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Veterinary Parasitology 180 (2011) 389– 393

Contents lists available at ScienceDirect

Veterinary Parasitology

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hort communication

irulence of Beauveria bassiana, Metarhizium anisopliae andaecilomyces lilacinus to the engorged female Hyalomma anatolicumnatolicum tick (Acari: Ixodidae)

ing Sun, Qiaoyun Ren, Guiquan Guan, Zhijie Liu, Miling Ma, Huitian Gou, Ze Chen,ouquan Li, Aihong Liu, Qingli Niu, Jifei Yang, Hong Yin ∗, Jianxun Luo ∗∗

tate Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Grazing Animal Diseases MOA, Key Laboratory of Veterinary Parasitology of Gansurovince, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou 730046, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 1 December 2010eceived in revised form 11 March 2011ccepted 24 March 2011

eywords:iological controlirulenceyalomma anatolicum anatolicumeauveria bassianaetarhizium anisopliae

aecilomyces lilacinus

a b s t r a c t

The tick is a common ectoparasite of livestock and humans, and is responsible for the trans-mission of pathogens among hosts. Direct and indirect impacts of ticks include limitingthe sustainable development of the animal husbandry industry and detrimental effectson human health. Despite these negative effects, the main method of controlling ticksremains the application of chemical acaricides, which can lead to ambient pollution andthe development of tick resistance to them. The biocontrol of ticks is one of the alterna-tive control methods that has received recent research attention. The present study usedTenebrio moliter bait methods to collect 13 species of entomopathogenic fungi from dif-ferent areas in China that were then tested to observe their effects on engorged femaleHyalomma anatolicum anatolicum ticks. The results showed that more than half of the iso-lates had some pathogenic effects on the ticks; in particular, two Beauveria bassiana strains(B.bAT01, B.bAT17) and one Metarhizium anisopliae strain (M.aAT26) were highly virulent,

causing up to 90% mortality. In addition, H. anatolicum anatolicum females were treated withB. bassiana B.bAT17 using different concentrations of the fungus. Results revealed that B.bassiana B.bAT17 is highly pathogenic against engorged H. anatolicum anatolicum females.This is the first report of the pathogenic effect of entomopathogenic fungi on engorged H.anatolicum anatolicum females. However, studies of the efficiency of this fungus against

e requi

ticks in the field ar

. Introduction

Hyalomma anatolicum anatolicum (Koch, 1844) (Acari:

xodidae) is a three-host tick species that transmits dif-erent species of the parasitic protozoan genera Theileria,ncluding Theileria ovis, Theileria annulata and Theileria equimong others (Ahmed et al., 2006; Yin et al., 2007; Li

∗ Corresponding author. Tel.: +86 931 8342515; fax: +86 931 8342515.∗∗ Corresponding author. Tel.: +86 931 8342551; fax: +86 931 8340977.

E-mail addresses: yinhong@caas.net.cn (H. Yin),ectorparasit@126.com (J. Luo).

304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.03.027

red before it can be used for tick management in practice.© 2011 Elsevier B.V. All rights reserved.

et al., 2010) and Babesia (Luo et al., 2003; Guan et al.,2009) in China. It is one of the most widely distributedtick species infesting cattle, sheep and goat in XinjiangProvince. Although the ticks are known to suck the blood of,and irritate, their animal hosts, as well as acting as trans-mission vectors of protozoan parasites, the losses causedby them are difficult to calculate. However, what is clear isthat being able to control ticks would also greatly improve

the control of tick-borne diseases. Tick control worldwideis based mainly on the repeated use of chemical acari-cides, which has resulted in various issues, including thedevelopment of resistance in ticks, toxicity, contaminationof food products and environmental pollution (Onofre et al.,

390 M. Sun et al. / Veterinary Parasitology 180 (2011) 389– 393

Table 1Origin of entomopathogenic fungal strains.

Fungus Strain Original host Origin

Beauveria bassiana B.bAT01 Soil Anhui, ChinaBeauveria bassiana B.bAT03 Soil Guizhou, ChinaBeauveria bassiana B.bAT11 Soil Guangxi, ChinaBeauveria bassiana B.bAT14 Soil Zhejiang, ChinaBeauveria bassiana B.bAT17 Soil Jiangsu, ChinaBeauveria bassiana B.bAT25 Forficula scudderi Gansu, ChinaMetarhizium anisopliae M.aAT07 Soil Guizhou, ChinaMetarhizium anisopliae M.aAT14 Soil Guangdong, ChinaMetarhizium anisopliae M.aAT15 Soil Jiling, China

Metarhizium anisopliae M.aAT25

Metarhizium anisopliae M.aAT26

Metarhizium anisopliae M.aAT27

Paecilomyces lilacinus P.lAT01

2001; Graf et al., 2004; Ostfeld et al., 2006). These disad-vantages of acaricide use have stimulated the search foralternative tick control methods, particularly through theuse of biological control methods using natural predators,such as ants, spiders, birds, and so on (Sutherst et al., 2000).

Entomopathogenic fungi have been used widely for thecontrol of agricultural and forest pests (Kaaya et al., 1996).Recently, efforts have been made to evaluate the potentialof fungi as biocontrol agents against important arthropodvectors of human and animal diseases. Under laboratoryconditions, six out of the 57 major entomopathogenic fungi(Eumycota, Deuteromycotina) genera were found to bepathogenic to ticks (Tanada and Kaya, 1993). In addition,many studies have demonstrated the high level of efficacyof fungal biopesticides against ticks (Kaaya and Hassan,2000; Onofre et al., 2001; Benjamin et al., 2002; Samishet al., 2004; Polar et al., 2005; Leemon and Jonsson, 2008).

The effectiveness of using entomopathogenic fungias biological control agents has already been demon-strated against several tick species, including Rhipicephalus(Boophilus) microplus, Rhipicephalus sanguineus, Anocen-tor nitens and Amblyomma cajennense (Fernandes andBittencourt, 2008). Among the entomopathogenic fungalspecies, Beauveria bassiana and Metarhizium anisopliae havebeen found to be the most pathogenic against ticks underboth laboratory and field conditions (Samish et al., 2004;Ostfeld et al., 2006). In addition, B. bassiana has already beenfound to cause natural infections in the ticks Hyalommaanatolicum and Ixodes ricinus (Samsinakova, 1956). How-ever, its effects on H. anatolicum anatolicum are currentlyunknown. In the present study, the virulence of three ento-mopathogenic fungal species, B. bassiana, M. anisopliae andPaecilomyces lilacinus, was tested as a possible biocontrolagent of engorged females of H. anatolicum anatolicum. Theresults form the basis of fungal strain screening for futuretick biocontrol studies.

2. Materials and methods

Adult H. anatolicum anatolicum were collected from the

bodies of naturally infected cattle, goats and sheep in theKashi region, Xinjiang Uygur Autonomous Region in north-western China in 2001, as described by Guan et al. (2001).All off-host stages were maintained in an incubator at 27 ◦Cwith nearly 80% relative humidity (RH); the on-host stages

Soil Gansu, ChinaSoil Gansu, ChinaSoil Shanxi, ChinaSoil Henan, China

were fed on either sheep or rabbits. All animal experimentswere performed according to the approved InstitutionalAnimal Care and Use Committee guidelines.

The origins of the three entomopathogenic fungalstrains were collected from different areas in China usingthe Tenebrio molitor bait methods of Jia et al. (2006) and aredescribed in Table 1. Thirteen isolates were cultured on PDA(potato dextrose agar) in Petri plates for 2 weeks at 26 ◦C.Conidia were harvested by washing the plates with ster-ile aqueous 0.05% Tween-80 solution. A hemocytometerand microscope were used to determine the concentra-tion of conidia and the final concentration was adjustedto 108 conidia/ml.

2.1. Bioassay 1

The virulence of each fungal strain against engorged H.anatolicum anatolicum females was tested by immersing inconidial suspensions (108 conidia/ml) for 5 min, followingthe method previously described by Posadas and Lecuona(2009). A control group was immersed in the same volumeof sterile aqueous 0.05% Tween-80 solution. A total of 420engorged female ticks were used and the initial weight ofthe females is homogeneous. The ticks were divided into14 groups, each containing 30 engorged females; one groupwas used as control, the others were immersed in fungi (seeTable 2). After treatment, the ticks were placed individuallyin Petri dishes (50 mm × 15 mm) for incubation in a con-trolled environment chamber (26 ◦C and RH ≥ 80 ± 10%) for21 days. Tick mortality was recorded daily until the endof the experiment (at 21 days) (Hedimbi et al., 2008). Acorrected mortality was calculated using the formula rec-ommended by Food and Agriculture Organization of theUnited Nations (FAO) (Abbott, 1925; FAO, 2004):

corrected mortality

= %treated mortality − %control mortality100 − %control mortality

2.2. Bioassay 2

Conidia of B. bassiana B.bAT17 were collected as forBioassay 1, and suspensions of conidia with concentrationsof 1 × 107, 1 × 108, 1 × 109 conidia/ml were prepared using

M. Sun et al. / Veterinary Parasitology 180 (2011) 389– 393 391

Table 2Mortality and corrected mortality of H. anatolicum anatolicum treated with 108 conidia/ml of fungus, under laboratory condition (26 ◦C and RH ≥ 80 ± 10%).

Fungus Strain The number ofticks

The number ofdead ticks

Mortality (%) Correctedmortality (%)

Beauveria bassiana B.bAT01 30 30 100 100Beauveria bassiana B.bAT03 30 23 76.67 75.87Beauveria bassiana B.bAT11 30 11 36.67 34.49Beauveria bassiana B.bAT14 30 27 90 89.66Beauveria bassiana B.bAT17 30 30 100 100Beauveria bassiana B.bAT25 30 23 76.67 75.87Metarhizium anisopliae M.aAT07 30 4 13.33 10.34Metarhizium anisopliae M.aAT14 30 2 6.67 3.46Metarhizium anisopliae M.aAT15 30 1 3.33 0Metarhizium anisopliae M.aAT25 30 22 73.33 72.41

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Metarhizium anisopliae M.aAT26 30

Metarhizium anisopliae M.aAT27 30

Paecilomyces lilacinus P.lAT01 30

Control group 30

terile aqueous 0.05% Tween-80 solution (see Table 2).ighty engorged female ticks were divided into four groups,ne of which formed the control group. The remaininghree groups were submerged in the suspension of coni-ia for 5 min, whereas the control group was immersed in

sterile aqueous 0.05% Tween-80 solution under the sameonditions. After treatment, the ticks were weighed andultivated as in Bioassay 1. The females were observed dailyo check for mortality and egg-laying until the last tick died.he egg masses were placed individually in 5-ml labeledyringes for cultivation under the same temperature andelative humidity conditions mentioned for Bioassay 1.he following biological parameters were recorded: ini-ial weight before oviposition (mg), final weight after theick died 3 days (mg), weight change (initial weight − finaleight, mg), egg mass weight (mg), pre-oviposition (days),

viposition (days) and survival period (days), egg produc-ion index (%EPI), nutritional index (%NI) (Bennett, 1974).

.3. Statistical analyses

Statistical analyses were performed using the soft-are DPS (Data Processing System) for Windows. Theean values of the initial weight were analyzed using the

ukey test (p < 0.05) with a 5% significant level. The finaleight, weight change, egg mass weight, pre-oviposition,

viposition period, survival period, EPI and NI were not nor-ally distributed and were therefore analyzed using the

ukey test (p < 0.05) and the Student–Newman–Keuls testp < 0.05) with a 5% significant level.

. Results

This study investigated the susceptibility of engorged H.natolicum anatolicum females to infection by local strainsf the entomopathogenic fungi B. bassiana, M. anisopliaend P. lilacinus.

.1. Bioassay 1

The results of Bioassay 1 indicate that the infection effi-acy of these fungi varies according to the species andtrain. In the current study, all the given fungal isolates

28 93.33 93.101 3.33 03 10 6.901 3.33 0

were able to kill ticks except for M. anisopliae M.aAT15 andM.aAT27. The susceptibility of engorged H. anatolicum ana-tolicum females to the tested fungi is shown in Table 2. Thedegree of virulence of different strains of the fungi variedconsiderably. The corrected mortality of treated femalesranged from 34.49% to 100% and from 0% to 93.10%, 21 dayspost-inoculation, for different strains of B. bassiana and M.anisopliae respectively, whereas mortality in the controlgroup was zero for all strains. Notably, B. bassiana B.bAT01and B.bAT17 caused 100% mortality of ticks treated withthese strains.

In H. anatolicum anatolicum, B. bassiana B.bAT03,B.bAT14, B.bAT25 caused higher and more uniform mortal-ity rates than did B. bassiana B.bAT11. M. anisopliae strainsM.aAT25 and M.aAT26 also caused higher mortality ratesthan did the strains M.aAT07 and M.aAT14. However, P.lilacinus P.lAT01 only caused mortality rates of 10% (cor-rected mortality of 6.90%), with fewer entomopathogeniceffects on engorged females. Compared with the strains ofboth M. anisopliae and B. bassiana, P. lilacinus P.lAT01 hadlittle, if any, entomopathogenic effect on the engorged ticks(Table 2). Therefore, of the fungal genera and species tested,M. anisopliae and B. bassiana demonstrated the highest vir-ulence.

3.2. Bioassay 2

The results of Bioassay 2 are shown in Tables 3–5. Therewas no significant difference in the initial weight of thefemale ticks from the different groups before their expo-sure to B. bassiana B.bAT17. However, the final weight,weight change and egg mass weight of all the treatedgroups differed significantly (p < 0.05) from those of cor-responding control groups (Tables 3 and 5).

With regard to the reproductive and nutritional effi-ciency indexes, the pre-oviposition period was not affectedby infection with B. bassiana B.bAT17 at any conidial con-centration tested (p > 0.05) (Tables 4 and 5). The females

treated with B. bassiana B.bAT17 showed statistically sig-nificant differences in egg weight compared with thecontrol group. By contrast, the oviposition and survivalperiods of the treated groups were not significantly dif-ferent across the conidial concentrations tested (Table 4),

392 M. Sun et al. / Veterinary Parasitology 180 (2011) 389– 393

Table 3Mean weight of engorged females and egg mass of H. anatolicum anatolicum treated with different concentrations of B. bassiana B.bAT17, under laboratoryconditions (26 ◦C and RH ≥ 80 ± 10%).

Concentration of B. bassiana Initial weight (mg) Final weight (mg) Alteration weight (mg) Egg mass weight (mg)

0 332.50a ± 141.25(20) 99.33b ± 38.59(20) 235.75a ± 120.08(20) 154.75a ± 96.94(20)B.bAT17 107 314.66a ± 129.01(20) 275.84a ± 110.03(20) 40.17b ± 59.57(20) 17.76b ± 44.08(20)B.bAT17 108 317.03a ± 128.29(20) 290.17a ± 123.57(20) 28.66b ± 23.38(20) 3.89b ± 12.16(20)B.bAT17 109 324.92a ± 140.06(20) 292.18a ± 123.98(20) 38.88b ± 48.20(20) 7.57b ± 22.57(20)

(n) sample size.Note: The difference between means with different small letters in a column is significant (p < 0.05).

Table 4Mean pre-oviposition, oviposition and survival periods of engorged females of H. anatolicum anatolicum treated with different concentrations of B. bassianaB.bAT17, under laboratory conditions (26 ◦C and RH ≥ 80 ± 10%).

Concentration of B. bassiana Pre-oviposition period (days) Oviposition period (days) Survival period (days)

0 7.85a ± 2.30(20) 22.85a ± 5.98(20) 36.65a ± 6.74(20)B.bAT17 107 6.80a ± 1.48(5) 5.20b ± 2.77(5) 7.95b ± 3.90(20)

8 a 4.67b ± 2.89(3) 6.80b ± 3.09(20)2.75b ± 1.50(4) 7.15b ± 3.75(20)

mn is significant (p < 0.05).

Table 5Reproductive efficiency index (REI) and nutritional index (NI) of engorgedfemales of H. anatolicum anatolicum treated with different concentra-tions of B. bassiana B.bAT17, under laboratory conditions (26 ◦C andRH ≥ 80 ± 10%).

Concentration of B.bassiana

Reproductiveefficiencyindex-REI (%)

Nutritionalindex-NI (%)

0 42.91a ± 14.48(20) 60.79a ± 17.35(20)B.bAT17 107 4.58b ± 10.74(20) 12.53b ± 25.05(20)B.bAT17 108 1.16b ± 3.88(20) 5.38b ± 14.23(20)B.bAT17 109 1.53b ± 4.00(20) 5.48b ± 13.26(20)

B.bAT17 10 7.33 ± 2.08(3)

B.bAT17 109 9.75a ± 3.86(4)

(n) sample size.Note: The difference between means with different small letters in a colu

but were lower than those of the control group. B. bassianaB.bAT17 infection induced significantly different alter-ations in most of the parameters analyzed, with deleteriouseffects on the oviposition process of the engorged of H. ana-tolicum anatolicum females, even killing them before theystarted ovipositioning in some cases.

4. Discussion

Entomopathogenic fungi, in general, are important nat-ural enemies of arthropods and can be used as biologicalcontrol agents (Chandler et al., 2000). Fungi have long beenconsidered potential pathogens of ticks owing to their widedispersal, wide host range and their ability to internalizein ticks via the cuticle. It is therefore unsurprising thatthe high efficacy of fungal biocontrol on ticks has beendemonstrated using in vitro techniques (Fernandes andBittencourt, 2008; Polar et al., 2008).

Until the current study, there had been no pub-lished report on the effect of entomopathogenic fungi onHyalomma ticks, except that the tetranactin liuyang mycinat a concentration of 100 ppm could kill 98% of Hyalommadetritum larvae (Tian, 1984). The current study demon-strates that spore suspensions containing 108 conidia/mlare highly efficient in the biological control of engorgedH. anatolicum anatolicum females (Table 5). Gindin et al.(2002) showed that Rhipicephalus (Boophilus) annulatusfemales were infected with strains of M. anisopliae and B.bassiana before egg laying, and that the highest mortalitywas 90% at 7 days post-inoculation; by contrast, our resultsshowed the highest mortality rate of engorged females tobe 100% by 5.71–9.89 days post-inoculation for B. bassianaB.bAT17 at a concentration of 107–109 conidia/ml. The pre-oviposition period of engorged H. anatolicum anatolicum

females was not affected by B. bassiana B.bAT17 infectionwhen aqueous conidial suspensions were tested. Similarresults were observed by Bittencourt et al. (1997) andAngelo et al. (2010). Results of the present study revealedthat B. bassiana B.bAT17 is highly efficient in the biologi-

(n) sample size.Note: The difference between means with different small letters in a col-umn is significant (p < 0.05).

cal control of engorged H. anatolicum anatolicum females.Kaaya and Hassan (2000) also demonstrated high mortalityin the tick species Amblyomma variegatum and Rhipi-cephalus appendiculatus in experiments using B. bassianaand M. anisopliae isolates.

The onset of infection also differs depending on theability of the fungal strains to penetrate the tick directlythrough its cuticle. In our study, the treated dead ticksappeared swollen, with brown areas on their body on thesecond day after artificial infection. By the third day afterinfection, white fungal mycelium started to emerge andsporulate on the surface of the tick, especially in the legs.As a whole, the mean mortality rates of ticks resulting frominfection with B. bassiana were significantly higher than ofticks infected with M. anisopliae or P. lilacinus. B. bassianaB.bAT17 strain completed its full life cycle within the ticks,resulting in fertile conidiophores forming on the H. ana-tolicum anatolicum females. These results suggest that themain form of penetration of entomopathogenic fungi inticks is through the cuticle, as has been described for other

tick species (Bittencourt et al., 1995).

In conclusion, the data presented here show thatnative entomopathogenic fungi can infect the engorgedfemales of H. anatolicum anatolicum, but that their effi-

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iency varies according to the fungal species and strain.aranga and colleagues reported a reduction of the A.

ariegatum tick population in experimental field plots fol-owing application of the mitosporic entomopathogenicungi M. anisopliae and B. bassiana (Maranga et al., 2005;

aranga et al., 2006). Our laboratory studies illustratedhat, of all the strains studied, B. bassiana B.bAT17 showedhe highest pathogenicity against engorged H. anatolicumnatolicum females. This study is the first report of theathogenic effects of entomopathogenic fungi on engorged. anatolicum anatolicum females. However, evaluation of

he biocontrol potential of these fungal strains under fieldonditions is needed before they can be used in tick man-gement. Therefore, studies are required to investigate andalidate the methods for possible use of this fungus in theeld.

cknowledgments

This study was financially supported by the Support-ng Plan (2007BAD40B06), “948”(2010-S04), Key Projectf Gansu Province (1002NKDA035 and 0801NKDA033),eef and Yak Production System Programme, MOA, Spe-ific Fund for Sino-Europe Cooperation, MOST, China, andhe State Key Laboratory of Veterinary Etiological Biologyroject (SKLVEB2008ZZKT019). The research was also facil-tated by EPIZONE (FOOD-CT-2006-016236), ASFRISK (No.11691), ARBOZOONET (No. 211757) and PIROVAC (KBBE--245145) of European Commission, Brussels, Belgium.

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