Journal of Invertebrate Pathology 116 (2014) 13–17

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Journal of Invertebrate Pathology

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Differential susceptibility to the Withering Syndrome agent and renalcoccidia in juvenile Haliotis rufescens, Haliotis discus hannaiand the interspecific hybrid

0022-2011/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jip.2013.12.002

⇑ Corresponding author.E-mail addresses: rgo006@ucn.cl (R. González), klohrman@ucn.cl (K.B. Lohrmann),

jps009@ucn.com (J. Pizarro), kbrokord@ucn.cl (K. Brokordt).

Roxana González a,⇑, Karin B. Lohrmann b, Javiera Pizarro b, Katherina Brokordt a

a Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chileb Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 June 2013Accepted 2 December 2013Available online 12 December 2013

Keywords:Withering SyndromeCandidatus Xenohaliotis californiensisCoccidium Margolisiella haliotisHaliotis rufescensHaliotis discus hannaiAbalone hybrid

Withering Syndrome (WS) is a pathogenic chronic disease caused by the intracellular rickettsial-like bac-terium ‘‘Candidatus Xenohaliotis californiensis’’ (WS-RLOs), which affects many abalone species. Therenal coccidium (Margolisiella haliotis) has often been observed concurrent with WS infection. The redabalone Haliotis rufescens is a very susceptible species to WS and is also infected by the coccidium M. hal-iotis. In contrast, the Japanese abalone Haliotis discus hannai is not infected by these parasites. Interspe-cific hybridization is a method for improving important traits in animal husbandry. The objective of thisstudy was to determine susceptibility to WS-RLO and M. haliotis infection in the hybrid generated from across between red and Japanese abalones. Juveniles from both species and the interspecific hybrid werechallenged by exposure to effluent from red abalone adults infected with both parasites. The animalswere analyzed by histology at 130 days post-challenge. A 33% prevalence WS-RLOs was observed inthe red abalone H. rufescens, whereas a 20% prevalence was observed in the hybrid. Infections weregraded on a scale of 0–3. Of these red abalones infected, 53% presented grade 1 infection intensity,10% had grade 2 infections, and 50% had grade 3 infections. However, the hybrids only presented inten-sities at the extremes of the scale; of those infected 33% showed grade 1 infections and 66% had grade 3infections. The coccidium prevalence was 7% in red abalone individuals and 13% in the hybrid abalone. Incontrast, the Japanese abalone did not present infections with either parasite. As with the prevalence, theinfection intensities for the coccidium were higher in the hybrid abalone; of those infected 25% had grade2 infections, and 75% had grade 3 infections, but the red abalone presented only grade 2 infection inten-sities. Therefore, the hybrid did not inherited non-susceptibility or resistance characteristics of the paren-tal H. discus hannai and possessed biological conditions that could foster development of both parasites.Development of a culture based on this hybrid abalone should consider its susceptibility to infection bycoccidian, WS-RLOs and the potential for developing the WS disease.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Withering Syndrome (WS) is a pathogenic chronic diseasecaused by the intracellular bacterium ‘‘Candidatus Xenohaliotis cal-iforniensis’’, which is a rickettsial-like organism (WS-RLO). Thisbacterium forms inclusions in the gastrointestinal epithelium ofseveral abalone species (Haliotis spp.) both in natural populationsand in culture. Thus far, such infections have been observed in H.cracherodii, H. corrugata, H. walallensis, H. sorenseni, H. fulgens, H.diversicolor supertexta and H. rufescens (Friedman et al., 2002).WS is manifested by morphological changes in the digestive gland(metaplasia), which yields a loss of functionality; the animal enters

starvation, which forces it to catabolize its energy reserves and al-ters the energy balance with a subsequent decline in foot mass andultimately death (Friedman et al., 2000; Braid et al., 2005; Mooreet al., 2009; Gonzalez et al., 2012). Like many gastrointestinalpathogens, transmission is direct and horizontal through the fe-cal-oral route (Friedman et al., 2002). Renal coccidia are often ob-served in parallel with ‘‘Candidatus Xenohaliotis californiensis’’ invarious abalone species (Friedman et al., 1993; 2002). The abalonerenal coccidium, Margolisiella haliotis, has been found in H. cracher-odii, H. corrugata, H. fulgens, H. kamschatkana and H. rufescens(Friedman et al., 1993). Its transmission is direct and horizontal,and it develops its entire life cycle in a single host (Friedmanet al., 1993). Although it has not been associated with mortalityin abalone and is not considered a high risk parasite, infection bythis coccidium often damages much of the renal tissue (pers.obs.); therefore, it could affect its functionality.

14 R. González et al. / Journal of Invertebrate Pathology 116 (2014) 13–17

In Chile, two abalone species are cultured: the red abalone Haliotisrufescens from California and the Japanese abalone Haliotis discus han-nai. The red abalone is highly susceptible to WS, and is also suscepti-ble to infection by the coccidium M. haliotis. In contrast, the Japaneseabalone is not susceptible to infection with WS-RLO (Gonzalez et al.,2012), and no infections of M. haliotis have been recorded for this aba-lone species (pers. obs.). Despite such infections in red abalone, theproduction of this species is highly superior to that of Japanese aba-lone in Chile [450 vs. 6.3 tons per year, respectively (Aqua 2011)];however, the latter has a higher market value [USD 37 vs. USD 50per kg for red abalone and Japanese abalone, respectively (Aqua,2012)]. More red abalone is produced because it is more adaptableto intensive farming conditions and has a higher growth rate whichis reflected in high production. Furthermore, less Japanese abaloneis produced because it does not adapt well to the intensive cultivationsystem and shell–boring ectoparasite infections affect its growth(genus Polydora spp.) (Enríquez and Villagrán, 2008).

Interspecific hybridization has been used to improve traits, suchas growth, survival rates and disease resistance in many fish andmollusks (Lafarga-De la Cruz and Gallardo-Escárate, 2011; Elliot,2001). Certain abalone hybrids have a high potential for use inaquaculture because they show positive heterosis or hybrid vigorfor traits such as growth, survival rate and/or a greater temperaturetolerance range compared with the parental species (Hulata, 2001;Lafarga-De la Cruz and Gallardo-Escárate, 2011). Additionally,interspecific hybrids are a good model for understanding host-par-asite relationships, and the results demonstrate a large variabilityin the hybrid response to parasitism. A study on monogenean par-asite incidence (Dactylogyrus) in natural hybrid populations fromthe fish Barbus meridionalis and B. haasi, showed that the monoge-nean parasites are specific to B. meridionalis and the hybrids be-tween this species and B. haasi are not infected, which suggestsphysiological compatibility between monogenean parasites andtheir hosts (Moulia, 1999). Hybrid populations between B. barbusand B. meridionalis present a more complex scenario. In such spe-cies, the Diplozoon gracile (monogenean) population follows bothdistribution and introgression for the host populations. The para-sites are common in pure B. meridionalis populations, uncommonfor pure B. Barbus, present on intermediate introgressed populationand increase with B. meridionalis introgression levels (Le Brun et al.,1992). Hybrids with intermediate genomic introgression levelsshow an intermediate behavior and thus intermediate parasitismlevels (Le Brun et al., 1992). The mussel-trematode model generatessimilar observations. Natural hybrids from Mytilus edulis, which issusceptible to parasitism by the trematode Prosorhynchus squama-tus, and M. galloprovincialis, which is resistant to the trematode, aremore or less susceptible to P. squamatus infection in accordancewith their introgression levels (Coustau et al., 1991). As there isno general rule of thumb of the hybrids in relation to parasites, thissituation has to be evaluated for each case.

In a recent study, the fertilization rate, hatching, settlement,growth and survival for the hybrid from H. rufescens and H. discushannai were evaluated, and it was concluded that the hybrid couldbe grown on a commercial scale (Lafarga-De la Cruz et al., 2012). Inaddition, this hybrid presented advantageous morphological andphysiological characteristics, such as a thicker shell than its paren-tal species H. discus hannai (which could reduce susceptibility toectoparasites, such as Polydora spp.) and higher thermal tolerancecompared with both parental species. For the latter trait, the studyconcluded that the temperature for maximum expression of theHsp70 gene (thermal stress response) was 24 �C for red abalone,26 �C for Japanese abalone and 28 �C for the hybrid. Such observa-tions suggest a new genomic conformation, and could be indicativeof heterosis on this hybrid (Lafarga-De la Cruz et al., 2012).

Red abalone is not cultured in the far north of Chile because theseawater temperatures are higher [15–23 �C (Pizarro et al., 1994)]

than in the north central (12–21 �C) and southern areas (7–16 �C),wherein cultures have already been conducted (Enríquez and Vil-lagrán, 2008). It is thought that the high temperatures in the farnorth are unfavorable to abalone culture. However, the preferredtemperature for H. rufescens is 18.8 �C (Díaz et al., 2000), so a high-er growth rate should be expected at this temperature. Recentstudies show that high temperatures are detrimental to red aba-lone infected with WS-RLOs, which grow three times slower com-pared with uninfected animals at 20 �C (0.87 and 2.6 mm/month,respectively) (Avellanal, unpublished data). In addition to the ef-fects from infection on abalone growth, clinical signs of the diseasetypically develop in the red abalone when temperatures exceed18 �C (Braid et al., 2005; Moore et al., 2000; 2009). Thus, WS is athreat to red abalone cultivation, especially in northern Chile.

The wide thermal tolerance likely presented by the hybrid from H.rufescens and H. discus hannai could allow the extension of abalonecultivation to the extreme north of the country. Nevertheless, thefeasibility of such extension depends, in part, on controlling infec-tious diseases under such conditions. Moreover, although the poten-tial physiological effects from renal coccidia infections in abalone areunknown, such effects are important to consider. Thus, the objectiveof this study was to determine the susceptibility of different abaloneto WS-RLO and the coccidium M. haliotis in the hybrid from red aba-lone (a species highly susceptible to WS-RLO and coccidia) and Jap-anese abalone (a species not susceptible to WS-RLO and coccidia).

2. Materials and methods

2.1. Animal source and maintenance

Juveniles of Haliotis rufescens and Haliotis discus hannai (15–23 mm length; n = 75/species) free of WS-RLOs- and coccidia wereobtained from the Abalone Culture Center of the Universidad Cató-lica del Norte located in Coquimbo, Chile. Seventy-five WS-RLOs-and coccidia-free specimens of the inter-specific hybrid (in thesame size range as H. rufescens and H. discus hannai) were donatedby a company in the Coquimbo Region. Furthermore, 50 H. rufes-cens adults infected with WS (�8 cm shell length) were donatedby an abalone aquaculture center from the Coquimbo Region.The animals were acclimated for 14 days at 18 �C in pond condi-tions and fed ad libitum with Macrocystis pyrifera algae.

2.2. Experimental design and sampling

Of the 75 juveniles from each species and interspecific hybrids,15 were used to verify the absence of coccidia and WS-RLOsthrough histological analyses (see method discussed below). Theremaining individuals were divided into 2 groups (n = 30): Group1exposed to WS-RLOs and coccidia horizontal transmission (i.e.,they were treated with such agents) by continuous exposure toeffluent from 50 infected red abalone adults (located in a headertank); and group 2 not exposed to infected abalones and supplieddirectly with microfiltered seawater (i.e., the control group). Eachtreatment and control group was divided into 3 10-L tanks(n = 10/tank), which were maintained with aeration, flow and aconstant temperature (18 �C) with micro-filtered (0.45 lm) seawa-ter as well as fed the alga M. pyrifera. The individuals were evalu-ated as control and treatment groups by histological observationsof their soft tissues, 130 days after the experiment began.

2.3. Histological analysis

The tissues sampled (gills, digestive gland, post-esophagus andnephridia) were fixed with Davidson’s for 24 h, dehydrated, clearedin a tissue processor, embedded in paraplast, cut to 5 lm and

R. González et al. / Journal of Invertebrate Pathology 116 (2014) 13–17 15

stained with Hematoxylin–Eosin for observation using lightmicroscopy.

Two quantitative descriptors of parasite populations were used,prevalence and intensity of infection (Bush et al., 1997). In thisstudy these descriptors are semi-quantitative, since they were re-corded from histological sections. Prevalence corresponds to per-centage of hosts displaying presence of a particular parasitespecies or condition. It was recorded independently for each ofthe three tanks. Intensity of infection corresponds to the numberof individuals of a particular parasite species or of a condition inan individual host. To assess the infection intensity the evaluationof the WS-RLO load in the digestive tract was performed in accor-dance with the scale in Friedman et al. (1997) (with modifications),which assigns three degrees of infection intensity using the num-ber of bacterial inclusions in a 200�magnification field: grade0 = absence; grade 1 = 1–5; grade 2 = 6–10; and grade 3P11 inclu-sions per field. The field with the highest numbers of parasites waschosen for counting them. The coccidium Margolisiella haliotis inthe abalone nephridia was quantified in accordance with the scalein Friedman et al. (1993), which assigns four degrees of infectionbased on the number of parasites in a 200�magnification field:grade 0 = absence of coccidia; grade 1 = 1–10 per field; grade2 = 11–100 per field; grade 3 = 101–1000 per field; and grade4 = P 1000 parasites per field.

2.4. Statistical analyses

The levels of prevalence for WS-RLOs and the coccidium M. hal-iotis were compared between abalone groups (i.e., each species andinterspecific hybrids) through an analysis of variance (ANOVA).The prevalence levels were transformed using an arcsine function,and the infection intensities were normalized using thex0 =p

(x + 3/8) transformation (Anscombe, 1948). A Tukey’s multi-ple comparison test was applied (SPSS, 2008) to determine signif-icant differences between the groups.

3. Results

Characteristic basophilic and homogeneous ‘‘Candidatus Xeno-haliotis californiensis’’ inclusions were observed (Fig. 1) in the gas-trointestinal epithelial cells, from both the red abalone H. rufescensand the hybrid. Histological analysis also showed Margolisiella hal-iotis coccidia only in the red abalone and in the hybrids (Fig. 2).

The infection prevalence of WS-RLOs in the red abalone was33%, whereas in the hybrids a 20% prevalence was observed. How-ever, these differences in prevalence were not significant(P = 0.374; F = 14.891). In contrast, WS-RLOs inclusions were notobserved in the Japanese abalone (Fig. 3).

Fig. 1. (a) ‘‘Candidatus Xenohaliotis californiensis’’ (WS-RLO) (arrows) in the stomach ephomogeneous staining at the cytoplasmic vacuole inclusions and apical position inside thWS-RLO (arrow) infection in the stomach epithelium for the hybrid from Haliotis rufesce

The infection intensity in the red abalone was divided into threegrades (Fig. 4a). Of those infected, 40% of the individuals presentedgrade 1 infections, 10% showed grade 2 infections, and 50% hadgrade 3 infections. The hybrids only showed intensities at both ex-tremes of the scale; 33% showed grade 1 infections, and 66%showed grade 3 infections. However, intensity differences betweenthe red abalone and hybrids were not significant (P = 0.382;F = 5.247).

Ninety-one percent of the red abalone infected with WS-RLOspresented the RLOv variant (with bacteriophages, Fig. 1b) de-scribed by Friedman and Crosson (2012); however, this form waspresent only in 65% of the hybrids with WS-RLOs (Fig. 3). Further-more, the RLOv infection intensities were higher in the infected hy-brids because they presented grade 3 infections, while the redabalone showed three intensity levels: grade 1 (44%), grade 2(11%) and grade 3 (44%) (Fig. 4a).

In red abalone, coccidial infections did not trigger a histologi-cally evident defensive response; however, they did elicit high lev-els of hemocytic infiltration in the hybrid (Fig. 2b). The coccidiumprevalence level was 7% in the red abalones and 13% in the hybrids;however, such differences were not significant (P = 0.306;F = 3.945). The Japanese abalone did not present infections withthis parasite (Fig. 3). As with the prevalence levels, the coccidiuminfection intensities were also higher in the hybrid, wherein 25%of the individuals presented grade 2 infections, and 75% presentedgrade 3 infections. The red abalone only presented grade 2 intensi-ties (Fig. 4b); however, such differences were not statistically sig-nificant (P = 0.388; F = 2.482).

4. Discussion

The hybrid from the red abalone Haliotis rufescens and the Jap-anese abalone Haliotis discus hannai presented similar prevalenceand infection intensity levels for WS-RLOs and coccidia (Margolisi-ella haliotis) compared with the parental species H. rufescens.Therefore, this hybrid is susceptible to developing both infectionsand did not appear to have inherited WS resistance from its H. dis-cus hannai parental species. Thus the hybrid from H. rufescens andH. discus hannai facilitates development of these intracellular par-asites, and the hybrid would not be advantageous for cultivationbased on such traits.

Furthermore, the RLOv variant was detected in both red abaloneand the hybrid. This form is morphologically distinct from ‘‘Candid-atus Xenohaliotis californiensis’’ and has been reported in severalabalone species, including red abalone (Friedman and Crosson,2012). The RLOv variant corresponds to this bacterium infectedwith a bacteriophage (hyperparasite). This phage could affect thebacterial physiology, such as through virulence and pathogenicity

ithelium of hybrids from Haliotis rufescens and H. discus hannai abalones. Note thee host cells. (b) RLOv variant inclusion (i.e., with the bacteriophage) (arrowhead) andns and H. discus hannai abalones.

Fig. 2. Coccidium Margolisiella haliotis (arrows), infecting cells from the left nephridium in (a) Haliotis rufescens and (b) interspecific hybrid abalone. Note the stronghemocytic infiltrations in the hybrid abalone (asterisks).

Fig. 3. Infection prevalences for ‘‘Candidatus Xenohaliotis californiensis’’ (WS-RLOs), the RLOv variant and the coccidium Margolisiella haliotis in Haliotis rufescensand H. discus hannai as well as the interspecific hybrid abalone.

Fig. 4. Infection intensities for (a) ‘‘Candidatus Xenohaliotis californiensis’’, (b) theRLOv variant and (c) the coccidium Margolisiella haliotis in Haliotis rufescens and H.discus hannai as well as the interspecific hybrid abalone.

16 R. González et al. / Journal of Invertebrate Pathology 116 (2014) 13–17

loss (Friedman and Crosson, 2012). Thus, it is important to deter-mine whether it is present because it could yield attenuated bacte-ria unable to develop WS in the host.

Fritz et al. (1994) proposed four alternative hypotheses thatsummarize the varying responses of hybrid organisms to parasite

infection. These authors suggested the following hypotheses: (1)additive hypothesis: the hybrids do not differ from the parents’average resistance (i.e., the hybrid produces an intermediate re-sponse); (2) dominance hypothesis: the hybrid resistance is similarto the parents’ resistance; (3) hybrid susceptibility hypothesis: theparasite infection is higher in the hybrid than the parents; and (4)hybrid resistance hypothesis (contrary to the above hypothesis):the hybrids are more resistant than the parents. Although suchhypotheses were proposed for the plant-parasite model, certainarguments can be extrapolated to the animal-parasite model(Moulia, 1999). Our results show that the susceptibility for the hy-brid from H. rufescens and H. discus hannai to WS-RLOs and Margol-isiella haliotis coccidium infections is not different from the H.rufescens parental species. Therefore, the dominance hypothesiswould best explain this scenario, because the hybrid resistanceto these infections is similar to the less resistant or more suscepti-ble parent (in this context, dominance refers to the phenotypicsimilarities between parents and hybrids).

Experimental challenges using hybrids have shown differencesin their susceptibility and/or resistance depending on the para-site-hybrid system studied. For example, both monogenean para-sites (D. gracile) in the hybrid B. barbus-meridionalis (Le Brunet al., 1992) and trematode (P. squamatus) in the hybrid M. edu-lis-galloprovincialis (Coustau et al., 1991) support the additivehypothesis because the hybrid prevalence was intermediate com-pared with that of the parental species. On the other hand, certainstudies support the hybrid susceptibility hypothesis (negative het-erosis or hybrid depression). For example, both the prevalence andabundance for the monogenean parasite Diplozoon sp. and thetrematode Bolbophorus confusus were higher in the hybrid fromthe cyprinid fishes Alburnus alburnus and Rutilus rutilio comparedwith both parental species. The acanthocephalan parasite Pomp-horhynchus bosniacus was also more abundant in this hybrid’sintestine (Dupont and Crivelli, 1988). An example that supportsthe hybrid resistance hypothesis is a study on columnaris diseasecaused by the flavobacterium Flavobacterium columnare, wherethe hybrid catfish from Ictalurus punctatus and I. furcatus, was sig-nificantly more resistant to the disease (31% cumulative mortality)than its parents, who had an 81% average mortality after exposureto the pathogen (Arias et al., 2012). The varying results associatedwith disease resistance in hybrids highlights the importance andneed for pathogen-specific animal studies before developing a cul-ture system based on producing a particular hybrid.

Few studies have evaluated the effect of hybridization on dis-ease resistance in cultured mollusks. In one study, Dang et al.(2011) compared antiviral and antibacterial activity for H. laevigataand H. rubra abalone, as well as their interspecific hybrid, to deter-mine potential resistance differences for viral ganglioneuritis andvibriosis between them. Antibacterial activity differences betweenthe groups were not observed, but increased antiviral activity wasobserved in the hybrid compared with H. laevigata.

R. González et al. / Journal of Invertebrate Pathology 116 (2014) 13–17 17

Genetic diversity plays an important role in defense against par-asitic infections (Beardmore and McConnel, 1998). For example,genetic factors may determine the probability of a pathogen-hostencounter (e.g., through behavior and microhabitat use) as wellas developing the infection (physiological compatibility) (Coustauet al., 1991). The differential susceptibility to WS-RLOs and renalcoccidia among the red, Japanese and interspecific hybrid abalonecould be due to defensive-related mechanisms or to specific hostcharacteristics that facilitate pathogen infection. Recent studieshave demonstrated a defensive response associated with WS, be-cause red abalone with WS have a greater number of circulatinghemocytes in the hemolymph compared with uninfected animals(Avellanal, unpublished data). Furthermore, using transcriptomicanalyses, genes involved in innate immunity that are actively ex-pressed in animals resistant to WS have been discovered and char-acterized (Crosson et al., 2012). These approaches indicate that thesource of intra-species resistance may be immunological. Further,the presence or activity of such genes may also be due to suscep-tibility differences among abalone species.

For M. haliotis infections, no studies have investigated an asso-ciated immune response. However, this study demonstrates a sig-nificant hemocytic infiltration response to coccidial infection in thehybrid abalone. In the red abalone, no such defense mechanismagainst this parasite was observed. This could indicate that the hy-brid may have an immunological advantage against the coccidium.

Our results suggest that susceptibility to WS-RLO infection andthe potential for WS disease development should be considered forcultures based on a hybrid from H. rufescens and H. discus hannai. Inaddition, despite the lack of information on the physiological ef-fects from coccidial infections, such infections and their potentialeffect should be considered.

Acknowledgments

We are grateful to the Centro de Producción de Abalón de laUniversidad Católica del Norte (CPA-UCN) for their invaluablecontribution in providing all the necessary facilities required toundertake this study under controlled conditions. We also thankWilliam Farías and Mauricio Soler for their valuable support duringwork.

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