Patrick Lassus

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.

Patterns of experimental contamination by Protogonyaulax tamarensis in some French commercial shellfish.

As a result of the proliferation of toxic marine dinoflagellates along European coasts and the recent discovery of paralytic poisons in French shellfish, experimental studies were conducted on four species of shellfish from the Brittany coasts. Contamination rates of a culture of toxic Protogonyaulax tamarensis, were determined for Mytilus edulis, Crassostrea gigas, Pecten maximus and Ruditapes philippinarum. Mussels and scallops were very rapidly contaminated showing high toxin accumulation rates, whereas rates for oysters and clams were low. During the decontamination phase, two stages were observed in mussels and scallops: a fast decrease in toxin, of the same order of magnitude as the accumulation, followed by a slow decrease, with the toxic rate remaining above the quarantine level of 80 micrograms/100 g. Toxin analysis, both in the culture and in the shellfish, was performed using high performance liquid chromatography. GTX3 and GTX8/epiGTX8 were the dominant toxins in the early stage of the decontamination phases, whereas GTX2 was the predominant compound during the slow phase of decontamination.

High performance liquid chromatography analysis of diarrhetic toxins inDinophysis spp. from the French coast

Diarrhetic shellfish poisoning has been a recurrent problem along the Brittany coast (France) since 1983. Okadaic acid (O.A.), the main toxin detected in mussels, is generally associated with the presence ofDinophysis cells in sea water. We report here the results of okadaic acid analyses by high performance liquid chromatography, on planktonic samples collected during the summer of 1991.D. sacculus, the majorDinophysis species, throughout this period, showed low okadaic acid content in raw extract, whereas toxic levels were 3- to 12-fold higher in sorted samples than in raw ones. A maximum O.A. concentration of 29.6 pg cell-1 was found inD. sacculus/D. cf. acuminata extract. Similarly, higher O.A. levels were noted in raw samples when these two species were associated. Generally speaking, variations in raw samples were similar to those in sorted samples. Nevertheless, whenD. rotundata reached a concentration equal to that ofD. sacculus in sorted samples, the O.A. level was lower.

Effect of the PSP-causing dinoflagellate, Alexandrium sp. on the initial feeding response of Crassostrea gigas

Abstract The effects of toxic and non-toxic strains of Alexandrium sp. on the initial feeding responses of the Pacific oyster Crassostrea gigas were determined by continuous monitoring of the clearance rate of individual oysters. This was determined fluorometrically by loss of fluorescence of live microalgae introduced as food. Feeding oysters with toxic or non-toxic A. tamarense and toxic A. fundyense resulted in stop/start clearance behaviour. In contrast, control oysters fed a reference microalga, Isochrysis sp., known to support their growth, did not exhibit this behavior. Pacific oysters acclimated to Isochrysis sp., fed mixtures of Alexandrium/ Isochrysis, showed further evidence of stop/start clearance. Exposure for periods up to 48 hours with non-toxic A. tamarense did not enhance clearance of either Alexandrium or Isochrysis.

Bioaccumulation of mycotoxins by shellfish: Contamination of mussels by metabolites of a Trichoderma koningii strain isolated in the marine environment

To determine whether toxic metabolites produced by fungi could cause shellfish toxicities, mussels were contaminated in laboratory conditions by sterile filtrates of a liquid culture of a strain of the fungus Trichoderma koningii previously isolated from a shellfish, the cockle (Cerastoderma edule). Mussels were kept in aerated natural seawater and fed with a culture of the microalga Isochrysis galbana, to which a filtrate of liquid fungal culture was added. Mussels were exposed to contamination for 7 days at 16 or 20 degrees C and extractions were then performed and their activity tested on blowfly larvae. The same toxicity was found in the fungal filtrate and the shellfish, indicating bioaccumulation. The digestive gland was the most toxic part of the mussel, confirming contamination by filtration. Treated mussels produced a mucus which appeared to be a means of eliminating toxic metabolites.

Paralytic shellfish poison accumulation yields and feeding time activity in the Pacific oyster (Crassostrea gigas) and king scallop (Pecten maximus)

Pacific oysters and king scallops placed individually in a recirculating flume were fed for 2 weeks with a constant concentration (120 cell ml ‐1 ) of a toxic strain of Alexandrium minutum. Fluorescence at the outlet of each experimental unit was measured continuously, and biodeposits were recovered twice daily to evaluate feeding time activity (FTA) and rates of organic filtration (OFR), ingestion (OIR) and organic absorption (OAR). Ion-pairing high performance liquid chromatography (IP-HPLC) was performed concurrently to quantify paralytic phycotoxins both (i) individually in shellfish at the end of the contamination period and (ii) in A. minutum cultures to estimate cellular toxin concentration. These data allowed comparison of the actual tissue toxin concentration (TOX) with the theoretical toxin accumulation rate (TAR) calculated from the linear relations between OAR, cell number, fluorescence and cell toxicity. The results show high FTA/TAR and FTA/TOX correlations (R 2 = 0.78) for both oysters and scallops. The TAR/TOX relation, though not yet clearly defined, suggests the minimum quantity of absorbed toxin at which the accumulation process is induced.

Influence of Crassostrea gigas (Thunberg) sexual maturation stage and ploidy on uptake of paralytic phycotoxins.

The purpose of this study was to assess paralytic phycotoxin uptake in diploid and triploid oysters at two stages of their sexual cycle corresponding to their status in early summer (June) and winter (November). Samples of diploid and triploid oysters were exposed to a toxic culture of Alexandrium minutum for 4 days in each season. No significant differences in filtration or clearance rates were observed during either November or June experiments. When diploid oysters were at resting stage (November), toxin uptake showed no significant difference between the ploidy classes. In contrast, when the diploid oysters were at the peak of their sexual maturation (June), the triploid oysters were seen to accumulate almost double the amount of paralytic toxins as the diploid ones.

Variations des zymogrammes estérasiques chez les larves de Palaemon serratus soumises à différentes durées de choc thermique

Abstract Palaemon serratus at larval stage 2, acclimatized at 16°, were subjected to a temperature increase of 15° for periods of 5, 20 or 40 min with or without return to the initial temperature for 12 h. The soluble protein concentration of larvae subjected to a thermal shock of 20 min duration is lower than in control larvae. Total esterase-2C activity per mg of soluble protein after a thermal shock of 20 min duration is less than in control larvae but, in contrast with larvae subjected to a shock of 40 min duration, the initial activity is restored after 12 hours. Esterase-2C activity zymograms, after polyacrylamide gradient gel electrophoresis, show twelve isozymes. The activity of each isozyme examined varies according to the duration of the thermal shock.

Storage and Detoxification of Bivalve Molluscs as a Tool in a Marketing Strategy

This study fostered partnership between scientists, shellfish growers, water treatment engineers, economists and regulators. The objective was to optimise land-based post-harvest treatments in such a way that oyster and mussel marketing could continue during toxic blooms of Dinophysis acuminata (DSP) and Alexandrium minutum (PSP). Among the different methods capable of eliminating toxic algae, sand filters reduced microalgae by 90–99 %, depending on sand grain size used, whereas immersed membranes (0.2 μm) retained 99 % of particles. However, equipment and operating costs were higher for immersed membranes. Experiments using disrupted algal cells showed that PSP and DSP toxins remained stable and soluble for 15 days following release from the cell. The soluble fractions were practically unavailable to mussels. The storage of oysters for 35 days required a re-circulating system at 16 °C, with a continuous algal food supply (Skeletonema costatum). These culture conditions provided a good balance between algal supply and sea water turnover, allowing for the conservation of mussel body condition and water quality, with regard to dissolved forms of nitrogen. The efficiency of detoxification was correlated with the ingestion of food particles. When oxidizing agents (H2O2) were used, detoxification kinetics followed a different course from those observed during natural decontamination processes. From an economic point of view, analysis of the type of shellfish farm provided a way to identify farms that were likely to purchase, either safe storage or detoxification equipment. Plant size was simulated as a function of the average quantity of shellfish processed.