Eric Mialhe

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

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.

Isolation and purification of the protozoan Bonamia ostreae (Pichot et al. 1980), a parasite affecting the flat oyster Ostrea edulis L.

The isolation and purification of Bonamia ostreae (Ascetospora), an intrahemocytic protozoan parasite of Ostrea edulis, have been achieved according to an original protocol. The purified cells showed good retention of infectivity and ultrastructural morphology. They could also be used to prepare specific immune sera for indirect immunofluorescence.

Prevention of IHHNV vertical transmission in the white shrimp Litopenaeus vannamei

Abstract The infectious hypodermal and hematopoietic necrosis virus (IHHNV) is very pathogenic for Litopenaeus stylirostris whereas infection in Litopenaeus vannamei is known to induce development and growth abnormalities and cause economic losses that range between 10% and 50% (Lightner and Redman, 1998). In the present work, on the basis of nested-PCR analysis, IHHNV prevalences were determined to be between 47% and 63% in Ecuadorian wild and domesticated broodstocks and around 95% in Panamanian domesticated broodstock. IHHNV was regularly detected in the ovaries of infected females whereas sperm from infected males was generally free of virus. IHHNV vertical transmission from infected females was clearly established. In the case of highly infected females, embryo development may abort. IHHNV-free nauplii and larvae were produced from females and males that were found to be free of virus on the basis of nested PCR performed in the case of females after eyestalk ablation and the first spawning. The reliability of this testing process was shown to be very high since about 87% of females was confirmed IHHNV-free through a second nested-PCR analysis performed after some additional spawnings. The nauplius productivity was higher for IHHNV-free females than for the infected ones. Virus-free verification of L. vannamei broodstock may become a general routine hatchery procedure in the near future to prevent the disease from spreading through vertical transmission.

Interactions between Bonamia ostreae (Protozoa: Ascetospora) and hemocytes of Ostrea edulis and Crassostrea gigas (Mollusca: Bivalvia): Entry mechanisms

Entry mechanisms of the intrahemocytic parasite Bonamia ostreae (Ascetospora) into hemocytes of the sensitive (Ostrea edulis) and a resistant (Crassostrea gigas) oyster species have been analyzed. The study was based upon the development of a B. ostreae-hemocyte in vitro system. Ultrastructural features and Cytochalasin B sensitivity of the phenomenon demonstrate that B. ostreae enters into the two oyster species hemocytes by host-specified phagocytosis, although parasite contribution to entry into O. edulis hemocytes cannot be discarded. Possible receptors and the postphagocytic fate of the parasite are discussed.

Interactions between Bonamia ostreae (Protozoa: Ascetospora) and hemocytes of Ostrea edulis and Crassostrea gigas (Mollusca: Bivalvia): in vitro system establishment

Abstract Based on parasite purification and hemocyte primary culture, an in vitro system was established for studying early interactions between Bonamia ostreae and the hemocytes of sensitive Ostrea edulis and resistant Crassostrea gigas. Infections were observed whichever the hemocytic types and the oyster species. The advantages and disadvantages of the system are discussed with regard to the study of recognition, entry, and survival mechanisms of the parasite.

Strategy for research and international cooperation in marine invertebrate pathology, immunology and genetics

During the last 10 years, marine invertebrate pathology has moved from morphological description and microscopic diagnosis of pathogens to molecular characterization of these pathogens and probebased diagnostics. Studies of host-pathogen interactions were undertaken to understand the immunity of molluscs and shrimps with a special new emphasis on immune gene characterization. Recently, genetic transformation has been considered for selecting resistant strains because of the numerous successes obtained with transgenic plants and vertebrates. The production of transgenic molluscs and shrimps, with genes or antisense sequences conferring resistance to specific pathogens, certainly constitutes a new priority for aquaculture. The quick development of research from pathology to immunology and genetics has been made possible partially by developing international cooperation to compensate for the limited manpower, on one hand inside the network of the pathologists, and on the other hand by removing barriers between topics. Regular meetings appear useful for regularly managing research in pathology-immunology-genetics of molluscs and shrimps, for analysing the strategy according to advances in similar fields related to other animal or plant groups, and for improving international cooperation between all scientists concerned from developing and developed countries.

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.

Development of new methodologies for diagnosis of infectious diseases in mollusc and shrimp aquaculture

Abstract The development of new methodologies for diagnosis of infectious diseases of molluscs and shrimps is related to progress in biotechnology and molecular biology. The different kinds of immunoreagents and nucleic-acid probes and the respective advantages and disadvantages of immunological and nucleic-acid hybridization-based methods are described and analysed in terms of developments and applications in marine invertebrates aquaculture.

Mussel (Mytilus edulis) treatment against the red copepod Mytilicola intestinalis

Abstract Mytilicola intestinalis , a copepod parasite of the gut of mussels, is endemic along European coasts. It has been responsible for heavy mortalities, especially in Mont Saint-Michels Bay (Northern Brittany) between 1982 and 1984. From laboratory screening of seven drugs, an organophosphorate Dichlorvos appears to be the most efficient. Dichlorvos treatment for 2 h at 30 mg/l concentration frees mussels from Mytilicola without any mortality. Later field trials showed an equal efficiency if the drug is used properly.