Fish & Shellfish Immunology 44 (2015) 232e240

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Resistance of Black-lip learl oyster, Pinctada margaritifera, to infection by Ostreid herpes virus 1mvar under experimental challenge may be mediated by humoral antiviral activity Terence L.S. Tan a, Ika Paul-Pont b, Olivia M. Evans b, Daniel Watterson c, Paul Young c,
Bichet d, Andrew C. Barnes a, *, Richard Whittington b, Angelique Fougerouse d, Herve a , 1
cile Dang Ce a

The University of Queensland, School of Biological Sciences and Centre for Marine Science, Brisbane, Queensland 4072, Australia The University of Sydney, Faculty of Veterinary Science, Camden, New South Wales 2570, Australia c Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia d Direction des Ressources Marines, Papeete, French Polynesia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 June 2014 Received in revised form 19 January 2015 Accepted 14 February 2015 Available online 21 February 2015

Ostreid herpesvirus 1 (OsHV-1) has induced mass mortalities of the larvae and spat of Pacific oysters, Crassostrea gigas, in Europe and, more recently, in Oceania. The production of pearls from the Black-lip pearl oyster, Pinctada margaritifera, represents the second largest source of income to the economies of French Polynesia and many Pacific Island nations that could be severely compromised in the event of a disease outbreak. Coincidentally with the occurrence of OsHV-1 in the southern hemisphere, C. gigas imported from New Zealand and France into French Polynesia tested positive for OsHV-1. Although interspecies viral transmission has been demonstrated, the transmissibility of OsHV-1 to P. margaritifera is unknown. We investigated the susceptibility of juvenile P. margaritifera to OsHV-1 mvar that were injected with tissue homogenates sourced from either naturally infected or healthy C. gigas. The infection challenge lasted 14 days post-injection (dpi) with sampling at 0, 1, 2, 3, 5, 7 and 14 days. Mortality rate, viral prevalence, and cellular immune responses in experimental animals were determined. Tissues were screened by light microscopy and TEM. Pacific oysters were also challenged and used as a positive control to validate the efficiency of OsHV-1 mvar infection. Viral particles and features such as marginated chromatin and highly electron dense nuclei were observed in C. gigas but not in P. margaritifera. Mortality rates and hemocyte immune parameters, including phagocytosis and respiratory burst, were similar between challenged and control P. margaritifera. Herpesvirus-inhibiting activity was demonstrated in the acellular fraction of the hemolymph from P. margaritifera, suggesting that the humoral immunity is critical in the defence against herpesvirus in pearl oysters. Overall, these results suggest that under the conditions of the experimental challenge, P. margaritifera was not sensitive to OsHV-1 mvar and was not an effective host/carrier. The nature and spectrum of activity of the humoral antiviral activity is worthy of further investigation. Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.

Keywords: Ostreid herpesvirus Humoral immunity Hemocytes Pinctada Herpes

1. Introduction

* Corresponding author. E-mail address: [email protected] (A.C. Barnes). 1 Present address: Department of Fisheries, Government of Western Australia, Perth, WA, Australia. http://dx.doi.org/10.1016/j.fsi.2015.02.026 1050-4648/Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.

Herpesvirus infections in bivalve molluscs were initially reported in 1972 in the Eastern oyster Crassostrea virginica in the USA [1]. Since then, numerous reports of infections by herpesviruses have emerged from countries all over the world including France [2,3], New Zealand [4], Australia [5], Mexico, Ireland [6e8], Spain [9], China and Japan [10]. The host range of Ostreid herpesvirus

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includes Pacific Oyster Crassostrea gigas [2,4,11], European flat oyster Ostrea edulis [12,13], Portuguese oyster Crassostrea angulata [12], Chilean oyster Tiostrea chilentis [14], Suminoe oyster Crassostrea rivularis [12,15], Manila clam Ruditapes philippinarum [12,16,17], carpet shell clam Ruditapes decussatus [18] and French scallop, Pecten maximus, [19]. Interspecies viral transmission has been demonstrated at the larval stage [12,15]. Infections by herpes or herpes-like viruses in bivalves appear to be ubiquitous and are often associated with sporadic episodes of high mortalities in the larvae and spat of susceptible species [20,21]. Outbreaks occur predominantly during the summer period, concomitant with the rapid increase of seawater temperature [20,21]. Infected bivalves do not exhibit any detectable gross pathological signs associated with the infection prior to death [2,22]. While the virus is detectable in all developmental stages of susceptible species, adults are less sensitive to the disease than spat and juveniles [5,15,23]. Several genotypic variants of the OsHV-1 virus (i.e. OsHV-1mvar, OsHV-1 D9 and OsHV-1 D15) have gradually replaced the OsHV-1 genotype as a cause of mortalities in bivalves. OsHV-1 mvar appeared during the summer of 2008 and subsequently replaced the OsHV-1 genotype in episodic summer outbreaks in C. gigas in France [3]. The virus has expanded its geographical range to the southern hemisphere during the spring-summer of 2010e2011 [6,24,25]. While previous herpesvirus infections in the Oceania region were not associated with mass mortalities of infected bivalves [4,5,26], OsHV-1 mvar was responsible for mass mortalities of C. gigas for three consecutive years since 2010/2011 in Australia and New Zealand [24,27,28]. Recently in French Polynesia, several batches of C. gigas imported from France and New Zealand for human consumption tested positive for OsHV-1 mvar (H. Bichet and D. Saulnier pers. comm., 2012). The culture of the Black-lip pearl oysters (Pinctada margaritifera) represents the second largest source of income for French Polynesia, contributing more than US$ 120 million per annum to the local economy [29,30]. An accidental release of Pacific oyster wastes into the lagoon environment in which pearl oysters reside is conceivable naturally raising concern that the virus can be transmitted to local pearl oysters. The present study investigated the susceptibility of P. margaritifera to OsHV1 mvar by determining mortality rate, viral prevalence, histopathological features, and cellular immune responses of juvenile P. margaritifera during an infection challenge experiment. 2. Materials and methods 2.1. Animal husbandry Hatchery-reared P. margaritifera (
12 months old, 4e5 cm length) were purchased in September 2012 from a commercial farm in Western Australia. It was not possible to import pearl oysters from French Polynesia due firstly to biosecurity restrictions but also because this oyster species is fragile and cannot stay out of the water for the prolonged period of time required for transit between the two countries. Oysters were transferred into a holding tank containing 300 L of artificial seawater (35 ppt) maintained at 23  C, with aeration and bio-filtration. Seawater was renewed at a rate of 120 L per hour. This process allowed the depuration of wastes accumulated during transportation. After 48 h, the oysters were transferred to 12 x 25 L tanks containing recirculating 35 ppt artificial seawater with bio-filtration and UV sterilisation in a Physical Containment level 2 certified containment facility (http:// www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/ certifications-1). Oysters were acclimated for 7 days and the temperature was gradually increased to 25  C at a rate of 0.3  C per day. P. margaritifera were fed daily with a phytoplankton mix containing

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Shellfish diet 1800™ and Nanno 3600™. Feeding ceased 1 day prior to the challenge experiment. Triploid OsHV-1 mvar -free C. gigas (5 months old) were collected in August 2012 from a commercial lease on the Hawkesbury River, New South Wales (NSW) and were used as a positive control during the experiment. The population of origin was free of clinical signs of disease and tested negative (n ¼ 30) for OsHV-1 mvar using a TaqMan assay adapted from a previously published protocol developed by Refs. [28,31]. Oysters were acclimatised following the protocol described above. 2.2. Challenge design The experiment lasted for 14 days post-injection (dpi) and consisted of two treatments: oysters injected with the control inoculum (control group) and oysters injected with the infected inoculum (challenged group). Each treatment group comprised 6 x 25 L experimental aquaria with 20 oysters per tank. Two aquaria were dedicated for monitoring of mortality rates while 4 were used for active sampling during the time course of the experiment. All aquaria were fitted with lids to prevent cross contamination by aerosols. For the inocula preparation, infected C. gigas were collected from the Georges River, NSW, in November 2011 (infected inoculum) at the same location where infected oysters were sampled previously [24] and healthy C. gigas (control inoculum) were collected from the OsHV-1 mvar free Hawkesbury River in December 2011. Both batches of oysters were stored at 80  C. Five oysters from each batch (control and infected) were removed from storage 1 day prior to commencement of the experiment and were allowed to thaw at 4  C overnight. Batches were processed separately and workbenches were disinfected with 1% Trigene® between batches to prevent cross contamination. Gills and mantles of oysters were dissected, pooled and processed as described in Ref. [32]. An aliquot (200 mL) of the purified homogenate was stored at 80  C for viral quantification by real-time PCR [31]. Remaining purified homogenates were maintained on ice prior to injection. Oysters were anesthetised by immersion in 1:4 filtered sterile seawater (FSSW) and distilled water containing MgCl2 (50 g/L). Once opened, oysters were rinsed with 35 ppt FSSW prior to being injected into the adductor muscle with 100 mL of infected or control inocula using 1 mL insulin syringes fitted with 25-gauge needles. The infected inoculum contained 1.3  106 viral DNA copies per 100 mL. Oysters were held out of water for 30 min with humidification before being returned to the experimental aquaria. Oysters were maintained for the duration of the experiment without feeding or water change. Mortalities were monitored twice daily. Dead oysters were removed and cumulative mortality was calculated. Oysters were sampled at 0, 1, 2, 3, 5, 7 and 14 dpi. At each sampling point, 10 oysters/species/treatment (total 40) were randomly chosen. A random number generator was used to determine the location and number of oysters sampled in each aquarium. Shell length of selected oysters was measured using a caliper. An aliquot of 500 mL of hemolymph per oyster was withdrawn from the adductor muscle using 1 mL insulin syringes fitted with a 25-gauge needle, through a notch ground in the oyster shell. Debris were removed by filtration through 80 mm nylon mesh and the hemolymph was immediately placed on ice to retard clumping of hemocytes [33]. Aliquots (150 mL) were stored at 80  C in 2 mL screw-capped tubes containing 0.1 mm dia zirconia/silica beads for further assessment of viral prevalence and quantification. Tissues were preserved in 10% v/v formalin seawater for histopathology. Workbenches and tools were disinfected with 1% Virkon® between each oyster.

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2.3. DNA extraction and viral quantification Viral enumeration was undertaken according to [27]. The hemolymph was chosen as suitable target to detect the virus because several authors report the presence of OsHV-1 in hemocytes, which travel throughout the animal in hemolymph, a fluid that permeates all tissues of the oyster through myriad vascular spaces. Briefly, 150 mL of hemolymph was disrupted by bead beating with a FastPrep-24® tissue and cell homogeniser and subsequently centrifuged at 3000 rpm for 10 min. The supernatant was transferred into 1.5 mL centrifuge tubes and stored at e 20  C. DNA extraction was performed on 50 mL of supernatant using a MagMAX™ FFPE total nucleic acid isolation kit and a MagMAX™ Express-96 magnetic particle processor. Purified DNA was kept in elution buffer in sealed MagMAX™ express- 96 deep well plates and stored at 20  C. 5 mL of the purified DNA was quantified in duplicates by real-time PCR as described by Refs. [34], using a MX3000P™ Real-time PCR cycler system. Every PCR experiments included positive and negative controls. Results were analysed using MxPro™ qPCR software. A threshold was automatically established by the software based on the exponential increase in fluorescence of the data curves of all reactions in the plate. Standards were created from a plasmid containing the amplicon region (University of Sydney, Camden, Australia) with a ten-fold dilution series used to create a standard curve that was included on each plate. A valid PCR run was defined as one exhibiting no amplification of negative controls, amplification of both replicates of the positive control with a Ct within the range of the standard curve, a standard curve with r2 > 0.95 and efficiency between 90 and 110%. The fluorescence threshold for each plate was calculated using the amplification-based threshold algorithm (Stratagene) for the standard curve, and applied to all samples on the plate. Quantification of OsHV-1 mvar DNA copies was performed using Ct values and absolute quantification of OsHV-1 DNA copies was determined by comparing Ct values obtained from samples with the standard curve. Results were expressed as a log10 of the viral DNA copy number per mL of hemolymph. 2.4. Histopathology Transverse sections from formaldehyde-fixed oysters were dissected, transferred to tissue embedding cassettes and processed for routine histology. Five mm sections were cut, stained with hematoxylin and eosin (H&E) and examined by light microscopy. The infiltration of hemocytes in the different tissues was determined and categorised according to the following scoresheet: 0: no infiltration; 1: light infiltration; 2: moderate infiltration; and 3: heavy infiltration. 2.5. Electron microscopy Pacific oysters were used as a positive control to demonstrate that viruses were present in oyster cells. For that purpose, gill tissues from two control Pacific oysters at 0 and 2 dpi and from two injected oysters at 2 and 3 dpi were observed. Tissues (mainly gills and mantle) from one pearl oyster of the control group were observed for each sampling time. 5 pearl oysters at 1 dpi, 7 pearl oysters at 2 dpi, 6 pearl oysters at 3 dpi, and 4 pearl oysters at 5, 7 and 17 dpi were examined under a transmission electron microscope for signs of viral presence. The gills, adductor muscle, digestive gland and mantle from pearl oysters and gills from Pacific oysters preserved in formaldehyde were processed using a microwave tissue processor. Briefly, the tissues were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, in 0.1 mM sodium cacodylate buffer, infiltrated by means of an ascending Epon resin acetone

series, and finally embedded and cured in Epon resin. Ultrathin sections were then cut, stained in 5% uranyl acetate in 50% ethanol and Reynolds lead citrate and examined in a JEM 1010 transmission electron microscope at 80 kV. This method was firstly used to demonstrate that our infection challenge was a success if viruses could be observed in the nucleus and cytoplasm of the C. gigas cells. 2.6. Cellular immunity Differences in hemocyte viability, morphology, and functions between challenged and control oysters were investigated by flow cytometry using a FACScalibur flow cytometer following the methods of [35]. Total and differential hemocyte counts, and hemocyte mortality were measured following the methods of [36] and [35]. Briefly, 100 mL of hemolymph were mixed with 200 mL of anti-aggregate solution [37] and 100 mL of FSSW. SYBR Green and propidium iodide were added and samples were incubated in the dark at room temperature (RT) for 60 min before analysis. Phagocytic rate of the hemocytes was determined as their ability to phagocytose fluorescent beads [38]. Aliquots (100 mL) of hemolymph were diluted with 200 mL of FSSW and mixed with 30 mL of a solution of 2 mm diameter latex fluorescent beads. Tubes were incubated in the dark for 2 h at 18  C before analysis. Phagocytosis rate was calculated as the percentage of hemocytes that have engulfed three or more fluorescent beads [38]. Phagocytosis capacity was defined as the average number of fluorescent beads engulfed per hemocyte ingesting at least 3 beads. Hemocyte oxidative activity (OA) was evaluated by adding a solution of DCFH-DA to 100 mL of hemolymph and 200 mL of FSSW [35]. Mixtures were incubated in the dark for 2 h at 18  C before analysis. DCF green fluorescence, directly linked to the oxidative activity within cells, was measured. Flow cytometry results were expressed as the mean geometric fluorescence per cell (in arbitrary units, A.U.). 2.7. Antiviral assays Antiviral activity was assessed using a heterologous Vero/Herpes simplex virus 1 (HSV-1) model as an homologous viral model for OsHV-1 is not available [39]. Frozen hemolymph from both oyster species were thawed and centrifuged at 2000  g for 15 min at 4  C. The supernatant was recovered to obtain the acellular fraction of the hemolymph. Total protein in extracts was determined using the Qubit® protein assay kit and fluorometer. Samples were adjusted to 1500 mg/mL total protein with FSSW and kept at 80  C prior to use. Plaque reduction neutralisation tests (PRNT) were performed with African green monkey kidney cells (Vero, ATCC CCL-81) seeded at 4  104 cells/well in 96-well flat-bottom plates containing 100 mL Opti-MEM® supplemented with 3% foetal calf serum. Cells were grown at 37  C, 5% CO2 in a humidified incubator overnight to achieve 90% confluence. Oyster hemolymph (1500 mg/mL total protein) and controls (FSSW and Opti-MEM® media) was serially diluted twofold from 2- to 128-fold in pre-warmed serumfree Opti-MEM® in a 96-well U-bottom plate. HSV-1 was thawed and diluted in SF Opti-MEM®. An equal volume of virus was added to the samples on the titration plate. Media was removed and cells were washed twice with phosphate-buffered saline (PBS). Cells were then inoculated with the virus/sample solution (50 mL/well, protein concentration: 750 mg/mL e 23.4 mg/mL, HSV-1 titre: 2000 PFU/mL, MOI: 0.001 PFU/cell) in duplicate and incubated for 2 h at 37  C, 5% CO2 to allow infection. After incubation, the inoculum was removed, replaced with 100 mL/well of M199 supplemented with 1.5% carboxymethylcellulose, 2.5% fetal calf serum and

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penicillin-streptomycin (100 units and 100 mg respectively per mL medium) and incubated at 37  C, 5% CO2 for 30 h in a humidified incubator. After the 30 h incubation period, the media overlay was removed, the monolayer was washed with PBS and cells were fixed with ice-cold 80% acetone in PBS (100 mL/well) for 20 min at 20  C. Fixative was removed and plates were allowed to dry at RT. Plates were then blocked with 5% skim milk protein in PBS with 0.05% Tween 20 (150 mL/well) for 30 min at RT, washed once in PBS-T, incubated in 1:1000 of primary antibody (mouse monoclonal anti-HSV-1 glycoprotein-C, MicroTrak® HSV-1/HSV-2 direct identification/typing kit) in blocking solution (50 mL/well) for 1 h at 37  C in the dark. Plates were then washed 3 times in PBS-Tween 20 and dried for 10 min at RT. Plaques were visualised on the Odyssey® Imager at a resolution of 42 mm, medium quality and level 5 intensity in the 800 nm channel. Plaque numbers were counted by eye. The 50% effective inhibitory concentration (IC50) was calculated by regression analysis of the dose response curves (three variables log (inhibitor) vs. response) obtained using Graphpad Prism 6 software. Potential cytotoxic effects of the hemolymph were assayed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Briefly, Vero cells grown to 90% confluence in 96-well plates were incubated with serial titrations of hemolymph or controls for 48 h at 37  C, 5% CO2. Media was then removed, replaced with 400 mg/mL MTT in Opti-MEM® (100 mL/ well) and incubated for 2 h at 37  C. Subsequently, medium was removed and DMSO (50 mL/well) was added to solubilise the formazan precipitate. Plates were then read on the micro-plate reader at 562 nm. The concentration of 50% cellular toxicity (CC50) was derived from dose response curves using Graphpad Prism 6 software. 2.8. Statistical analysis Differences in cumulative mortality were investigated using percentage comparison tests. Differences in the viral prevalence, viral DNA copies, flow cytometry data between treatments and sampling times were assessed using a two-way ANOVA. Difference in the viral assays between treatments was evaluated using oneway ANOVA. Prior to analysis, homogeneity of variance was checked using Cochran's test. Data were log or arcsin√p-transformed to meet homogeneity requirements. Where ANOVA was significant, differences were separated by a Tukey's test. When variances were not homogeneous, the non-parametric ManneWhitney U or KruskaleWallis tests were used. The maximum type I error was set at a ¼ 0.05. Analyses were performed on Statistica 10 and GraphPad Prism 6 softwares. 3. Results 3.1. Mortality rates, prevalence and intensity Mortalities of P. margaritifera and C. gigas reached 6% and 0% respectively during the acclimation period (n ¼ 240). Oyster sizes were similar between treatments and sampling times (2-way ANOVA, p > 0.05). Average shell height of P. margaritifera sampled was 56.0 ± 0.8 mm (±SE). OsHV-1 mvar was detected in the infected inoculum but not in the control inoculum. OsHV-1 mvar infection in Pacific oysters was successful as demonstrated by the high prevalence (>100%) and viral loads (>109 copies/mL of hemolymph) from 1 dpi as well as the high mortality rates (>90%) from 4 dpi (Paul-Pont et al. in prep). high mortality rates (>90%) from 4 dpi (Paul-Pont et al. in prep). In contrast to Pacific oysters that displayed 90% and 0% mortality for the challenged and control groups respectively, cumulative

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mortality of P. margaritifera was similar between the two groups (Percentage comparison test, p > 0.05; Fig. 1). At the completion of the experiment, mortalities reached 62.5% and 57.5% for the challenged and control groups, respectively (Fig. 1). OsHV-1 mvar was present in 80% and 90% of challenged P. margaritifera at 1 and 2 dpi respectively (Fig. 2A). The virus was not detected in control oysters and at T0. A significant decrease in prevalence was noticed (1 way-Anova, p < 0.05, Fig. 2A). From 1 dpi, Pacific oysters presented significant higher viral DNA copies (>109 DNA copies per mL), (Paul-Pont et al. in prep) than pearl oysters (6.1  104 DNA copies per mL) (Fig. 2B, p < 0.05, 2 wayAnova). At 3 dpi, values were still low for P. margaritifera and the viral load decreased during the experiment until reaching 1.12  103 DNA copies per mL at completion (Fig. 2B, p < 0.05, 2 way Anova). 3.2. Histopathology No obvious pathological alterations or signs of disease were detected under light microscopy. Pearl oysters presented a low infiltration of hemocytes in tissues, which increased from 0 to 3 dpi. Then, levels stayed high until 14 dpi. However, no significant differences were noticed over time for the hemocyte infiltrations 1, 2 and 3 (p > 0.05, 2-way Anova). No differences were observed between treatments for level of infiltration 1 and 3, but challenged oysters had more hemocyte infiltrations 2 than control oysters (p < 0.05, 2-way Anova). 3.3. Electron microscopy In TEM, in comparison to the control C. gigas (Fig. 3A), the positive control (challenged C. gigas) exhibited marginated chromatin (Fig. 3B) and highly electron dense nuclei (Fig. 3C). OsHV1 mvar particles were observed in gill cells of infected C. gigas. Some nuclear particles appeared empty, assumed to be capsids while nucleocapsids contained an electron-dense core (Fig. 3C). Extracellular (Fig. 3D) and intracytoplasmic viruses (Fig. 3E) were also observed. Nuclear particles were circular or polygonal in shape, averaging 95 ± 1.5 nm (n ¼ 84) in diameter. Intracytoplasmic and extracellular viruses were enveloped, averaging 105.4 ± 5.7 nm (n ¼ 8) in diameter (Fig. 3, C-E).These features and OsHV-1 mvar particles were not observed in the tissues of challenged P. margaritifera. 3.4. Cellular immunity Cytometric measurement using size (FSC) and internal complexity (SSC) allowed for discrimination of 3 subpopulations of hemocytes for P. margaritifera: granulocytes, hyalinocytes, and small agranulocytes. Hyalinocytes were the most numerous cells in the hemolymph, accounting for 59.1% of the hemocytes at T0 whereas granulocytes represented 14.5% and small agranulocytes 26.4%. Total hemocyte count (THC) of P. margaritifera varied significantly between times (2-way ANOVA, p < 0.05) but was similar between treatments (2-way ANOVA, p > 0.05). THC increased for the control oysters at 6 dpi and decreased for the challenged oysters at 3 dpi. Proportions of granulocytes and hyalinocytes were similar between treatments (2-way ANOVA, p > 0.05) although varied significantly between times (p < 0.05, 2-way ANOVA). The percentage of granulocytes was significantly higher at 0 dpi, decreased at 1 dpi, remained low until 7 dpi, and then increased at 14 dpi (p < 0.05, Tukey test). The percentage of hyalinocytes was significantly different at 2 dpi from 0, 7, and 14 dpi (p < 0.05, Tukey test).

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Fig. 1. Cumulative mortalities (±SE) for each treatment during the course of the experiment. Cont: Control; Chall: challenged; PM: P. margaritifera.

Proportions of small agranulocytes were constant with time (KruskalleWallis and U tests, p > 0.05) but comprised significantly lower proportions in challenged oysters (2-way ANOVA, p < 0.05). Total hemocyte mortality varied significantly between times (KruskalleWallis test, p < 0.05) but was similar between both treatments (U test, p > 0.05). Hemocyte mortality appeared to be quite high at the start of the experiment with 19.8%, and then decreased significantly over time, reaching 6.5% in both treatments at 14 dpi (p < 0.05, KruskalleWallis test). Phagocytic rate and capacity of P. margaritifera hemocytes were similar between treatments (U test, p > 0.05) but varied significantly with time (KruskalleWallis test, p < 0.05). The phagocytosis rate decreased from 50% at 0 dpi to approximately 27% at 2 dpi, and then increased until completion of the experiment. At completion

of the experiment, values were similar to the start. For the phagocytosis capacity, oysters appeared to engulf fewer beads at 2, 3 and 5 dpi than at 0, 1, 7 and 14 dpi. Among different sub-populations of hemocytes, mean OA per granulocyte were similar between times and treatments (2-way ANOVA, p > 0.05). OA per hyalinocyte was also similar between treatments though it varied significantly between times but without a clear trend (2-way ANOVA, p < 0.05). In small agranulocytes, OA per cell varied significantly between treatments and times (KruskalleWallis and U tests, p < 0.05). OA per small agranulocyte was observed to be significantly higher in challenged P. margaritifera, especially at 2 and 3 dpi (Tukey test, p < 0.05). 3.5. Antiviral assays HSV-1 inhibiting activity was detected in the hemolymph of P. margaritifera (IC50 ¼ 77.73 ± 20.88 mg/mL) and was higher than in the hemolymph of C. gigas (IC50 ¼ 473.45 ± 48.94 mg/mL) (1-way ANOVA, p < 0.05). Cytotoxicity was not detected in C. gigas (CC50  750 mg/mL; Table 1) and was noticed in 3 pearl oysters (Table 1). Anti-HSV-1 activity of P. margaritifera were similar between OsHV-1 mvar challenged (n ¼ 5) and control (n ¼ 5) animals (t-test, p > 0.05, Table 1). 4. Discussion

Fig. 2. Prevalence (A) and viral DNA copy per mL of hemolymph (B) of challenged P. margaritifera.

The pearl industry has on numerous occasions borne the catastrophic social and economic consequences of mass mortality events in cultivated oysters [40]. Recently, severe mortalities were reported in cultivated P. margaritifera in Fiji (Southgate, pers. comm.). However, the causes of these mortalities were not well understood [40,41]. French Polynesia imported C. gigas for human consumption, which tested positive for OsHV-1 (H. Bichet and D. Saulnier pers. comm., 2012.). Hence, there is a potential risk for transmission of OsHV-1 by accidental co-habitation of P. margaritifera with infected shellfish. Indeed, some French Polynesia residents used to immerse C gigas in the lagoon between their purchase and their consumption, and to throw away the shells in the lagoon after their degustation. Pearl oysters have experienced diseases and mortality caused by viruses or virus-like particles in French Polynesia and Japan [23,40e42], and the possibility that these incidents were caused by OsHV-1 has been suggested [41]. Considering its virulence and the recent massive mortalities of

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Fig. 3. Transmission electron micrograph of Crassostrea gigas gill tissue. Healthy control (A) and challenged C. gigas gill tissue section at 2 dpi (BeE). A fibrocyte infected with OsHV1. There is margination of chromatin in the nucleus (*), and there are numerous empty capsids (arrow heads) and complete nucleocapsids (arrows) (B). High power view of a peripheral (marginal) area of the nucleus. There is condensation of heterochromatin within which lie empty capsids and complete nucleocapsids. The nuclear membrane is intact (C). Extracellular enveloped virus particles (arrows) within connective tissues of the gills (D). Intracytoplasmic non-enveloped nucleocapsids (arrows) and empty capsids (arrowheads) observed within a mucous cell in the gill. Some nucleocapsids are enveloped (*) (E).

Table 1 Cytotoxicity and antiviral activity of acellular oyster hemolymph. IC50: Inhibitory concentration to 50% of cells; CC50: cytotoxic concentrations for 50% of cells; CI: confidence level; FASW: filter-sterile artificial seawater. Sample

Species

Treatment

IC50 (mg/mL) Mean

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera P.magaritifera C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas FASW -ve control

Control Control Control Control Control Injected Injected Injected Injected Injected NA NA NA NA NA NA NA NA NA NA NA NA

43.8 98.1 73.9 5.9 33.8 45.4 237.9 35.2 80.3 123.1 471 640.8 620.8 644.3 640.1 613.3 360.7 472 278.6 292.9 1104 22,603

CC50 (mg/mL) 95% CI

Mean

þ

e

14.5 32.5 23.5 0.9 8.3 11.8 132.8 10.1 26.7 53 418.9 762.4 1031.9 790.2 870.7 776.6 339.2 453.7 396.2 500.8 1211.3 880,613

471 640.8 620.8 613.3 360.7 644.3 640.1 472 278.6 292.9 221.7 348.2 387.6 354.9 368.9 342.7 174.8 231.3 163.6 184.8 577.9 22,037.3

>750 >750 252.6 276.3 >350 222.5 >750 >350 >350 >350 >750 >750 >750 >750 >750 >750 >750 >750 >750 >750 >750 >750

95% CI þ

e

22.5 33.4

766.9 705.7

27.7

24.6

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C. gigas reported in Australia and New Zealand [24,25,28], the potential for infection of P. margaritifera by OsHV-1 mvar was of major concern to the South Pacific pearl industry. Mortality of challenged C. gigas, used as a positive control in the present study, was similar to that reported by Ref. [21] and the prevalence reached 100% in the challenged animals. Replication of OsHV-1 mvar occurs in the nucleus of susceptible cells where viral DNA is packaged into immature nucleocapsids [43]. Infectious viral particles are generated upon acquiring the tegument and envelop before being transported to the cell surface and subsequently released [43]. Viral replication was apparent in infected C. gigas gills. Nuclei exhibited marginated chromatin and were highly electron dense, which are ultra-structural changes typical of OsHV1 infection [2,17,22]. Thus, the inoculum was capable of inducing disease resulting in high mortalities of healthy C. gigas. This infection challenge model is therefore valid for interpretation of the susceptibility of P. margaritifera to the Australian isolate of OsHV1 mvar. The OsHV-1 mvar-infected homogenate did not appear to trigger disease in P. margaritifera. OsHV-1 infections are characterised by brief and severe mortalities in susceptible bivalves [21,44,45], but OsHV-1 mvar did not induce significantly higher mortality in challenged P. margaritifera than in controls. Several hypotheses could explain the high mortality rates observed in both control and challenged groups of P. margaritifera during the experiment. First, it could be due to the husbandry of the animals, as these oysters are very fragile and sensitive to manipulation. Secondly, P. margaritifera is amongst the world's most energetic bivalve species; with exceptionally high respiration rates, excretion rates, and energy demands [46]. P. margaritifera were not fed throughout the infection challenge and this could have contributed to a decrease of the metabolism and then, to mortalities. Furthermore, considering the energetic nature of P. margaritifera, toxic waste products might have accumulated if the filtration systems were not sufficiently optimised to efficiently neutralise the high quantities of metabolic waste in the system produced by P. margaritifera. Finally, Pearl oysters were also subjected to wounding as the inocula were injected into the adductor muscle, and wound healing is an energetically expensive process [47]. Mounting immune responses to combat infection is similarly a very energetically expensive process [48]. Thus, P. margaritifera used during the experiment were likely to have been compromised by a lack of energy input. The decline in the prevalence and intensity of OsHV-1 mvar over time suggested the presence of anti- OsHV-1 mvar mechanisms, which inhibited the replication of OsHV-1 mvar in P. margaritifera. This was supported by electron microscopy of challenged P. margaritifera, as the different tissue types observed did not reveal any obvious ultra-structural changes (i.e. marginated chromatin and highly electron dense nuclei), that are typical of OsHV-1 infections [2,22,23]. In the present experiment, OsHV-1 mvar could not replicate and cause disease and mortality in pearl oysters. Several hypotheses may explain the resistance of the pearl oyster to OsHV-1 mvar. The fact that no significant difference between challenged and control oysters was observed for the cellular and humoral immune parameters analysed in the study acted in favour of a defence factor constitutively present in the oyster. Overall, no significant differences were noted in the cellular immune parameters between infected and control oysters but these parameters varied over time. It seemed that the three first days are critical for the oysters to begin the process of elimination of the virus, as it is after 3 days that the prevalence decreased. These modifications were associated with a decrease of the total hemocyte count from 1 to 3 dpi. Variations of circulating hemocyte

concentration correspond either to movements of hemocytes between the circulatory system and tissues or to hematopoietic processes for the production of young cells [49]. This decrease of circulating hemocytes was associated with an increase of infiltrating hemocytes in tissues, as revealed by light microscopy. The decrease of the proportion of granulocytes between 0 and 1 dpi indicated that it is this sub-population that was mostly mobilised in tissues. Granulocytes are the cells with the highest ability to engulf particles and eliminate infectious agent by enzymatic or oxidative degradation [36,50]. Then, after 3 dpi, the THC and the proportion of granulocytes increased, suggesting a recruitment of new circulating hemocytes and granulocytes. At completion of the experiment, values returned to their initial state, before the injection. These modifications of the hemocyte characteristics over time were associated with some changes in hemocyte functions such as a decrease of the phagocytosis capacity at 2, 3 and 5 days after injection and an increase of the total oxidative activity at 3 dpi. However, this could not explain how the pearl oyster defends against the virus. Despite testing in a heterologous system, herpesvirus-inhibiting activity was observed in the acellular fraction of the hemolymph, which suggested that the anti-OsHV-1 mechanism involves humoral components of the immune system. Humoral immunity is based on production of soluble effector molecules that increase and regulate anti-microbial activities [51,52]. The presence of anti-viral substances has been well established in several bivalve species [53,54]. Anti-viral substances present in the hemolymph of bivalves could inhibit viral replication [53]. However, little is known about the mechanisms involved in triggering such anti-viral defences in molluscs [55]. The humoral anti-viral substances secreted by P. margaritifera are not induced by infections but are constitutively secreted in the hemolymph, as no difference was observed between treatments. In contrast, the hemolymph of C. gigas had less or no herpesvirus-inhibiting activity. This observation supports the hypotheses that the presence of these anti-viral substances in adult C. gigas that are otherwise absent in the larvae and juveniles may explain the adults being less sensitive to OsHV-1 mvar despite continuously being exposed to the virus as previously proposed [39,54]. It is also conceivable that, conversely to C. gigas, cells of P. margaritifera may lack receptors recognisable by OsHV-1 mvar, and thus the replication process is not initiated. Indeed, replication of OsHV-1 mvar can only occur following binding of the viral envelope to the plasma membrane in a process mediated by viral glycoproteins and specific surface receptors on the plasma membranes of susceptible cells [43,56]. To conclude, under the conditions of the experimental challenge, juveniles P. margaritifera were not sensitive to OsHV-1 mvar and was unlikely to be a long-term host/carrier. Despite the rapid spread of OsHV-1 worldwide and potential for accidental introduction of OsHV-1 to the lagoons of French Polynesia, OsHV-1 may not represent a risk to P. margaritifera cultivators. However, it is unclear if the susceptibility of P. margaritifera to OsHV-1 mvar will remain low in view of predicted global climate change, of the evolution of the virus, evidenced by the occurrence of novel microvariants [3,25], and of the hatchery selection program that is occurring now to select oysters according to their color or growth rate. Hemocyte physiology in bivalves is influenced by fluctuations in environmental conditions that could increase susceptibility to diseases [57e59]. Outbreaks of OsHV-1 mvar are highly correlated with physiological and immunological weakening, prevalence of pathogens, and favorable environmental conditions, particularly high water temperatures [20,44]. With the predicted increase in sea-surface temperatures and rainfall over the majority of pearl oyster cultivation regions, it is likely that climate change will have

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significant impact on P. margaritifera culture; particularly the weakening of its immune defences that may result in increased susceptibility to OsHV-1 and other infectious agents [41,60].

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