Extracellular products (ECPs) of the pathogenic Vibrio aestuarianus 01/32 were previously reported to display lethality in Crassostrea gigas oysters and to cause morphological changes and immunosuppression in oyster hemocytes. To identify the source of this toxicity, biochemical and genetic approaches were developed. ECP protease activity and lethality were shown to be significantly reduced following incubation with metal chelators, suggesting the involvement of a zinc metalloprotease. An open reading frame of 1836 bp encoding a 611-aa metalloprotease (designated Vam) was identified. The deduced protein sequence showed high homology to other Vibrio metalloproteases reported to be involved in pathogenicity. To further confirm the role of this enzyme in ECP toxicity, a plasmid carrying the vam gene under the control of an araC-P(BAD) expression cassette was transferred to a Vibrio splendidus related strain, LMG20012(T), previously characterized as non-pathogenic to oysters. Expression of Vam conferred a toxic phenotype to LMG20012(T) ECPs in vivo and cytotoxicity to oyster hemocytes in vitro. Collectively, these data suggest that the Vam metalloprotease is a major contributor to the toxicity induced by V. aestuarianus ECPs and is involved in the impairment of oyster hemocyte functions.

Coccolithus braarudii and Calcidiscus leptoporus are 2 coccolithophores (Prymnesiophyceae: Haptophyta) known to possess a complex heteromorphic life cycle, with alternation between a motile holococcolith-bearing haploid stage and a non-motile heterococcolith-bearing diploid stage. The ecological implications of this type of life cycle in coccolithophores are currently poorly known. The nutritional preferences of each stage of both species, and their growth response to conditions of turbulence were investigated by varying their growth conditions. Of the different culture media tested, only the synthetic seawater medium did not support the growth of both stages of C. braarudii and C. leptoporus. With natural seawater-based media, the growth rate of the haploid phase of both coccolithophores was stimulated by the addition of soil extract (K/2: 0.23 ± 0.02 d -1 and K/2 with soil extract 0.35 ± 0.01 d -1 for the C. braarudii haploid stage), while the diploid phase was not, indicating that the motile stage is capable of utilizing compounds present in soil extract or ingesting bacteria that are activated in enriched media. The addition of sodium acetate to the medium also stimulated the haploid phase of C. braarudii, and further experiments using labeled bacteria demonstrated the capacity for phagotrophy of this motile stage. The effect of nutrient concentrations on the growth rates of both species was evaluated, showing clear differences between the 2 phases of the life cycle, with higher growth rates for the diploid stage in nutrient-rich media (K/2: 0.34 ± 0. leptoporus).Responses of the 2 phases of both coccolithophores to physical turbulence were also different, with a haploid flagellate stage sensitive to mixing and a more resistant non-motile diploid stage. The results of these experiments strongly indicate that each morphological stage of C. braarudii and C. leptoporus corresponds to a different ecological niche, the motile haploid stage exploiting a more stable oligotrophic niche than the diploid non-motile stage, in accordance with field observations. The 2-dimensional phytoplankton niche space model of Margalef (1978; Oceanol Acta 1:493-509), defined by nutrients and turbulence, was amended to integrate this hypothesis.

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen3a analogue was determined by using two-dimensional 1 H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29 -58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys 32 -Cys 47 and Cys 48 -Cys 55 ). These two coils are in turn linked together by the third disulfide bond (Cys 36 -Cys 54 ). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg 45 and Arg 50 , which belong to the helix, and side chains of Arg 37 and Arg 53 , which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.Antimicrobial peptides are major elements of host-defense systems represented in all species from plants to vertebrate and invertebrate animals. Among these antimicrobial molecules, cysteine-rich peptides are the most widespread. They are structurally classified into (i) peptides with a ␤-sheet structure such as the mammalian defensins (1), (ii) peptides with a ␤-hairpin-like fold such as tachyplesins from horseshoe crabs (2), thanatin (3), porcine protegrins (4, 5), androctonin (6), or gomesin (7), and (iii) peptides adopting the cystine-stabilized ␣␤-motif, including invertebrate and plant defensins (8Ϫ10).Only recently, such effectors of innate immunity were isolated from crustaceans, whereas numerous peptides have been characterized from other arthropods, both insects and chelicerates. Three peptides, named penaeidins (Pen), 1 were initially purified in their active form (5.48Ϫ6.62 kDa) from the hemocytes of the shrimp Litopenaeus (Penaeus) vannamei, and they were fully characterized at the amino acid level (Pen-1, Pen-2, Pen3a) and by cDNA cloning from a hemocyte library (pen-2, -3a, -3b, and-3c) (11). Penaeidins are an original p...