Effects of an environmentally relevant concentration of diuron on oyster genitors during gametogenesis: responses of early molecular and cellular markers and physiological impacts.

Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris.

The present study reports the characterization of Ls-Stylicin1, a novel antimicrobial peptide from the penaeid shrimp, Litopenaeus stylirostris. The predicted mature peptide of 82 residues is negatively charged (theoretical pI=5.0) and characterized by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. The recombinant Ls-Stylicin1 has been isolated in both monomeric and dimeric forms. Both display strong antifungal activity against Fusarium oxysporum (1.25 microM

Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.

Molecular characterization of two isoforms of defensin from hemocytes of the oyster Crassostrea gigas.

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. We previously characterized the first AMP from an oyster, a defensin, that was shown to be continuously expressed in the mantle of Crassostrea gigas. In this study, we report the cDNA cloning of two new isoforms of the defensin AMP family (Cg-defh1 and Cg-defh2) from the hemocytes of the oyster. The deduced amino acid sequences reveal two peptides of 73 amino acid residues with a mature portion consisting of 43 amino acid residues. Cg-Defh1 and Cg-Defh2 share 86% amino acid identity and belong to the "arthropod-molluscs defensin family". qRT-PCR analyses indicate that Cg-defh2 is continuously expressed in the hemocytes of C. gigas. In addition, after a bacterial challenge, the level of Cg-defh2 transcripts decreases dramatically in the circulating hemocyte population and this decrease can be correlated with an increase of Cg-defh2 transcripts in the gill and the mantle tissue, suggesting a possible migration of the hemocytes expressing Cg-defh2 towards the tissues implicated in the first defense barrier of the oyster. These results would suggest an important role of Cg-Defh2 in the oyster response to a microbial challenge.

Involvement of penaeidins in defense reactions of the shrimp Litopenaeus stylirostris to a pathogenic vibrio.

The present study reports for the first time the involvement of an antimicrobial peptide in the defense reactions of a shrimp infected by a pathogenic Vibrio, Vibrio penaeicida. New members of the penaeidin family were characterized in the shrimp Litopenaeus stylirostris by RT-PCR and RACE-PCR from hemocyte total RNAs, and by mass spectrometry detection and immunolocalization of mature peptides in shrimp hemocytes. In infected shrimps, bacteria and penaeidin distribution colocalized in the gills and the lymphoid organ that represented the main infected sites. Moreover, the shrimp immune response to infection involved massive hemocyte recruitment to infection sites where released penaeidin may participate in the isolation and elimination of the bacteria, We show that the ability of the shrimps to circumvent shrimp infections is closely related to a recovery phase based on the hematopoietic process.

Crustacean immunity. Antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge.

We report here the isolation from plasma of two penaeid shrimp species of novel peptides/polypeptides with exclusive antifungal activities. A set of three molecules was purified with molecular masses at 2.7 kDa (Penaeus vannamei), 7.9 kDa, and 8.3 kDa (Penaeus stylirostris). Primary structure determination was performed by a combination of Edman degradation and mass spectrometry. The peptides display 95-100% sequence identity with a C-terminal sequence of hemocyanin, indicating that they are cleaved fragments of the shrimp respiratory protein. Specific immunodetection of the hemocyanin-derived (poly)peptides revealed that experimental microbial infections increase their relative concentration in plasma as compared with nonstimulated animals. Thus, the production of antifungal (poly)peptides by limited proteolysis of hemocyanin could be relevant to a shrimp immune reaction that would confer a new function to the multifunctional respiratory pigment of crustaceans.

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (Crustacea, Decapoda).

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.

Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge.

Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules. The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating haemocytes in the first 3 hours following stimulation and (2) an increase in plasma penaeidin concentration occurs after microbial challenge, together with (3) a penaeidin immunoreactivity in cuticular tissue, which can be related to the chitin-binding activity we demonstrate here for penaeidins.

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.

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, 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 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 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.

In Vitro Activity of the Limulus Antimicrobial Peptide Tachyplesin I on Marine Bivalve Pathogens

Tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crab Tachypleus tridentatus. We studied the in vitro activity of tachyplesin I against bivalve pathogens: the oyster parasites Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis and Perkinsus marinus, the histozoic parasite of the Eastern oyster Crassostrea virginica, and the bacterium Vibrio P1, pathogenic for the clam Tapes philippinarum. Viability of the protozoans was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to tachyplesin I, B. ostreae and P. marinus viabilities were reduced in a dose-dependent manner, up to, respectively, 94 and 62% within a 500 µg/ml peptide concentration. The fine structure of P. marinus was highly altered by the peptide. Tachyplesin I also displayed a potent activity against marine vibrios, with a MIC of 0.4-0.8 µg/ml against Vibrio P1. We examined the morphology of oyster hemocytes treated by tachyplesin I, together with the cell functional capabilities to produce chemiluminescence. No effect of the peptide was found on bivalve host cells. As transgenic technology is currently being applied to marine invertebrates, these results indicate that tachyplesin I may provide effective gene sequences to be manipulated in order to produce disease-resistant bivalves.

Future of biotechnology-based control of disease in marine invertebrates.

Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.

Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies.

Various haemolymph components of the shrimp Penaeus japonicus were identified and characterised by monoclonal antibodies. Three groups of monoclonal antibodies were raised. Their reactivity to haemocyte types and/or secreted molecules was determined by immunofluorescence and the molecular masses of the antigens were analysed by western-blotting. A 170 kDa protein, in reducing conditions, was recognized by four panhaemocytic monoclonal antibodies from group 1. This protein was present both in the plasma and in the haemocytes from which it appears to be secreted. The shrimp haemocytes were separated by isopycnic centrifugation on a Percoll gradient and the different subpopulations were antigenically analysed using the two monoclonal antibodies, 40E2-2A and 40E10-2B, from group 2. The granular cells were labelled by 40E2-2A which was specific for a protein of 142 kDa also present in plasma. By comparison, the 40E10-2B monoclonal antibody was assumed to be the marker for small hyaline and semigranular cells since the granular ones were not labelled. Moreover, the antigen immunoprecipitated by this monoclonal antibody was shown to have different molecular masses of 250, 150, 66 and 27 kDa under nonreducing conditions. It appeared to be secreted by the haemocytes. Some plasma proteins were recognized by the third group of monoclonal antibodies. The antibodies, designated 41D11-3A, 42C11-3B and 42E8-3C, all immunoprecipitated a protein with an apparent molecular mass of 180 kDa under reduced conditions. The 44E6-3D antibody was specific for a 75 kDa protein under reduced conditions and was shown to be immunoreactive against P. japonicus haemocyanin extract.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro activity of the antimicrobial peptide magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis.

Magainins are peptide antibiotics with broad antibacterial and antiparasitic activities, originally extracted from the skin of Xenopus laevis. We investigated the effects of magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis. Viability of purified protozoa was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to magainin 1, Bonamia viability was reduced in a dose-dependent manner. Within the peptide concentration of 500 micrograms/ml, the parasite viability was reduced by 94%. Electron microscopy showed membrane damage and release of cytoplasmic organelles in the injured Bonamia. The study of magainin 1 activity against Ostrea edulis hemocytes did not show any morphological change in the host cells, and the peptide did not impair the capabilities of hemocytes to produce chemiluminescence when stimulated to phagocytize zymosan particles. The possibility to genetically transform molluscs to generate disease-resistant organisms is currently under investigation. Antimicrobial peptides such as magainins may provide effective gene sequences to be manipulated.

Phagocytosis associated chemiluminescence of hemocytes in Mytilus edulis (Bivalvia).

The respiratory burst associated with phagocytosis by Mytilus edulis hemocytes was investigated by measurement of luminol-dependent chemiluminescence (LDCL). After experimental parameters (number of cells, quantity of stimulus) were determined, the biochemical mechanisms involved in the chemiluminescent process were investigated using inhibitors of oxygen radicals and enzymes. In particular, catechol-like phenols suggested the involvement of NADPH-oxidase and peroxidase in oxidative metabolism of mussel hemocytes. The variability of LDCL response observed among individuals and separated hemocyte subpopulations strongly suggests a variable immunocapacity depending on hemogram composition. Using a specific monoclonal antibody to discriminate different hemocyte types, the eosinophilic granulocytes appeared to exhibit the highest LDCL activity.

Evidence of neutralizing activity against T3 coliphage in oyster Crassostrea gigas hemolymph.

To investigate defense reactions of bivalve molluscs against viruses, experimental in vitro assays have been developed using T3 coliphage as a test virus. A native neutralizing factor in oyster Crassostrea gigas serum showed high individual variability and was enhanced significantly by repeated sampling of hemolymph from the same oysters. The responsible factor is apparently thermolabile and sensitive to EDTA treatment. Because of an inhibitory effect by the enzymatic inhibitor, phenylmethylsulphonyl fluoride (PMSF), the T3-neutralizing factor may be related to serine protease.

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.