Réjean Tremblay

Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids

SUMMARY We compared lipid dynamics and the physiological responses of blue mussels Mytilus edulis, a cold-adapted species, and oysters Crassostrea virginica, a warmer-water species, during simulated overwintering and passage to spring conditions. To simulate overwintering, animals were held at 0°C, 4°C and 9°C for 3 months and then gradually brought to and maintained at 20°C for 5 weeks to simulate spring–summer conditions. Changes in lipid class and fatty acid composition were related to clearance rate and oxygen consumption. We found major differences between species in triglyceride (TAG) metabolism during overwintering. Mussels used digestive gland TAG stores for energy metabolism or reproductive processes during the winter, whereas oysters did not accumulate large TAG stores prior to overwintering. Mussel TAG contained high levels of 20:5n-3 compared to levels in oysters and in the diet. This may help to counteract the effect of low temperature by reducing the melting point of TAG and thus increasing the availability of storage fats at low temperature. Mussels seemed better able to mobilise 20:5n-3 and 18:4n-3 than other fatty acids. We also found that both bivalves underwent a major remodelling of membrane phospholipids. The unsaturation index decreased in the gills and digestive glands of both species during the early stages of warming, principally due to decreases in 22:6n-3 and 20:5n-3. In digestive glands, the unsaturation index did not increase with decreasing temperature beyond a threshold attained at 9°C whereas a perfect negative relationship was observed in gills, as predicted by homeoviscous adaptation. The presence of digestive enzymes and acids in the digestive gland microenvironment may lead to specific requirements for membrane stability. That oysters had lower metabolic rates than mussels coincides with a lower unsaturation index of their lipids, as predicted by Hulberts theory of membranes as metabolic pacemakers. Both species showed increased 20:4n-6 levels in their tissues as temperature rose, suggesting an increasing availability of this fatty acid for eicosanoid biosynthesis during stress responses. The contrast between the species in TAG dynamics and the similarity of their phospholipid remodelling emphasises the essential functional role of membrane phospholipid structure and the contrasting use of TAG by oysters and mussels during overwintering.

Variation of lipid class and fatty acid composition of Chaetoceros muelleri and Isochrysis sp. grown in a semicontinuous system

The lipid class and the fatty acid composition of microalgae are of primary importance in feeding filter-feeding animals properly and variations of those parameters have never been investigated in a cultivation system applied to hatcheries. The objective of this study was to document the lipid class and fatty acid composition of the diatom Chaetoceros muelleri (CHGRA) and the flagellate Isochrysis sp. (Clone TISO) during the entire process of hatchery cultivation in relation to the period of the year (summer vs. winter). At the beginning of the cultivation process of CHGRA, in a small volume of culture, triacylglycerol (TAG), sterol, saturated, monounsaturated, and polyunsaturated fatty acids (particularly 20:5n3) were high, sharply dropping when volume was increased. An inverse relationship has been observed for acetone mobile polar lipids (AMPLs; pigment) and phospholipids (PLs), thus leading to no significant effect of the volume of culture on the total lipid (TL) content of CHGRA. The total lipid content of TISO increased with the volume of culture due to AMPL and PL classes and saturated and polyunsaturated fatty acids. The short- and long-term variability of lipid class composition has been observed for both species. Such results highlight the high variability of marine microalgae lipid content during the culture process and season as well. Hatchery implication is discussed. D 2003 Elsevier Science B.V. All rights reserved.

Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae

Background Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus. Findings Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene–interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides). Conclusion Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.

Physiological and biochemical traits correlate with differences in growth rate and temperature adaptation among groups of the eastern oyster Crassostrea virginica.

SUMMARY We tested two hypotheses in this study: first, that intraspecific growth variations in a marine bivalve are correlated with physiological (basal metabolic rate and scope for growth) and biochemical (membrane lipids) characteristics, and, second, that this bivalve shows intraspecific variations in physiological and biochemical adaptations to temperature. To test these hypotheses, five genetically distinct groups of juvenile oysters Crassostrea virginica that showed differences in their growth rates were maintained in the laboratory (1) for further measurements of growth and standard metabolic rates and (2) subjected to acclimation at 4°C, 12°C and 20°C and further examined for scope for growth and determination of membrane lipid composition. Our results show that a lower basal metabolic rate and lower unsaturation index of membrane lipids coincides with higher growth rates and a higher scope for growth in oysters. We provide evidence that intraspecific differences in basal metabolic rate in oysters are related to membrane unsaturation as predicted by Hulberts theory of membranes as metabolic pacemakers. Furthermore, our results suggest that the theory of membranes as metabolic pacemakers is related to intraspecific differences in growth. A perfect negative relationship was observed between the acclimation temperature and the unsaturation index of membrane lipids in oysters, as predicted by the homeoviscous adaptation theory. However, changes in the unsaturation index in response to temperature were mainly due to variations in the eicosapentaenoic (20:5n-3) fatty acid in fast-growing oysters, whereas slow-growing animals changed both docosahexaenoic acid (22:6n-3) and 20:5n-3. Thus, the pattern of biochemical compensation in response to temperature in this species shows intraspecific variation.

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry.

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.

Lipid remodeling in wild and selectively bred hard clams at low temperatures in relation to genetic and physiological parameters

SUMMARY A temperature decrease usually induces an ordering effect in membrane phospholipids, which can lead to membrane dysfunction. Poikilotherms inhabiting eurythermal environments typically counteract this temperature effect by remodeling membrane lipids as stipulated in the homeoviscous adaptation theory (HVA). Hard clams, Mercenaria mercenaria, can suffer high overwintering mortalities in the Gulf of St Lawrence, Canada. The selectively bred M. mercenaria var. notata can have higher overwintering mortalities than the wild species, thus suggesting that the two varieties have different degrees of adaptation to low temperatures. The objective of this study was to investigate the changes in lipid composition of soft tissues in wild and selected hard clams in relation to their metabolic and genetic characteristics. Clams were placed at the northern limit of their distribution from August 2003 to May 2004; they were exposed to a gradual temperature decrease and then maintained at <0°C for 3.5 months. This study is the first to report a major remodeling of lipids in this species as predicted by HVA; this remodeling involved a sequential response of the phospholipid to sterol ratio as well as in levels of 22:6n-3 and non-methylene interrupted dienoic fatty acids. Hard clams showed an increase in 20:5n-3 as temperature decreased, but this was not maintained during overwintering, which suggests that 20:5n-3 may have been used for eicosanoid biosynthesis as a stress response to environmental conditions. Selectively bred hard clams were characterized by a higher metabolic demand and a deviation from Hardy-Weinberg equilibrium at several genetic loci due to a deficit in heterozygote frequency compared with wild clams, which is believed to impose additional stress and render these animals more vulnerable to overwintering mortality. Finally, an intriguing finding is that the lower metabolic requirements of wild animals coincide with a lower unsaturation index of their lipids, as predicted by Hulberts theory of membranes as pacemakers of metabolism.

Effect of the Tidal Cycle on Lysosomal Membrane Stability in the Digestive Gland of Mya arenaria and Mytilus edulis L.

Abstract For the correct use of a stress index in the assessment of disturbances due to pollution, it is indispensable to know whether the response investigated exhibits natural fluctuations which could subsequently bring biases to the interpretations. Stability of the lysosomal membrane was measured at short time intervals during two full tidal cycles in the digestive gland of Mytilus edulis L. and Mya arenaria . In both species, destabilisation of the lysosomal membrane was observed during emersion periods. There was no indication of membrane destabilisation in the soft-shelled clams when they were submerged. In mussels, the destabilisation of the lysosomal membrane was observed occasionally during the immersion period. Air exposure and the associated hypoxia appeared to be the factors explaining the lysosomal responses in these two species. The physiological importance of these findings seems closely linked to the role of lysosomes in the well known process of proteolysis via controlled autophagy and to phasic digestive rhythms, two strategies for a maximal utilisation of limited food resources during the tidal cycle. These results demonstrate for the first time to our knowledge, under natural conditions, the differences of lysosomal responses between two bivalve species inhabiting the intertidal zone. It appeared finally, that the infaunal bivalve M. arenaria is most suitable as a sentinel species when the lysosomal membrane destabilisation is chosen as a biomarker of stress in field conditions.

Identification of lipid and saccharide constituents of whole microalgal cells by 13 C solid-state NMR☆

Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater (Chlamydomonas reinhardtii) and marine (Pavlova lutheri and Nannochloropsis oculata) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by ¹³C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as ¹³C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR.

Effect of biofilm age on settlement of Mytilus edulis

Biofilm ageing is commonly assumed to improve mussel settlement on artificial substrata, but the structure and taxonomic composition of biofilms remains unclear. In the present study, multi-species biofilms were characterized at different ages (1, 2, and 3 weeks) and their influence on settlement of the blue mussel, Mytilus edulis, was tested in the field. As biofilms can constitute a consistent food resource for larvae, the lipid quality, defined as the proportion of related essential fatty acids, may be a selection criterion for settlement. Overall mussel settlement increased on biofilms older than 1 week, and the enhanced settlement corresponded to the abundance and composition of the biofilm community, rather than to essential fatty acid levels. However, during a pulse of phytoplankton, the positive influence of biofilm was not detected, suggesting that pelagic cues overwhelmed those associated with biofilms. The influence of biofilms on mussel settlement could be more crucial when planktonic resources are limited.

Solid-State NMR Structure Determination of Whole Anchoring Threads from the Blue Mussel Mytilus edulis

The molecular structure of the blue mussel Mytilus edulis whole anchoring threads was studied by two-dimensional (13)C solid-state NMR on fully labeled fibers. This unique material proves to be well ordered at a molecular level despite its heterogeneous composition as evidenced by the narrow measured linewidths below 1.5 ppm. The spectra are dominated by residues in collagen environments, as determined from chemical shift analysis, and a complete two-dimensional assignment (including minor amino acids) was possible. The best agreement between predicted and experimental backbone chemical shifts was obtained for collagen helices with torsion angles (-75°, +150°). The abundant glycine and alanine residues can be resolved in up to five different structural environments. Alanine peaks could be assigned to collagen triple-helices, β-sheets (parallel and antiparallel), β-turns, and unordered structures. The use of ATR-FTIR microscopy confirmed the presence of these structural environments and enabled their location in the core of the thread (collagen helices and antiparallel β-sheets) or its cuticle (unordered structures). The approach should enable characterization at the molecular level of a wide range of byssus macroscopic properties.