Edouard Kraffe

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.

Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: roles of membrane proteins, phospholipids and their fatty acid compositions

SUMMARY Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5°C to 15°C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15°C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5°C to 15°C, the maximal oxidative capacity of mitochondria measured at 15°C increased slightly, but rates at both assay temperatures (5°C and 15°C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15°C were expressed relative to cytochrome a and c1 levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15°C decreased the ratio of diacylphosphatidylethanolamine/diacylphosphatidylcholine (diacylPE/diacylPC), whereas warm acclimation led to restructuring of fatty acids (FA) and to increases of plasmalogen forms of PE and PC. Modification of overall membrane unsaturation did not appear to be the primary aim of restructuring membrane FA during warm acclimation, as total mitochondrial phospholipids and the major phospholipid classes only showed slight shifts of their acyl composition with warm acclimation. On the other hand, natural lysophosphatidylcholine (LysoPC) showed dramatic changes in FA content, as 16:0 and 18:1n-9 doubled whereas 22:6n-3 decreased from around 50% to 32% in warm acclimated trout. Similarly, in cardiolipin (CL), the levels of 16:0 and 18:1n-7 halved while 18:2n-6 increased to over 20% of the FA with warm acclimation. Given the central role of CL in modulating the activity of CCO, F0F1-ATPase and ANT, these changes suggest that specific compositional changes in CL are important modulators of mitochondrial capacities. The many structural changes in membrane lipids contrast with the limited modifications of the membrane protein components examined and support the concept of lipid structure modulating mitochondrial capacities.

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψm) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψm in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψm and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

Rapid mitochondrial adjustments in response to short-term hypoxia and re-oxygenation in the Pacific oyster Crassostrea gigas

SUMMARY As oxygen concentrations in marine coastal habitats can fluctuate rapidly and drastically, sessile marine organisms such as the oyster Crassostrea gigas can experience marked and rapid oxygen variations. In this study, we investigated the responses of oyster gill mitochondria to short-term hypoxia (3 and 12 h, at 1.7 mg O2 l−1) and subsequent re-oxygenation. Mitochondrial respiratory rates (states 3 and 4 stimulated by glutamate) and phosphorylation efficiency [respiratory control ratio (RCR) and the relationship between ADP and oxygen consumption (ADP/O)] were measured. Cytochrome c oxidase (CCO) activity and cytochrome concentrations (a, b, c1 and c) were measured to investigate the rearrangements of respiratory chain subunits. The potential implication of an alternative oxidase (AOX) was investigated using an inhibitor of the respiratory chain (antimycin A) and through gene expression analysis in gills and digestive gland. Results indicate a downregulation of mitochondrial capacity, with 60% inhibition of respiratory rates after 12 h of hypoxia. RCR remained stable, while ADP/O increased after 12 h of hypoxia and 1 h of re-oxygenation, suggesting increased phosphorylation efficiency. CCO showed a fast and remarkable increase of its catalytic activity only after 3 h of hypoxia. AOX mRNA levels showed similar patterns in gills and digestive gland, and were upregulated after 12 and 24 h of hypoxia and during re-oxygenation. Results suggest a set of controls regulating mitochondrial functions in response to oxygen fluctuations, and demonstrate the fast and extreme plasticity of oyster mitochondria in response to oxygen variations.

Evidence of a tetradocosahexaenoic cardiolipin in some marine bivalves

Separation of phospholipid classes in lipid extracts from the scallop Pecten maximus, the Pacific oyster Crassostrea gigas, and the blue mussel Mytilus edulis was conducted using HPLC. An isolated polar lipid fraction was found to contain a very high level of DHA, up to 80 mol% of the total FA. MS with electrospray ionization in the positive-ion mode, tandem MS (MS-MS) and multidimensional NMR spectroscopy were used to analyze the detailed chemical structure of this polar lipid fraction. The isolated fraction contained exclusively cardiolipin (CL) molecules, predominantly in a form with four docosahexaenoyl chains (Do4CL). To the best of our knowledge, this is the first time that such a CL form has been analytically characterized and described in these three bivalve species. This tetradocosahexaenoic CL is presumed to reflect a specific adaptation in bivalves that enhances the structural and functional mechanisms of biomembranes in response to variations in environmental conditions (temperature, salinity, emersion).

Prevalence, intensity, and aneuploidy patterns of disseminated neoplasia in cockles (Cerastoderma edule) from Arcachon Bay: Seasonal variation and position in sediment

The present report presents the first evidence of disseminated neoplasia (DN) in cockles Cerastoderma edule from Arcachon Bay (France). Aneuploidy of neoplastic cells allowed the use of flow cytometry (FCM) to diagnose and stage DN. A 1year survey (2007) of the prevalence and intensity (% of aneuploid circulating cells in neoplastic cockles) was conducted. Prevalences ranged from 2.2% (June) to 13.6% (May), and disease intensity ranged from 18.7% (June) to 95.5% (September). These percentages were not correlated with seawater temperature, but rather showed unexplained oscillations over the year. Prevalence and intensity of DN were higher in cockles found at the surface of sediment compared to those buried normally (11.8% vs. 6.7% and 53.0% vs. 40.6%, respectively, p<0.05). DN could thus be one mechanism leading to unexplained presence of cockles at the surface of the sediment in Arcachon Bay. Ploidy characteristics of neoplastic cells were also investigated using FCM, revealing an unusual, broad continuum of ploidy distribution from 1.6 to 9.6n. Ploidy values were not in whole numbers in contrast to the rounded values reported in other studies. Ploidy varied according to DN intensity, with the ploidy distribution of neoplastic cells from lightly-diseased cockles being unimodal (3.7n median). In contrast, highly-diseased cockles showed a bimodal ploidy distribution (3.0n and 4.7n medians). This suggests that, in cockles from Arcachon Bay, mechanisms leading to aneuploidy are complex, developing during disease progression.

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum.

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl+alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl+alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.

Effect of day length on oxidative capacities of mitochondria from red muscle of rainbow trout (Oncorhynchus mykiss)

In nature, seasons may be more reliably announced by changes in photoperiod than in temperature. To evaluate the role of day length in setting oxidative capacities of trout muscle mitochondria, we acclimated trout to summer (15 degrees C, 16L:8D), winter (5 degrees C, 8L:16D) and mixed conditions (15 degrees C, 8L:16D). Maximal oxidative capacities of isolated mitochondria at 5 and 15 degrees C were higher in mixed than summer conditions and higher again in winter conditions. At 5 degrees C, state 4 rates changed little with acclimation state whereas at 15 degrees C state 4 rates were lower in summer than in mixed or winter conditions. Using concentrations of the adenylate nucleotide translocase as the denominator for these rates gave much the same conclusions. By using inhibitors to block flux at specific points in the electron transport chain, we found that flux through Complexes II-IV was lowest in summer acclimated trout, increased upon acclimation to mixed and to winter conditions. Flux through complex IV was similar in trout acclimated to summer and mixed conditions, but increased significantly with acclimation to winter conditions. Flux through complex IV was 1.5 fold higher than state 3 rates for summer-acclimated trout but was similar to state 3 rates in trout acclimated to mixed or winter conditions. Our results indicate that a reduction in day length initiates increases in mitochondrial oxidative capacity typically associated with cold acclimation and that acclimation to both cold temperatures and short day lengths enhanced these changes. The overall similarity of the responses of state 3, of flux through complexes II-IV and of flux through complex IV suggests that a generalised mechanism such as changes in the phospholipid composition of the inner mitochondrial membrane may coordinate these changes.

Docosahexaenoic acid-and eicosapentaenoic acid-enriched cardiolipin in the manila clam Ruditapes philippinarum

The FA composition of cardiolipin (CL) from the Manila clam Ruditapes philippinarum was investigated in whole body and individual organs. CL was isolated by HPLC and its chemical structure characterized using NMR spectroscopy. Two prominent FA, EPA and DHA, were found in approximately equal proportions, contributing together up to 73 mol% of the total FA. The FA composition of CL is presumed to reflect a specific synthesis pathway independent of diet and of total glycerophospholipid FA composition. To the best of our knowledge, this is the first time that a CL dominated by the two PUFA 22∶6n−3 and 20∶5n−3 has been characterized and described. This EPA+DHA specificity of the CL in the Manila clam is though to reflect a functional and structural modification of mitochondrial membranes of this bivalve species compared with scallops, oysters, and mussels that possess a CL dominated by DHA. The FA composition and levels of CL differed little between separated organs, and the large pool of DHA and EPA was found fairly equally distributed in gills, mantle, foot, siphon, and muscle. However, whereas DHA and PUFA levels were most stable between organs, EPA and arachidonic acid were significantly more variable and seemed to be interrelated.

A Striking Parallel Between Cardiolipin Fatty Acid Composition and Phylogenetic Belonging in Marine Bivalves: A Possible Adaptative Evolution?

Thirty-five species of marine mollusk bivalves were analyzed for their fatty acid (FA) composition of cardiolipin (Ptd2Gro). All species showed a Ptd2Gro with strong selectivity for only a few polyunsaturated fatty acids, but three characteristic FA profiles emerged, with clear parallels to bivalve phylogeny. A first group of 12 species belonging to the Eupteriomorphia subgroup (Filibranchia) was characterized by a Ptd2Gro almost exclusively composed of 22:6n-3, whereas in the four Filibranchia Pteriomorph species analyzed, this FA was combined with substantial proportions of 18:2n-6 and 18:3n-3. Finally, a third group of 20 species, all belonging to the Heterodonta subclass, possessed Ptd2Gro containing predominantly both 22:6n-3 and 20:5n-3. Polyunsaturated FA moieties and arrangements in the Ptd2Gro of some marine species investigated in other classes of the mollusk phylum (Gastropoda, Polyplacophora) were found to be different. The present results suggest that the specific Ptd2Gro FA compositions in bivalves are likely to be controlled and conserved in species of the same phylogenetic group. Functional significances of the evolution of this mitochondrial lipid structure in bivalves are discussed.