Christophe Lambert

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.

Effect of a mono-specific algal diet on immune functions in two bivalve species - Crassostrea gigas and Ruditapes philippinarum

SUMMARY The impact of diets upon the fatty acid composition of haemocyte polar lipids and consequently upon immune parameters has been tested in the oyster Crassostrea gigas and the clam Ruditapes philippinarum. Oysters and clams were fed each of three cultured algae: Chaetoceros calcitrans, which is rich in 20:5(n-3) and 20:4(n-6) and poor in 22:6(n-3) fatty acids; T-Iso (Isochrysis sp.), which is rich in 22:6(n-3) and deficient in 20:5(n-3) and 20:4(n-6); and Tetraselmis suecica, which is deficient in 22:6(n-3) and contains only small amounts of 20:5(n-3) and 20:4(n-6). Fatty acid composition of haemocyte polar lipids was greatly affected by the diet. Oysters and clams fed C. calcitrans maintained a higher proportion of 20:5(n-3) and 20:4(n-6) in their haemocyte polar lipids, while these polyunsaturated fatty acids decreased drastically for animals fed T-Iso. However, the T-Iso diet maintained 22:6(n-3) in haemocyte polar lipids of both species. Higher 20:5(n-3) and 20:4(n-6) contents in diets appeared to have a positive effect upon total haemocyte count, granulocyte percentage, phagocytic rate and oxidative activity of clam haemocytes. Similarly, a positive effect of 20:5(n-3) on oxidative activity of oyster haemocytes was observed but to a lesser extent than in clams. Interestingly, when oyster haemocytes are submitted to a stressful condition, a positive effect of a higher dietary 22:6(n-3) content on the phagocytic rate was noticed.

Roseobacter gallaeciensis sp. nov., a new marine bacterium isolated from rearings and collectors of the scallop Pecten maximus

Four bacterial strains were isolated from larval cultures and collectors of the scallop Pecten maximus. They showed a high level of intragroup genomic relatedness (84-95%) as determined by DNA-DNA hybridization. The cells were Gram-negative, strictly aerobic, motile, ovoid rods. They grew at temperatures from 15 to 37 degrees C and from pH 7.0 to 10, but did not grow in the absence of NaCl and required growth factors. They had the ability to use a wide variety of compounds as sole carbon source: D-mannose, D-galactose, D-fructose, D-glucose, D-xylose, melibiose, trehalose, maltose, cellobiose, sucrose, mesoerythritol, D-mannitol, glycerol, D-sorbitol, meso-inositol, succinate, propionate, butyrate, gamma-aminobutyrate, DL-hydroxybutyrate, 2-ketoglutarate, pyruvate, fumarate, glycine, L-alpha-alanine, beta-alanine, L-glutamate, L-arginine, L-lysine, L-ornithine and L-proline. They exhibited oxidase and catalase activities but no denitrification activity. The isolates did not contain bacteriochlorophyll a. The G + C content ranged from 57.6 to 58 mol%. Phylogenetic analyses of the 16S rRNA sequence revealed that these isolates belong to the genus Roseobacter. On the basis of quantitative hybridization data, it is proposed that these isolates should be placed in a new species, Roseobacter gallaeciensis. The type strain is Roseobacter gallaeciensis BS107T (= CIP 105210T).

Comparative analysis of Vibrio splendidus-related strains isolated during Crassostrea gigas mortality events.

French mollusc production is based mainly on the Pacific cupped oyster, Crassostrea gigas. Since 1991, annual mass mortality of juveniles has been reported during summer months. These recurring episodes concern professionals who fear that like Portugese oyster, C. angulata, C. gigas could in turn disappear following one of these epizooties. Previously, bacteriological analysis of moribund oyster juveniles yielded an isolate of a Vibrio splendidus biovar II strain, named TNEMF6. This isolate was demonstrated to be pathogenic to Crassostrea gigas spat by experimental challenge. To study the association between summer oyster mortality and presence of TNEMF6 cluster strains, Vibrionaceae fauna were isolated from infected spat along the French Atlantic coast between 1997-1998. Strains related to V. splendidus biovar II were selected. Comparison with TNEMF6 was performed by classical biochemical tests and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) of SSU rDNA, rpoD, and gyrB genes. Genomic similarities were confirmed by DNA/DNA hybridization. Only one strain out of 14, TNNIII7, was found to be closely related to the pathogenic bacteria. Neither the phenotypic nor the genotypic markers used in this study were able to distinguish pathogenic from non-pathogenic strains of the widespread V. splendidus. However, future genetic comparisons of TNEMF6 and TNNIII7 is likely to reveal genes involved in pathogenicity.

Vibrio pectenicida sp. nov., a pathogen of scallop (Pecten maximus) larvae.

Five strains were isolated from moribund scallop (Pecten maximus) larvae over 5 years (1990-1995) during outbreaks of disease in a hatchery (Argenton, Brittany, France). Their pathogenic activity on scallop larvae was previously demonstrated by experimental exposure. The phenotypic and genotypic features of the strains were identical. The G + C content of the strains was in the range 39-41 mol%. DNA-DNA hybridization showed a minimum of 73% intragroup relatedness. Phylogenetic analysis of small-subunit rRNA sequences confirmed that these strains should be affiliated within the family Vibrionaceae and that they are closely related to Vibrio tapetis and Vibrio splendidus. Phenotypic and genotypic analyses revealed that the isolates were distinct from these two vibrios and so constitute a new species in the genus Vibrio. They utilized only a limited number of organic substrates as sole carbon sources, including betaine and rhamnose, but did not utilize glucose and fructose. In addition, their responses were negative for indole, acetoin, decarboxylase and dihydrolase production. The name Vibrio pectenicida is proposed for the new species; strain A365 is the type strain (= CIP 105190T).

Anthropogenic vs. lithogenic origins of trace elements (As, Cd, Pb, Rb, Sb, Sc, Sn, Zn) in water column particles: northwestern Mediterranean Sea

Abstract The distribution of heavy metal was analyzed in water column particles collected in autumn (October 1985) and spring (March 1986) by two series of sediment traps from a mooring located in the northeastern Mediterranean Sea continental slope. Four traps were set, at 50, 100, 300, 600 m depths on the mooring in 645 m deep water in the Lacaze-Duthier canyon. The total metal concentrations were determined by ICP-MS. Results show that Rb and Sc contents display typical shale values. As, Cd, Pb, Sb, Zn (normalized to Sc) display high enrichment factors (up to 50) over shale compositions. Distinctive temporal variability as well as the respective contributions of local (Tet, Aude) and remote (Rhone) rivers and Sahara-derived aerosols have been identified. Fluxes of most elements observed in the upper 100 m can be attributed to atmospheric fluxes. In the deepest traps (300 and 600 m) these fluxes are, however, mostly dominated by riverine particles advected from the continental shelf. Most of the trace-element enrichments are more likely to be related to the anthropogenic input rather than to biological cycling. Isotopic composition of lead determined by ICP-MS enabled to evaluate that the proportion of anthropogenic lead derived from European gasoline consumption ranged between 50 and 100%.

Impact of season and rearing site on the physiological and immunological parameters of the Manila clam Venerupis (=Tapes, =Ruditapes) philippinarum

Juvenile clams were distributed in four rearing sites selected for their varied ecological characteristics to assess the effects of environmental conditions on the physiological and immunological parameters, and Brown Ring Disease (BRD) status. Clams were sampled every 3 months for 15 months at each site. Brittany rearing sites, especially the Bay of Brest, showed the worst performances in terms of immunological and physiological indices and disease status, while the best were obtained in Marennes ponds. When the health of the clams was compared to assess seasonal effects, the winter clearly was a stressful period. A combination of bad rearing site and winter conditions led to major mortalities in the Bay of Brest in February. In other sites, winter mortalities were low. Condition index, total haemocyte count and haemocyte size were greatly affected by seasonal variation whereas haemocyte complexity and lysozyme content were more affected by the location of the site. Growth and haemolymph protein content were affected by both season and sites. Linear regressions between and within the physiological and immunological parameters indicated that large haemocyte size was related to low total haemocyte count (THC) and low haemocyte mortality. This relationship suggests a reduction in the cell division rate. Total haemocyte count and protein and lysozyme concentrations were positively correlated to the condition index. Seeded clams showed very low BRD prevalence in all sites and for all seasons; however, high prevalence was observed in natural stocks from one of the studied sites (Gulf of Morbihan), suggesting that hatchery-seeded clams may be more resistant to BRD and may be worthy of further studies.

Immunological responses of the Manila clam (Ruditapes philippinarum) with varying parasite (Perkinsus olseni) burden, during a long-term exposure to the harmful alga, Karenia selliformis, and possible interactions.

The present study evaluated the possible effects of a toxic dinoflagellate, Karenia selliformis, upon immunological hemocyte functions of the Manila clam Ruditapes philippinarum, and on the progression of infection by Perkinsus olseni. Clams with variable levels of perkinsosis were exposed for 6 weeks to simulated blooms of cultured the K. selliformis (10(2) and 10(3)cell ml(-1)). Samples were collected after 0, 2, 3, and 6 weeks of exposure. The following hemocyte parameters were measured by flow cytometry: percentage of dead cells, cell size and complexity, apoptosis, phagocytosis, and production of reactive oxygen species. Agglutination activities of K. selliformis on horse erythrocytes, serum protein concentration, and condition index of clams were also assessed. The harmful alga K. selliformis caused a significant decrease in hemocyte size and percentage of apoptotic cells. In contrast, P. olseni did not affect clams strongly; the only significant effect was an increase in hemocyte size in heavily infected clams. After 2 and 3 weeks, the prevalence and burden of P. olseni decreased in clams exposed to K. selliformis, but after 6 weeks, and a diminution in K. selliformis cell density in the exposure, this effect disappeared. In vitro tests exposing P. olseni to K. selliformis showed direct algal toxicity to the parasite (increased percentage of dead cells and altered morphology). Initial exposure of P. olseni-infected clams to K. selliformis appeared to modify the host-parasite interaction by causing effects in both organisms.

Effects of Alexandrium minutum exposure upon physiological and hematological variables of diploid and triploid oysters, Crassostrea gigas.

The effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon physiological parameters of the Pacific oyster, Crassostrea gigas, were assessed. Diploid and triploid oysters were exposed to cultured A. minutum and compared to control diploid and triploid oysters fed T. Isochrysis. Experiments were repeated twice, in April and mid-May 2007, to investigate effects of maturation stage on oyster responses to A. minutum exposure. Oyster maturation stage, Paralytic Shellfish Toxin (PST) accumulation, as well as several digestive gland and hematological variables, were assessed at the ends of the exposures. In both experiments, triploid oysters accumulated more PSTs (approximately twice) than diploid oysters. Significant differences, in terms of phenoloxidase activity (PO) and reactive oxygen species (ROS) production of hemocytes, were observed between A. minutum-exposed and non-exposed oysters. PO in hemocytes was lower in oysters exposed to A. minutum than in control oysters in an early maturation stage (diploids and triploids in April experiment and triploids in May experiment), but this contrast was reversed in ripe oysters (diploids in May experiment). In the April experiment, granulocytes of oysters exposed to A. minutum produced more ROS than those of control oysters; however, in the May experiment, ROS production of granulocytes was lower in A. minutum-exposed oysters. Moreover, significant decreases in free fatty acid, monoacylglycerol, and diacylglycerol contents in digestive glands of oysters exposed to A. minutum were observed. Concurrently, the ratio of reserve lipids (triacylglycerol, ether glycerides and sterol esters) to structural lipids (sterols) decreased upon A. minutum exposure in both experiments. Also, several physiological responses to A. minutum exposure appeared to be modulated by maturation stage as well as ploidy of the oysters.

Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses.

Extracellular products (ECPs) of the pathogenic Vibrio aestuarianus 01/32 were previously reported to display lethality in Crassostrea gigas oysters and to cause morphological changes and immunosuppression in oyster hemocytes. To identify the source of this toxicity, biochemical and genetic approaches were developed. ECP protease activity and lethality were shown to be significantly reduced following incubation with metal chelators, suggesting the involvement of a zinc metalloprotease. An open reading frame of 1836 bp encoding a 611-aa metalloprotease (designated Vam) was identified. The deduced protein sequence showed high homology to other Vibrio metalloproteases reported to be involved in pathogenicity. To further confirm the role of this enzyme in ECP toxicity, a plasmid carrying the vam gene under the control of an araC-P(BAD) expression cassette was transferred to a Vibrio splendidus related strain, LMG20012(T), previously characterized as non-pathogenic to oysters. Expression of Vam conferred a toxic phenotype to LMG20012(T) ECPs in vivo and cytotoxicity to oyster hemocytes in vitro. Collectively, these data suggest that the Vam metalloprotease is a major contributor to the toxicity induced by V. aestuarianus ECPs and is involved in the impairment of oyster hemocyte functions.