Maxime Lafont

Priscila Goncalves

Ximing Guo

Caroline Montagnani

David Raftos

Timothy Green
email: timothy.green@viu.ca

Timothy Greena

Transgenerational plasticity and antiviral immunity in the Pacific oyster (Crassostrea gigas) against Ostreid herpesvirus 1 (OsHV-1)

1-Introduction

Invertebrates can mount sophisticated immune responses with the potential to exhibit a form of innate immune memory [START_REF] Chang | What vaccination studies tell us about immunological memory within the innate immune system of cultured shrimp and crayfish[END_REF][START_REF] Contreras-Garduno | Insect immune priming: ecology and experimental evidences[END_REF][START_REF] Milutinovic | Immune memory in invertebrates[END_REF]. After exposure to certain stimuli or parasite infections, the immune system of some invertebrate species can be primed to respond more vigorously upon a secondary infection caused by the same type of parasite [START_REF] Contreras-Garduno | Insect immune priming: ecology and experimental evidences[END_REF]. This long-lasting, groupspecific immune response in invertebrates is called 'immune-priming' [START_REF] Kurtz | Evidence for memory in invertebrate immunity[END_REF]. In some incidences, immune priming occurs not only within a generation, but also across generations with offspring from primed parents also having improved survival to parasite exposure, a state called 'transgenerational immune-priming' [START_REF] Little | Maternal transfer of strainspecific immunity in an invertebrate[END_REF][START_REF] Sadd | Trans-generational immune priming in a social insect[END_REF]. Immune priming has been reported in different groups of invertebrates, including ctenophores, sponges, mollusks and arthropods [START_REF] Milutinovic | Immune memory in invertebrates[END_REF]. Studies investigating the phenomenon of immune-priming in invertebrates are quite heterogeneous and largely differ in terms of host-parasite combination, experimental design, elicitors used for priming (i.e. non-lethal dose of parasite or PAMP) and route of priming (i.e. oral or injection) (reviewed by [START_REF] Contreras-Garduno | Insect immune priming: ecology and experimental evidences[END_REF]. This heterogeneous array of experiments makes it complicated to provide a mechanistic explanation for this phenomenon.

Ostreid herpesvirus 1 (OsHV-1) is responsible for serious economic losses of the edible Pacific oyster, Crassostrea gigas [START_REF] Burge | Mortality and herpesvirus infections of the pacific oyster Crassostrea gigas in tomales bay, California, USA[END_REF][START_REF] Jenkins | Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 u-var) in Crassostrea gigas (Pacific oysters) in Australia[END_REF][START_REF] Keeling | New Zealand juvenile oyster mortality associated with ostreid herpesvirus 1 -an opportunisitic longitudinal study[END_REF][START_REF] Renault | Herpes-like virus infecting Japanese oyster (Crassostrea gigas) spat[END_REF][START_REF] Segarra | Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008[END_REF]. OsHV-1 can cause 100% mortality rate of C. gigas in less than one week [START_REF] Paul-Pont | Descriptive epidemiology of mass mortality due to Ostreid herpesvirus-1 (OsHV-1) in commercially farmed Pacific oysters (Crassostrea gigas) in the Hawkesbury River estuary, Australia[END_REF], with the commercial production of C. gigas having now ceased entirely in several affected estuaries within Australia [START_REF] Whittington | Further observations on the influence of husbandry practives on OsHV-1 uVar mortality in Pacific oysters Crassostrea gigas: age, cultivation structures and growing height[END_REF]. Urgency to mitigate the impacts of OsHV-1 has led to a closer examination of the antiviral responses of C. gigas to OsHV-1 infection (reviewed by [START_REF] Green | Antiviral defense and innate immune memory in the oyster[END_REF]. Observational studies suggest C. gigas are capable of adapting to OsHV-1 infection. C. gigas that have survived a mortality event appeared to be more resistant later in life to OsHV-1 [START_REF] Evans | Detection of Ostreid herpesvirus-1 microvariants in healthy Crassostrea gigas following disease events and their possible role as reservoirs of infection[END_REF][START_REF] Pernet | Mass mortalities of Pacific oysters Crassostrea gigas reflect infectious diseases and vary with farming practices in the Mediterranean Thau lagoon, France[END_REF] and female C. gigas infected with OsHV-1 can transfer this protection to their offspring [START_REF] Barbosa-Solomieu | Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas)[END_REF].

Experimental studies using a heterologous immune-priming model provide evidence of immune plasticity in C. gigas to OsHV-1 infection [START_REF] Green | Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 μvar)[END_REF]Green et al., 2015b;[START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF][START_REF] Pauletto | Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication[END_REF]. The immune system of C. gigas can be primed with synthetic virus-associated molecular patterns (i.e. poly(I:C)) to induce an antiviral response that hampers subsequent infection with OsHV-1 [START_REF] Green | Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 μvar)[END_REF]Green et al., 2014b). This protection to OsHV-1 can be longlasting, persisting for at least 5 months [START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF]. Furthermore, this protection appears to be transmitted to offspring. C. gigas larvae produced from parents stimulated with poly(I:C) have improved survival to OsHV-1 infection [START_REF] Green | Primed for success: oyster parents treated with poly(I:C) produce offspring with enhanced protection against Ostreid herpesvirus type I infection[END_REF]. Discovering the mechanism used by C. gigas to transmit antiviral immunity to the next generation would be highly beneficial to the aquaculture industry. This knowledge could motivate the development of practical and cost-effective treatments for improving oyster health [START_REF] Contreras-Garduno | Insect immune priming: ecology and experimental evidences[END_REF][START_REF] Wang | Maternal immune transfer in mollusc[END_REF].

Transgenerational immune priming in invertebrates can arise from both maternal and paternal sources [START_REF] Mcnamara | The effect of maternal and paternal immune challenge on offspring immunity and reproduction in a cricket[END_REF][START_REF] Roth | Paternally derived immune priming for offspring in the red flour beetle, Tribolium castaneum[END_REF][START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF]. Maternal immune priming appears to be mediated by at least three non-exclusive mechanisms. Mothers exposed to pathogens or certain stimuli may provision their eggs with antimicrobial compounds [START_REF] Yue | Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge[END_REF]. This antimicrobial activity declines as the antimicrobial compounds are metabolized in the developing embryos [START_REF] Benkendorff | Chemical defense in the egg masses of benthic invertebrates: an assessment of antibacterial activity in 39 mollusks and 4 polychaetes[END_REF]. Mothers may also transmit signals to enhance the immune response of their offspring [START_REF] Barribeau | Royal decree: gene expression in transgenerationally immune primed bublebee workers mimics a primary immune response[END_REF][START_REF] Hernandez Lopez | Transgenerational immune priming in honeybees[END_REF][START_REF] Zanchi | Differential expression and costs between maternally and paternally derived immune priming for offspring in an insect[END_REF]. These signals include the transfer of microbial degradation products (i.e. PAMPs) to their developing embryos to prime their immune response [START_REF] Freitak | The maternal transfer of bacteria can mediate trans-generational immune priming in insects[END_REF], or via epigenetic mechanisms, such as DNA chromatin modification or small RNA, to elevate the constitutive expression of immune effector genes [START_REF] Castro-Vargas | Methylation on RNA: a potential mechanism related to immune priming within but not across generations[END_REF][START_REF] Norouzitallab | In: Probing the Phenomenon of Trained Immunity in Invertebrates during a Transgenerational Study, Using Brine Shrimp Artemia as a Model System[END_REF]. Paternal immune priming can also be transmitted by epigenetic mechanisms via the sperm or by compounds transferred with the seminal fluid [START_REF] Eggert | Different effects of paternal transgenerational immune priming on survival and immunity in step and genetic offspring[END_REF].

Here, we investigated the mechanisms that underpin transgenerational immune priming in C. gigas against OsHV-1, using the immune elicitor poly(I:C). This study aimed to (i) investigate the effect of time between parental exposure to poly(I:C) and spawning on the resistance of larvae to OsHV-1 infection, (ii) quantify the contribution of maternal and paternal provisioning to offspring response, and (iii) determine if parental exposure to poly(I:C) reconfigures the constitutive expression of immune-related genes in unchallenged C. gigas larvae.

2-Materials and methods

2-1 Oysters, immune challenge & mating trials

Adult Crassostrea gigas were collected from Port Stephens estuary (NSW, Australia) and held in conditioning tanks at the Sydney Institute of Marine Science (Sydney, Australia). OsHV-1 DNA has not been detected in C. gigas cultivated in Port Stephens estuary [START_REF] Go | Mass mortalities of unknown aetiology in pacific oysters Crassostrea gigas in Port Stephens, New South Wales, Australia[END_REF]. Adult C. gigas were held in 60 L tanks at 21 °C with supplemental feeding with live microalgae (Isochrysis galbana,, Chaetoceros muelleri) and microalgae concentrate (Shellfish Diet, 1800; Reed Mariculture). Prior to spawning, C. gigas had a notch filed in their shells and were injected with 100 μl of poly(I:C) (Sigma, 5 mg ml -1 in seawater) or sterile seawater (control) in the adductor muscle. C. gigas were stripped spawned by making small incisions in the gonad with a scalpel blade, and washing gametes into a plastic beaker with 1 μm filtered seawater. Gametes from each parent were kept separate until fertilization. Fertilization strategies are outlined below. Fertilized eggs were transferred to individual 20 L tanks filled with 5 μm filtered seawater (temperature 21 °C, salinity 35 ppt) and D-veliger larvae were harvested at 24 h postfertilization by draining tanks through a 40 μm nylon sieve.

Experimental infection of C. gigas D-larvae with OsHV-1 was conducted according to the protocol outlined in [START_REF] Burge | Quantifying ostreid herpesvirus (OsHV-1) genome copies and expression during transmission[END_REF]. Experiments involving OsHV-1 were conducted in a physical containment level 2 (PC2) facility at the Sydney Institute of Marine and all waste arising from experiments was decontaminated by heat sterilization (autoclaved at 121 °C for 15 min [START_REF] Hick | Stability of Ostreid herpesvirus-1 (OsHV-1) and assessment of disinfection of seawater and oyster tissues using a bioassay[END_REF]. Briefly, gill and mantle tissue from OsHV-1 infected and non-infected adult C. gigas was homogenized in 10-vol of sterile seawater containing 1000 units ml -1 of penicillin and 1 mg ml -1 of streptomycin, clarified by centrifugation and 0.2 μm filtered to prepare OsHV-1 and control homogenates, respectively [START_REF] Burge | Quantifying ostreid herpesvirus (OsHV-1) genome copies and expression during transmission[END_REF][START_REF] Renault | Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1[END_REF]. D-veliger larvae (24 h post-fertilization) from each family were placed in duplicate 500 ml Erlenmeyer flasks containing 200 ml of sterile seawater, 100 units ml -1 of penicillin and 0.1 mg ml -1 of streptomycin. Larvae density was 30 larvae ml -1 and cultures were fed daily with 10 7 cells of live I. galbana. For each family, one Erlenmeyer flask was inoculated with OsHV-1 homogenate and the other flask was inoculated with the control tissue homogenate [START_REF] Burge | Quantifying ostreid herpesvirus (OsHV-1) genome copies and expression during transmission[END_REF][START_REF] Renault | Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1[END_REF]. Flasks inoculated with OsHV-1 received the equivalent of 10 9 OsHV-1 genome copies. Cultures were sampled at 48 h post inoculation and the assessment of live/dead larvae was performed using a compound microscope and Sedgewick rafter slide. Aliquots of 10 3 larvae from each culture were pelleted by centrifugation (1000 g, 5 min) and stored at -80 °C for subsequent nucleic acid extraction.

To address the specific aims of this study, different fertilization strategies were adopted, as follows: (i) Experiment 1: effect of time between parental immune stimulation and spawning on offspring immunity. Two cohorts of adult oysters (both males and females) were injected with poly(I:C) at 10 or 3 days prior to spawning. A third cohort of adult oysters were injected with sterile seawater at 3 days prior to spawning as a control. Up to six pair-mated families were produced for each treatment (Fig. 1). Offspring from the 11 pair-mated families were challenged with OsHV-1 as described above. In addition to the assessment of survival rates following OsHV-1 inoculation, unchallenged offspring from the pair-mated families generated in this experiment was analyzed by RNA-seq and qPCR (sections 2.4 and 2.5). Molecular analyses were performed in order to investigate whether poly(I:C) treatment alters offspring transcriptional responses.

(ii) Experiment 2: contribution of maternal and paternal immune stimulation to offspring performance. The role of maternal immune stimulation on offspring immunity to OsHV-1 was examined by mating a single male oyster with five poly (I:C)-treated females and five control (seawater-injected) females to produce 10-half sibling families that share the same father (Fig. 2). Likewise, the role of paternal immune stimulation on offspring immunity was tested by mating a single female oyster with five poly(I:C)treated males and five seawater-injected males to produce 10 families that share the same mother (Fig. 2). Offspring from each family was challenged with OsHV-1 as detailed above.

2-2 Nucleic acid purification

Total RNA and DNA was isolated using TriReagent ® (Sigma-Aldrich) and Isolate II Genomic DNA Kit (Bioline), respectively. Purity and yield of nucleic acids were evaluated using NanoDrop™ 2000 Spectrophometer (Thermo Scientific™). Total RNA (0.5 μg) was reverse transcribed using the Tetro cDNA synthesis kit (Bioline) with random hexamers.

2-3 OsHV-1 DNA detection and quantification

Absolute quantification of OsHV-1 DNA was determined by quantitative polymerase chain reaction (qPCR) according to [START_REF] Pepin | Rapid and sensitive detection of ostreid herpesvirus1 in oyster samples by real-time PCR[END_REF] using SensiFAST™ SYBR ® No-ROX (Bioline) and the C9/C10 primer pair (Table 1). The concentration of OsHV-1 DNA was estimated from a standard curve generated from the C9/C10 amplicon product cloned into the pCR4-TOPO vector (Thermo Scientific™) according to the protocols and calculations outlined in the Applied Biosystems manual of absolute real-time RT-PCR quantification (Applied Biosystems, 2003). The plasmid was diluted in distilled water (Standard Curve: PCR efficiency = 95%, R 2 = 0.995) and the dynamic range of the qPCR assay was 10 8 to 10 3 copies.

2-4 Transcriptome sequencing and differential gene expression

Six larval RNA samples were selected for high-throughput mRNA sequencing (RNA-seq). These samples were from experiment 1 and consisted of three pools (families) of larvae produced from C. gigas treated with poly(I:C) and three pools of control larvae. These pools of larvae had not been inoculated with OsHV-1 as we were trying to determine whether unchallenged larvae from poly(I:C)-stimulated parents have a different transcriptome to control larvae. The TruSeq protocol (Illumina), including a poly(A) isolation step, was followed for cDNA synthesis and library preparation. Samples were barcoded and sequenced by the Australian Genome Research Facility (AGRF, Brisbane) on a HiSeq2500 instrument (Illumina) using a 50 bp single end, strandspecific run. The raw sequence reads are available at the National Centre for Biotechnology Information (NCBI) Short Read Archive (NCBI BioProject: PRJNA494182).

Nucleotide reads were quality filtered using Trimmomatic (version 0.32) [START_REF] Bolger | Trimnomatic: a flexible trimmer for Illumina sequence data[END_REF] and the quality of the trimmed reads was visualized using fastQC (version 0.10.1). Processed reads from each library were individually mapped back to the GigaTON reference transcriptome for C. gigas [START_REF] Riviere | GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the Pacific oyster Crassostrea gigas[END_REF]. The GigaTON transcriptome assembly encompasses a total of 56,621 contigs (median length = 1659; N50 = 2238) generated by the combination of 114 RNA-seq libraries, which include an extensive range of developmental stages (e.g. unfertilized eggs, two-cell embryos to two-year-old adults), tissues (e.g. wholeembryos, whole-larvae, whole-spat, gill, hemocyte, mantle, adductor muscle, gonad, digestive tract and labial palp) and physiological conditions (e.g. oysters at ambient conditions and exposed to disturbed temperature, salinity and heavy metals). In addition, mapping of our sequencing reads to the GigaTON transcriptome produced higher alignment rates than the mapping against the C. gigas genome (version 101; 79.4 vs. 66.4% overall mapping, respectively). The draft genome sequence for C. gigas is known to have a number of assembly errors in genome scaffolds [START_REF] Hedgecock | Secondgeneration Linkage Maps for the Pacific Oyster Crassostrea gigas Reveal Errors in Assembly of Genome Scaffolds[END_REF]. Therefore, given its complexity and broad genetic diversity, the GigaTON assembly was used as a reference transcriptome in the current study. Mapping of processed reads to the GigaTON assembly was performed using Bowtie2, with strict parameters (-score-minL,-0.1,-0.1, -no-mixed, -no-discordant, -fr -nofw). Assembled contigs were then clustered based on the proportion of shared reads and expression patterns using Corset (default settings) [START_REF] Davidson | Corset: enabling differential gene expression analysis for de novo assembled transcriptomes[END_REF]. The cluster-level count data were processed using the edgeR Bioconductor package [START_REF] Robinson | edgeR: a bioconductor package for differential expression analysis of digital gene expression data[END_REF], testing for differences in gene expression between larvae produced from C. gigas treated with poly(I:C) or seawater. Clusters with non-zero counts in at least half of the analyzed samples (3 out of 6 samples) were kept for downstream analyses. Data were normalized for sequencing depth (library size) and RNA composition (TMM normalization). Differential expression was calculated using the quantile-adjusted conditional maximum-likelihood method, followed by the exact test. Contigs were considered to be differentially expressed at p < 0.05 with false detection rate (FDR) lower than 5%. Differentially expressed genes were annotated using BlastX and the molluca non-redundant (nr) database.

2-5 RT-qPCR

To validate the transcriptome data, we evaluated the relative expression profiles of 10 genes identified as differentially expressed by RNA-seq (section 2.4). Annotation could not be assigned to these 10 differentially expressed genes because they matched uncharacterized proteins in the NCBI databases. Genes were chosen for RT-qPCR based on fold change (up-or down-regulated) and function (known or unknown). Another 3 antiviral genes (IRF, Viperin and ADAR-L) were included in this analysis. These genes were not identified to be differentially expressed by RNA-seq, but their expression has been shown to be altered by poly(I:C) treatment and OsHV-1 infection (Green et al., 2015a). The relative expression of these 13 target genes were quantified in cDNA samples from unchallenged eggs and larvae produced from parents stimulated with poly(I:C) or seawater. RT-qPCR was performed in a CFX96 Touch™ Real-Time PCR Detection System (BIO-RAD), as described previously by [START_REF] Green | Primed for success: oyster parents treated with poly(I:C) produce offspring with enhanced protection against Ostreid herpesvirus type I infection[END_REF], using the primers in Table 1, which included the internal reference gene eEF1α. Amplification efficiency of each primer pair was validated using a serial dilution of cDNA.

2-6 Statistical analysis

Survival and qPCR data were analyzed for statistical differences using analysis of variance (ANOVA) in the SPSS (IBM) version 22.0. Tukey's method for multiple comparisons was used to compare means if significant differences were found (p < 0.05). Data that did not meet Levene's test of equal variances was arcsine transformed. Data are presented as mean ± standard deviation.

3-Results

3-1 Effect of time between parental immune stimulation and spawning on offspring immunity

The time between parental immune stimulation and spawning had a significant effect on the survival of offspring to OsHV-1 inoculation (F5,28 = 10.99, p < 0.01). At 48 h post-inoculation with OsHV-1, average cumulative mortality of larvae produced from parents stimulated with poly(I:C) at 3 and 10 days prior to spawning was 14.4 ± 7.2% and 37.5 ± 12.2%, respectively. Larvae produced from control parents (injected with seawater at 3 days pre-spawning) had a cumulative mortality of 45.3 ± 21.7% at 48 h post-inoculation with OsHV-1 (Fig. 1). Priming parents with poly(I:C) at 3 days prior to spawning did significantly reduce mortality of larvae compared to controls (p < 0.05), but no improvement was observed in larvae generated from parents treated with poly(I:C) at day 10 prior to spawning (p > 0.05). Priming parents with poly(I:C) had no effect on OsHV-1 replication in larvae. No difference in the amount of OsHV-1 DNA in the tissue of larvae was observed between the three treatments (p > 0.05). The mean concentration of OsHV-1 DNA in larvae was 1.2 × 10 5 and 1.61 × 10 5 genome copies larva -1 at 48 h post-inoculation for larvae produced from parents primed with poly(I:C) at 3 and 10 days prior spawning, respectively. Control larvae had 1.1 × 10 5 genome copies larva -1 .

We also investigated whether poly(I:C) stimulation of parents had an effect on survival of nonchallenged (NC) larvae. Parental immune stimulation prior to spawning did not effect survival of offspring (Fig. 1, p > 0.05). The cumulative mortality of larvae at 48 h post inoculation with the control homogenate was 9.7 ± 6.9% and 7.9 ± 5.3% for larvae produced from parents stimulated with poly(I:C) at 10 and 3 days prior to spawning, respectively. The cumulative mortality of control larvae was 14.8 ± 7.5% at 48 h post inoculation with the control homogenate. No OsHV-1 DNA was detected in the tissue of larvae exposed to the control homogenate.

3-2 Effect of maternal and paternal immune stimulation on offspring immunity to OsHV-1

Maternal immune stimulation prior to spawning resulted in C. gigas offspring (larvae) with improved immunity to OsHV-1 infection (p < 0.05, Fig. 2). The average cumulative mortality of larvae produced from poly(I:C) stimulated mothers was 8.3 ± 5.3%, whereas control larvae had a cumulative mortality 2.3 times higher (18.9 ± 6.3%) at 48 h post-inoculation with OsHV-1. Larvae produced from poly(I:C) stimulated mothers also had significantly lower amounts of OsHV-1 DNA in their tissues compared to control larvae (p < 0.05). The average concentration of OsHV-1 DNA in larvae produced from poly(I:C) stimulated mothers was 4.0 × 10 4 genome copies per larva, while control larvae had 2.3-fold more OsHV-1 (9.3 × 10 4 copies larva -1 ) at 48 h post inoculation.

Paternal immune stimulation prior to spawning had no significant effect on the survival of C. gigas offspring to OsHV-1 (p > 0.05, Fig. 2). The average cumulative mortality of larvae produced from poly(I:C) stimulated fathers was 20.6 ± 4.6%, whereas control larvae had a cumulative mortality of 31.5 ± 3.4% at 48 h post-inoculation with OsHV-1. There was also no difference in the amount of OsHV-1 DNA in the tissue of D-larvae produced from poly(I:C) or control stimulated fathers (p > 0.05).

Paternal or maternal immune stimulation with poly(I:C) prior to spawning did not affect the survival of non-challenged larvae (i.e. offspring exposed to the control inoculum) (p > 0.05, Fig. 2). Average mortality of larvae in these control treatments was lower than 4% and OsHV-1 DNA was not detected in their tissue.

3-3 Differential gene expression in unchallenged larvae following parental immune challenge

RNA-seq was used to investigate whether parental immune stimulation alters the transcription of immune-related genes in offspring. Six cDNA libraries were generated with mRNA from unchallenged Dlarvae (24 h post-fertilization) produced from parents stimulated with poly (I:C) (N = 3, 3 days prior to spawning) or seawater (N = 3). Illumina sequencing yielded more than 203 million reads with an average PHRED quality of 37. The average number of reads was 34 M per library (SD = 3.3 M; max = 37 M; min = 28 M). Overall, 79.4% of the total output reads mapped to the GigaTON reference transcriptome (SD = 7.2%; max = 87.8%; min = 70.8%). Reads mapped to contigs were clustered into 22,450 gene clusters using Corset. A total of 14,479 (64.5%) of these gene clusters were present in at least half of the samples sequenced (3 out of 6 RNA-seq libraries) and were used for downstream analysis.

Analysis of expression levels for the 14,479 gene clusters revealed that larvae produced from immune-stimulated parents have very similar gene expression profiles to control larvae (Fig. 3a). Of the 14,479 gene clusters, only 47 were putatively identified to be differentially expressed (FDR-adjusted, p < 0.05) in D-larvae produced by immunestimulated parents. D-larvae produced from immune-stimulated parents exhibited 22 up-regulated genes (fold-change between 2.7 and 302.6) and 25 down-regulated genes (fold-change between -2.9 and -153.1). Blast analysis showed that 87% of the differentially expressed genes (DEGs; 41 gene clusters) had a significant match against the Mollusca non-redundant (nr) and/or the NCBI nucleotide (nt) databases (e-value < 1 -10 ). Of the DEGs with Blast hits, only 39% were annotated with a putative gene function. Differentially expressed genes were found to be associated with immune and stress responses (e.g. caspase14, sacsin, zinc metalloproteinase, F-box only protein 22), cellular signaling and communication (e.g. regulator of G-protein signaling protein, integrin beta pat-3, tenascin and fibrocystin-L), regulation of the cell cycle and cellular division (e.g. nibrin and baculoviral IAP repeatcontaining protein 2) (Fig. 3b). We have also identified a number of differentially expressed genes encoding uncharacterized proteins. A few of these uncharacterized proteins exhibited conserved domains, including integrase, reverse transcriptase and recombinase motifs (Table 2).

3-4 Validation and expression pattern analysis

A total of 47 DEGs were identified by RNA-seq to be differentially expressed between larvae produced from parents treated with poly(I:C) or seawater (controls) prior to spawning. To validate the RNA-seq analysis, we chose 10 candidate DEGs (25%) for RT-qPCR analysis. The expression of three known antiviral genes (interferon regulatory factor, Viperin and adenosine deaminase RNA-specific) were also evaluated. RTqPCR did not identify differences in the expression level of these ten DEGS in groups of larvae produced from parents treated with poly(I:C) or seawater. These samples analyzed by RT-qPCR included cDNA sequenced by RNA-seq (experiment I) and cDNA samples from experiment II. Based on these results, we were unable to validate the RNA-seq analysis by RT-qPCR.

The antiviral genes of IRF, viperin and ADAR-L had higher relative expression in eggs from mothers stimulated with poly(I:C) at 3 or 10 days prior to spawning (p < 0.05, Fig. 4a). Higher expression of these genes were also observed in eggs produced by mothers treated with poly(I:C) at 3 days prior to spawning (p < 0.05, Fig. 4b) but no difference was observed in unchallenged D-larvae produced from parents stimulated with poly(I:C) or seawater (p > 0.05).

4-Discussion

Heterogeneous immune priming experiments showed that Crassostrea gigas primed with poly(I:C) have improved survival to OsHV-1 infection [START_REF] Green | Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 μvar)[END_REF]Green et al., 2015b;[START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF], and this protection can be passed to the next generation [START_REF] Green | Primed for success: oyster parents treated with poly(I:C) produce offspring with enhanced protection against Ostreid herpesvirus type I infection[END_REF]. Despite the physiological, metabolic and immunological response of C. gigas to OsHV-1 infection being well characterized [START_REF] Corporeau | Proteomic signatures of the oyster metabolic response to herpesvirus OsHV-1 μvar infection[END_REF][START_REF] Green | Antiviral defense and innate immune memory in the oyster[END_REF][START_REF] Martenot | Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis[END_REF][START_REF] Tamayo | Physiological changes in Pacific oyster Crassostrea gigas exposed to the herpesvirus OsHV-1 uvar[END_REF][START_REF] Young | Differential expression of novel metabolic and immunological biomarkers in oysters challenged with a virulent strain of OsHV-1[END_REF], relatively little is known regarding the molecular mechanisms underpinning the phenomenon of immune priming. A core set of genes expressed in response to OsHV-1 infection has been identified in adults [START_REF] He | Transcriptome analysis reveals strong and complex antiviral response in a mollusc[END_REF][START_REF] Rosani | Dual analysis of host and pathogen transcriptomes in Ostreid herpesvirus 1 -positive Crassostrea gigas[END_REF] and larvae [START_REF] Zhang | Massive expansion and functional divergence of innate immune genes in a protostome[END_REF], and differences in antiviral gene expression in C. gigas primed with poly(I:C) [START_REF] Green | Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 μvar)[END_REF]Green et al., 2015b), and their progeny [START_REF] Green | Primed for success: oyster parents treated with poly(I:C) produce offspring with enhanced protection against Ostreid herpesvirus type I infection[END_REF] following inoculation with OsHV-1 have been characterized. Stimulation with double-stranded RNA, such as poly(I:C), induces the expression of antiviral effector genes (Green et al., 2014b), for at least seven days (Green et al., 2014a), and this response appears to inhibit OsHV-1 replication [START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF][START_REF] Pauletto | Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication[END_REF]. Do C. gigas offspring produced from parents stimulated with poly(I:C) also have elevated expression of antiviral effector genes?

In the current study, oyster larvae had higher survival to OsHV-1 when their mothers, but not their fathers, were administered poly(I:C) prior to spawning (Fig. 2). This improved survival could not be explained by reconfiguration of the constitutive expression of antiviral genes in unchallenged larvae. Transcriptomic analysis revealed that non-challenged larvae produced from parents primed with polyI:C have similar transcriptional profiles to control larvae. Despite this overall similarity in transcriptome response, a small subset of 47 genes was found to be differentially regulated between offspring of polyI:Ctreated and non-treated parents (Fig. 3). However, validation of RNA-seq data by RT-qPCR did not identify any differentially expressed genes between the two offspring cohorts (21% of the genes identified by RNAseq were tested by RT-qPCR). This discrepancy might be caused by the small sample sizes (N = 3) used in the current study. Alternatively, our results imply that the improved survival of C. gigas larvae might occur via maternal provisioning of antiviral compounds in the eggs. In support of maternal provisioning is the upregulation of antiviral effector genes, including viperin, in the eggs of C. gigas following stimulation with poly (I:C) (Fig. 4). Viperin has been shown to be induced by poly(I:C) treatment via a hemolymph cytokine and to play a direct role in oyster antiviral defense (Green et al., 2015c). Maternal provisioning is consistent with a previous study on the scallop Chlamys farreri, where mothers stimulated with heat-killed Vibrio transfer antibacterial proteins to their offspring via the egg [START_REF] Yue | Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge[END_REF]. Crosses involving OsHV-1 infected C. gigas mothers produce progengy (larvae and spat developmental life-stages) have survival rates statistically higher than other types of crosses, suggesting OsHV-1 infection mothers transmit some form of protection to their offspring [START_REF] Barbosa-Solomieu | Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas)[END_REF].

Maternal provisioning of antiviral compounds to C. gigas larvae has the potential advantage that it is the mother, not the developing embryo, who invests resources into its offspring's antiviral defense. Immunity is a life-history trait that can be expected to be traded off with other physiological processes, such as growth, reproduction and self-maintenance [START_REF] Rauw | Immune response from a resource allocation perspective[END_REF]. Immune activation entails a significant energetic cost, revealed by raised metabolic rates between 8 and 28% in a range of terrestrial insects following PAMP inoculation or injury [START_REF] Ardia | Costs of immunity in insects: an induced immune responses increases metabolic rate and decreases antimicrobial activity[END_REF][START_REF] Freitak | Immune response is energetically costly in white cabbage butterfly pupae[END_REF]. The metabolic requirement of immunity has not been quantified for marine bivalves, but it is likely to have a similar high cost. During early larval development of C. gigas, activating an immune response can compromise other physiological processes. In ideal environmental conditions, C. gigas larvae allocate ∼75% of their total metabolic energy budget to protein synthesis (∼55%) and ion transport (∼20%) [START_REF] Lee | Metabolic cost of protein synthesis in larvae of the Pacific oyster (Crassostrea gigas) is fixed across genotype, phenotype, and environmental temperature[END_REF][START_REF] Pan | Predicting phenotypic variation in growth and metabolism of marine invertebrate larvae[END_REF]. Thus, maternal provisioning of immunity represents a beneficial investment from mother to offspring, reducing the cost of producing and maintaining an expensive antiviral response. It now needs to be determined if this antiviral immunity persists or declines during C. gigas embryonic developments, as the antiviral compounds are metabolized.

Selective breeding programs in many countries have developed improved C. gigas stocks, which are better suited for aquaculture purposes [START_REF] De Melo | Improvements in desirable traits of the Pacific oyster, Crassostrea gigas, as a result of five generations of selection on the West Coast, USA[END_REF]Degremont et al., 2015a;[START_REF] Swan | Genotype x environment interactions for weight in Pacific oysters (Crassostrea gigas) on five Australian farms[END_REF]. Indeed, survival to OsHV-1 infection is a trait that has received considerable scientific attention given the benefits it could provide to oyster production worldwide [START_REF] Camara | Assessment of heat shock and laboratroy virus challenges to selectively breed for ostreid herpesvirus 1 (OsHV-1) resistance in the Pacific oyster, Crassostrea gigas[END_REF][START_REF] Dégremont | Evidence of herpesvirus (OsHV-1) resistance in juvenile Crassostrea gigas selected for high resistance to the summer mortality phenomenon[END_REF]Degremont et al., 2015b). To access genetically improved C. gigas stocks, shellfish farms have to source spat from aquaculture hatcheries [START_REF] Robert | Bivalve hatchery technology: the current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France[END_REF]. However, aquaculture hatchery supply of C. gigas spat is inconsistent due to larval mortality events [START_REF] Robert | Bivalve hatchery technology: the current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France[END_REF], which can be caused by OsHV-1 (Barbosa-Solomieu et al., 2005;[START_REF] Hine | Herpesvirus associated with mortalities among hatcheryreared larval Pacific oysters Crassostrea gigas[END_REF][START_REF] Renault | Concomitant herpes-like virus infections in hatchery-reared larvae and nursery-cultured spat Crassostrea gigas and Ostrea edulis[END_REF]. Our results demonstrate that it is possible to utilize transgenerational immune priming to reduce mortalities in hatchery production of C. gigas, thus challenging the concept that such therapies cannot be adapted for oyster aquaculture [START_REF] Pernet | Infectious disease in oyster aquaculture require a new integrated approach[END_REF][START_REF] Rodgers | A literature review as an aid to identify strategies for mitigating ostreid herpesvirus 1 in Crassostrea gigas hatchery and nursery systems[END_REF]. This study also showed that transgenerational immune priming against OsHV-1 is consistently reproducible, alleviating the concerns surrounding the existence of immune priming in shellfish [START_REF] Hauton | Adaptive immunity in invertebrates: a straw house without a mechanistic foundation[END_REF]. Future research should focus on characterizing the transfer of antiviral compounds from mother to offspring. The application of this knowledge can have great potential for improving oyster health and welfare in aquaculture.

FIGURE 1

Schematic diagram of experimental design for investigating the effect of time between parental immune stimulation and spawning on offspring immunity to OsHV-1. Two cohorts of adult Crassostrea gigas were injected with poly(I:C) at 3 and 10 days prior to spawning. A third cohort of C. gigas were injected with seawater. Up to 6 pair mated families were produced for each treatment. D-larvae from each family were inoculated with OsHV-1 or control homogenate (NC). Cumulative mortality for each treatment was determined at 48 h post-inoculation (mean ± standard deviation). Different letters indicate significant differences (p < 0.05) between treatments.

FIGURE 2

Schematic diagram of experimental design for investigating the contribution of maternal and paternal immune stimulation to offspring performance. D-larvae from each family were inoculated with OsHV-1 or control homogenate (NC). Cumulative mortality for each treatment was determined at 48 h post-inoculation (mean ± standard deviation). Different letters indicate significant differences (p < 0.05) between treatments.

FIGURE 3

The expression of interferon regulatory factor (IRF), viperin and double-stranded RNAspecific adenosine deaminase (ADAR) in unfertilized eggs of Crassostrea gigas that were stimulated with poly(I:C) or seawater (control). a) experiment i: effect of time between parental immune stimulation and spawning on offspring immunity to OsHV-1. b) experiment ii: maternal effect on offspring immunity to OsHV-1. Expression is presented as the mean ± standard deviation. Different letters indicate significant differences (p < 0.05) between treatments.

TABLE 1

Primer pairs used for qPCR analysis. Genes in bold are known internal reference genes or antiviral proteins selected from the scientific literature. The closest match in the NCBI mollucan database (BlastX) and its annotation is provided.

FIGURE 4

 4 FIGURE 4 Transcriptional response of unchallenged D-larvae of C. gigas produced from parents stimulated with poly(I:C) or seawater (control) prior to spawning. (a) Non-metric multidimensional scaling (NMDS) plot summarizing the expression level of 14,479 gene clusters identified by Trinity and Corset in pooled Dlarvae samples produced from pair-mated families. (b) Heat map of gene identified to be differentially expressed by RNA-seq between unchallenged D-larvae.

Acknowledgement

The authors acknowledge funding provided to T. Green from the Macquarie University postdoctoral research scheme (MQ Grant 9201300681), Canada Research Chair Program (Grant No. 950231856) and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN-2018-06761). The authors are grateful for a travel bursary to M. Lafont from IFREMER.

Conflicts of interest

The authors declare no competing interests.

TABLE 2

Genes identified by RNA-seq to be differentially expressed between larvae produced from broodstock stimulated with poly(I:C) or seawater. Genes highlighted in bold were investigated by RT-qPCR.