Physiological and pathological changes in the eastern oyster Crassostrea virginica infested with the...

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Journal of Invertebrate Pathology xxx (2015) xxx–xxx

YJIPA 6639 No. of Pages 13, Model 5G

11 February 2015

Contents lists available at ScienceDirect

Journal of Invertebrate Pathology

journal homepage: www.elsevier .com/ locate/ j ip

Physiological and pathological changes in the eastern oyster Crassostreavirginica infested with the trematode Bucephalus sp. and exposedto the toxic dinoflagellate Alexandrium fundyense

http://dx.doi.org/10.1016/j.jip.2015.01.0110022-2011/� 2015 Published by Elsevier Inc.

⇑ Corresponding author.E-mail address: [email protected] (M. Lassudrie).

Please cite this article in press as: Lassudrie, M., et al. Physiological and pathological changes in the eastern oyster Crassostrea virginica infested wtrematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandrium fundyense. J. Invertebr. Pathol. (2015), http://dx.doi.org/1j.jip.2015.01.011

Malwenn Lassudrie a,⇑, Gary H. Wikfors b, Inke Sunila c, Jennifer H. Alix b, Mark S. Dixon b,Doriane Combot a, Philippe Soudant a, Caroline Fabioux a, Hélène Hégaret a

a Laboratoire des Sciences de l’Environnement Marin (LEMAR), Institut Universitaire Européen de la Mer (IUEM), UBO/CNRS/IRD/IFREMER, rue Dumont d’Urville,technopôle Brest-Iroise, 29280 Plouzané, Franceb Northeast Fisheries Science Center, NOAA National Marine Fisheries Service, 212 Rogers Avenue, Milford, CT 06460, USAc State of Connecticut, Department of Agriculture, Bureau of Aquaculture, P.O. Box 97, Milford, CT 06460, USA

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

323334353637383940414243

Article history:Received 27 June 2014Revised 21 January 2015Accepted 29 January 2015Available online xxxx

Keywords:Crassostrea virginicaAlexandrium fundyenseBucephalus sp.Perkinsus marinusHost–pathogen interaction

44454647484950515253545556

Effects of experimental exposure to Alexandrium fundyense, a Paralytic Shellfish Toxin (PST) producerknown to affect bivalve physiological condition, upon eastern oysters, Crassostrea virginica with a variablenatural infestation of the digenetic trematode Bucephalus sp. were determined.

After a three-week exposure to cultured A. fundyense or to a control algal treatment with a non-toxicdinoflagellate, adult oysters were assessed for a suite of variables: histopathological condition, hemato-logical variables (total and differential hemocyte counts, morphology), hemocyte functions (ReactiveOxygen Species (ROS) production and mitochondrial membrane potential), and expression in gills ofgenes involved in immune responses and cellular protection (MnSOD, CAT, GPX, MT-IV, galectin CvGal)or suspected to be (Dominin, Segon).

By comparing individual oysters infested heavily with Bucephalus sp. and uninfested individuals, wefound altered gonad and digestive gland tissue and an inflammatory response (increased hemocyteconcentration in circulating hemolymph and hemocyte infiltrations in tissues) associated with trematodeinfestation. Exposure to A. fundyense led to a higher weighted prevalence of infection by the protozoanparasite Perkinsus marinus, responsible for Dermo disease. Additionally, exposure to A. fundyense in trem-atode-infested oysters was associated with the highest prevalence of P. marinus infection. These observa-tions suggest that the development of P. marinus infection was advanced by A. fundyense exposure, andthat, in trematode-infested oysters, P. marinus risk of infection was higher when exposed to A. fundyense.These effects were associated with suppression of the inflammatory response to trematode infestation byA. fundyense exposure. Additionally, the combination of trematode infestation and A. fundyense exposurecaused degeneration of adductor muscle fibers, suggesting alteration of valve movements and catch state,which could increase susceptibility to predation. Altogether, these results suggest that exposure oftrematode-infested oysters to A. fundyense can lead to overall physiological weakness that decreaseoyster defense mechanisms.

� 2015 Published by Elsevier Inc.

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1. Introduction marinus (Dermo), Haplosporidium nelsoni (MSX) and Haplosporidi- 66
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The eastern oyster Crassostrea virginica, native to the east coastsof North and Central America, is a major commercial species with144,012 tons produced in 2011 in the USA, which accounted for75% of worldwide production of this species (FAO, 2014). Infectiousdiseases of eastern oysters caused by protozoan parasites Perkinsus

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um costale (SSO) are responsible for economic loss from mortalityduring epizootics (Ford and Tripp, 1996). The protozoan parasiteP. marinus has been the primary cause of mortality in cultured C.virginica on the east coast of the US (Ford and Smolowitz, 2007).Other parasites also may present a serious risk to the oysterindustry. Among these are the digenetic trematodes, referred toas bucephalids, that in advanced infestations affect gonadal folli-cles causing sterility (Lauckner, 1983). Marine bivalves are the firstintermediary hosts for bucephalids, fish being the final hosts(Olsen, 1974).

ith the0.1016/

http://dx.doi.org/10.1016/j.jip.2015.01.011

mailto:[email protected]


http://www.sciencedirect.com/science/journal/00222011

http://www.elsevier.com/locate/jip



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Diseases caused by parasites imply an imbalance in the host-parasite relationship; for example, the host immune response isinadequate to control proliferation. In bivalves, internal defenseis based upon an innate immune system composed primarily ofhemocyte-mediated cellular mechanisms and humoral factorssuch as lectins, lysins, and antimicrobial peptides (Cheng, 1996;Hine, 1999; Song et al., 2010; Soudant et al., 2013). The best knownimmune responses to microbial pathogens involve phagocytosis byindividual hemocytes followed by intracellular degradation(Cheng, 1996; Janeway and Medzhitov, 2002; Song et al., 2010;Soudant et al., 2013). Encapsulation by masses of hemocytes isanother mechanism of isolation and destruction of pathogens toolarge for phagocytosis (Cheng, 1981; Feng, 1988; Fisher, 1986,1988). Cellular immune response to bucephalid trematodes inbivalves usually is low or not detected (Cheng and Burton, 1965;da Silva et al., 2002) and consists mainly, when existent, ofhemocyte infiltration (Cheng and Burton, 1965); however, humoralfactors seem also to be involved (Feng and Canzonier, 1970).

The immune systems of marine bivalves may be affected byenvironmental factors, such as harmful algal blooms (HABs)(Hégaret et al., 2011). Toxic dinoflagellates, such as Alexandriumspecies that produce neurotoxins, were reported to decrease phago-cytosis, production of Reactive Oxygen Species (ROS), and adhesionby hemocytes of many bivalve species in in vitro assays (Hégaretet al., 2011; Mello et al., 2013), suggesting a direct immuno-suppressive effect. Such an effect was also hypothesized in vivo afterobservation of decreased total hemocyte count (THC) in the bluemussel Mytilus edulis (Galimany et al., 2008) and increase in hemo-cyte mortality in Crassostrea gigas (at 18 �C) (Hégaret et al., 2007)exposed to Alexandrium spp. A positive effect of A. minutum expo-sure (depending upon the season and the oyster ploidy) or of toxinaccumulation, however, was reported upon THC in C. gigas(Haberkorn et al., 2010a; Hégaret et al., 2007), possibly stimulatingthe immune system. Modifications of cellular pro- and anti-oxidantmechanisms, which may participate in the immune response andare involved in cellular protection, also have been reported(Fabioux et al., in preparation; Lassudrie et al., 2014). Whether ornot these effects upon immune-related variables are direct conse-quences of Alexandrium sp. exposure could not be assessed, becauseAlexandrium spp. also affect general physiological processes (Cucciet al., 1985; Gainey and Shumway, 1988a, 1988b; Landsberg,2002; Shumway, 1990) and tissue integrity (Galimany et al.,2008; Haberkorn et al., 2010b; Lassudrie et al., 2014; Medhioubet al., 2012), that could indirectly affect hemocytes.

A hypothesis is that toxic dinoflagellate exposure could increasebivalve susceptibility to disease by altering both defense andphysiological condition. HAB intensity and frequency are reportedto be increasing (Van Dolah, 2000), and HAB co-occurrence withpathogen infections in marine bivalves is recurrent. Only fewstudies, however, have reported the effects of these interactionswhich could lead to modified host–pathogen relationships(Bricelj et al., 2011; da Silva et al., 2008; Hégaret et al., 2009).One such interaction was reported by Bricelj et al. (2011), whoshowed that exposure to Alexandrium tamarense increased mortal-ity of juvenile clams challenged with Vibrio tapetis, the causativeagent of Brown Ring Disease.

The present study was undertaken to investigate possibleinteractions between parasitic diseases and toxic Alexandrium incompromising the health of the eastern oyster C. virginica. Thisstudy was conducted in spring, when Alexandrium fundyenseblooms may occur along the northeastern coast of the USA(Anderson et al., 2005; Hattenrath et al., 2010; Lopez et al., 2014)and when P. marinus intensity and Haplosporidium spp. prevalenceand intensity in oysters are at the lowest (Ford and Smolowitz,2007; Ford, 1985). Experimental oysters were found to containhigh infestations of the trematode Bucephalus sp. We hypothesized

Please cite this article in press as: Lassudrie, M., et al. Physiological and patholtrematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandj.jip.2015.01.011

that: (1) oyster exposure to A. fundyense could modify susceptibil-ity to parasitic infestations, and (2) the interaction of A. fundyenseand parasites could lead to combined effects upon oyster physio-logical condition. To test these hypotheses, we examined parasiticinfestations and histopathological changes in oysters exposed tocultured A. fundyense. In addition, we assessed immune status bydetermining hematology (total and differential hemocyte counts)and hemocyte functions (ROS production and mitochondrialmembrane potential), as well as gene expression of sequencescoding for proteins associated with immune responses andcellular protection: a galectin, CvGal, involved in recognition ofmicro-organisms prior to phagocytosis and also implicated inproliferation of P. marinus (Tasumi and Vasta, 2007); the twonewly-described C. virginica major plasma proteins Dominin (Itohet al., 2011) and Segon (Xue et al., 2012), for which specific rolesare still under investigation; antioxidant enzymes (MnSOD, CAT,GPX); and a metallothionein family (MT-IV) that responded toimmune challenge (Jenny et al., 2006; Wang et al., 2010) andwas isolated from hemocytes (Jenny et al., 2006). Gene expressionanalysis was conducted in the gill, an organ rich in hemocytes andin direct contact with the external milieu.

2. Materials and methods

2.1. Experimental oysters

Eastern oysters C. virginica of 9.3 ± 0.1 cm length (mean ± SE)were collected from Milford Harbor (CT, USA) on April 20th,2012, and placed the same day in tanks with running, unfilteredseawater.

2.2. Characteristics of the seawater supplied

Throughout the experiment, flow of 30–40 L h�1 of unfilteredseawater (containing natural phytoplankton) from Milford Harborwas distributed to the tanks. Temperature of the seawater was notcontrolled to provide the natural temperature of the local oysterhabitat and varied from 10.5 to 15.8 �C during the 23 days of theexperiment (12.9 ± 0.4 �C, mean ± SE). Salinity was 24.2 ± 0.5 ppt(mean ± SE) over the entire experiment. Light cycle in experimen-tal facilities was controlled, with L:D = 14:10, correspondingroughly to the natural cycle when the experiment was conducted.

Biodiversity of phytoplankton in the seawater supplied to thetanks was surveyed during the experiment. 150 mL was sampledevery 3 days and fixed with 3% Lugol’s solution. Sedimentationcolumns were used to concentrate the samples, and slides wereobserved under an inverted light microscope. Two diatoms,Skeletonema sp. and Thalassionema sp., dominated the phytoplank-ton community during the experiment, and no harmful algalspecies were detected.

Total particulate matter (TPM) and particulate organic matter(POM) in the water supplied to the tanks was quantified every4 days by filtering 300 mL in triplicate through Whatman GF/C fil-ters following the procedure described by Galimany et al. (2011).Briefly, TPM was determined by weighing dried filters (48 h at60 �C). Particulate inorganic matter (PIM) then was obtained byweighing ashed filters (4 h at 450 �C), and POM was calculated asthe difference between POM and TPM. TPM in the seawater sup-plied to the tanks was 13 ± 1 mg L�1 (mean ± SE) during the exper-iment, with POM accounting for 20 ± 1% of the TPM (mean ± SE).

2.3. Algal culture

The non-toxic dinoflagellate Scrippsiella lachrymosa (strainCCMP2666) was used as the control alga for the experiment, and

ogical changes in the eastern oyster Crassostrea virginica infested with therium fundyense. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/



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the toxic dinoflagellate A. fundyense (BF2 strain, isolated from theGulf of Maine, USA) was used for the toxic-algal exposure. Bothdinoflagellate species were cultured at 20 �C with 24 h light usingf/2-enriched filtered seawater from Milford Harbor (Guillard andRyther, 1962) in 20-L glass carboy assemblies using aseptic tech-nique (Ukeles, 1973). To maintain consistency in culture qualityduring the study, cultures were harvested semi-continuously inlate-log or early-stationary phase. The Paralytic Shellfish Toxins(PSTs) produced by the BF2 strain of A. fundyense, cultured and har-vested under these conditions, were previously reported to occurin the following mass proportions: GTX1,4 17.4%; NEO 29.0%;GTX2,3 5.4%; B1 6.2%; STX 6.6%; and C1,2 35.3% (Galimany et al.,2008). Cultured algal cell densities were determined by flowcytometry (FACScan, BD BioSciences, San Jose, CA, USA).

2.4. Experimental design and sampling

170 oysters were distributed haphazardly into ten 18-L tanks(17 oysters per tank). Oysters were acclimated for 3 days to labora-tory conditions with no addition of algal culture before beingexposed for 23 days (April 23rd to May 17th, 2012) to the two algaltreatments, S. lachrymosa or A. fundyense (5 replicates each). Fourpulses of algal culture per day were provided to each tank tosimulate tidal delivery. Each pulse of algal culture yielded a finalcell count in each tank of 103 cells mL�1 for the two first weeksand 2 � 103 cells mL�1 the last week (9–18 � 106 cells per oyster,per day after the T0 sampling). Water flow was stopped for 1 h fol-lowing algal distribution to allow feeding and prevent immediatewashout of the algae. This procedure added the dinoflagellates tothe existing, background plankton community described above.

80 oysters (8 oysters per tank) were sampled individually at theend of the acclimation period, prior to dinoflagellate addition (T0)and after 23 days exposure (T23). Hemolymph was withdrawn fromthe adductor muscle of each oyster with a syringe before oysterswere shucked, and soft tissues were weighed. Next, the anal-rectalregion was excised for P. marinus quantification, and a tissue cross-section and a transversal section were fixed for histological analy-sis. Remaining digestive gland and remaining gills were dissected,individually frozen immediately in liquid N2, and stored at �80 �Cfor toxin quantification and gene expression analysis, respectively.Oysters at T0 were analyzed for P. marinus only.

2.5. Condition index

Condition index (CI) was determined for 77 oysters at eachsampling time, T0 and T23, as follows (adapted from Bodoy et al.,1986):

CI ¼ wet flesh weightheight�width

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Table 1Semi-quantitative scale categorizing intensity of histopathological conditionsobserved (from Lassudrie et al., 2014).

Levelintensity

Occurrence of the pathological condition in the examined tissuearea

0.0 Absence0.5 Very low (1–5 total occurrence)1.0 Low (>5 occurrence/presence in all fields at magnification 10�)1.5 Moderate (presence in all fields at magnification 20�/covering

about 10% of the tissue)2.0 High (presence in all fields at magnification 40�/covering about

20% of the tissue)2.5 Very high (presence in all fields at magnification 60�/covering

about 30% or above of the tissue)

2.6. Analysis of hemocyte variables

77 oysters sampled at T23 were analyzed for hemocyte vari-ables. Hemolymph withdrawn from adductor muscle was storedtemporarily in Eppendorf microcentrifuge tubes held on ice beforeflow-cytometric analysis. Characteristics determined in live, circu-lating hemocytes – total (THC) and differential hemocyte counts(granulocytes and hyalinocytes) (in cell mL�1), size, and internalcomplexity (in arbitrary units a.u.), as well as mortality (percent-age of dead hemocytes) – were assessed following Hégaret et al.(2003). Functional responses, i.e. production of Reactive OxygenSpecies (ROS) (specifically H2O2 and O2

� �) by unstimulatedhemocytes and mitochondrial membrane potential (MMP) weredetermined as described in Delaporte et al. (2003) and Donaghy


et al. (2012), respectively. Hemocyte analyses were performedwith a FACScan flow-cytometer, and data were processed usingWinMDI 2.8.

2.7. Histopathological observations

54 oysters sampled at T23 were analyzed for histopathology. A5-mm cross section of soft tissues including gills, mantle, gonadand digestive gland, and a transversal section including adductormuscle, heart and kidney, were excised and fixed in Davidson’s fix-ative for 48 h at 4 �C (Shaw and Battle, 1957). Tissue sections thenwere rinsed in 50% ethanol in filtered seawater and transferred to70% ethanol, dehydrated in ascending ethanol solutions, clearedwith xylene, and embedded in paraffin wax. Six-lm sections werestained with Harris’ hematoxylin and eosin (Howard et al., 2004)and read blind under a light microscope.

Histopathological condition was examined per tissue. Intensityof each histopathological observation was rated using a six-levelsemi-quantitative scale ranging from 0 to 2.5 as described inTable 1, according to Lassudrie et al. (2014). Based upon this scale,mean weighted prevalences of each histopathological conditionwere calculated as the mean intensities of each histopathologicalcondition of all individuals.

In addition, the sum of mean weighted prevalences of eachhistopathological condition was calculated in each organ.

Gonadal maturation stage was determined following an 8-level,semi-quantitative scale (1: sexually undifferentiated, to 8:spawned) adapted from Ford and Figueras (1988) by Powell et al.(1993). Mean maturation stage was calculated and used forstatistical analysis.

2.8. Gene expression analysis in gills

77 oysters sampled at T23 were analyzed for gene expressionfollowing the procedure described by Béguel et al. (2013) andcomplying with Bustin et al. (2009) recommendations. Briefly,RNA was extracted from the gills and DNase-treated. The quantityand quality of total RNA were checked before reverse-transcrip-tion. The transcript levels of targeted genes were determined byqPCR relative to a constitutively-expressed reference gene, the18S rRNA (GenBank accession AB199895.1). Primers for 18S ribo-somal, Dominin (AB199895.1), Segon (JQ235755.1), MnSOD(EH646608.1), CAT (CV087224.1), GPX (CD650160.1) and all theknown MT-IV subtypes, A, B and C (DQ117912.1, DQ117913.1,DQ117914.1) were picked from the literature (Brulle et al., 2012;Itoh et al., 2011; Xue et al., 2012), or designed using Primer 3web v. 4.0.0 (Koressaar and Remm, 2007; Untergasser et al.,2012) from nucleotide sequences published in GenBank forCvGal (DQ779197.1) (forward: 50-GAACCGTGTAATGGCGAAGT-30,reverse: 50-TACATTCCCGAGGTCCAGTC-30). When non-annotatedsequences from C. virginica were used, gene identification was




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performed with BLASTX (Altschul et al., 1997), accepting the resultshowing the lowest E-value. Primer pairs amplified a singlesequence and sequencing of each amplicon (performed by GATCBiotech) ensured they corresponded to the expected nucleotidesequence. No Perkinsea and Bucephalidea nucleotide sequenceswas amplified according to sequence comparison in GenBankdatabase using BLASTN (Altschul et al., 1997). PCR efficiency ofall primer pairs, calculated based upon amplification of a standardcurve from a serial dilution of control cDNA, was constrainedbetween 95% and 105%. The relative expression ratio (R) of eachtarget gene was calculated following the Pfaffl (2001) formula.

2.9. Parasitic infestations

77 oysters for each sampling time, T0 and T23, were analyzed forP. marinus. A sample of the anal-rectal region from each oyster wasincubated for 8 days in the dark at room temperature in Ray’s FluidThioglycollate Medium (RFTM) as modified by Bushek et al. (1994).Hypnospores of P. marinus in tissue squashes then were stained withLugol’s solution and examined under the light microscope. Intensityof infection was determined using the Mackin scale (Mackin, 1962)(0: no parasite detected, to 5: heavy infection). Prevalence of infec-tion, mean weighted prevalence (intensity of the infection/totalnumber of oyster examined), and mean intensity (intensity of theinfection/number of infected oysters) were calculated.

Prevalence (n = 77) and intensity (n = 10) of trematode Buceph-alus sp. infestation were assessed by histological observations atT23. Intensity of infestation was determined as the mean percent-age of the area of Bucephalus sp. sporocyst occupation calculatedfrom 10 random fields per oyster (including gills, digestive gland,gonad and mantle) at total magnification 150�, using Image Jsoftware.

Other parasites were detected by examination of histologicalslides on 54 oysters at T23. Prevalence of Haplosporidium spp. wasdetermined, and weighted prevalence and intensity of the infectionwere assessed using a semi-quantitative scale followed by acomposite rating matrix as published by Kim et al. (2006) for afinal rating on a 4-level scale. Intensity of infestation with otherparasites was rated on the six-level semi-quantitative scale rang-ing from 0 to 2.5 described in Table 1. Mean weighted prevalenceof each infestation was calculated (intensity of the infestation/number of oyster examined) and used for statistical analysis.

2.10. Toxin detection

77 oysters sampled at T23 were analyzed for toxins. Digestivegland tissue was ground using a ‘‘Dangoumeau’’-like ball grinderand frozen in liquid nitrogen. Paralytic Shellfish Toxin (PST) accu-mulation in the digestive gland was estimated using a PSP ELISAkit (Abraxis), after being extracted from ground digestive glandin HCl 0.1 M (1:1, w:v) and boiled for 5 min, according to manufac-turer instructions, leading to acid hydrolysis that can induce chem-ical conversion of some PST analogues to STX (Vale et al., 2008).This ELISA assay recognizes mostly STX, and other PSTs only tovarying degrees (cross-reactivities of 100% for STX and from 29%to 0% for other PSTs). Consequently, toxicity was expressed aslg STX kg�1 of wet digestive gland weight. The principle of thisassay is different from bioassay or HPLC methods; therefore,results obtained with the Abraxis PSP ELISA kit should not becompared directly to toxicity obtained with other methods.

2.11. Statistical analysis

Considering the low number of oysters infected with Haplospo-ridium spp., no statistical analyses was performed to assess effectof this infection upon pathological and physiological variables.


Effects of algal treatment and trematode infestation were testedupon prevalence of P. marinus with non-parametric, Chi-squaretests performed independently in each Bucephalus sp.-infested orBucephalus sp.-uninfested group, to assess effect of A. fundyenseexposure. Differences in P. marinus infection weighted prevalencewere assessed using non-parametric tests: the Mann–Whitney test(effect of algal treatment or trematode infestation, independently)and the Kruskal–Wallis test (non-nested – comparison of fourgroups categorized by both algal treatment and trematode infesta-tion) followed by the Nemenyi–Damico–Wolfe–Dunn (NDWD)post hoc test, to detect differences between the four groups definedaccording to algal exposure and trematode status.

Effects of Bucephalus sp. and P. marinus infestation status uponPST accumulation were tested with a two-way ANOVA (the factorswere infestation with Bucephalus sp. and with P. marinus), andeffect of the intensity level of each infection upon PST accumula-tion was tested using the Kruskal–Wallis test.

Two-way ANOVA was used to analyze effects of trematodeinfestation, algal treatment, and the interaction, and effects ofP. marinus infection, algal treatment, and the interaction, uponhemocyte variables and gene expression, followed by the LeastSignificant Difference (LSD) post hoc test, to detect differencesbetween the four groups defined according to algal exposure andtrematode status, or algal exposure and P. marinus status.

For histopathological changes and other parasitic infestations,the Mann–Whitney test was used to assess the effect of algaltreatment, Bucephalus sp., and P. marinus infestation individually,and the Kruskal–Wallis test followed by the NDWD post hoc test,to detect differences between the four groups defined accordingto algal exposure and trematode status, or algal exposure andP. marinus status.

Data were log or 1/X transformed, when needed, to meetnormality and homoscedasticity assumptions, and percentage datawere transformed as Arc-sin of square root. Differences wereconsidered significant when p < 0.05. Statistics were performedwith Statgraphics Plus statistical software (Manugistics, Inc.,Rockville, MD, USA) and R version 2.15.1 (R Core Team, 2012).

3. Results

3.1. Parasitic infestations

Infestation with the trematode Bucephalus sp. was detected inhistological sections. The microscopic presentation of Bucephalussp. in the tissues consisted of sporocysts containing germ ballsand different developmental stages of cercariae (Fig. 2A, B, E).Although trematode sporocyst branches were present mainly inthe gonad, they could be observed in connective tissues in allorgans and in hemolymph vessels and were sometimes observedbreaking the hemolymph-vessel walls. After 23 days of algalexposure, 34% of experimental oysters were infested with thetrematode Bucephalus sp. (n = 77). Prevalence was not significantlydifferent at T23 between the A. fundyense-treated group (29%;n = 38) and the S. lachrymosa-treated group (38%; n = 39). Trema-tode sporocyst occupation accounted for 22 ± 2% of the tissues inoysters (mean ± SE, n = 10 oysters), and no significant differencebetween algal treatments was detected.

Prevalence of infection with P. marinus in oysters at the begin-ning of the experiment was 65% (n = 77) and decreased signifi-cantly to 38% after 23 days exposure to the control dinoflagellate(p < 0.01) (n = 39), whereas, it remained at 61% in A. fundyense-exposed oysters (n = 38) (Fig. 1A), but the difference between algaltreatments at T23 was not significant.

At the beginning of the experiment, P. marinus weighted preva-lence was 0.7 ± 0.1 (mean ± SE; n = 77) (Fig. 1A), and decreasedsignificantly after 23 days in S. lachrymosa-exposed oysters




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A B

Fig. 1. (A) Prevalence (bars) and weighted prevalence (lines) (mean ± SE) of Perkinsus marinus in oysters Crassostrea virginica at the beginning of the experiment (T0) and after23 days (T23) of algal exposure to the non-toxic Scrippsiella lachrymosa (dashed line and gray bar) or toxic Alexandrium fundyense (solid line and black bar). Letters indicatesignificant differences in prevalence (upper case; chi-square test, p < 0.01) and in weighted prevalence (lower case; Kruskal–Wallis test followed by NDWD post hoc test,comparing the four groups, p < 0.05). At T0, n = 77; at T23, n = 38–39 per group. (B) Prevalence of infection with P. marinus in oysters sampled at T23 according to algaltreatment and infestation with the trematode Bucephalus sp. ⁄ indicates significant difference among infested oysters between the two groups (chi-square test, p < 0.05). Nper group is indicated inside each bar.



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(p < 0.05) to 0.4 ± 0.1 (mean ± SE; n = 39). In A. fundyense-exposedoysters, weighted prevalence of P. marinus after 23 days exposure,however, did not vary significantly compared to day 0, remainingat 0.7 ± 0.1 (mean ± SE; n = 38), and was thus significantly highercompared to S. lachrymosa-exposed oysters (p < 0.05).

Trematode infestation (not determined at day 0) had no signif-icant effect upon prevalence and weighted prevalence of P. marinusat T23.

s

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Fig. 2. Histopathological conditions in experimental oysters Crassostrea virginica inducexposure for 23 days. (A) Bucephalus sp. sporocysts (s) in gill intraplical sinus inducing disassociated with hemocyte infiltration (⁄) and hemocytes (h) in gonaducts (G) and folliclfollicles (F) and visible in gonoducts (G); (D) heavy hemocyte infiltration (⁄) associated wduct and tubule degeneration in a digestive gland infested with Bucephalus sp. sporocystsand hemocyte infiltration (⁄) and presence of ceroid bodies (c); (F) normal digestive ductducts and undergoing diapedesis (h) associated with ceroid bodies (c) in the digestive glanBucephalus sp.-infested oyster exposed to A. fundyense; (I) normal adductor muscle.


A significant effect of algal exposure was found upon prevalenceof P. marinus among Bucephalus sp.-infested oysters at T23. Indeed,82% of Bucephalus sp.-infested oysters exposed to A. fundyense wereinfected with P. marinus (n = 11), which was significantly highercompared to prevalence of Bucephalus sp.-infested oysters exposedto the control dinoflagellate (40%; n = 15) (p < 0.05) (Fig. 1B). Such adifference was not detected within oysters uninfested withBucephalus sp. (45% of P. marinus prevalence).

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ed by the trematode Bucephalus sp. or its interaction with Alexandrium fundyenseruption of normal gill structure; (B) gonad infested with Bucephalus sp. sporocysts (s)es (F) lumina; (C) normal gonad of an uninfested oyster, with gametes maturing inith destruction of Bucephalus sp. sporocysts (s) in the digestive gland; (E) digestive(s): epithelium atrophy (a) associated with epithelium sloughing into the lumen (sl)s and tubules in an uninfested oyster; (G) hemocyte infiltrated (⁄) around digestived of Bucephalus sp.-infested oysters; (H) degenerated adductor muscle fibers (d) in a




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Intensity of P. marinus infection (calculated for infected oysters)was 1.02 ± 0.08 (mean ± SE; n = 88) and did not vary significantlyduring the experiment or with algal treatment or trematode status.

Haplosporidium spp. was observed in 9% of the examined oys-ters (5 of 54 oysters), at intensities ranging from 1 (4 oysters) to3 (1 oyster) and with a weighted prevalence of 0.13/4. Other micro-organisms and parasites were observed on histological slides:Nematopsis ostrearum oocysts in all examined organs, RLO (Rickett-sia-like organisms) in digestive tract epithelium, platyhelmintheTurbellaria sp. in intestinal lumen, and ciliates attached to the epi-thelia of pericardium, gills, mantle or in digestive tract lumina. Allof these organisms were observed at very low intensities and prev-alence: mean weighted prevalence per condition was less than 0.6/2.5, and no statistical difference was detected attributable to algalexposure, Bucephalus sp. or P. marinus infection (n = 54 oysters).

3.2. PST accumulation in the digestive gland

PST accumulation, as measured with the Abraxis ELISA assay,was 1,030 ± 124 lg STX kg�1 of wet digestive gland (mean ± SE,n = 38) in A. fundyense-exposed oysters after 23 days of exposure;no PST was detected in the control group. No significant differencein PST measurement was detected according to Bucephalus sp. orP. marinus infection status or level.

3.3. Condition index

No significant effect upon condition index after 23 days ofexperimental exposure was detected according to algal treatmentor P. marinus infection; whereas, infestation with the trematodeBucephalus sp. was associated with a significantly higher(p < 0.01) condition index (0.28 ± 0.01, mean ± SE; n = 26)compared to uninfested oysters (0.24 ± 0.01, mean ± SE; n = 51).

3.4. Histopathology

Statistical differences related to A. fundyense exposure, trema-tode Bucephalus sp., and the algal treatment – Bucephalus sp. inter-action, P. marinus, and the algal treatment – P. marinus interaction,upon each histopathological observation in experimental oystersafter 23 days of algal exposure, are presented in Table 2.

Intact and partially-lysed cells of A. fundyense or S. lachrymosaand of other algae from the seawater supplied were observed inintestine lumina of the oysters, regardless of trematode- or P. mari-nus-status.

Growth of Bucephalus sp. sporocysts in intraplical sinuses andconnective tissue of gills caused disruption of the normal configu-ration of gill filaments in all infested oysters (Fig. 2A). In addition,in the digestive gland hemolymph sinus of one oyster (of 54oysters analyzed), heavy focal infiltration with hemocytes wasobserved around the trematode sporocysts, associated with tissuedestruction (Fig. 2D).

Oysters infested with the trematode Bucephalus sp. had moreintense hemocyte infiltration in the mantle (p < 0.001), gills(p < 0.001), gonads (p < 0.001) (Fig. 2B, C) and digestive gland(p < 0.001), compared to oysters with no visible trematodes.Bucephalus sp. infestation also caused higher weighted prevalenceof ceroid bodies in gonadal tissues (p < 0.01) and in digestive-glandconnective tissue (p < 0.01) (Fig. 2E, G). In the digestive gland,trematode infestation was associated also with increased epithe-lium sloughing into digestive ducts (p < 0.05) and tubules(p < 0.001) (Fig. 2E, F), mucus in digestive duct lumina(p < 0.001), and hemocytes in digestive-tubule lumina (p < 0.05).Atrophy of the digestive-tubule epithelium (Fig. 2E, F) was associ-ated with both trematode infestation (p < 0.01) and P. marinusinfection (p < 0.05).


A reduction in the average gonadal maturation stage (p < 0.001)was detected in Bucephalus sp.-infested oysters (Fig. 2B, C, andFig. 3), with a mean maturation stage of 3.0 ± 0.2 in Bucephalussp.-uninfested oysters, corresponding to a mid-gonadal develop-ment stage, vs. 1.8 ± 0.1 in infested oysters, corresponding to anearly developmental stage.

Some histopathological conditions were observed more inten-sely in response to trematode infestation, but only in oystersexposed to the control alga S. lachrymosa: hemocyte infiltrationaround digestive ducts and tubules (p < 0.05) (Fig. 2E, F, G, andFig. 4B) or within gonoduct and follicle lumina (p < 0.05) (Fig. 2B,C, and Fig. 4D), and degeneration of gametes within follicles(p < 0.01) (Fig. 2B, C, and Fig. 4E).

In the kidney, hemocyte infiltration was lower in control oys-ters (exposed to S. lachrymosa and without trematodes) than inoysters exposed to A. fundyense or with Bucephalus sp. (p < 0.01)(Fig. 4C).

Trematode infestation (p < 0.01), P. marinus infection (p < 0.05)and A. fundyense-exposure (p < 0.05) led to higher weighted preva-lence of hemocyte diapedesis in digestive ducts (Fig. 2G). As aconsequence, trematode-infested oysters exposed to A. fundyensehad the highest weighted prevalence of hemocyte diapedesis indigestive ducts (p < 0.05) (Fig. 4A) compared to the other algaltreatment – Bucephalus sp. groups. The same result was observedin P. marinus-infected oysters exposed to A. fundyense comparedto the other algal treatment – P. marinus status groups (p < 0.05).

Degeneration of muscle fibers in the adductor muscle was sig-nificantly more intense in trematode-infested oysters also exposedto A. fundyense (p < 0.01) than in oysters in other groups (Fig. 2H, Iand Fig. 4F).

Other histopathological conditions observed did not vary signif-icantly with algal treatment, trematode or P. marinus status(Table 2).

The sum of the mean weighted prevalence for each type ofhemocyte response (infiltration, diapedesis, presence in lumina),per organ, is presented in Fig. 5A. The following general patternis apparent: A. fundyense exposure, and even more so Bucephalussp. infestation, increased hemocyte responses. Hemocyteresponses to the interaction, however, were lower compared tothe response to Bucephalus sp. alone.

The global pattern for ceroidosis in each organ is presented inFig. 5B as the sum of the mean, weighted prevalence per organ,illustrating that trematodes Bucephalus sp. infestation causedhigher intensity of ceroidosis.

The sum of the mean weighted prevalence for each type ofdegenerative condition (sloughing epithelium, cellular degenera-tions, mucus in digestive lumina) per organ shows that degenera-tion was associated principally with Bucephalus sp. (Fig. 5C).

3.5. Hemocyte variables

Percentage of dead hemocytes was lower in trematode-infestedoysters than in uninfested oysters (p < 0.05). Total hemocyte countwas significantly higher in oysters with trematodes and alsoexposed to the control alga S. lachrymosa (p < 0.05) (Fig. 6). Otherhemocyte variables were not affected significantly by algal treat-ment, trematodes, or the interaction. When considering the inter-action of algal treatment with P. marinus infection, MMP of bothgranulocytes and hyalinocytes was significantly higher in circulat-ing hemolymph of uninfected oysters exposed to the control alga(p < 0.05), but other hemocyte variables were not affected.

3.6. Gene expression

A significantly lower MnSOD gene expression level (p < 0.05)was found in trematode-infested oysters exposed to the control




Table 2Statistical differences in weighted prevalence of histopathological conditions in experimental oysters Crassostrea virginica according to exposure to either toxic Alexandriumfundyense or the control dinoflagellate Scrippsiella lachrymosa for 23 days (Mann–Whitney test), infestation with trematodes Bucephalus sp. (Mann–Whitney test), between thefour conditions defined by algal treatment and Bucephalus sp. infestation (Kruskal–Wallis test), according to Perkinsus marinus infection (Mann–Whitney test), and between thefour conditions defined by algal treatment and P. marinus infection (Kruskal–Wallis test).

Histopathologicalobservations

Algaltreatment(n = 26–28)

Bucephalus sp.infestation (n = 22–32)

Difference between the four algal –Bucephalus sp. conditions (n = 9–18)

Perkinsus marinusinfection (n = 27)

Difference between the four algal –P. marinus conditions (n = 9–18)

Pericardium–Ceroid bodies in

auricleNS NS NS NS NS

Kidney–Hemocyte infiltration ** NS ** NS NS–Ceroid bodies NS NS NS NS NS

Adductor muscle–Hemocyte infiltration NS NS NS NS NS–Muscle fiber

degeneration

* * ** NS NS

–Ceroid bodies NS NS NS NS NS

Gills–Hemocyte infiltration NS *** *** NS NS–Ceroid bodies NS NS NS NS NS

Mantle–Hemocyte infiltration NS *** ** NS NS–Ceroid bodies NS NS NS NS NS

Gonad–Maturation NS *** *** NS NS–Hemocyte infiltration NS *** *** NS NS–Ceroid bodies NS ** * NS NS–Gamete degeneration

in gonoductsNS NS NS NS NS

–Gamete degenerationin follicles

NS ** ** NS NS

–Hemocytes ingonoduct andfollicle lumina

NS * * NS NS

Digestive gland connective tissue–Hemocyte infiltration NS *** *** NS NS–Ceroid bodies NS ** * NS NS

Stomach–Epithelium sloughing

into the lumenNS NS NS NS NS

–Hemocytes indiapedesis

NS NS NS NS NS

–Hemocytes in lumen NS NS NS NS NS–Dinoflagellate cells in

lumenNS NS NS NS NS

–Other microalgal cellsin lumen

NS NS NS NS NS

–Hemorhage NS NS NS NS NS

Intestine–Hemocytes in

diapedesisNS NS NS NS NS

–Hemocytes in lumen NS NS NS NS NS–Dinoflagellate cells in

lumenNS NS NS NS NS

–Other microalgal cellsin lumen

NS NS NS NS NS

Digestive ducts and tubules–Hemocyte infiltration

surroundingNS ** * NS NS

Digestive ducts–Epitheliumsloughing into thelumen

NS * NS NS NS

–Hemocytediapedesis

* ** ** * *

–Hemocytes inlumen

NS NS NS NS NS

–Mucus in lumen NS * ** NS NS

(continued on next page)



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Please cite this article in press as: Lassudrie, M., et al. Physiological and pathological changes in the eastern oyster Crassostrea virginica infested with thetrematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandrium fundyense. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.01.011



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Table 2 (continued)

Histopathologicalobservations

Algaltreatment(n = 26–28)

Bucephalus sp.infestation (n = 22–32)

Difference between the four algal –Bucephalus sp. conditions (n = 9–18)

Perkinsus marinusinfection (n = 27)

Difference between the four algal –P. marinus conditions (n = 9–18)

Digestive tubules–Epithelium atrophy NS ** * * NS–Epitheliumsloughing into thelumen

NS *** *** NS NS

–Hemocytes inlumen

NS * NS NS NS

–Mucus in lumen NS NS NS NS NS

NS: no significant difference.* Significant differences were indicated by p < 0.05.

** Significant differences were indicated by p < 0.01.*** Significant differences were indicated by p < 0.001.

Bucephalus sp. : ScripsTr0 ScripsTr1 AlexTr0 AlexTr10

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Fig. 3. Percentages of oysters Crassostrea virginica at each gonadal maturation stagefollowing a semi-quantitative scale from 1 (sexually undifferentiated) to 8(spawned) (adapted from Ford and Figueras, 1988, by Powell et al., 1993) accordingto algal treatment (toxic Alexandrium fundyense or control Scrippsiella lachrymosafor 23 days) and trematode Bucephalus sp. infestation. N in each group: 13 inS. lachrymosa – Uninfested; 13 in S. lachrymosa – Infested;.18 in A. fundyense –Uninfested; 10 in A. fundyense – Infested.



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alga S. lachrymosa (R = 0.978 ± 0.006; mean ± SE; n = 15) comparedto uninfested oysters fed the same diet (R = 1.000 ± 0.009;mean ± SE; n = 24). Other gene transcript levels did not vary withalgal treatment, trematodes, or the interaction. No significanteffect of P. marinus infection and its interaction with algal treat-ment upon gene expression was detected.

4. Discussion

Finding a population of eastern oysters, C. virginica, with a highprevalence and severity of infestation with the trematode parasiteBucephalus sp. provided an unanticipated opportunity to describephysiological responses of oysters to multiple infectious agents.Additional parasitic infections with Perkinsus marinus and Haplos-poridium spp. were detected, but at lower intensity (and low prev-alence for Haplosporidium spp.) typical of the season. Both parasitescan cause extensive tissue disruption that may persist after infec-tions have declined; however, intensities of P. marinus and Haplos-poridium spp. at the study site have been low for years prior thisexperiment (Sunila, 2013), consistent with the few pathologicaleffects related to P. marinus in this study. Thus, the availability ofthis oyster population provided a model for experimental investi-gation of interactions between parasitism with Bucephalus sp.and harmful-algal-bloom exposure in affecting oyster health andsusceptibility to the other parasitic infections.

Results highlight the principle that harmful algal exposureinteracts significantly with both host and parasite, and such


interactions underscore the difficulty in diagnosing oyster healthin the natural environment where simultaneous, multiple stimulimay invoke oyster responses at tissue, cellular, and molecularlevels.

4.1. Effects of parasitic infestations

Infestation with the trematode Bucephalus sp. affected experi-mental oysters from the tissue to the molecular level. Connectivetissue in the gonad was the main site of infestation, suggesting thatthis species was Bucephalus cuculus, as observed previously in C.virginica by Hopkins (1954), McCrady (1874) and Tennent (1906)(NB: Tennent misidentified it as Bucephalus haimeanus at first,see Lauckner, 1983). The tissue occupation by the trematodeswas high enough to increase condition index. The main conse-quence of infestation in the gonad, the most severely affectedorgan, was reduced gonadal maturation compared to uninfestedoysters. In fact, bucephalid sporocysts are known to perturbgonadal development in bivalves, including C. virginica (Calvo-Ugarteburu and McQuaid, 1998; da Silva et al., 2002; Hopkins,1954; McCrady, 1874; Tennent, 1906), because the parasite partlysubstitutes for the gonadal tissue and uses the host’s reproductiveenergy (Calvo-Ugarteburu and McQuaid, 1998; Lafferty and Kuris,2009; Minguez et al., 2012). Energy uptake from the host by thetrematodes probably also explains the increased gamete degener-ation in gonadal follicles compared to uninfested oysters (in thenon-toxic dinoflagellate-exposed oysters). Observation of gilldeformation resulting from trematode sporocyst growth suggestedthat infestation could affect respiratory and feeding performance.

Digestive glands of oysters infested with Bucephalus sp. showeddegradation of digestive ducts and tubules, as indicated byepithelium sloughing into the lumen, atrophy of digestive tubuleepithelium, and presence of mucus in digestive duct lumen. Disar-rangement and degeneration of digestive diverticula and gut wereobserved also by Cheng and Burton (1965) in C. virginica infestedwith Bucephalus sp.; both mechanical and physiological causes,such as impaired nutrient uptake, were mentioned as possiblemechanisms (Cheng and Burton, 1966; Cheng, 1965). In addition,such degeneration can be typical with P. marinus and H. nelsoniinfections (Farley, 1968; Ford, 1985; Smolowitz, 2013), as shownby digestive tubule epithelium atrophy significantly associatedalso with P. marinus infection. These degenerations could thus alsopartly result from other, earlier, parasitic infections.

Hemocyte infiltration into several tissues was observed in oys-ters infested with Bucephalus sp., indicating an inflammatoryresponse, as also observed in mussels P. perna (da Silva et al.,2002). In our study, however, only one oyster showed focal, heavyhemocyte infiltration associated with partial destruction of trema-tode sporocyst branches. Effective destruction of Bucephalus sp. by




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Fig. 4. Mean weighted prevalences of histopathological conditions modified by the interaction of algal treatment (toxic Alexandrium fundyense or control Scrippsiellalachrymosa) and infestation with trematodes Bucephalus sp. in oysters Crassostrea virginica after 23 days of experimental algal exposure. (A) hemocyte diapedesis in digestiveducts; (B) hemocyte infiltration surrounding digestive ducts and tubules; (C) hemocyte infiltration in kidney; (D) hemocytes in gonoduct and follicle lumina; (E) degenerationof gametes in gonadal follicles; (F) muscle fiber degeneration in the adductor muscle. Letters indicate significant differences between conditions (Kruskal–Wallis test followedby post hoc NDWD test, p < 0.05). Error bars represent standard errors. N per group: 13 in S. lachrymosa – Uninfested; 13 in S. lachrymosa – Infested; 18 in A. fundyense –Uninfested; 10 in A. fundyense – Infested.



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hemocytic defense responses is usually not observed in bivalvesand has only been reported rarely (Cheng and Burton, 1965; daSilva et al., 2002; Douglass, 1975); whereas, other digenetic trema-todes, such as Proctoeces maculatus infesting mussels Mytilus gallo-provincialis can be encapsulated (Villalba et al., 1997). In addition,hemocytes also migrated in response to lesions caused by the par-asitic infestations. Indeed, degeneration of gametes in follicles wasassociated with infiltration and presence of hemocytes in follicleand gonaduct lumina, probably to resorb degenerated material. Indigestive glands of oysters exposed to the non-toxic dinoflagellate,degeneration caused by parasitic infections was associated withmore intense hemocyte infiltration. This also was associated withmore intense hemocyte diapedesis in the digestive-duct epitheliaof P. marinus- and in Bucephalus sp.-infested oysters, and presenceof hemocytes in digestive tubule lumina of Bucephalus sp.-infestedoysters. In hemolymph, the increase in hemocyte concentration intrematode-infested oysters exposed to the non-toxic alga wasaccompanied by a decrease in dead hemocytes within trematode-infested oysters. This may result from enhancement of de novohemocyte production, as proposed in previous studies (Hégaretand Wikfors, 2005; Hégaret et al., 2004, 2007; Soudant et al.,2004) and may help to sustain the inflammatory response.


In the two most infested tissues, gonad and digestive gland,presence of Bucephalus sp. also was associated with more numer-ous ceroid bodies (also referred to as brown cells, serous cells, porecells, or rhogocytes in the literature), as previously observed byCheng and Burton (1965). Ceroid, which is believed to play a rolein detoxication and in the excretory system in bivalves(Haszprunar, 1996; Zaroogian and Voyer, 1995), is constituted ofnon-degradable material resulting mainly from the accumulationof oxidized proteins and lipids (Yin, 1996). The increased intensityof ceroidosis in affected oysters may indicate oxidative stressinduced by the inflammatory response upon trematode infestation.In hemocytes of P. marinus-infected oysters exposed to the controldinoflagellate, MMP was lower compared to uninfected oysters,suggesting that P. marinus affects cellular metabolic functions ofits host. This effect could be a consequence of antioxidantcompounds produced by P. marinus that protect the parasite fromthe oxidative burst associated to phagocytosis and enables it todevelop within C. virginica hemocytes (Anderson, 1999; Schottet al., 2003; Smolowitz, 2013; Volety and Chu, 1995; and seereview of Soudant et al., 2013) or be associated with hemocyteapoptosis repression by P. marinus (Goedken et al., 2005; Hugheset al., 2010; Ly et al., 2003).




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Fig. 5. Sum of the mean weighted prevalences of each different histopathological conditions, per organ, corresponding to the following categories: (A) hemocyte responses;(B) ceroidosis; (C) degenerations in experimental oysters Crassostrea virginica after 23 days of experiment according to algal treatment (toxic Alexandrium fundyense or controlScrippsiella lachrymosa) and infestation with trematodes Bucephalus sp. N per group: 13 in S. lachrymosa – Uninfested; 13 in S. lachrymosa – Infested; 18 in A. fundyense –Uninfested; 10 in A. fundyense – Infested.

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Fig. 6. Total hemocyte count (THC) from circulating hemolymph of oysterCrassostrea virginica after exposure to toxic Alexandrium fundyense or controlScrippsiella lachrymosa for 23 days, and trematode Bucephalus sp. infestation. Lettersindicate significant differences (2-way ANOVA followed by LSD post hoc test,p < 0.05). N per group: 24 in S. lachrymosa – Uninfested; 15 in S. lachrymosa –Infested; 27 in A. fundyense – Uninfested; 11 in A. fundyense – Infested.



11 February 2015

4.2. Effect of A. fundyense exposure

In all oysters exposed for three weeks to A. fundyense, histolog-ical observations of intact or lysed A. fundyense cells in intestinelumen, as well as PST accumulation in the digestive gland, demon-strated that oysters ingested and digested this alga. In oystersuninfested by trematodes, increased hemocyte infiltration inresponse to A. fundyense exposure also was detected in connectivetissues of kidneys, which form an excretory complex together withthe pericardial gland (Morse, 1987). This inflammatory response,which was specific to this organ, may indicate that algal toxinsare carried, presumably by hemocytes, toward kidneys to beexcreted. Galimany et al. (2008) also suggested that hemocytescould transport PST from tissues to feces by diapedesis throughthe digestive epithelium. In fact, hemocyte diapedesis is a typicalresponse of bivalves exposed to toxic Alexandrium spp.(Haberkorn et al., 2010b; Lassudrie et al., 2014; Medhioub et al.,2012). Similarly, we observed that A. fundyense exposure induceddiapedesis of hemocytes through the digestive duct epithelia inoysters uninfested by trematodes.

In addition, A. fundyense exposure affected oyster susceptibilityto Dermo disease, caused by P. marinus. Indeed, althoughweighted prevalence decreased during the time course of theexperiment in oysters exposed to the control alga, no decreasewas observed in A. fundyense-exposed oysters, resulting in ahigher weighted prevalence than in control oysters. In contrast,da Silva et al. (2008) described lower intensity of infection withPerkinsus olseni in Manila clams exposed to the toxic dinoflagellateKarenia selliformis, which was attributed to direct toxicity of thealga to the parasite. Similarly, another dinoflagellate, Prorocentrum


minimum, was reported to be toxic to P. olseni (Hégaret et al.,2009). Such direct interaction between A. fundyense and P. marinuswould not explain the results of the present study. Thus, inabilityto repress P. marinus infection could have been caused by physio-logical alteration to the host induced by A. fundyense exposure,either by decreasing P. marinus excretion rate, or by decreasingdefense mechanisms. The last hypothesis was suggested byGalimany et al. (2008) after observation of increased trematode(Gymnophallidae) prevalence after A. fundyense exposure inmussels. In the present study, effects of A. fundyense alone uponhemocytes were minimal, suggesting that other mechanisms areinvolved.

4.3. Effects of the interaction between parasitic infestations andA. fundyense exposure

In oysters infested with the trematode Bucephalus sp., theexposure to A. fundyense was associated with a higher prevalenceof P. marinus, suggesting that the combined effects of A. fundyenseand trematodes may promote P. marinus infection or at leastprevent regression.

The responses of hemocytes to the trematodes that were sup-pressed by A. fundyense exposure (THC, infiltrations in kidneyand in digestive gland, hemocytes in gonoduct and follicle lumina)may have impaired immune functions. In fact, deficiencies inimmune functions could explain why infection and proliferationof P. marinus was enhanced.

Another response to combined Bucephalus sp. infestation andA. fundyense exposure was degeneration of adductor muscle fibers.Paralytic Shellfish Toxins (PSTs) and bucephalids individually areknown to affect adductor muscles of bivalves (Calvo-Ugarteburuand McQuaid, 1998; Haberkorn et al., 2010b; Hégaret et al.,2012; Howell, 1967); however, in the present experiment, onlythe interaction of both led to a significant increase in myodegener-ation. Again, suppression of hemocyte responses could have a rolein this pathology. A first hypothesis considers the potential role ofhemocytes in carrying and detoxifying PSTs, which would be lessefficient in parasitized oysters. Although the PST content in thedigestive gland remained unchanged between oysters with orwithout trematodes, PST concentration in other tissues may havevaried. Another hypothesis considers the role of hemocytes innutrient digestion and transport, combined with trematode energyuptake, resulting in depressed nutrient supply to the muscle andenergy depletion. The degeneration of adductor muscle fiberscould have deleterious consequences upon valve movements andthe catch state. Adductor muscle dysfunction also could affectfiltration, interfering with respiration and nutrition functions,and could increase susceptibility of oysters to predation andexposure to environmental variations that otherwise could bemoderated by shell closure.




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11 February 2015

5. Conclusions

Infestation with trematodes, Bucephalus sp., affected oyster, C.virginica, physiological functions including reproduction andimmunity. In addition, considering histopathological lesions ingills and digestive tubules, respiration and digestion could bealtered. Neither A. fundyense blooms nor Bucephalus sp. infestationhave been reported to be lethal for C. virginica. The results of thepresent study suggest, however, that an A. fundyense bloom couldintensify the severity of a potentially-lethal disease, Dermo,especially in trematode-infested oysters that would be more sus-ceptible to Dermo infections. In addition, oysters with trematodeinfestations exposed to blooms of PST-producing microalgae maybe more susceptible to predation as a consequence of muscle fiberdegeneration that could compromise valve-closure movementsand the catch state of the adductor muscle. There is increasingevidence of HAB relationships with climate change (temperature,eutrophication, etc.) (Anderson et al., 2008; Hallegraeff et al.,2003), and HABs can modify host–pathogen interactions, for exam-ple by aggravating disease status as demonstrated in the presentstudy. In this context, a better understanding of complex bioticinteractions is needed to predict and possibly prevent new diseaseoutbreaks in bivalve populations.

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Acknowledgments

This work was supported by Université de Bretagne Occiden-tale, the NOAA Fisheries Service Aquaculture Program, and from‘‘Laboratoire d’Excellence’’ LabexMER (ANR-10-LABX-19), whichwas co-funded by a grant from the French government under theprogram ‘‘Investissements d’Avenir’’. Authors are grateful to EveGalimany, Barry Smith, Yaqin Li, Christophe Lambert, Nelly LeGoïc, Anne-Laure Cassone and Marc Long for technical assistanceand advice, to Milford laboratory staff for their participation indissections, to Joseph DeCrescenzo for help with the RFTM assayand to Sébastion Hervé for graphical abstract realization. Authorsare also grateful to the anonymous reviewers who helped improvethe manuscript.

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