OsHV-1 countermeasures to the Pacific oyster’s anti-viral response

The host-pathogen interactions between the Pacific oyster (Crassostrea gigas) and Ostreid herpesvirus type 1 (OsHV-1) are poorly characterised. Herpesviruses are a group of large, DNA viruses that are known to encode gene products that subvert their host’s antiviral response. It is likely that OsHV-1 has also evolved similar strategies as its genome encodes genes with high homology to C. gigas inhibitors of apoptosis (IAPs) and an interferon-stimulated gene (termed CH25H). The first objective of this study was to simultaneously investigate the expression of C. gigas and OsHV-1 genes that share high sequence homology during an acute infection. Comparison of apoptosis-related genes revealed that components of the extrinsic apoptosis pathway (TNF) were induced in response to OsHV-1 infection, but we failed to observe evidence of apoptosis using a combination of biochemical and molecular assays. IAPs encoded by OsHV-1 were highly expressed during the acute stage of infection and may explain why we didn’t observe evidence of apoptosis. However, C. gigas must have an alternative mechanism to apoptosis for clearing OsHV-1 from infected gill cells as we observed a reduction in viral DNA between 27 and 54 h post-infection. The reduction of viral DNA in C. gigas gill cells occurred after the up-regulation of interferon-stimulated genes (viperin, PKR, ADAR). In a second objective, we manipulated the host’s anti-viral response by injecting C. gigas with a small dose of poly I:C at the time of OsHV-1 infection. This small dose of poly I:C was unable to induce transcription of known antiviral effectors (ISGs), but these oysters were still capable of inhibiting OsHV-1 replication. This result suggests dsRNA induces an anti-viral response that is additional to the IFN-like pathway.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

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

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.

Mass mortality in bivalves and the intricate case of the Pacific oyster, Crassostrea gigas

Massive mortality outbreaks in cultured bivalves have been reported worldwide and they have been associated with infection by a range of viral and bacterial pathogens. Due to their economic and social impact, these episodes constitute a particularly sensitive issue in Pacific oyster (Crassostrea gigas) production. Since 2008, mortality outbreaks affecting C. gigas have increased in terms of intensity and geographic distribution. Epidemiologic surveys have lead to the incrimination of pathogens, specifically OsHV-1 and bacteria of the Vibrio genus, in particular Vibrio aestuarianus. Pathogen diversity may partially account for the variability in the outcome of infections. Host factors (age, reproductive status…) including their genetic background that has an impact on host susceptibility towards infection, also play a role herein. Finally, environmental factors have significant effects on the pathogens themselves, on the host and on the host-pathogen interaction. Further knowledge on pathogen diversity, classification, and spread, may contribute towards a better understanding of this issue and potential ways to mitigate the impact of these outbreaks.

Global change and climate-driven invasion of the Pacific oyster (Crassostrea gigas) along European coasts: a bioenergetics modelling approach

Aim The spread of non-indigenous species in marine ecosystems world-wide is one of today's most serious environmental concerns. Using mechanistic modelling, we investigated how global change relates to the invasion of European coasts by a non-native marine invertebrate, the Pacific oyster Crassostrea gigas. Location Bourgneuf Bay on the French Atlantic coast was considered as the northern boundary of C. gigas expansion at the time of its introduction to Europe in the 1970s. From this latitudinal reference, variations in the spatial distribution of the C. gigas reproductive niche were analysed along the north-western European coast from Gibraltar to Norway. Methods The effects of environmental variations on C. gigas physiology and phenology were studied using a bioenergetics model based on Dynamic Energy Budget theory. The model was forced with environmental time series including in situ phytoplankton data, and satellite data of sea surface temperature and suspended particulate matter concentration. Results Simulation outputs were successfully validated against in situ oyster growth data. In Bourgneuf Bay, the rise in seawater temperature and phytoplankton concentration has increased C. gigas reproductive effort and led to precocious spawning periods since the 1960s. At the European scale, seawater temperature increase caused a drastic northward shift (1400 km within 30 years) in the C. gigas reproductive niche and optimal thermal conditions for early life stage development. Main conclusions We demonstrated that the poleward expansion of the invasive species C. gigas is related to global warming and increase in phytoplankton abundance. The combination of mechanistic bioenergetics modelling with in situ and satellite environmental data is a valuable framework for ecosystem studies. It offers a generic approach to analyse historical geographical shifts and to predict the biogeographical changes expected to occur in a climate-changing world.

Involvement of Mitochondrial Activity and OXPHOS in ATP Synthesis During the Motility Phase of Spermatozoa in the Pacific Oyster, Crassostrea gigas

In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied 1) changes in spermatozoal mitochondrial activity, that is, mitochondrial membrane potential (MMP), and intracellular ATP content in relation to motion parameters and 2) the involvement of OXPHOS for spermatozoal movement using carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoal ATP content decreased steadily during the first 9 h postactivation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that the ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or from potential phosphagens during the first 2 h of movement as deduced by the decoupling by CCCP of OXPHOS. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyze intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.

Intraplate versus ridge volcanism on the Pacific-Antarctic Ridge near 37°S -111°W

Exploration of the Foundation Volcanic Chain (33 degrees S-131 degrees W; 37 degrees S-111 degrees W) revealed the existence of different magmatic provinces with relation to their geological settings. (1) The Pacific-Antarctic Ridge (PAR) is made up of several en echelon segments where both glassy midocean ridge basalts (MORBs) with low incompatible elements (K2O<200 ppm, Zr<120 ppm and Ce <20 ppm) as well as andesites and dacites have erupted, (2) Oblique Ridges located up to 300 lan from the PAR axis are topped with seamounts made up essentially of transitional (T) and enriched (E) MORBs with intermediate incompatible elements (K2O=0.11-0.40 %, Zr=70-140 ppm and Ce=15-30 ppm), (3) the Foundation Seamounts (FS) consisting essentially of isolated volcanoes which have erupted alkalic lavas (alkali basalt, trachybasalt and trachyandesite) with high incompatible elements (K2O (0.50-1.1 %, Zr (>150 ppm) and Ce (>48 ppm)) at about 306-1300 km from the PAR axis, (4) The Old Pacific Seamounts built on a crust older than 23 m. y. located west of longitude 124 degrees W (> 1300 km from the PAR axis) consist of T and EMORB. On the PAR axis, extensive crystal fractionation (>65%) produced the silicic lavas. On the basis of Pacific plate reconstruction using a half spreading rate of about 50 mm/yr and integrating the observed compositional changes with respect to the structural settings, it is inferred that the last volcanic events giving rise to the FS took place at about 110 km from the PAR axis about 5 m. y. ago. The Oblique Ridges built between 5 m. y. and <1 m. y. are believed to represent ancient leaky transforms and/or large discontinuities between accreting ridge segments filled by volcanic cones during the interaction (mixing) of the enriched plume components of the FS with PAR depleted (MORB type) magmatism. The Old Pacific Seamounts built on ancient crust (>23 m. y.) with MORB volcanics comparable to those of the the Oblique Ridge-PAR provinces, could also have been formed by an interaction between the Foundation Seamount (dredge site 28) hotspot magmatism and that of an ancient accreting ridge magmatism precursor of the PAR.