A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.

Noradrenaline and alpha-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells.

Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults. A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling. In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells. Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various alpha- and beta-adrenoceptor agonists and antagonists. Results show that NA and alpha-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes. Furthermore, exposure of hemocytes to NA or to the alpha-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein. Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca(2+)-dependent PKC inhibitor Gö 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter. These results suggest that alpha-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca(2+)-dependent PKC and PI 3-kinase. Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells.

Noradrenaline modulates oyster hemocyte phagocytosis via a beta-adrenergic receptor-cAMP signaling pathway.

Catecholamines (CA) regulate several physiological processes in molluscs. Experiments have been conducted to determine the effects of noradrenaline (NA), the principal CA circulating in bivalve hemolymph, on oyster hemocytephagocytosis. Results show that NA had a dose-dependent inhibitory effect on phagocytosis at physiological concentrations of 0.1 microM and above. The beta-adrenoceptor agonist isoproterenol mimicked the inhibitory effects of NA on phagocytosis, whereas the alpha-adrenoceptor agonist phenylephrine had no significant effect. Furthermore, the beta-adrenoceptor antagonist propanolol, but not the alpha-adrenoceptor antagonist prazosin, prevented the inhibition of phagocytosis by NA. The type IV phosphodiesterase inhibitor rolipram acted synergistically with a suboptimal concentration of isoproterenol to inhibit phagocytosis, and the protein kinase A inhibitor H-89, but not the protein kinase C inhibitor calphostin C, attenuated the effect of isoproterenol. These results show that NA can modulate oyster hemocyte phagocytosis via a beta-adrenergic receptor/cAMP/protein kinase A signaling pathway.

Stress-induced catecholamine changes in the hemolymph of the oyster Crassostrea gigas.

The stress response is a series of coordinated physiological reactions increasing an organism's capacity to maintain homeostasis in the presence of threatening agents. This fundamental process is known to involve hormonal signaling to rapidly modulate key physiological functions in vertebrates, but data are lacking concerning neuroendocrine responses to stress in invertebrates. The present study examined circulating catecholamine (CA) responses to stress in oysters. Mechanical disturbances (consisting of shaking the animals) and temperature or salinity variations were applied to the animals because these three types of stressors are commonly encountered by oysters in aquaculture or in their natural habitat. Results show that both circulating noradrenaline (NA) and dopamine (DA) concentrations increased in response to stress. The catecholaminergic response to acute mechanical stressors was rapid (less than 5 min), transient (a return to basal CA levels was observed after 60-90 min), and reflected both the intensity and duration of the perturbation. In contrast, responses to temperature and salinity variations were long lasting (up to 72 h). CA concentrations varied from 1.61 +/- 0.30 ng NA/ml and 0.41 +/- 0.05 ng DA/ml to maximal values of 22.07 +/- 0.97 ng NA/ml and 2.24 +/- 0.19 ng DA/ml. Such CA concentrations are known to induce physiological responses in bivalves, suggesting that stress-induced NA and DA changes exert a regulatory function in oysters.

Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus.

Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms. Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption. In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen Vibrio splendidus. Oysters (Crassostrea gigas) were challenged with a low dose of a pathogenic V. splendidus strain and subjected to a mechanical stress 3 days later. Both mortality and V. splendidus loads increased in stressed oysters, whereas they remained low in unstressed animals. Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of V. splendidus in challenged oysters. These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile C. gigas vulnerability to Vibrio splendidus.

New evidence of the copepod maternal food effects on reproduction.

Failure of female reproductive capacity in the copepod Calanus helgolandicus was related to number and combination of the phytoplankton species in the diets. The maternal food effects were detectable at different levels: fecundity, oogenesis and hatching. Fecundity and hatching were normal with two single (ca. Isochrysis galbana and Prorocentrum minimum) and one mixed (Phaeodactylum tricornutum+Dunaliella tertiolecta+Pavlova lutherii+I. galbana+P. minimum) diets. With the single P. lutherii diet, fecundity decreased, but hatching remained optimal. The daily egg production and hatching rates decreased significantly in females fed the other single P. tricornutum, D. tertiolecta and mixed (P. tricornutum+D. tertiolecta+P. lutherii+I. galbana) diets, or starved. The fecundity decrease coincided with gonad atresia, which was reversible when P. tricornutum and P. lutherii diets were replaced by P. minimum diet. It was irreversible when D. tertiolecta was replaced by P. minimum, leading to female sterilization expressed by the deterioration of OS3 and OS2 oocytes, as a function of the feeding duration. We assume that atresia of female gonads was caused by the limitation of essential nutrients in food, such as fatty acids, which induced catabolism and recycling of yolk reserves and thus, maintenance of gonad integrity and low spawning rates. With the D. tertiolecta diet, abnormally high increase of ornithine concentrations in eggs showed that the ornithine metabolism and polyamine pathway were affected during oogenesis, leading atresia of oocytes to be deeply disturbed and followed up by necrosis of the gonads.

Evidence for a form of adrenergic response to stress in the mollusc Crassostrea gigas.

Catecholamines and pro-opiomelanocortin (POMC)-derived peptides, some of the central regulators of the stress-response systems of vertebrates, are also present in invertebrates. However, studies are needed to determine how these hormones participate in the organisation of neuroendocrine stress-response axes in invertebrates. Our present work provides evidence for the presence of an adrenergic stress-response system in the oyster Crassostrea gigas. Noradrenaline and dopamine are released into the circulation in response to stress. Storage and release of these hormones take place in neurosecretory cells presenting morphological and biochemical similarities with vertebrate chromaffin cells. Both in vivo and in vitro experiments showed that applications of the neurotransmitters acetylcholine or carbachol caused no significant release of noradrenaline or dopamine. Moreover, the nicotinic antagonists hexamethonium and &agr; -bungarotoxin and the muscarinic antagonist atropine caused no significant inhibition of catecholamine release in stressed oysters. Adrenocorticotropic hormone (ACTH) induced a significant release of noradrenaline, but the release of dopamine in response to ACTH was not significant. These results suggest that, unlike that of vertebrates, the adrenergic stress-response system of oysters is not under the control of acetylcholine and that other factors, such as the neuropeptide ACTH, might control this system.

Noradrenaline modulates hemocyte reactive oxygen species production via beta-adrenergic receptors in the oyster Crassostrea gigas.

Catecholamines (CA) are known to be present in the microenvironment of molluscan immunocytes. In the present study, experiments were conducted to determine the effects of noradrenaline (NA), the principal CA circulating in bivalve hemolymph, on the luminol-dependent chemiluminescence (CL) of oyster Crassostrea gigas hemocytes. Results show that NA had a dose-dependent inhibitory effect on the CL-response at the physiological concentration of 0.1 microM and above. The alpha-adrenoceptor agonist phenylephrine had no significant effect on the CL-response whereas the beta-adrenoceptor agonist isoproterenol mimicked the inhibitory effects of NA on the CL-response. The beta-adrenoceptor antagonist propanolol, but not the alpha-adrenoceptor antagonist prazosin, prevented the negative effects of NA on the CL-response. Taken together, these results show that beta-adrenergic receptors are present at the surface of oyster hemocytes and allow NA to down-regulate the CL-response.

Chemosensory grazing by marine calanoid copepods (arthropoda: crustacea).

In laboratory experiments, mixed populations of two marine copepods (Acartia clausi and Eurytemora herdmani) when fed artificial food particles consisting of microcapsules that were either enriched with an encapsulated homogenate of naturally occurring phytoplankton or nonenriched preferentially ingested the enriched capsules. Beads or nonenriched capsules were either seldom ingested or not ingested at all. The observations demonstrate that filter-feeding in these species is a behavioral process, under sensory control, and that the copepods are able to discriminate between enriched and nonenriched food particles.