We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimps Penaeus 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 NH 2 -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 COOHterminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

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.

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 Õannamei. It 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 2 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. harÕeyi failed to elicit O y production in the haemocytes. It is discussed that this may explain this strain's 2 capability of evading the host's oxidative microbicidal activity, which would be a virulence factor in these bacteria. Heat-killed bacteria hardly induced NBT reduction in the cells. The fungicide propiconazole or Tilt w , found as a pollutant in the aquatic environment where the shrimp are reared, was tested for its effect on NBT reduction by the haemocytes. In haemocytes that did not receive an experimental stimulant, Tilt w induced the reduction of NBT in a dose-dependent manner. In experimentally stimulated haemocytes, however, Tilt w strongly reduced the reaction upon the stimulant PMA. Probable explanations for these seemingly controversial effects of Tilt w are discussed, as are possible consequences of this sort of pollutants for shrimp aquaculture. This easy to perform and relatively cheap and simple quantitative assay for measuring the activity of an oxidative microbicidal mechanism in shrimp haemocytes, appears quite reliable and may therefore prove to be a valuable tool for monitoring shrimp health and immunologic status. q

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase CgEcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.T he oyster pathogen, Vibrio splendidus strain LGP32 was isolated from massive mortality events in the production of Crassostrea gigas oysters (1). However, up to now, little has been known about the route of infection and pathogenic processes of LGP32 (2, 3). A metalloprotease has been associated with toxicity (4, 5) and the outer membrane protein (OMP) OmpU was shown to be a major determinant of LGP32 virulence (6).As bacterial surface components, OMPs are both used by hosts for pathogen recognition and by pathogens for interaction with and invasion of host cells, serving as adhesion proteins (adhesins)

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Penaeidins are 5.5-to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.

Penaeidins are a family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp. In response to microbial stimulation, they are released into the blood circulation and they further attach to shrimp cuticle surfaces through a chitin-binding property. In the present paper, we have analysed their expression, regulation and distribution in shrimp tissues in response to experimental microbial challenge. We have shown that penaeidin mRNA and protein are restricted to granular haemocytes and that their expression and distribution are regulated through dramatic changes in haemocyte populations, both circulating and infiltrating shrimp tissues. Two distinct phases in the immune reactions were evidenced: (a) a migration of haemocytes towards the infection site within the first 12 h following microbial injection, with a local and massive release of peptides; (b) the appearance into the blood circulation and tissues of a haemocyte population displaying increased penaeidin-transcriptional activity, which may correspond to a systemic reaction involving haemocyte proliferation process. Finally, in vitro confrontation of haemocytes and bacteria revealed that penaeidins are released from granular haemocytes by a novel phenomenon of intracellular degranulation, probably followed by the lysis of the cells. Furthermore, penaeidins were shown covering bacterial surfaces suggesting that the peptides could be involved in opsonic activity. Penaeidin-positive bacteria were observed to be phagocytosed mainly by hyaline cells, a population that does not express penaeidins.