Dominique Ansquer

Bacterial flora associated with a trophic chain consisting of microalgae, rotifers and turbot larvae: Influence of bacteria on larval survival

Abstract In order to increase knowledge about the bacteria in cultures and their influence on the culture performance, bacterial analyses and physical and chemical measures were carried out for different biotopes of two similar trophic chains (A and B). These trophic chains consisted of microalgae ( Pavlova lutheri or Platymonas suecica ), rotifers ( Brachionus plicatilis ) and turbot larvae ( Scophthalmus maximus ). About 10 7 bacteria/ml were regularly observed by direct counts in the culture water of the algae and rotifers, and from 10 4 to 10 5 bacteria/individual in rotifers and turbot larvae. In general, the composition of the bacterial flora presented some elements of stability, since some taxa were found in successive samples of the same biotope. On the other hand, there were very few similarities between the bacterial flora of different biotopes, except between the gut of the rotifers and their medium. Nevertheless, among the Vibrionaceae, which were the chief constituent of the bacterial flora in the gut of the turbot larvae, some were probably introduced by the rotifers. The numbers of Vibrionaceae in both rotifer cultures increased over the course of the experiments. The turbot larvae B refused to ingest prey which contained large quantities of bacteria and died on days 7 and 8, whereas 40–60% of larvae A remained alive at day 10. The conclusions drawn from this experiment can be applied to improving the survival and growth of larvae as well as to developing sanitary procedures for hatcheries.

Experimental infection models for shrimp vibriosis studies: a review

Vibrio species have become a major source of concern for shrimp culture because of their close association with low survival rates in hatcheries or growout ponds. New shrimp pathogens belonging to the Vibrio genus have been described although their virulence is not yet fully understood. Indeed, they may act as opportunistic agents in secondary infections or be true pathogens. This review presents the usefulness of infection models with vibriosis pathogens for pathogenicity experiments, testing of curative or prophylactic treatments and the study of host-factors influencing bacterial virulence. Furthermore, some guidelines for experimental trials are given to evaluate the in vivo virulence of Vibrio isolates.

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits.

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin’, is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.

Vibrio nigripulchritudo monitoring and strain dynamics in shrimp pond sediments.

Aims:  A description of bacterial pathogens in shrimp ponds is necessary to understand their pathogenesis. Vibrio nigripulchritudo was shown to contain saprophytic and pathogenic strains among New Caledonian isolates. We established a method to map the development of V. nigripulchritudo in pond sediments at three different genetic levels: the species level, then at the pathogenic cluster level and finally at the plasmid level, present only in all highly pathogenic isolates.

Ontogeny of osmoregulation in the Pacific blue shrimp, Litopenaeus stylirostris (Decapoda, Penaeidae): Deciphering the role of the Na(+)/K(+)-ATPase.

The role of the main ion transporting enzyme Na+/K+-ATPase in osmoregulation processes was investigated in Litopenaeus stylirostris. The development and localization of the osmoregulation sites were studied during ontogenesis by immunodetection of Na(+)K(+)-ATPase using monoclonal antibodies and transmission electron microscopy (TEM). Osmoregulation sites were identified as the pleurae and branchiostegites in the zoeae and mysis stages. In the subsequent post-metamorphic stages the osmoregulatory function was mainly located in the epipodites and branchiostegites and osmotic regulation was later detected in the gills. The presence of ionocytes and microvilli in these tissues confirmed their role in ionic processes. The complete open reading frame of the mRNA coding for the α-subunit of Na+K+-ATPase was characterized in L. stylirostris. The resulting 3092-bp cDNA (LsNKA) encodes a putative 1011-amino-acid protein with a predicted molecular mass of 112.3kDa. The inferred amino acid sequence revealed that the putative protein possesses the main structural characteristics of the Na+K+-ATPase α-subunits. Quantitative RT-PCR analyses indicated that LsNKA transcripts did not significantly vary between the different developmental stages. The number of transcripts was about 2.5-fold higher in the epipodites and gills than in any other tissues tested in juveniles. A reverse genetic approach was finally implemented to study the role of LsNKA in vivo. Knockdown of LsNKA expression by gene-specific dsRNA injection led to an increase of shrimp mortality following an abrupt salinity change compared to control animals. These data strongly suggest that LsNKA plays an important role in osmoregulation when the shrimp are challenged by changing salinities.

Nigritoxin is a bacterial toxin for crustaceans and insects

The Tetraconata (Pancrustacea) concept proposes that insects are more closely related to aquatic crustaceans than to terrestrial centipedes or millipedes. The question therefore arises whether insects have kept crustacean-specific genetic traits that could be targeted by specific toxins. Here we show that a toxin (nigritoxin), originally identified in a bacterial pathogen of shrimp, is lethal for organisms within the Tetraconata and non-toxic to other animals. X-ray crystallography reveals that nigritoxin possesses a new protein fold of the α/β type. The nigritoxin N-terminal domain is essential for cellular translocation and likely encodes specificity for Tetraconata. Once internalized by eukaryotic cells, nigritoxin induces apoptotic cell death through structural features that are localized in the C-terminal domain of the protein. We propose that nigritoxin will be an effective means to identify a Tetraconata evolutionarily conserved pathway and speculate that nigritoxin holds promise as an insecticidal protein.The Tetraconata concept suggests that insects and crustaceans may share evolutionarily conserved pathways. Here, the authors describe the animal tropism and structure-function relationship of nigritoxin, showing that this protein is lethal for insects and crustaceans but harmless to other animals.