009. Variable extent of hybridization between invasive sika (Cervus nippon) and native red deer (C. elaphus) in a small geographical area. Molecular Ecology, 18(5): 862-876.

Steffen A, Eggena A, Dietzj AB, Womack JE, Tranzinger G, Fries S. 1993. Isolation and mapping of polymorphic microsatellites in cattle. Animal Genetic, 24(2): 121-124.

Templeton AR. 1986. Coadaptation and outbreeding depression. In: Soule ME. Conservation Biology: the Science of Scarcity and Diversity. Massachussets: Sinauer Associates Inc., 105-116.

Valière N. 2002. GIMLET: a computer program for analysing genetic individual identification data. Molecular Ecology Notes, 2(3): 377-379.

Wang YN, Bao YX, Liu J, Lin JJ, Zhang X, Zheng WC, Pan CC. 2012.

536 HE, et al.


Genetic diversity of black muntjac (Muntiacus crinifrons) in Jiulongshan National Nature Reserve by feces DNA. Acta Theriologica Sinica, 32(2): 101-109.(in Chinese)

Weir BS, Cockerham CC. 1984. Estimating F-statistics for the analysis of population structure. Evolution, 38(6): 1358-1370.

Wilson PJ, White BN. 1998. Sex identification of elk (Cervus elaphus canadensis), moose (Alces alces), and white-tailed deer (Odocoileus virginianus) using the polymerase chain reaction. Journal of Forensic Sciences, 43(3): 477-482.

Wright S. 1978. Evolution and the genetics of populations. In: Variability Within and Among Natural Populations. Chicago: University of Chicago Press, 4.

Wu H. 2004. Conservation Genetics of Chinese Sika Deer (Cervus Nippon). Ph.D. thesis, Zhejiang University. (in Chinese)

Wu H, Hu J, Wan QH, Fang SG, Liu WH, Zhang SY. 2008. Microsatellite polymorphisms and population genetic structure of sika deer in China. Acta Theriologica Sinica, 28(2): 109-116. (in Chinese)

Xu JH, Wang LL, Xue HL, Wang YS, Xu LX. 2009. Microsatellite variation and genetic diversity in Marmota himalayana. Chinese Journal of Zoology, 44(2): 34-40. (in Chinese)

Yang J, Jiang ZG. 2011. Genetic diversity, population genetic structure and demographic history of Przewalski’s gazelle (Procapra przewalskii): implications for conservation. Conservation Genetics, 12(6): 1457-1468.

Zachos FE, Althoff C, Steynitz Yv, Eckert I, Hartl GB. 2007. Genetic

analysis of an isolated red deer (Cervus elaphus) population showing

signs of inbreeding depression. European Journal of Wildlife Research,

53(1): 61-67.

Zeng Y, Jiang ZG, Li CW. 2007. Genetic variability in relocated Père

David’s deer (Elaphurus davidianus) populations—Implications to

reintroduction program. Conservation Genetics, 8(5): 1051-1059.

Zhou FD, Yue BS, Xu L, Zhang YZ. 2005. Isolation and character-

rization of microsatellite loci from forest musk deer (Moschus

berezovskii). Zoological Science, 22(5): 593-598.



Complete mitochondrial genome of yellow meal worm (Tenebrio molitor)

Li-Na LIU1, Cheng-Ye WANG2,*

1. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

2. Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Research Institute of Resource Insects, Chinese Academy

of Forestry, Kunming 650224, China

Abstract: The yellow meal worm (Tenebrio molitor L.) is an important resource insect typically used as animal feed additive. It is also widely used for biological research. The first complete mitochondrial genome of T. molitor was determined for the first time by long PCR and conserved primer walking approaches. The results showed that the entire mitogenome of T. molitor was 15 785 bp long, with 72.35% A+T content [deposited in GenBank with accession number KF418153]. The gene order and orientation were the same as the most common type suggested as ancestral for insects. Two protein-coding genes used atypical start codons (CTA in ND2 and AAT in COX1), and the remaining 11 protein-coding genes started with a typical insect initiation codon ATN. All tRNAs showed standard clover-leaf structure, except for tRNASer (AGN), which lacked a dihydrouridine (DHU) arm. The newly added T. molitor mitogenome could provide information for future studies on yellow meal worm.

Keywords: Tenebrionidae; Mitogenome; Evolution; Resource insect; Yellow meal worm

Mitochondrial genes have been widely used in studies of phylogenetics, phylogeography and molecular diagnostics (Kocher et al, 1989; Simmons & Weller, 2001; Wolstenholme, 1992). The full-length mitochon-drial genome (mitogenome) has become an important tool for studies of genome architecture, population gene-tics, primer design, and molecular evolution (Kim et al, 2009; Lee et al, 2009; Liao et al, 2010; Yukuhiro et al, 2002). Furthermore, mitogenome sequences provide im-portant information for pest control. For example, insec-ticide resistance in arthropod pest Tetranychus urticae is controlled by mtDNA (Van Leeuwen et al, 2008).

Tenebrionidae is a large family of Coleoptera and more than 20 000 species have been described worldwide (Bouchard et al, 2011). Despite this large taxonomic diversity, information about the Tenebrionidae mito-genome is still limited. To date, only two complete mit-ogenomes are available in GenBank (NC_003081: Trib-olium castaneum and NC_013554: Adelium sp. NCS-2009). Due to the importance of mitogenomes in popul-ation studies and pest control, studies on the mitogenome of tenebrionids are required.

The yellow meal worm (Tenebrio molitor L.) is an

important resource insect, which has great value as an animal feed additive because of its high protein content. It has also been used in numerous experimental studies (Gomez et al, 2013; Simon et al, 2013). Its relatively large body-size and ease of rearing make it attractive for biological research. An increasing number of researchers have used this beetle as a model system for studies in biology, biochemistry, evolution, immunology and physiology (Dobson et al, 2012; Goptar et al, 2013; Ohde et al, 2013; Prokkola et al, 2013; Yan et al, 2013; Zhu et al, 2013). However, the genetic background of this beetle, in both nuclei and organelles, has not yet been determ-ined, and information is urgently needed. 1

In this study, we sequenced the whole mitogenome of T. molitor for the first time and analyzed its nucleotide composition and major characteristics. The entire mitogenome was found to be 15 785 bp long and harbo-red 13 protein-coding genes (PCGs), 22 tRNA genes, and

Received: 27 March 2014; Accepted: 25 July 2014

Foundation item: This work was supported by grant from the Science

and Technology Committee of Yunnan Province (2011FB141)


538 LIU &WANG


2 rRNA genes. The gene arrangement in the T. molitor mitogenome was identical to the ancestral insect mitog-enome arrangement (Boore et al, 1998). The mitogenome of T. molitor is an important addition to the continuing efforts in developing T. molitor as a model system for biological studies.


Sample and DNA extraction An adult T. molitor was collected in Kunming,

Yunnan Province, China, on July 29, 2012. The adult insect was one of the descendants from our inbred line, which has been established and maintained in our laboratory since May 2008. All members of this inbred line have one common ancestral mitochondrial geno-me background. After collection, the fresh materials were immediately preserved in 100% ethanol and stored in a −20 °C refrigerator before genomic DNA extraction. Total genomic DNA was extracted with the WizardTM Genomic DNA Purification Kit, in accor-dance with the manufacturer's instructions (Promega, USA).

PCR amplification and sequencing

To sequence the complete mitogenome, long PCR primers and some short PCR primers were designed by multiple sequence alignments of all available complete Coleoptera mitogenomes using ClustalX1.8 (Thompson et al, 1997) and Primer Premier 5.0 software. Primer sequence information can be obtained from the authors upon request. Long PCRs were performed using TaK-aRa LA Taq polymerase with the following cycling parameters: initial denaturation for 5 min at 95 °C, followed by 30 cycles at 95 °C for 50 s, 50 °C for 50 s, 68 °C for 2 min and 30 s; and a final extension step of 68 °C for 10 min. Short fragments were amplified with TaKaRa Taq polymerase: initial denaturation for 5 min at 94°C, followed by 35 cycles at 94 °C for 1 min, 45−53 °C for 1 min, 72 °C for 2 min, and a final extension step of 72 °C for 10 min. The PCR products were detected via electrophoresis in 1.5% agarose gel, and purified using the QIAquick PCR Purification Kit (QIAGEN, USA). Sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Electrophoresis of purified sequencing products was performed on an ABI-3730 DNA Analyzer (Applied Biosystems).

Gene identification and tRNA structures Sequences from overlapping fragments were assem-

bled with the neighboring fragments using the SeqMan program included in the Lasergene software package (DNAStar Inc., Madison, Wisc.). Comparison of the DNA or amino acid sequences with homologous regions of known full-length insect mitogenome sequences indicated 13 protein-coding genes (PCGs) and two rRNA genes based on MEGA 5.0 software (Tamura et al, 2011). The nucleotide sequences of the PCGs were translated on the basis of the invertebrate mtDNA genetic code. Transfer RNA gene analysis was conducted based on tRNAscan-SE software v.1.21 (Lowe & Eddy, 1997), and the folding of the predicted tRNA sequences were further confirmed by visual inspection. Finally, the T. molitor mitogenome sequence was deposited in GenBank with accession number KF418153.

Sequence analysis

The A+T-content of the whole genome was calcul-ated based on the EditSeq program included in the Lasergene software package. The nucleotide composition at each codon position of the PCGs and codon usage were calculated based on MEGA 5.0. Gene overlaps and intergenic-space sequences were hand-counted.

Nucleotide composition skew was calculated for the PCGs and the whole genome base on the EditSeq prog-ram included in the Lasergene software package (DN-AStar Inc., Madison, Wisc.), using the following formula proposed by Perna & Kocher (1995): GC-skew=(G–C)/(G+C) and AT-skew=(A–T)/(A+T), where C, G, A and T are the frequencies of the four bases.

RESULTS AND DISCUSSION

General features of Tenebrio molitor mitogenome The complete mitogenome of T. molitor was 15 785

bp in length, and consisted of 2 rRNAs (srRNA and lrRNA), 13 PCGs (ATP6, ATP8, COI-III, ND1-6, ND4L, CytB), and 22 tRNAs (Figure 1). Like many other insect mitogenomes, the major strand (J strand) carried most of the genes (9 PCGs and 14 tRNAs), whereas the remaining genes were on the minor strand (N strand) (4 PCGs, 8 tRNAs and 2 rRNA genes) (Figure 1). Gene order and orientation were the same as the most common type suggested as ancestral for insects [18, 23] (Boore et al, 1998; Taanman, 1999).

All genes were closely assembled and only nine intergenic spacers were observed. These intergenic

Complete mitochondrial genome of yellow meal worm (Tenebrio molitor) 539


Figure 1 Circular map of the Tenebrio molitor mitogenome COⅠ, COⅡ and COⅢ refer to the cytochrome C oxidase subunits; CytB refers to cytochrome B; ATP6 and ATP8 refer to subunits 6 and 8 of F0 ATPase;

ND1-6 refer to components of NADH dehydrogenase. tRNAs are denoted as one-letter symbols consistent with the IUPAC-IUB single letter amino acid codes.

Gene names not underlined indicate a clockwise transcriptional direction, whereas underlines indicate a counter-clockwise transcriptional direction. L* and S*

denote tRNALeu(UUR) and tRNASer(UCN), respectively.

spacers were 53 bp in total, with individual sizes ranged from 1 to 22 bp. In addition, there were 36 bp overl-apping sequences totally in 15 overlaps between different genes, ranging from 1−7 bp (Table 1).

Comparative analysis with two other published Tenebrionidae mitogenomes of Tribolium castaneum and Adelium sp. NCS-2009 exhibited highly conserved geno-me architectures including gene order, nucleotide compo-sition, codon usage, and amino acid composition (Friedr-ich & Muqim, 2003; Sheffield et al, 2009).

Protein-coding genes (PCGs)

Eleven of the 13 PCGs of T. molitor used standard ATN (Met) start codon, while the remaining two PCGs used atypical start codons (CTA in ND2 and AAT in COX1) (Table 1). ATP6, ATP8, ND4L, ND6, and CytB genes had the common stop codon TAA; ND1 and ND3 had the stop codon TAG; while ND2, COX1, COX2, COX3, ND5, and ND4 terminated with incomplete stop codon T or TA (Table 1). Similar cases have been found in other insect mitogenomes (Yin et al, 2012). For example, a single T residue was deemed as the stop

codon for COⅠ, COⅡ, ND5 and CytB, and TA was deemed as the stop codon for ATP6, ND4, ND4L and ND6 in Coreana raphaelis (Kim et al, 2006); similarly, a single T was considered as the stop codon for COⅠ, COⅡ and ND4, and TA residue as the stop codon for ATP6 in Hyphantria cunea (Liao et al, 2010). Incomplete stop codons also exist in vertebrate mitochondrial genomes such as in human mitochondria (Andrews et al, 1999). It has been demonstrated that incomplete stop codons can produce functional stop codons in polycistronic transcription cleavage and polyadenylation processes (Ojala et al, 1981).

Transfer RNA and ribosomal RNA genes

Twenty-two tRNA genes (one specific for each amino acid and two for leucine and serine) were identi-fied within the T. molitor mitogenome. The 22 tRNA genes were interspersed throughout the coding region and ranged from 60 to 70 bp in length (Table 1). All tRNAs, except tRNASer (AGN), were folded into typical cloverleaf secondary structures (Figure 2). The unusual tRNASer (AGN) lacked a dihydrouridine (DHU) arm, which was replaced by a simple loop (Figure 2). Another

540 LIU &WANG


unusual feature was the use of TCT as the tRNASer (AGN) anticodon instead of GCT, which is used in most other arthropods. This incomplete tRNASer structure has also been detected in other insect groups (Kim et al, 2006; Li

et al, 2011; Liao et al, 2010; Salvato et al, 2008; Wolstenholme, 1992; Yang et al, 2011).

Twenty-two non-Watson-Crick base pairs were observed in the T. molitor mt tRNAs, including 17 G-U

Table 1 Summary of Tenebrio molitor mitogenome

Gene Direction Nucleotide number Size (bp) Anticodon Start codon Stop codon

tRNAIle F 1–64 64 29–31 GAT – –

tRNAGln R 62–130 69 98–100 TTG – –

tRNAMet F 131–198 68 160–162 CAT – –

ND2 F 204–1206 1003 – CTA(M) T(AA)#

tRNATrp F 1207–1272 66 1240–1242 TCA – –

tRNACys R 1295–1355 61 1324–1326 GCA – –

tRNATyr R 1356–1421 66 1388–1390 GTA – –

COX1 F 1423–2956 1534 – AAT(M) T(AA)#

tRNALeu(UUR) F 2957–3021 65 2986–2988 TAA – –

COX2 F 3022–3709 688 – – –

tRNALys F 3710–3779 70 3740–3742 CTT – –

tRNAAsp F 3779–3843 65 3809–3811 GTC – –

ATP8 F 3844–3999 156 – ATA(M) TAA

ATP6 F 3993–4664 672 – ATG(M) TAA

COX3 F 4664–5448 785 – ATG(M) TA(A)#

tRNAGly F 5448–5509 62 5478–5480 TCC − −

ND3 F 5510–5863 354 – ATT(M) TAG

tRNAAla F 5862–5927 66 5891–5893 TGC – –

tRNAArg F 5927–5990 64 5957–5959 TCG – –

tRNAAsn F 5990–6054 65 6021–6023 GTT – –

tRNASer(AGN) F 6055–6114 60 6077–6079 TCT – –

tRNAGlu F 6116–6177 62 6146–6148 TTC – –

tRNAPhe R 6176–6239 64 6206–6208 GAA – –

ND5 R 6240–7953 1714 – ATA(M) T(AA)#

tRNAHis R 7951–8013 63 7981–7983 GTG - -

ND4 R 8014–9346 1333 – ATG(M) T(AA)#

ND4L R 9340–9627 288 – ATG(M) TAA

tRNAThr F 9630−9692 63 9660−9662 TGT – –

tRNAPro R 9693−9758 66 9726−9728 TGG – –

ND6 F 9761−10258 498 – ATT(M) TAA

CytB F 10258−11394 1137 – ATG(M) TAA

tRNASer(UCN) F 11393−11458 66 11423−11425TGA − −

ND1 R 11476−12429 954 − ATA(M) TAG

tRNALeu(CUN) R 12427−12491 65 12460−12462TAG – –

lrRNA R 12491–13771 1281 − – –

tRNAVal R 13774−13842 69 13810-13812 TAC – –

srRNA R 13844−14615 772 – – –

A+T-rich region R 14616−15785 1170 – – –

tRNA abbreviations follow the IUPAC-IUB three letter code.

#: TAA stop codon is completed by the addition of 3' A residues to the mRNA.



542 LIU &WANG


Figure 2 Predicted secondary clover-leaf structure for the 22 tRNA genes of the Tenebrio molitor mitogenome The tRNAs are labeled with abbreviations of their corresponding amino acids. Point () indicate Watson-Crick base-pairing. Arms of tRNAs (clockwise from

top) are amino acid acceptor (AA) arm, TψC (T) arm, anticodon (AC) arm and dihydrouridine (DHU) arm.



pairs, which form a weak bond in the tRNAs. The remaining five were atypical pairings: one mismatch in tRNALeu (CUN) (U-U), one in tRNALeu (UUR) (U-U), two in tRNATyr (2 U-U), and one in tRNATrp (A-G) (Figure 2). These mismatches found in tRNAs can be corrected through RNA-editing mechanisms (Lavrov et al, 2000).

The lrRNA and srRNA genes of the T. molitor mitogenome were 1 281 and 772 bp in length, respec-tively. As observed in other insects (Boore et al, 1998; Kim et al, 2009), these two genes were located between tRNALeu (CUN) and tRNAVal as well as between tRNAVal and the A+T-rich region, respectively (Figure 1). The A+T contents of the lrRNA and srRNA genes were 78.92% and 77.33%, respectively.

A+T-rich region

The 1 170 bp A+T-rich region of the T. molitor mitogenome was located between the srRNA and tRNAIle genes (Table 1). Like the two other Tenebrionidae mitogenomes, the A+T-rich region also exhibited the highest A+T content (85.38%) in the T. molitor mitogenome. Unlike some insect species (Kim et al, 2006; Yin et al, 2012), the region did not have large tandem repetitive sequences; however, it did have some microsatellite-like repeats (for example, (T)15, (AT)7, (A)20, and (G)8). The poly-T stretch (15 bp) has been suggested to function as a possible recognition site for the initiation of replication of the mtDNA minor strand

(Andrews et al, 1999; Kim et al, 2009). The A+T-rich region in insect mitogenomes is equivalent to the control region of vertebrate mitogenomes, which harbor the origin sites for transcription and replication (Andrews et al, 1999; Yukuhiro et al, 2002; Zhu et al, 2013).

Nucleotide composition and codon usage

The A+T content of the whole T. molitor mitogen-ome coding region was 72.35%, showing an obvious AT mutation bias (Eyre-Walker, 1997), as observed in two other published Tenebrionidae species (Friedrich & Muqim, 2003; Sheffield et al, 2009). The AT-skew value of the major strand was 0.204, indicating the occurrences of As in the major strands were greater than those of Ts. The mean value of the A+T content of the 13 PCGs was 69.27%, with a strong A+T bias. The A+T content at the third codon position (76.30%) was higher than the first (64.75%) and second position (66.77%), indicating that the third codon position was most susceptible to AT mutation bias (Eyre-Walker, 1997).

The relative synonymous codon usage (RSCU) in the T. molitor mitochondrial PCGs was investigated and the results are summarized in Table 2. The four most frequently used codons were ATT (Ile), ATA (Met), TTT (Phe), and TTA (Leu), accounting for 25.7% of all codons in the T. molitor mitogenome. These four codons were composed of A or T nucleotides, indicating that their biased usage resulted from strong AT mutation bias

Table 2 Codon number and relative synonymous codon usage in Tenebrio molitor mitochondrial protein coding genesa)

Codon Count RSCU Codon Count RSCU Codon Count RSCU Codon Count RSCU

UUU(F) 227 1.34 UCU(S) 99 2.28 UAU(Y) 113 1.47 UGU(C) 25 1.61

UUC(F) 113 0.66 UCC(S) 23 0.53 UAC(Y) 41 0.53 UGC(C) 6 0.39

UUA(L) 198 2.29 UCA(S) 101 2.33 UAA(*) 0 0 UGA(W) 74 1.61

UUG(L) 73 0.84 UCG(S) 11 0.25 UAG(*) 0 0 UGG(W) 18 0.39

CUU(L) 90 1.04 CCU(P) 46 1.39 CAU(H) 31 0.87 CGU(R) 26 1.82

CUC(L) 9 0.1 CCC(P) 18 0.55 CAC(H) 40 1.13 CGC(R) 2 0.14

CUA(L) 131 1.51 CCA(P) 61 1.85 CAA(Q) 50 1.54 CGA(R) 26 1.82

CUG(L) 18 0.21 CCG(P) 7 0.21 CAG(Q) 15 0.46 CGG(R) 3 0.21

AUU(I) 274 1.52 ACU(T) 53 1.07 AAU(N) 116 1.21 AGU(S) 26 0.6

AUC(I) 86 0.48 ACC(T) 40 0.8 AAC(N) 75 0.79 AGC(S) 6 0.14

AUA(M) 252 1.67 ACA(T) 98 1.97 AAA(K) 78 1.46 AGA(S) 66 1.52

AUG(M) 49 0.33 ACG(T) 8 0.16 AAG(K) 29 0.54 AGG(S) 15 0.35

GUU(V) 91 1.69 GCU(A) 48 1.22 GAU(D) 42 1.2 GGU(G) 50 0.94

GUC(V) 14 0.26 GCC(A) 24 0.61 GAC(D) 28 0.8 GGC(G) 20 0.38

GUA(V) 92 1.7 GCA(A) 81 2.05 GAA(E) 53 1.43 GGA(G) 102 1.92

GUG(V) 19 0.35 GCG(A) 5 0.13 GAG(E) 21 0.57 GGG(G) 40 0.75

a) RSCU, relative synonymous codon usage. Letters in brackets are the single-letter amino acid codes. Total number of codons, excluding the stop codons

(*)=3696.

544 LIU &WANG


(Powell & Moriyama, 1997; Rao et al, 2011). The total number of non-stop codons (CDs) of

PCGs was 3 696. Among all amino acids encoded by the 13 PCGs, Leu (14.04%), Ile (9.74%), Ser (9.38%), and Phe (9.20%) were the four most abundant amino acids, three of which were encoded by the AT-rich codons (see above), suggesting that the AT bias affected amino acid composition (Foster et al, 1997; Min & Hickey, 2007).

SUMMARY

Recently, T. molitor has been used as a model to

study intoxication response. However, analyzing the transcriptome of T. molitor is time-consuming work due to the lack of a sequenced genome (Oppert et al, 2012). A known T. molitor mitogenome sequence could accelerate contig assembly and annotation, as well as help to study informative rearrangements in mitochondrial genomes (Timmermans & Vogler, 2012) and perform high resolution phylogenetic analysis of Coleoptera (Kim et al, 2009).

Acknowledgements: We thank Dr. Zhong-Bao ZHAO

for helpful suggestions during manuscript preparation.

References

Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. 1999. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nature Genetics, 23(2): 147.

Boore JL, Lavrov DV, Brown WM. 1998. Gene translocation links insects and crustaceans. Nature, 392(6677): 667-668.

Bouchard P, Bousquet Y, Davies AE, Alonso-Zarazaga MA, Lawrence JF, Lyal CHF, Newton AF, Reid CAM, Schmitt M, Adam Ślipiński S, Smith ABT. 2011. Family-group names in Coleoptera (Insecta). ZooKeys, 88: 1-972.

Dobson AJ, Johnston PR, Vilcinskas A, Rolff J. 2012. Identification of immunological expressed sequence tags in the mealworm beetle Tenebrio molitor. Journal of Insect Physiology, 58(12): 1556-1561.

Eyre-Walker A. 1997. Differentiating between selection and mutation bias. Genetics, 147(4): 1983-1987.

Friedrich M, Muqim N. 2003. Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum. Molecular Phylogenetics and Evolution, 26(3): 502-512.

Foster PG, Jermiin LS, Hickey DA. 1997. Nucleotide composition bias affects amino acid content in proteins coded by animal mitochondria. Journal of Molecular Evolution, 44(3): 282-288.

Gomez A, Cardoso C, Genta FA, Terra WR, Ferreira C. 2013. Active site characterization and molecular cloning of Tenebrio molitor midgut trehalase and comments on their insect homologs. Insect Biochemistry and Molecular Biology, 43(8): 768-780.

Goptar IA, Shagin DA, Shagina IA, Mudrik ES, Smirnova YA, Zhuzhikov DP, Belozersky MA, Dunaevsky YE, Oppert B, Filippova IY, Elpidina EN. 2013. A digestive prolyl carboxypeptidase in Tenebrio molitor larvae. Insect Biochemistry and Molecular Biology, 43(6): 501-509.

Kim KG, Hong MY, Kim MJ, Im HH, Kim MI, Bae CH, Seo SJ, Lee SH, Kim I. 2009. Complete mitochondrial genome sequence of the yellow-spotted long-horned beetle Psacothea hilaris (Coleoptera: Cerambycidae) and phylogenetic analysis among coleopteran insects. Molecules and Cells, 27(4): 429-441.

Kim MI, Baek JY, Kim MJ, Jeong HC, Kim KG, Bae H, Han YS, Jin BR, Kim I. 2009. Complete nucleotide sequence and organization of

the mitogenome of the red-spotted apollo butterfly, Parnassius bremeri (Lepidoptera: Papilionidae) and comparison with other lepidopteran insects. Molecules and Cells, 28(4): 347-363.

Kim I, Lee EM, Seol KY, Yun EY, Lee YB, Hwang JS, Jin BR. 2006. The mitochondrial genome of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae). Insect Molecular Biology, 15(2): 217-225.

K ocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, Wilson AC. 1989. Dynamics of mitochondrial DNA evolution in

animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America, 86(16): 6196-6200.

Lavrov DV, Brown WM, Boore JL. 2000. A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus. Proceedings of the National Academy of Sciences of the United States of America, 97(25): 13738-13742.

Lee EM, Hong MY, Kim MI, Kim MJ, Park HC, Kim KY, Lee IH, Bae CH, Jin BR, Kim I. 2009. The complete mitogenome sequences of the palaeopteran insects Ephemera orientalis (Ephemeroptera: Ephemeridae) and Davidius lunatus (Odonata: Gomphidae). Genome, 52(9): 810-817.

Li H, Gao J, Liu H, Liu H, Liang A, Zhou X, Cai W. 2011. The architecture and complete sequence of mitochondrial genome of an assassin bug Agriosphodrus dohrni (Hemiptera: Reduviidae). International Journal of Biological Sciences, 7(6): 792-804.

Liao F, Wang L, Wu S, Li YP, Zhao L, Huang GM, Niu CJ, Liu YQ, Li MG. 2010. The complete mitochondrial genome of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae). International Journal of Biological Sciences, 6(2): 172-186.

Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research, 25(5): 955-964.

Min XJ, Hickey DA. 2007. DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins. DNA Research, 14(5): 201-206.

Ohde T, Yaginuma T, Niimi T. 2013. Insect morphological diversi-fication through the modification of wing serial homologs. Science, 340(6131): 495-498.



Ojala D, Montoya J, Attardi G. 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature, 290(5806): 470-474.

Oppert B, Dowd SE, Bouffard P, Li L, Conesa A, Lorenzen MD, Toutges M, Marshall J, Huestis DL, Fabrick J, Oppert C, Jurat-Fuentes JL, Palli SR. 2012. Transcriptome profiling of the intoxication response of Tenebrio molitor larvae to Bacillus thuringiensis Cry3Aa protoxin. PLoS One, 7(4): e34624.

Perna NT, Kocher TD. 1995. Unequal base frequencies and the estimation of substitution rates. Molecular Biology and Evolution, 12: 359-361.

Prokkola J, Roff D, Karkkainen T, Krams I, Rantala MJ. 2013. Genetic and phenotypic relationships between immune defense, melanism and life-history traits at different temperatures and sexes in Tenebrio molitor. Heredity, 111(2): 89-96.

Powell JR, Moriyama EN. 1997. Evolution of codon usage bias in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 94(15): 7784-7790.

Rao Y, Wu G, Wang Z, Chai X, Nie Q, Zhang X. 2011. Mutation bias is the driving force of codon usage in the Gallus gallus genome. DNA Research, 18(6): 499-512.

Salvato P, Simonato M, Battisti A, Negrisolo E. 2008. The complete mitochondrial genome of the bag-shelter moth Ochrogaster lunifer (Lepidoptera, Notodontidae). BMC Genomics, 9: 331.

Sheffield NC, Song H, Cameron SL, Whiting MF. 2009. Nonstationary evolution and compositional heterogeneity in beetle mitochondrial phylogenomics. Systematic Biology, 58(4): 381-394.

Simmons RB, Weller SJ. 2001. Utility and evolution of cytochrome b in insects. Molecular Phylogenetics and Evolution, 20(2): 196-210.

Simon E, Baranyai E, Braun M, Fábián I, Tóthmérész B. 2013. Elemental concentration in mealworm beetle (Tenebrio molitor L.) during metamorphosis. Biological Trace Element Research, 154(1): 81-87.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10): 2731-2739.

Taanman JW. 1999. The mitochondrial genome: structure, transcription, translation and replication. Biochimica et Biophysica Acta, 1410(2): 103-123.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24): 4876-4882.

Timmermans MJ, Vogler AP. 2012. Phylogenetically informative rearrangements in mitochondrial genomes of Coleoptera, and monophyly of aquatic elateriform beetles (Dryopoidea). Molecular Phylogenetics and Evolution, 63(2): 299-304.

Van Leeuwen T, Vanholme B, Van Pottelberge S, Van Nieuwenhuyse P, Nauen R, Tirry L, Denholm I. 2008. Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-Mendelian inheritance in action. Proceedings of the National Academy of Sciences of the United States of America, 105(16): 5980-5985.

Wolstenholme DR. 1992. Animal mitochondrial DNA: structure and evolution. International Review of Cytology, 141: 173-216.

Yan QH, Yang L, Wang Q, Zhang HR, Shao Q. 2013. Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor. Molekuliarnaia Biologiia, 46(4): 576-583.

Yang F, Du YZ, Wang LP, Cao JM, Yu WW. 2011. The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii. Gene, 485(1): 7-15.

Yin H, Zhi YC, Jiang HD, Wang PX, Yin XC, Zhang DC. 2012. The complete mitochondrial genome of Gomphocerus tibetanus Uvarov, 1935 (Orthoptera: Acrididae: Gomphocerinae). Gene, 494(2): 214-218.

Yukuhiro K, Sezutsu H, Itoh M, Shimizu K, Banno Y. 2002. Significant levels of sequence divergence and gene rearrangements have occurred between the mitochondrial genomes of the wild mulberry silkmoth, Bombyx mandarina, and its close relative, the domesticated silkmoth, Bombyx mori. Molecular Biology and Evolution, 19(8): 1385-1389.

Zhu JY, Yang P, Zhang Z, Wu GX, Yang B. 2013. Transcriptomic immune response of Tenebrio molitor pupae to parasitization by Scleroderma guani. PLoS One, 8(1): e54411.

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ZOOLOGICAL RESEARCH · 2014-12-16 · Zoological Research 2011; Pound et al, 2014), to name just a few. The key advantage of using NHPs is that NHPs are phylogenetically the most