Evelyne Bachère

Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas

Summary:  Research on innate immunity of the penaeid shrimps and the oyster Crassostrea gigas is motivated greatly by economical necessities. Indeed, the aquaculture of these organisms is now limited by the development of infectious diseases. Studying anti‐microbial peptides/proteins (AMPs), which are effector molecules of the host defense, is particularly attractive not only for progressing basic knowledge on immunity but also because they offer various possible applications for disease management in aquaculture. AMPs are explored with a global approach, considering their structure, properties, function, gene expression, and tissue distribution during the response to infections. In shrimp, investigations of the penaeidins, which are constitutively expressed peptides, have highlighted the importance of hemocytes and hematopoiesis as major elements of the immune response, providing both local and systemic reactions. The activation of hematopoiesis must be regarded as a regulatory way for the expression and distribution of constitutively expressed immune effectors. As complementary approaches, genomics and gene profiling are promising to deepen our understanding of the anti‐microbial defense of the oyster and the shrimp. However, real progress will depend also on the characterization of hemocyte lineages and hematopoiesis of these marine invertebrates as well as on the ontogenesis of their immune systems.

Penaeidins, a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)

We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimpsPenaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by a pyroglutamic acid at the first position. Comparison of the data obtained from the cDNA clones and mass spectrometry showed that two of these peptides are probably COOH-terminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei.

Ž. A spectrophotometric nitroblue tetrazolium NBT reduction assay was used to demonstrate the Ž y . production of superoxide anions O by haemocytes of the white shrimp Penaeus Oannamei .I t 2 was found that haemocytes, without receiving an experimental stimulant, showed a rather high Ž background activity. Therefore, optimal parameters number of haemocytes, type of incubation . medium, type and concentration of stimulants were first established, in order to obtain a reliable and reproducible quantitative assay. With this optimized assay, and using specific inhibitors, it was shown that it is indeed the production of O y that was measured. Activities varied strongly 2 among individual shrimp specimens. Live bacteria, among these Vibrio strains, induced O y production in the haemocytes, in a dose-dependent manner. Whereas Vibrio anguillarum and a probiotic strain of V. alginolyticus evoked clear reactions, a pathogenic strain of V. harOeyi failed to elicit O y production in the haemocytes. It is discussed that this may explain this strains 2

Shrimp immunity and disease control

Abstract The sustainability and development of shrimp aquaculture are largely at stake as significant ecological and pathological problems are increasing in the vast majority of the shrimp producing countries. Prevention and control of diseases are now the priority for the durability of this industry. Within the past decade, intensification of the shrimp production, based on progress in zootechnology, has increased but with little corresponding increase in scientific knowledge of shrimp physiology. Within this field, shrimp immunology is a key element in establishing strategies for the control of diseases in shrimp aquaculture. Research needs to be directed towards the development of assays to evaluate and monitor the immune state of shrimp. The establishment of regular immune checkups will permit the detection of shrimp immunodeficiencies but also to help monitor and improve environment quality. For this, immune effectors must be first identified and characterised. In the end, however, the assumption may be made that the sustainability of aquaculture will depend on the selection of disease-resistant shrimp, i.e. to develop research in immunology and genetics at the same time. The development of strategies for prophylaxis and control of shrimp diseases could be aided by the establishment of a collaborative network to contribute to progress in basic knowledge of penaeid immunity. However, to improve efficiency, it appears essential also to open this network to complementary research areas related to shrimp pathology, physiology, genetics and environment.

Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas

An expressed sequence tag program was undertaken to isolate genes involved in defense mechanisms of the Pacific oyster, Crassostrea gigas. Putative function could be assigned to 54% of the 1142 sequenced cDNAs. We built a public database where all EST information are accessible through numerous search profiles (http://www.ifremer.fr/GigasBase). Based on sequence similarities we identified 20 genes that may be implicated in immune function. We investigated the expression of four of these genes during bacterial challenge of oysters. Three of them were induced in response to challenge lending support to their involvement in oyster immunity. Moreover, four other genes were highly homologous to components of the NF-kappa B signaling pathway which is involved in innate immune response in Drosophila and mammals. Altogether, our results open a new way to investigate the immune response in mollusks.

Characterization of a Defensin from the Oyster Crassostrea gigas RECOMBINANT PRODUCTION, FOLDING, SOLUTION STRUCTURE, ANTIMICROBIAL ACTIVITIES, AND GENE EXPRESSION

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized α-β motif (CS-αβ) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (crustacea, decapoda)

Abstract. The production of antimicrobial peptides represents a first-line host defense mechanism of innate immunity that is widespread in nature. Only recently such effectors were isolated in crustacean species, whereas numerous antimicrobial peptides have been characterized from other arthropods, both insects and chelicerates. This review presents findings on a family of antimicrobial peptides, named penaeidins, isolated from the shrimp Penaeus vannamei. Their structure and antimicrobial properties as well as their immune function will be discussed through analyses of penaeidin gene expression and peptide distribution upon microbial challenge.

Anti-infectious immune effectors in marine invertebrates: potential tools for disease control in larviculture

Abstract The aquaculture of molluscs and crustaceans is an economical activity that is threatened by the repeated appearance of new diseases, non-infectious and infectious, which affect cultured species. Difficulties in controlling diseases in marine aquaculture come mainly from the differences in susceptibility of the animals according to their developmental stage (from larvae to adults) and from the diversity of pathogens that affect them. Among the causative agents encountered in marine aquaculture, viral and bacterial problems as well as fungal diseases dominate the larval production. Methods have been proposed for the control of disease in aquaculture, such as the use of probiotic bacteria, immunostimulants or the improvement of the larval quality via the nutrition, and zootechnical progress has been made. So far, antibiotics have been intensively used as preventive and curative measures, but such practices are now questioned because regular use of these chemical products has led to the appearance of drug-resistant bacteria and can also have harmful effects on the environment. In this context, alternative treatments as well as animal health monitoring have to be established as preventive measures. In the long term, the most effective way for sustainable aquaculture production will certainly rely upon the production of animals selected for increased resistance to disease, but until now, few papers can be found on this topic for marine invertebrates. Today, immune criteria as enhancement of non-specific defence responses are being considered. Investigation of the innate immune systems of molluscs and crustaceans may give new insights into the management and control of diseases in aquaculture. In particular, antimicrobial peptides present various application possibilities by their use as therapeutic agents, as tools for monitoring the health status of cultured animals, and by the use of encoding genes as selection markers for improving resistance to infections.

Knowledge and research prospects in marine mollusc and crustacean immunology

Abstract In the context of infectious diseases in mollusc and shrimp aquaculture, research must be focused on diagnosis for zoosanitary controls but also on obtaining resistant animals. This last strategy depends heavily on the development of knowledge about marine invertebrate immunology. With the establishment of purification protocols for the main invertebrate pathogens, progress has been made in the study of host-pathogen interactions at cellular and molecular levels and in identifying immune effectors involved in the destruction of pathogens. Recent information on molluscs and crustaceans is presented, concerning both hemocyte studies and cellular defence functions and humoral effectors, with special reference to their application to selection of pathogen-resistant animals. With this aim, research prospects will essentially be devoted to the identification and characterization of immune genes, either specific or heterologous, which could be candidates for mollusc and shrimp genetic transformation.

Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation.

A protocol is described to separate several subpopulations of hemocytes in a unique medium which avoids cell aggregation and retains cell-viability. Isopycnic centrifugation in Percoll followed by counterflow centrifugal elutriation provides large quantities of separated granulocyte and hyalinocyte subpopulations.