Elodie Fleury

Microarray Analysis Highlights Immune Response of Pacific Oysters as a Determinant of Resistance to Summer Mortality

Summer mortality of Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. A high heritability was estimated for resistance to summer mortality, which provided an opportunity to develop lines of oysters that were resistant (R) or susceptible (S) to summer mortality. Previous genome-wide expression profiling study of R and S oyster gonads highlighted reproduction and antioxidant defense as constitutive pathways that operate differentially between these two lines. Here, we show that signaling in innate immunity also operates differentially between these lines, and we hypothesize that this is at the main determinant of their difference in survival in the field. A reanalysis of our published microarray data using separate ANOVAs at each sampling date revealed a specific “immune” profile at the date preceding the mortality. In addition, we conducted additional microarray profiling of two other tissues, gills, and muscle, and both showed an overrepresentation of immune genes (46%) among those that are differentially expressed between the two lines. Eleven genes were pinpointed to be simultaneously differentially expressed between R and S lines in the three tissues. Among them, ten are related to “Immune Response.” For these genes, the kinetics of R mRNA levels between sampling dates appeared different just before the morality peak and suggests that under field conditions, R oysters had the capacity to modulate signaling in innate immunity whereas S oysters did not. This study enhances our understanding of the complex summer mortality syndrome and provides candidates of interest for further functional and genetics studies.

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.

A Functional Study of Transforming Growth Factor-Beta from the Gonad of Pacific Oyster Crassostrea gigas

The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.

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.

Determination of risk factors for herpesvirus outbreak in oysters using a broad-scale spatial epidemiology framework

Marine diseases have major impacts on ecosystems and economic consequences for aquaculture and fisheries. Understanding origin, spread and risk factors of disease is crucial for management, but data in the ocean are limited compared to the terrestrial environment. Here we investigated how the marine environment drives the spread of viral disease outbreak affecting The Pacific oyster worldwide by using a spatial epidemiology framework. We collected environmental and oyster health data at 46 sites spread over an area of 300 km2 along an inshore-offshore gradient during an epizootic event and conducted risk analysis. We found that disease broke out in the intertidal farming area and spread seaward. Mortalities and virus detection were observed in oysters placed 2 km from the farming areas, but oysters of almost all sites were subclinically infected. Increasing food quantity and quality, growth rate and energy reserves of oyster were associated with a lower risk of mortality offshore whereas increasing turbidity, a proxy of the concentration of suspended particulate matter, and terrestrial inputs, inferred from fatty acid composition of oysters, were associated with a higher risk of mortality. Offshore farming and maintenance of good ecological status of coastal waters are options to limit disease risk in oysters.