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

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.

A sandwich enzyme linked immunosorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn, Macrobrachium rosenbergii (de Man).

A sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed to improve diagnosis of white tail disease of the giant freshwater prawn, Macrobrachium rosenbergii, caused by the nodavirus, MrNV. Polyclonal antibodies were produced by immunization of Balb/C mice using a purified suspension of the virus and IgG anti-MrNV were purified from ascitic fluid. A sandwich method was successfully developed, coating first with unlabelled antibody and detecting trapped antigens with a second biotinylated antibody. Reaction was demonstrated using an avidin-peroxidase conjugate. Tissue extracts from M. rosenbergii infected with MrNV or purified viral extracts (control) were successfully identified in an individual ELISA, thus confirming the validity of the method. This S-ELISA should be the technique of choice for epidemiological studies of this disease and is a rapid and inexpensive assay with high specificity and sensitivity.

Protease inhibitors and haemagglutinins associated with resistance to the protozoan parasite, Perkinsus marinus, in the Pacific oyster, Crassostrea gigas.

Perkinsus marinus is a protozoan responsible for dramatic mortality in the Eastern oyster, Crassostrea virginica, but not in the Pacific oyster, C. gigas. To understand the host-parasite relationship, we inoculated P. marinus trophozoites into the shell cavity of C. gigas and measured, over 2 months, (i) intensity of infection, (ii) protease inhibitory activities against P. marinus proteases and against bovine z-chymotrypsin, (iii) plasma haemagglutinin titre, (iv) plasma protein concentration, (v) plasma lysozyme activity and (vi) total haemocyte count. We observed that the highest protease inhibitory activities and haemagglutinin titres (3-10 days post-challenge) preceded parasite elimination (initiated 7 days post-challenge). In contrast, plasma protein concentration, lysozyme activity and total haemocyte count showed no significant modification following the challenge. It is hypothesized that the capacity of C. gigas to increase its protease inhibitors represents the key event in resistance to parasite infection by neutralizing the proteases secreted by P. marinus, thus preserving the oyster haemagglutinins from degradation. Such haemagglutinins will be ready to act as opsonins stimulating phagocytosis of parasites.

In vitro production of peroxynitrite by haemocytes from marine bivalves: C-ELISA determination of 3-nitrotyrosine level in plasma proteins from Mytilus galloprovincialis and Crassostrea gigas.

BACKGROUND: Peroxynitrite is increasingly proposed as a contributor to defence system in marine bivalve. It can be formed by combination of superoxide and nitric oxide, and can react with tyrosine residues of proteins giving rise to 3-nitrotyrosine. RESULTS: The present article describes a competitive ELISA for the measurement of 3-nitrotyrosine contents of plasma proteins from marine bivalves by means of a monoclonal anti 3-nitrotyrosine antibody mouse IgG. CONCLUSIONS: This assay is sensitive enough to determine the amounts of 3-nitrotyrosine in plasma proteins from one animal only. Using the C-ELISA, we have shown that the phagocytosis of zymosan particles increased the 3-nitrotyrosine levels of plasma proteins from mussel M. galloprovincialis and oyster C. gigas 5.8 and 7.5 times respectively.

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis.

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.

Differential distribution and defence involvement of antimicrobial peptides in mussel.

In previous papers, we characterised 3 types of 4-kDa, cysteine-rich, cationic antimicrobial peptides: MGDs (for Mytilus galloprovincialis defensins), mytilins and myticins, which are abundant in the mussel hemocytes. In the present work, we revealed a differential distribution of the cells expressing the different genes. In addition, using confocal and electron microscopy, we confirmed that defensins and mytilins were partially located in different sub-types of circulating hemocytes although the peptides can be located in the same cell, and even in the same granule. We also demonstrated that mytilins exert their microbicidal effect within the cells through the process of phagosome-mytilin granule fusion leading to the co-location of ingested bacteria and mytilins.

Ribosomal DNA sequences of Glugea anomala, G. stephani, G. americanus and Spraguea lophii (Microsporidia): phylogenetic reconstruction.

The microsporidian species Glugea anomala, G. stephani, G. americanus and Spraguea lophii were compared by using sequence data derived from their small subunit rDNA genes which were amplified by polymerase chain reaction and directly sequenced. These sequence data and published data of G. atherinae were analyzed and were used to infer a phylogenetic tree. The 5 microsporidian fish parasites appeared to be closely related. The higher sequence similarities demonstrated among G. anomala, G. stephani and G. atherinae suggest that these 3 parasites are in fact only 1 species of Glugea. Moreover, the higher sequence similarities between S. lophii and G. americanus support the transfer of the latter Glugea species into the genus Spraguea.

Insulin during the early postembryonic development of the gilt-head sea bream, Sparus aurata: ultrastructural, immunohistochemical, and biochemical studies.

Insulin was sought during the early postembryonic development of gilt-head sea bream, Sparus aurata, using ultrastructural, immunohistochemical and biochemical techniques. The endocrine pancreas appeared at hatching (Day 0) as a single cluster of morphologically similar cells. Secretory granules formed from Day 1 onwards but the cells could only be identified as insulin-producing B cells at the end of the endotrophic period (Day 3-Day 4). Insulin-immunoreactive cells were detected in the pancreatic primordium from hatching onwards and their number increased after the end of the endo-exotrophic period. Early insulin production was also found using an ELISA method on homogenates of prelarvae and larvae. Insulin levels were fairly high during the endotrophic period, decreased strongly at mouth opening, and then increased at the end of the endo-exocrine period. The origin and role of the large amount of hormone detected during the strictly endotrophic phase of ontogenesis are discussed in light of data on other vertebrates.

Identification and phylogenetic relationships of microsporidia by riboprinting.

The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish (Chloroscombrus chrysurus) is related although not identical to Microgemma ovoidea, a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei, are not related to the fish isolates.

Development, characterization and future prospects of monoclonal antibodies against spores of Glugea atherinae (protozoa-microsporidia-fish parasites).

Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV+ patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae. The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV+ patients.

Use of cross-reactive antigens of the microsporidian Glugea atherinae for the possible detection of Enterocytozoon bieneusi by western blot.

The microsporidia Enterocytozoon bieneusi is reported in 10-30% of those infected with the human immunodeficiency virus. The parasite appears to be a cause of gastralgia, malabsorption, and diarrhea. A Western blot technique using another microsporidian species, Glugea atherinae, has demonstrated an antigenic similarity between this parasite and E. bieneusi. Preliminary results show the variability of the antigenic profiles obtained from the sera of immunodeficient patients infected with E. bieneusi and also of the cross-reactivity to Glugea sp. antigens of some sera from patients with cryptosporidiosis. The origin of this cross-reactivity is undetermined. The possibility of coinfection with undetected microsporidia is not excluded. These results raise questions concerning the interpretation of serologic data and of the potential immunodiagnostic value of microsporidian antigens.

Direct, simplified, and sensitive assay of angiotensin II in plasma extracts performed with a high-affinity monoclonal antibody.

A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.

Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies.

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA. The efficiency of the anti-N system in detecting purified and crude viruses as well as the virus in infected-organ extracts and infected blood was compared with that of a recently described antigen capture ELISA based on the detection of viral envelope glycoprotein Gp (anti-G system). For the detection of purified virus, the anti-N system was found to be as sensitive as the anti-G system (detection limit, 1 ng of total viral protein per ml), but the anti-N system was much more sensitive than the anti-G system for the detection of crude VHSV I (detection limits, 1 x 10(4) PFU/ml versus 5 x 10(5) PFU/ml). In organ extracts, VHSV I could be detected by both systems 3 days postinfection. The signal for the assay of VHSV I in blood 24 h postinfection was higher with the anti-N system than the anti-G system. Furthermore, VHSV I could be detected in 80% of the brain samples of surviving trout by the anti-N system and also by the anti-G system, but with a lower signal. In conclusion, we have developed a highly sensitive immunoassay for VHSV I that is more rapid and easier to perform than the currently used plaque assay.

[The Bothriocephalus scorpii complex (Mueller, 1776). Differentiation of species of parasites of the turbot (Psetta maxima) and the brill (Scophthalmus rhombus). Study of protein fractions and antigen complexes]. / Le complexe Bothriocephalus scorpii (Mueller, 1776). Différenciation des espèces parasites du Turbot (Psetta maxima) et de la Barbue (Scophthalmus rhombus). Etude des fractions protéiques et des complexes antigéniques.

Previous electrophoretic studies (Renaud et al., 1983) carried out on the Cestode Bothriocephalus scorpii (Mueller, 1776) showed that the populations parasiting the Turbot and the Brill on the Mediterranean coast, are two distinct species: Bothriocephalus (supra sp. scorpii) gregarius and Bothricephalus (supra sp. scorpii) barbatus respectively. This work has been done with the purpose of identifying these two species by using biochemical tests. The study of whole body proteins by electrophoresis of zones of acrylamide gel shows the existence of nineteen distinct protein fractions in both populations. Among those different protein fractions, two of them (Rf 53 and Rf 87) are present only in the Cestode of the Brill and one (Rf 42) seems to be specific of the Turbot. On the other hand, the fraction of Rf 27 is present only in the Cestodes of the Brill. The immunoelectrophoretic study of antigenic components shows seven or eight major fractions depending on the source of protein, five of them being common to both. The reactions of absorption of antiserum permitted characterization of three arcs of precipitation proper in the Cestodes of the Brill corresponding to protein fractions of Rf (27, 53, 87) and three arcs in the Cestodes of the Turbot, corresponding to protein fractions of Rf (42, 21, 66).

[Cymothoadian parasitosis of the sea-dace (Dicentrarchus labrax Linnaeus 1758) during breeding. II. Parasitic ecophysiology in the Diana pond (upper Corsica)]. / Parasitoses à cymothoadien chez le loup (Dicentrarchus labrax Linnaeus, 1758) en élevage. II--Ecophysiologie parasitaire dans le cas de l'étang de Diana (Haute-Corse).

This study deals with the problem of some aspects of the influence of infestation by an hematophagous parasite Nerocila orbignyi (Crustacean, Isopoda, Cymothoidae) on sea-bass (Dicentrarchus Labrax, L. 1758) ecophysiology, reared in the pond Diana (Corsica). We can summarise results as: decrease in body condition, in weight, in levels blood protein, blood lipids and triglycerides; but increase in levels blood urea. We too observe hypochromic macrocytic anemia with increase in eosinophils, neutrophils and a decrease in lymphocytes. When parasitism decrease, we remark improvement of body condition and biometry characteristics; an increase in level blood protein, lipid, cholesterol and a decrease in the level blood urea. We remark erythropoiesis stimulation.

[Some aspects of nutritional mechanisms in the isopod Ceratothoa oestroides Risso, 1826 parasite of fishes (author's transl)]. / Quelques aspects des mécanismes nutritionnels chez l'isopode Cymothoidae: Ceratothoa oestroides (Risso, 1826).

Some nutritional mechanisms of the isopod Cymothoidae Ceratothoa oestroides, haematophagic parasite of the teleostean sparid fish Boops boops have been studied in female sexual phase individuals. The study of qualitative (hepatopancreas repletion, different colouration) and quantitative variations (weight variations-) of digestive tract and its appendages has permitted to observe that blood absorption is close related to sexual developmental stages and intermolt cycle. The general pattern seems as follows: food intake is made prior or just before vitellogenesis and after releasing of "pulli". The use of ingested nutriments and reserves allow vitellogenesis and intramarsupium larval development. We have also tried to investigate if it does exist a mechanism starting blood absorption and attempted to elucidate its nature. A such mechanism seems indeed to exist, it might have been partially dependent upon a stimulus such as osmotic pressure or ionic composition of host blood. Moreover, chemoreceptors activity is envisaged those could likely occur as bucal appendages, bucal cavity or even the oesophagian region of parasites.

[Water transfer in relation to the salinity of the medium in two isopods Cymothoidae: Meinertia oestroides (Risso, 1862) and Anilocra physodes (L., 1758) (parasites of marine fishes)]. / Les transferts d'eau fonction de la salinité du milieu chez deux isopodes Cymothoidae: Meinertia oestroides (Risso, 1862) et Anilocra physodes (L., 1758) (parasites de poissons marins).

Meinertia oestroides and Anilocra physodes (Isopoda Cymothoidae) respectively buccal and skin parasites of the teleostean Fish Boops boops are isosmotic with the surrounding medium in sea water (SW), hyperosmotic and hyperionic in diluted media. The drinking rate of meinertia is 0.73 microliter in SW and 5.30 microliter h-1 g-1 wet weight in 5/10 SW; Anilocra shows 0.31 microliter in SW and 0.21 microliter h-1 g-1 wet weight in 6/10 SW. The distribution space of [51Cr] EDTA in Meinertia reaches 32.7% in SW and 28.9% of wet weight in 5/10 SW; Anilocra indicates 29.6% in full SW and 19.2% of wet weight in 6/10 SW. The rate of primary urine production in Meineria evaluated from the biological period of [51Cr] EDTA is 130 mg in SW and 338 mg 24 h-1 g-1 wet weight in 6/10 SW. The area of pleopoda is 826 mm2 for Anilocra, 336 mm2 for Meinertia and 277 mm2/g wet weight for Emetha audouini (another buccal parasite); 92.6% for Anilocra, 84.1% for Meinertia and 81.8% for Emetha of the apparent water diffusion outflux take place through the pleopoda. The apparent permeability diffusion coefficient Pd in SW is 1.63 x 10(-4) cm/sec for Anilocra, 2.80 x 10(-4) cm/sec for Meinertia and 3.31 x 10(-4) cm/sec for Emetha. In dilute media Pd is 1.30 x 10(-4) cm/sec for Anilocra and 3.44 x 10(-4) cm/sec in Meinertia. The results are compared with those already obtained for other non parasitic Crustaceans and according to the parasitic localization of the species above mentionned. An hypothesis concerning the mechanisms of feeding (hameatophgia) is proposed. The accuracy of results is discussed.

[Metazoan parasites of Boops boops (Linnaeus, 1758) (Teleostean Sparidae) in the "Golfe du Lion", faunistic and ecology (author's transl)]. / Faunistique et ecologie des metazoaires parasites de Boops boops linnaeus (1758) (Teleosteen Sparidae) dans le Golfe du Lion.

A global study of the Metazoan parasites of Boops boops (L., 1758) have been made in the "golfe du Lion". Fourteen different species (eight Platyhelmintha, one Nematoda and five Crustacea) have been inventorized. Their respective localisation on the hosts, globals and specifics corresponding prevalences as well as their variations according to the size of the fish, and their abundance have been precised. Parasitics associations have also been examined.

[Influence of the cymothoid isopods Meinertia oestroides, Meinertia parallela, and Anilocra physodes (Crustacea; parasites of fish) on the growth of hosts, Boops boops and Pagellus erythrinus (Sparidae) (author's transl)]. / Influence des cymothoadiens Meinertia oestroides, Meinertia parallela et Anilocra physodes (Crustacés, Isopodes; parasites de poissons) sur la croissance des poissons hôtes Boops boops et Pagellus erthyrinus (Sparidés).

Studies of weight-size (W = aLb) and growth (L = aT + b) in fish (Boops boops and Pagellus erythrinus), potential hosts of some Cymothoid isopods (Meinertia oestroides, Meinertia parallela, and Anilocra physodes) have been performed. Infested fishes show a slight decrease in weight compared to normal fish. Cymothoid do not exert a significant influence on the weight-size ratio; on the other hand, a delay in growth does occur.