Lipid and fatty acid composition of diatoms revisited: rapid wound-activated change of food quality parameters influences herbivorous copepod reproductive success.

Lipid and fatty acid composition are considered to be key parameters that determine the nutritive quality of phytoplankton diets for zooplanktonic herbivores. The fitness, reproduction and physiology of the grazers are influenced by these factors. The trophic transfer of lipids and fatty acids from algal cells has been typically studied by using simple extraction and quantification approaches, which, as we argue here, do not reflect the actual situation in the plankton. We show that cell disruption, as it occurs during a predator's grazing on diatoms can drastically change the lipid and fatty acid content of the food. In some algae, a rapid depletion of polyunsaturated fatty acids (PUFAs) is observed within the first minutes after cell disruption. This fatty acid depletion is directly linked to the production of PUFA-derived polyunsaturated aldehydes (PUA); these are molecules that are thought to be involved in the chemical defence of the algae. PUA-releasing diatoms are even capable of transforming lipids from other sources if these are available in the vicinity of the wounded cells. Fluorescent staining reveals that the enzymes involved in lipid transformation are active in the foregut of copepods, and therefore link the depletion processes directly to food uptake. Incubation experiments with the calanoid copepod Temora longicornis showed that PUFA depletion in PUA-producing diatoms is correlated to reduced hatching success, and can be compensated for by externally added single fatty acids.

Survey of the chemical defence potential of diatoms: screening of fifty one species for alpha,beta,gamma,delta-unsaturated aldehydes.

In recent years a negative influence of diatom-derived alpha,beta,gamma,delta-unsaturated aldehydes (PUA) on the reproductive success of copepods and invertebrates has been suggested. Since adverse chemical properties of diatoms would question the traditional view of the marine food web, this defense mechanism has been investigated in detail, but the PUA-release by test organisms has only been determined in a few cases. The observed effects were nevertheless frequently discussed from a general point of view often leading to contradictory conclusions. We have examined the PUA-production of 51 diatom species (71 isolates) in order to provide a basis for the interpretation of laboratory and field results on the influence of diatom food on the reproductive success of their consumers. PUA-production is species and strain dependent. Thirty-six percent of the investigated species (38% of the cultivated isolates) release alpha,beta,gamma,delta-unsaturated aldehydes upon cell disruption in concentrations from 0.01 to 9.8 fmol per cell. Thalassiosira rotula and Thalassiosira pacifica, major spring-bloom forming diatoms isolated from Roscoff (Bretagne, English Channel, France) and Puget Sound (Washington, USA) were among the PUA-producing strains.

Determination and quantification of alpha,beta,gamma,delta-unsaturated aldehydes as pentafluorobenzyl-oxime derivates in diatom cultures and natural phytoplankton populations: application in marine field studies.

Reactive alpha,beta,gamma,delta-unsaturated aldehydes and oxo-acids produced by marine diatoms upon cell damage interfere negatively with the reproduction success of their grazers. A simple, sensitive and specific method based on gas-chromatography coupled to mass spectrometry (EI or CI/EC) was developed for the quantification of these deleterious substances in laboratory diatom cultures and in natural phytoplankton populations. For aldehyde quantification, diatom containing samples are damaged in the presence of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) which leads to an in situ derivatisation without inhibition of the biosynthesis of the aldehydes. The oxime derivates of oxo-acids were in addition reacted with N-tert-butyldimethylsilyl-N-methyl-trifluoracetamide (MTBSTFA).

Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom.

The growth cycle in nutrient-rich, aquatic environments starts with a diatom bloom that ends in mass sinking of ungrazed cells and phytodetritus. The low grazing pressure on these blooms has been attributed to the inability of overwintering copepod populations to track them temporally. We tested an alternative explanation: that dominant diatom species impair the reproductive success of their grazers. We compared larval development of a common overwintering copepod fed on a ubiquitous, early-blooming diatom species with its development when fed on a typical post-bloom dinoflagellate. Development was arrested in all larvae in which both mothers and their larvae were fed the diatom diet. Mortality remained high even if larvae were switched to the dinoflagellate diet. Aldehydes, cleaved from a fatty acid precursor by enzymes activated within seconds after crushing of the cell, elicit the teratogenic effect. This insidious mechanism, which does not deter the herbivore from feeding but impairs its recruitment, will restrain the cohort size of the next generation of early-rising overwinterers. Such a transgenerational plant-herbivore interaction could explain the recurringly inefficient use of a predictable, potentially valuable food resource--the spring diatom bloom--by marine zooplankton.

Evidence for a direct link between stress and immunity in the mollusc Haliotis tuberculata.

Stress is thought to cause increased disease outbreaks and mortality in a number of invertebrates but currently very little information is available on mechanisms linking physiological states of stress and reduced disease resistance in these organisms. In the present study, we examined the possibility that stress alters immune functions, the principal line of defense against pathogens, in a molluscan model, the abalone Haliotis turbeculata. Immune parameters were investigated in abalones subjected to a 15 min mechanical disturbance which, as indicated by noradrenaline and dopamine hemolymphatic levels, resulted in a transient state of physiological stress. During the application of the stressor, immune parameters such as the number of circulating hemocytes, the migratory activity, the phagocytic capacity and the respiratory burst responses of hemocytes, decreased significantly. All parameters returned to initial values within 15-30 min after the end of the disturbance and a transient period of immunostimulation occurred between 100 and 480 min after the stress for all immune parameters except intracellular superoxide anion production. These results indicate that in the abalone H. tuberculata, as in vertebrates, a link exists between stress and the immune system. This may begin to answer why stress and disease outbreaks are linked in shellfish.

P35-sensitive caspases, MAP kinases and Rho modulate beta-adrenergic induction of apoptosis in mollusc immune cells.

Apoptosis is an important mechanism for the preservation of a healthy and balanced immune system in vertebrates. Little is known, however, about how apoptotic processes regulate invertebrate immune defenses. In the present study, we show that noradrenaline, a catecholamine produced by the neuroendocrine system and by immune cells in molluscs, is able to induce apoptosis of oyster Crassostrea gigas hemocytes. The apoptosis-inducing effect of noradrenaline was mimicked by isoproterenol and blocked by propranolol, which indicates that noradrenaline triggers apoptosis via a beta-adrenergic signaling pathway. Exposure to the pan-caspase inhibitor Z-VAD-FMK or expression of the caspase inhibitor P35 under the transcriptional control of a mollusc hsp70 gene promoter reduced the number of apoptotic cells among noradrenaline-treated hemocytes. These results suggest that P35-sensitive caspases are involved in the apoptotic process triggered by beta-adrenergic signaling. Complementary experiments suggest that mitogen-activated protein kinases and Rho, a member of the Ras GTPase family, may be involved in antiapoptotic mechanisms that modulate the apoptotic effect of noradrenaline. Taken together, these results provide a first insight into apoptotic processes in mollusc immune cells.

Stress-induced immune changes in the oyster Crassostrea gigas.

Information concerning the effect of stress on invertebrate immune functions are scarce. The present study investigated the consequences of a 15-min mechanical disturbance on immune parameters in oysters Crassostrea gigas. As indicated by noradrenaline and dopamine measurements, the mechanical disturbance caused a transient state of stress in oysters. The number of circulating hemocytes, the migratory and phagocytic activities and reactive oxygen species production of hemocytes were measured before, during and after application of the stressor. Results show that all immune functions were significantly downregulated during stress and a transient period of immunostimulation was observed 30-240 min after the end of the disturbance. Taken together, these results suggest that stress can exert a profound influence on oyster immune functions and they may explain why stress and the outbreak of disease are often linked in shellfish culture. Furthermore, the present study strongly suggests that checking the stress status of animals may be necessary to avoid biases when studying oyster immune responses in vivo.