Philippe Soudant

Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst

Abstract The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters ( Crassostrea virginica ) were subjected to a sudden increase in temperature from 20 to 28 °C for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to: (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution; (2) assess haemocyte viability using propidium iodide (PI); (3) quantify haemocyte phagocytosis with fluorescent microbeads; and (4) measure the respiratory burst response of individual haemocytes using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS). The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan; granulocytes showed the highest induced fluorescence.

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.

Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process

Abstract Spawning individuals of the scallop Pecten maximus were conditioned on three test diets: Tahiti Isochrysis , a mixture (PTSC) and Chaetoceros calcitrans . The scallops fed T- Isochrysis showed a better hatching rate and lower atresia than those fed the other two diets. Proximate composition of the female gonads was not modified by the differences in the diets. Enrichment of gonads with sterol esters and triglycerides, characteristic of storage of lipids, observed with the broodstock fed the diatoms, did not result in successful gametogenesis and spawning. The monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) composition of neutral and polar lipids of gonads was related to the fatty acid composition of the diet. However, the 20 and 22 carbon PUFA were maintained at levels relatively independent of those of the diet; when these fatty acids were low in the diet, their concentration in the lipids of the gonads decreased but still remained significant. This effect was more pronounced in the polar than the neutral lipids. The preferential incorporation of 22:6 n − 3, 20:4 n − 6 and 20:5 n − 3 in the polar lipids indicates their role in gametogenesis and embryogenesis. The sterol composition of the gonads (free sterols and sterol esters) reflected that of the diet; however, the cholesterol, mainly found in the esterified fraction, was maintained at stable levels independent of dietary supply. Two sterols (22-dehydrocholesterol and 24-methylenecholesterol) that were either absent from, or present only in trace amounts in the diets, were found to occur at constant levels in the female gonads, regardless of the nature of the diet.

Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: I. Haemocyte types and morphology

In this report, we provide detailed protocols for flow-cytometric characterization of haemocytes from the eastern oyster, Crassostrea virginica, and report effects of sudden temperature elevation upon haemocyte characteristics. Haemocytes were differentiated from other particles in the haemolymph using a combination of the DNA-binding fluorochrome SYBR Green and internal cell complexity. In formalin-fixed haemolymph, four distinct subpopulations of haemocytes were found: small hyalinocytes, large hyalinocytes, small granulocytes, and granulocytes. Hyalinocytes were numerically dominant, small and large together accounting for 60% of the haemocytes; granulocytes accounted for 30–35%, and small granulocytes were least numerous at <5%. These percentages were the same for haemolymph analysed immediately after it was withdrawn from living oysters (without fixation) as for haemolymph fixed with formalin, although small and large hyalinocytes were not always clearly differentiated into two distinct populations. There was a general trend for the fixed cells to appear in the flow cytometer to be larger and more complex than the fresh ones (except for granulocyte complexity). Oysters subjected to a sudden increase in temperature from 20 to 28 °C for 1 week showed significant changes in haemocyte morphology. Sudden temperature elevation was associated with a general decrease in size of all haemocyte types. The ability to detect changes in individual haemocyte types for many oysters by these flow-cytometric methods should improve progress in understanding oyster mortality associated with combined effects of environmental stresses, disease, and parasites.

Comparison of the lipid class and fatty acid composition between a reproductive cycle in nature and a standard hatchery conditioning of the Pacific Oyster Crassostrea gigas

Abstract The lipid composition of Crassostrea gigas was analyzed during the reproductive phase in natural as well as under artificial conditions. The lipid content increased and accumulated in the gonads, but to a higher extend in the naturally conditioned animals. The percentage of neutral lipid in total lipid of the gonad plus mantle was stable, high (>70%) and equal under both conditions, underscoring that the lipid reserves were preferentially located in that organ. The composition of the polar lipid classes was stable with little variations. The fatty acid (FA) composition of the neutral lipids in all organs is influenced by diet. However, there is a different response according to organs. A high dietary impact occured in the digestive gland whereas the muscle was less affected. The polyunsaturated fatty acid (PUFA) level of the neutral and polar lipids in the gonads changed little despite the dietary conditions but the respective proportions of (n-3) and (n-6) PUFA differed drastically as a result of diet composition. There was clear evidence in all organs for a specific accumulation of 22:6(n-3) and 20:5(n-3) in the polar lipids under both conditioning diets. The proportions of 22:6(n-3) and 20:5(n-3) from neutral and polar lipids of oysters conditioned artificially were significantly lower than of those that were naturally conditioned.

Fatty acids of serine, ethanolamine, and choline plasmalogens in some marine bivalves

The FA composition of glycerophospholipid (GPL) classes and subclasses was investigated in whole animals of three marine bivalve mollusks: the Japanese oyster Crassostrea gigas, the blue mussel Mytilus edulis, and the Manila clam Ruditapes philippinarum. Individual organs (gills, mantle, foot, siphon, and muscle) of the Manila clam also were examined. The PS plasmalogen (PSplsm), PE plasmalogen (PEplsm), and PC plasmalogen (PCplsm) subclasses were isolated by HPLC, and their individual FA compositions were examined using GC. Plasmalogen forms of PS and PE, when compared to their respective diacyl forms, were found to be specifically enriched with non-methylene-interrupted (NMI) FA (7,15–22∶2, 7, 13–22∶2, and their precursors) and 20∶1n−11 FA. Such a clear specific association was not found for PCplsm. Interestingly, this trend was most apparent in PSplsm, and the above FA were found to be, respectively, the predominant PUFA and monounsaturated FA in the PSplsm isolated from the three species. This specificity was maintained in all the analyzed organs of the Manila clam but varied in proportions: The highest level of plasmalogens, NMI FA, and 20∶1n−11 was measured in gills and the lowest was in muscle. These results represent the first comprehensive report on a FA composition of the PSplsm subclass isolated from mollusks. The fact that NMI FA and 20∶1n−11, which are thought to be biosynthesized FA, were mainly associated with aminophospholipid plasmalogens (PE and PS) is likely to have a functional significance in bivalve membranes.

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.

Impact of the quality of dietary fatty acids on metabolism and the composition of polar lipid classes in female gonads of Pecten maximus (L.)

Abstract The impact of dietary deficiency of the essential fatty acids 20:5(n-3), 22:6(n-3) and 20:4(n-6) was studied at the level of different polar lipid classes that constitute the membrane structure of female gonads of Pecten maximus by using three microalgal diets. After 11 weeks of conditioning of the broodstock, the relative percentages of polar lipid classes were very little, or not at all, influenced by the composition of the microalgae. On the other hand, the imprint of dietary fatty acid composition was reflected in the relative composition of fatty acids specific to some classes but not all of them. The dietary fatty acid composition thus clearly influenced that of phosphatidylcholine (PC), the plasmalogens and to a smaller extent the phosphatidylserine (PS) and phosphatidylethanolamine (non plasmalogen PE), but not of phosphatidylinositol (PI) and the glycolipid (GLY). The PI showed a very stable composition and was rich in 20:4(n-6) (>30%) irrespective of the diet. Likewise, the 22:6(n-3) content was consistently higher than 76% in the glycolipid. The maintenance of the specific fatty acid composition of these classes is probably a physiological necessity for gametogenesis and embryogenesis.

Fatty acid composition of polar lipid classes during larval development of scallop Pecten maximus (L.)

The major phospholipid classes in the larvae of the scallop Pecten maximus were phosphatidylcholine (PC) and plasmalogens (PLSM) (35.5 and 32.1 mol%, respectively). The minor classes were glycosyldiacylglycerol-like (GLY), phosphatidylinositol (PI), phosphatidylserine (PS) and non-plasmalogen phosphatidylethanolamine (PE) at 6.7, 9.1, 8.5 and 8.1 mol%, respectively. Abundance of phospholipid classes and their content of saturated, monounsaturated and polyunsaturated fatty acids remained relatively conserved during the course of larval development. During larval development there was a decrease in the amount of 20-carbon polyunsaturated fatty acids (PUFA) in the polar lipids, while the amount of 22-carbon PUFA remained constant. These changes, which occurred mainly during the lecithotrophic phase, imparted a specificity in PUFA composition to the different classes of polar lipids. During this phase, 20:5(n 3) replaced 20:4(n 6) in PI but decreased sharply in PLSM and, to a lesser extent, in PC. After the initial phase of composition changes, the fatty acid composition of the polar lipid classes became stable, with specific associations of some of the PUFA with certain polar lipid classes: 22:6(n 3) with GLY, 20:5(n 3) with PE, 20:4(n6) with PI. The 22:6(n3):20:5(n3) ratio in PLSM during exotrophy was always twice as high as in the PC fraction.