Myrina Boulais

Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas

We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.

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

ABSTRACT 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.

Survival, growth and reproduction of cryopreserved larvae from a marine invertebrate, the Pacific oyster (Crassostrea gigas).

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf), early D-larvae (24±2 hpf) and late D-larvae (43±2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001) than those of the control (non cryopreserved larvae). Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool). In all but two crosses out of 13 tested (P<0.001), development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.

Flow cytometric assessment of morphology, viability, and production of reactive oxygen species of Crassostrea gigas oocytes. Application to Toxic dinoflagellate (Alexandrium minutum) exposure

The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques.

Application of Flow Cytometry to Assess Deepwater Horizon Oil Toxicity on the Eastern Oyster Crassostrea virginica Spermatozoa

ABSTRACT The Deepwater Horizon (DWH) oil spill in 2010 resulted in the release of millions of liters of oil into the Gulf of Mexico. Dispersants such as Corexit 9500A were used to disperse oil both at the well-head and at the surface. Polycyclic aromatic hydrocarbons and dispersants have been shown to deleteriously affect early life stages of organisms including oysters. This study examined the impacts of chemically enhanced water-accommodated fractions [CEWAF; 1.29–26.14 µg/l tPAH50 (a sum of 50 different polycyclic aromatic hydrocarbons)], high-energy water-accommodated fractions (HEWAF; 16.53–248.89 µg/l tPAH50), and dispersants (0.625–10 mg/l) on the cellular functions (viability, mitochondrial membrane potential (MMP), reactive oxygen species production (ROS), and acrosomal integrity) and resulting fertilization success of eastern oyster Crassostrea virginica spermatozoa. While viability of spermatozoa was not affected by CEWAF and HEWAF at concentrations tested, dispersant exposure caused significant decrease in viability at the highest concentration tested. Fertilization success as well as MMP and ROS production were significantly decreased upon exposure to CEWAF, HEWAF, and dispersants. Also, although not affected by HEWAF exposure, acrosomal integrity decreased upon exposure to CEWAF and dispersants at concentrations tested. The results of this study suggest that impaired fertilization and reduced viability observed after exposure to DWH oil spill contaminants may result, at least partially, from alterations of cellular functions of spermatozoa and contribute to negative effects on oyster populations, and thus the ecology and economy of the Gulf of Mexico.

pH controls spermatozoa motility in the Pacific oyster (Crassostrea gigas)

ABSTRACT Investigating the roles of chemical factors stimulating and inhibiting sperm motility is required to understand the mechanisms of spermatozoa movement. In this study, we described the composition of the seminal fluid (osmotic pressure, pH, and ions) and investigated the roles of these factors and salinity in initiating spermatozoa movement in the Pacific oyster, Crassostrea gigas. The acidic pH of the gonad (5.82±0.22) maintained sperm in the quiescent stage and initiation of flagellar movement was triggered by a sudden increase of spermatozoa external pH (pHe) when released in seawater (SW). At pH 6.4, percentage of motile spermatozoa was three times higher when they were activated in SW containing 30 mM NH4Cl, which alkalinizes internal pH (pHi) of spermatozoa, compared to NH4Cl-free SW, revealing the role of pHi in triggering sperm movement. Percentage of motile spermatozoa activated in Na+-free artificial seawater (ASW) was highly reduced compared to ASW, suggesting that change of pHi triggering sperm motility was mediated by a Na+/H+ exchanger. Motility and swimming speed were highest in salinities between 33.8 and 42.7‰ (within a range of 0 to 50 ‰), and pH values above 7.5 (within a range of 4.5 to 9.5). Summary: Oyster sperm motility is not triggered in an acidic environment and motility is controlled by internal pH alkalinization, mediated by a Na+/H+ exchanger.

The sensitivity of a deep‐sea fish species (Anoplopoma fimbria) to oil‐associated aromatic compounds, dispersant, and Alaskan North Slope crude oil

A predominant concern following oil spills is toxicity to aquatic organisms. However, few data are available on effects in deep-sea cold water fishes. The present study had 3 major objectives. The first was to investigate the relative sensitivity of the deep-sea species Anoplopoma fimbria (sablefish) to acute effects of 3 aromatic compounds (toluene, 2-methylnaphthalene, and phenanthrene), dispersant alone, and chemically enhanced water accommodated fractions (CEWAFs) of Alaskan North Slope crude oil. The second was to determine the critical target lipid body burden (CTLBB) for sablefish by fitting aromatic hydrocarbon toxicity data to the target lipid model (TLM), which then allowed expression of CEWAF exposures in terms of dissolved oil toxic units. The final aim was to apply a passive sampling method that targets bioavailable, dissolved hydrocarbons as an alternative analytical technique for improved CEWAF exposure assessment. The results indicate that sablefish exhibit sensitivity to Corexit 9500 (96-h median lethal concentration [LC50] = 72.2 mg/L) within the range reported for other fish species. However, the acute CTLBB of 39.4 ± 2.1 μmol/goctanol lies at the lower end of the sensitivity range established for aquatic species. The utility of both toxic units and passive sampling measurements for describing observed toxicity of dispersed oil is discussed. The present study is novel in that a new test species is investigated to address the uncertainty regarding the sensitivity of deep-sea fishes, while also employing modeling and measurements to improve exposure characterization in oil toxicity tests. Environ Toxicol Chem 2018;37:2210-2221.