Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing

Genomic Diversity of the Ostreid Herpesvirus Type 1 Across Time and Location and Among Host Species

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. This is particularly true for pathogens with low per-site mutation rates, such as DNA viruses, that do not exhibit a large amount of evolutionary change among genetic sequences sampled at different time points. However, whole-genome sequencing can reveal the accumulation of novel genetic variation between samples, promising to render most, if not all, microbial pathogens measurably evolving and suitable for analytical techniques derived from population genetic theory. Here, we aim to assess the measurability of evolution on epidemiological time scales of the Ostreid herpesvirus 1 (OsHV-1), a double stranded DNA virus of which a new variant, OsHV-1 μVar, emerged in France in 2008, spreading across Europe and causing dramatic economic and ecological damage. We performed phylogenetic analyses of heterochronous (n = 21) OsHV-1 genomes sampled worldwide. Results show sufficient temporal signal in the viral sequences to proceed with phylogenetic molecular clock analyses and they indicate that the genetic diversity seen in these OsHV-1 isolates has arisen within the past three decades. OsHV-1 samples from France and New Zealand did not cluster together suggesting a spatial structuration of the viral populations. The genome-wide study of simple and complex polymorphisms shows that specific genomic regions are deleted in several isolates or accumulate a high number of substitutions. These contrasting and non-random patterns of polymorphism suggest that some genomic regions are affected by strong selective pressures. Interestingly, we also found variant genotypes within all infected individuals. Altogether, these results provide baseline evidence that whole genome sequencing could be used to study population dynamic processes of OsHV-1, and more broadly herpesviruses

Synthesis of the “PLAN DE SAUVEGARDE” using selected all-triploid oysters to reduce the shortage of spat in France due to OsHV-1–associated mortality in Crassostrea gigas

Due to massive mortality of Crassostrea gigas spat in France since 2008, a “plan de sauvegarde” was set up from 2011 to 2014 (hereafter referred to as PS1 to PS4), in order to reduce the shortage of spat. This plan involved the participation of commercial hatcheries, the French Research Institute for Exploitation of the Sea (Ifremer), and the Direction des Pêches Maritimes et de l'Aquaculture (DPMA) of the French Ministry of Agriculture. It was based on selecting diploid lines of C. gigas for their higher resistance to the oyster herpesvirus OsHV-1 (2nR group), and one of these lines was subsequently tetraploidized (4nR group). Both the 2nR and 4nR groups were produced by Ifremer, and then transferred to commercial hatcheries that produced the selected triploids (3nR groups). We report here the mortality rates of the 3nR group for each of the four “plan de sauvegarde” campaigns and compare these with the mortalities of the classic production of commercial hatcheries (both 2n and 3n), benchmarks of selected (2nR group) and unselected (2n-control group) oysters produced by Ifremer, and wild-caught spat, representing a total of 104 diploid and triploids batches. For PS1, the 3nR group had a mean mortality of 67% and did not show any advantage over the 2n- and 3n-commercial groups, suggesting a lack of genetic progress in the 2nR and 4nR groups. For PS2, OsHV-1 resistance was increased in both the 2nR and 4nR groups and, consequently, the 3nR group exhibited a mean mortality of 52%, which was significantly lower than the mortality of the 2n- (87%) and 3n-(76%) commercial groups in 2012. Unfortunately, the mortality of the 3nR group reached 62% and 71% in PS3 and PS4, respectively, although it was expected to be lower than that in PS2. OsHV-1 DNA was quantified in the live oysters at deployment (1356 oysters) and at the endpoint (1171 oysters), as well as in moribund oysters sampled during peak mortality (539 oysters). The results strongly supported the involvement of this pathogen during the main mortality outbreak in May/June. Meanwhile, Vibrio aestuarianus was also suspected to cause unexpected mortality of PS3 oysters in August and September, and it was detected in moribund PS4 oysters during both the mortality events, in May and July. Despite genetic improvement for OsHV-1 resistance, this translated into variable commercial genetic gain. This could be explained by the limited genetic backgrounds of the 2nR and 4nR groups, the reemergence of V. aestuarianus in France since 2012, the changing levels of genetic improvement in both the 3nR group and the commercial groups, as well as the limited broodstock genetic variation where small numbers of males were used. Results on growth and yield are discussed

Enhancing resistance to Vibrio aestuarianus in Crassostrea gigas by selection

Ostreid herpesvirus 1 (OsHV-1) and Vibrio aestuarianus are the two main pathogens affecting the production of French oyster (Crassostrea gigas). The absence of genetic correlation between the two diseases is promising for the development of stocks with dual resistance. Using unselected and selected oysters concerning enhanced resistance to OsHV-1 infection, we investigated the first generation of mass selection and the response to selection to increase the resistance to V. aestuarianus for two stocks. For each stock, four groups were produced in June 2013 using either parents unchallenged with the bacteria or counterparts that survived experimental infections by the bacteria. Thus, groups were unselected oysters for both pathogens, selected for either the virus or the bacteria, and dually selected for both pathogens. All groups of each stock were evaluated at the spat and juvenile sizes following experimental infection by V. aestuarianus in May 2014. Regardless of their level of selection for OsHV-1, oysters produced from parents that survived V. aestuarianus showed similar mortalities (47% and 53% for stocks A and B, respectively) during the bacterial challenge than those produced from unchallenged parents (43% and 56%, respectively). Thus, no positive response to selection at the first generation to increase the bacterial resistance was found at the spat and juvenile sizes. At the adult stage and with experimental infection with V. aestuarianus, only stock B showed a positive response to selection for increasing the bacterial resistance with a decrease in mortality of 14% in comparison with unselected oysters. Similar results were observed when oysters were tested for 27 months in the field with the absence of response to selection for stock A, while a 13% decrease in mortality was observed for stock B. For stock B, mortality at endpoint after 27 months in the field reached 89% for the control not selected at all, decreased to 84% for oysters selected for their resistance to the bacteria, was 53% for the oysters selected for their resistance to OsHV-1, and was 32% for oysters selected for dual resistance. Realized heritability estimated at the first generation of mass selection for stock B was higher for oysters selected for dual resistance, ranging from 0.47 to 0.80, than those only selected for V. aestuarianus, ranging from 0.05 to 0.30. Selection for dual resistance in C. gigas could limit the impact of both OsHV-1 and V. aestuarianus on oyster production

Resistance to OsHV-1 Infection in Crassostrea gigas Larvae

The ostreid herpesvirus(OsHV-1) is one of the major diseases that affect the Pacific oyster Crassostrea gigas. Selective breeding programs were recently shown to improve resistance easily to OsHV-1 infections in spat, juvenile, and adult oysters. Nevertheless, this resistance has never been investigated in larvae, whereas this developmental stage has crucial importance for the production of commercial hatcheries, as well as explaining the abundance of spatfall. A first trial tested several viral suspensions at several concentrations using contaminated water with OsHV-1 in 4- and 10-day-old larvae that we reproduced from an unselected broodstock. In follow up on the results, one viral suspension at a final concentration of 10+6 OsHV-1 DNA copies per L was used to assess resistance to OsHV-1 infection in C.gigas larvae that we reproduced from selected and unselected broodstock. A second trial evaluated OsHV-1 resistance in larvae from both broodstocks intrials 2a, 2b, and 2c with 4,10, and 16-day-old larvae for 7 days, which corresponded to post D larvae, umbo larvae, and eyed larvae, respectively. The mortality of unchallenged larvae for both stocks were low (<15%) at day 7 intrials 2a and 2b, whereas it ranged from 48 to 56% in trial 2c. More interestingly, selected larvae had significantly lower mortality than unselected larvae when exposed to OsHV-1 in all of the trials. Thus, themortality was 11 and 49% for the selected larvae at day 7 post-exposure in trials 2a and 2c, respectively, in comparison with 84 and 97% for the unselected larvae. Although this difference in mortality was observed at day 5 in trial 2b, it was reduced at day 7, to 86 and 98% for the selected and unselected larvae, respectively. For the first time in the literature, the difference in mortality or the delayed on set of mortality between selected and unselected larvae have indicated a genetic resistance to OsHV-1 infection at the larval stage. Such finding should facilitate the selective breeding programs focusing on resistance to OsHV-1 infection by reducing the span of the genetic evaluation, and thus decreasing its cost

Susceptibility variation to the main pathogens of Crassostrea gigas at the larval, spat and juvenile stages using unselected and selected oysters to OsHV-1 and/or V. aestuarianus

French commercial hatcheries are massively producing Crassostrea gigas selected for their higher resistance to OsHV-1, and soon should also implement selection for increasing resistance to Vibrio aestuarianus. The first objective of this study was to optimize the breeding programs for dual resistance to OsHV-1 and V. aestuarianus to determine the earliest life stage for which oysters are able to develop disease resistance. Wild stocks and selected families were tested using experimental infections by both pathogens at the larval, spat and juvenile stages. Oyster families could be evaluated for OsHV-1 as soon as the larval stage by a bath method, but this only highlighted the most resistant families; those that showed the highest resistance to V. aestuarianus could be determined using the cohabitation method at the juvenile stage. The second objective of this study was to determine if selection to increase/decrease the resistance to OsHV-1 and V. aestuarianus could have an impact on other major pathogens currently detected in hatchery at the larval stage, and in nursery and field at the spat/juveniles stages (V. coralliilyticus, V. crassostreae, V. tasmaniensis, V.neptunius, V. europaeus, V. harveyi, V. chagasi). No relationship was found between mortality caused by V. aestuarianus/OsHV-1 and the mortality caused by the other virulent bacterial strains tested regardless the stages, except between OsHV-1 and V. tasmaniensis at the juvenile stage. Finally, miscellaneous findings were evidenced such as (1) bath for bacterial challenges was not adapted for spat, (2) the main pathogens at the larval stage were OsHV-1 and V. coralliilyticus using bath, while it was V. coralliilyticus, V. europaeus, and V. neptunius at the juvenile stage by injection, and (4) variation in mortality was observed among families/wild controls for all pathogens at larval and juvenile stages, except for V. harveyi for larvae

SNP-based parentage analyses over two successive generations demonstrates the feasibility of efficient production of inbred lines in the Pacific oyster (Crassostrea gigas) by self-fertilization of simultaneous hermaphrodites despite severe inbreeding depression

The Pacific oyster, Crassostrea gigas, is a species in which true male and true female well as hermaphrodite individuals have been reported. Among the latter, most are sequential, with one or more sex changes throughout their live, while a few (<2%) are simultaneous hermaphrodites (SH) which may self-fertilize. Sex determinism and its functional bases remain unclear is this species. In this study, one SH oyster was found among a mature broodstock and used to produce a progeny by self-fertilization. Several thousand offspring were obtained for this family (SF1) but only 73 SF1 oysters survived following an episode of mass mortality at the spat stage. Six of them were conditioned for reproduction and three were found to be SH, and one produced a limited but viable progeny by self-fertilization (SF2). Oysters were sampled and genotyped with 226 SNP markers, confirming that the SF1 and the SF2 oysters were produced by self-fertilization over two successive generations. To our knowledge, it is the second study reporting the feasibility of production on an inbred line of Pacific oysters by self-fertilization using a simultaneous hermaphrodite as primary genitor, and the first study to generate two successive generations of selfing. Observed mean inbreeding coefficient of SF2 oysters produced by selfing for two generations was 0.61, ranging from 0.46 to 0.80. This coefficient was lower than the expected theoretical value (0.8125), suggesting that the most homozygous offspring died at early stages due to inbreeding depression. Our study demonstrates that inbred lines of Pacific oysters can be produced by self-fertilization but may exhibit higher heterozygosity than theoretically expected due to better survival of the most heterozygous offspring

Monitoring Autophagy at Cellular and Molecular Level in Crassostrea gigas During an Experimental Ostreid Herpesvirus 1 (OsHV-1) Infection

Mortality outbreaks of young Pacific oysters, Crassostrea gigas, have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas, recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1