Virgile Quillien

Oyster reproduction is affected by exposure to polystyrene microplastics

Significance Plastics are a contaminant of emerging concern accumulating in marine ecosystems. Plastics tend to break down into small particles, called microplastics, which also enter the marine environment directly as fragments from a variety of sources, including cosmetics, clothing, and industrial processes. Given their ubiquitous nature and small dimensions, the ingestion and impact of microplastics on marine life are a cause for concern, notably for filter feeders. Oysters were exposed to polystyrene microparticles, which were shown to interfere with energy uptake and allocation, reproduction, and offspring performance. A drop in energy allocation played a major role in this reproductive impairment. This study provides ground-breaking data on microplastic impacts in an invertebrate model, helping to predict ecological impact in marine ecosystems. Plastics are persistent synthetic polymers that accumulate as waste in the marine environment. Microplastic (MP) particles are derived from the breakdown of larger debris or can enter the environment as microscopic fragments. Because filter-feeder organisms ingest MP while feeding, they are likely to be impacted by MP pollution. To assess the impact of polystyrene microspheres (micro-PS) on the physiology of the Pacific oyster, adult oysters were experimentally exposed to virgin micro-PS (2 and 6 µm in diameter; 0.023 mg·L−1) for 2 mo during a reproductive cycle. Effects were investigated on ecophysiological parameters; cellular, transcriptomic, and proteomic responses; fecundity; and offspring development. Oysters preferentially ingested the 6-µm micro-PS over the 2-µm-diameter particles. Consumption of microalgae and absorption efficiency were significantly higher in exposed oysters, suggesting compensatory and physical effects on both digestive parameters. After 2 mo, exposed oysters had significant decreases in oocyte number (−38%), diameter (−5%), and sperm velocity (−23%). The D-larval yield and larval development of offspring derived from exposed parents decreased by 41% and 18%, respectively, compared with control offspring. Dynamic energy budget modeling, supported by transcriptomic profiles, suggested a significant shift of energy allocation from reproduction to structural growth, and elevated maintenance costs in exposed oysters, which is thought to be caused by interference with energy uptake. Molecular signatures of endocrine disruption were also revealed, but no endocrine disruptors were found in the biological samples. This study provides evidence that micro-PS cause feeding modifications and reproductive disruption in oysters, with significant impacts on offspring.

In Vivo RNA Interference of a Gonad-Specific Transforming Growth Factor-β in the Pacific Oyster Crassostrea gigas

We investigated the role of oyster gonadal TGFβ (og-TGFβ) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFβ, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFβ dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFβ appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.

Association among growth, food consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas

To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.

Contrasted survival under field or controlled conditions displays associations between mRNA levels of candidate genes and response to OsHV-1 infection in the Pacific oyster Crassostrea gigas

Pacific oyster Crassostrea gigas suffers from chronic or sporadic mortality outbreaks worldwide, resulting from infectious diseases and/or physiological disorders triggered by environmental factors. Since 2008, ostreid herpesvirus OsHV-1 μVar has been identified as the main agent responsible for mass mortality of juvenile oysters in Europe. Previous studies of genome-wide expression profiling have provided candidate genes that potentially contribute to genetically-based resistance to summer mortality. To assess their value in determining resistance to the juvenile mass mortality that has occurred in France since 2008, we analyzed the expression of 17 candidate genes in an experimental infection by OsHV-1 μVar, and in an in vivo field experiment. Individual quantification of mRNA levels of 10 out of the 17 targeted genes revealed significant variation, of which 7 genes were showed differences between conditions that created significant differences in mortality, and 6 depended on the number of OsHV-1 genome copies individually quantified in mantle tissue. Complex SOD metalloenzymes known to be part of the antioxidant defense strategies may at least partly determine susceptibility or resistance to OsHV-1-associated mortality. Furthermore, inhibitor 2 of NF-κB, termed CgIκB2, exhibited highly significant variation of mRNA levels depending on OsHV-1 load in both experiments, suggesting its implication in the antiviral immune response of C. gigas. Our results suggest that CgIκB2 expression would make a good starting point for further functional research and that it could be used in marker-assisted selection.

Disruption of amylase genes by RNA interference affects reproduction in the Pacific oyster Crassostrea gigas

ABSTRACT Feeding strategies and digestive capacities can have important implications for variation in energetic pathways associated with ecological and economically important traits, such as growth or reproduction in bivalve species. Here, we investigated the role of amylase in the digestive processes of Crassostrea gigas, using in vivo RNA interference. This approach also allowed us to investigate the relationship between energy intake by feeding and gametogenesis in oysters. Double-stranded (ds)RNA designed to target the two α-amylase genes A and B was injected in vivo into the visceral mass of oysters at two doses. These treatments caused significant reductions in mean mRNA levels of the amylase genes: −50.7% and −59% mRNA A, and −71.9% and −70.6% mRNA B in 15 and 75 µg dsRNA-injected oysters, respectively, relative to controls. Interestingly, reproductive knock-down phenotypes were observed for both sexes at 48 days post-injection, with a significant reduction of the gonad area (−22.5% relative to controls) and germ cell under-proliferation revealed by histology. In response to the higher dose of dsRNA, we also observed reductions in amylase activity (−53%) and absorption efficiency (−5%). Based on these data, dynamic energy budget modeling showed that the limitation of energy intake by feeding that was induced by injection of amylase dsRNA was insufficient to affect gonadic development at the level observed in the present study. This finding suggests that other driving mechanisms, such as endogenous hormonal modulation, might significantly change energy allocation to reproduction, and increase the maintenance rate in oysters in response to dsRNA injection. Summary: Disruption of amylase gene expression reduces absorption efficiency and constrains reproduction in oysters; the reduction in energy intake is insufficient to affect reproduction, suggesting that dsRNA causes disturbances that increase maintenance costs.

Sex-Specific Regulation of AMP-Activated Protein Kinase (AMPK) in the Pacific Oyster Crassostrea gigas

ABSTRACT The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

A Microarray-Based Analysis of Gametogenesis in Two Portuguese Populations of the European Clam Ruditapes decussatus

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication

ABSTRACT Double-stranded RNA (dsRNA)-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve (Crassostrea gigas), as well as the specific role of the subunit 2 of the nuclear factor κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-IκB2 and green fluorescent protein genes were injected in vivo into oysters before being challenged by OsHV-1. Survival appeared close to 100% in both dsRNA-injected conditions associated with a low detection of viral DNA and a low expression of a panel of 39 OsHV-1 genes as compared with infected control. Long dsRNA molecules, both Cg-IκB2- and GFP-dsRNA, may have induced an anti-viral state controlling the OsHV-1 replication and precluding the understanding of the specific role of Cg-IκB2. Immune-related genes including Cg-IκB1, Cg-Rel1, Cg-IFI44, Cg-PKR and Cg-IAP appeared activated in the dsRNA-injected condition, potentially hampering viral replication and thus conferring a better resistance to OsHV-1 infection. We revealed that long dsRNA-mediated genetic interference triggered an anti-viral state in the oyster, emphasizing the need for new reverse genetics tools for assessing immune gene function and avoiding off-target effects in bivalves. Summary: Double-stranded ribonucleic acid (dsRNA) injection in the Pacific oyster induced an anti-viral state controlling ostreid herpesvirus 1 replication and precluding the understanding of the role of the inhibitor of nuclear factor-κB, Cg-IκB2.

Bioactive extracellular compounds produced by the dinoflagellate Alexandrium minutum are highly detrimental for oysters

Blooms of the dinoflagellate Alexandrium spp., known as producers of paralytic shellfish toxins (PSTs), are regularly detected on the French coastline. PSTs accumulate into harvested shellfish species, such as the Pacific oyster Crassostrea gigas, and can cause strong disorders to consumers at high doses. The impacts of Alexandrium minutum on C. gigas have often been attributed to its production of PSTs without testing separately the effects of the bioactive extracellular compounds (BECs) with allelopathic, hemolytic, cytotoxic or ichthyotoxic properties, which can also be produced by these algae. The BECs, still uncharacterized, are excreted within the environment thereby impacting not only phytoplankton, zooplankton but also marine invertebrates and fishes, without implicating any PST. The aim of this work was to compare the effects of three strains of A. minutum producing either only PSTs, only BECs, or both PSTs and BECs, on the oyster C. gigas. Behavioral and physiological responses of oysters exposed during 4 days were monitored and showed contrasted behavioral and physiological responses in oysters supposedly depending on produced bioactive substances. The non-PST extracellular-compound-producing strain primarily strongly modified valve-activity behavior of C. gigas and induced hemocyte mobilization within the gills, whereas the PST-producing strain caused inflammatory responses within the digestive gland and disrupted the daily biological rhythm of valve activity behavior. BECs may therefore have a significant harmful effect on the gills, which is one of the first organ in contact with the extracellular substances released in the water by A. minutum. Conversely, the PSTs impact the digestive gland, where they are released and mainly accumulated, after degradation of algal cells during digestion process of bivalves. This study provides a better understanding of the toxicity of A. minutum on oyster and highlights the significant role of BECs in this toxicity calling for further chemical characterization of these substances.

Whole transcriptome sequencing and biomineralization gene architecture associated with cultured pearl quality traits in the pearl oyster, Pinctada margaritifera

Background Cultured pearls are unique gems produced by living organisms, mainly molluscs of the Pinctada genus, through the biomineralization properties of pearl sac tissue. Improvement of P. margaritifera pearl quality is one of the biggest challenges that Polynesian research has faced to date. To achieve this goal, a better understanding of the complex mechanisms related to nacre and pearl formation is essential and can now be approached through the use of massive parallel sequencing technologies. The aim of this study was to use RNA-seq to compare whole transcriptome expression of pearl sacs that had producing pearls with high and low quality. For this purpose, a comprehensive reference transcriptome of P. margaritifera was built based on multi-tissue sampling (mantle, gonad, whole animal), including different living stages (juvenile, adults) and phenotypes (colour morphotypes, sex). Results Strikingly, few genes were found to be up-regulated for high quality pearls (n = 16) compared to the up-regulated genes in low quality pearls (n = 246). Biomineralization genes up-regulated in low quality pearls were specific to prismatic and prism-nacre layers. Alternative splicing was further identified in several key biomineralization genes based on a recent P. margaritifera draft genome. Conclusion This study lifts the veil on the multi-level regulation of biomineralization genes associated with pearl quality determination.