A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors.

The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase chain reaction with 16S specific primers and probes. A multiple-locus variable-number tandem-repeat analysis (VNTR) was conducted on all isolates based on a multiplex PCR-VNTR with a set of primer pairs designed from the V. tasmaniensis LGP32 genome. Preliminary validation of the primers on a set of collection strains from the V. splendidus clade confirmed that the former V. splendidus-related LGP32 and relative strains were related to V. tasmaniensis rather than to the type strain V. splendidus LMG 4042. The VNTR analysis was then successfully conducted on 335 isolates which led to the characterization of 87 different profiles. Our results showed that (1) the high diversity of VNTR did not enlighten significant correlation between a specific pattern and the origin of collected samples. However, populations isolated from animal samples tend to differ from those of the background environment; (2) oyster mortality events could not be linked to the clonal proliferation of a particular VNTR type. However, few different patterns seemed successively associated with samples collected during peaks of oyster's mortality. (3) Finally, no correlation could be seen between specific VNTR patterns and sequence phylogeny of the virulence factors vsm and ompU that were detected among strains isolated during as well as outside mortality events. These results, combined with incongruence observed between the ompU and vsm phylogenetic trees, suggested both large diffusion of strains and massive lateral gene transfer within the V. splendidus clade.

Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities: from RNA-seq to candidate genes.

The black-chinned tilapia Sarotherodon melanotheron heudelotii is an ecologically appealing model as it shows exceptional adaptive capacities, especially with regard to salinity. In spite of this, this species is devoid of genomic resources, which impedes the understanding of such remarkable features. De novo assembly of transcript sequences produced by next-generation sequencing technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. It also facilitates the development of quantitative real-time PCR (qPCR) assays for analysing gene expression under different environmental conditions. Nevertheless, obtaining accurate and reliable qPCR results from such data requires a number of validations prior to interpretation. The transcriptome of S. melanotheron was sequenced to discover transcripts potentially involved in the plasticity of male reproduction in response to salinity variations. A set of 54 candidate and reference genes was selected through a digital gene expression (DGE) approach, and a de novo qPCR assay using these genes was validated for further detailed expression analyses. A user-friendly web interface was created for easy handling of the sequence data. This sequence collection represents a major transcriptomic resource for S. melanotheron and will provide a useful tool for functional genomics and genetics studies.

Spatio-temporal analysis of cyprinid herpesvirus 3 genetic diversity at a local scale.

Cyprinid herpesvirus 3 (CyHV-3), the causative agent of koi herpesvirus disease, is a major threat for carp populations in many countries worldwide, including Indonesia. It has been shown that many genotypes circulate worldwide, all highly related to one of the two known lineages U/I and J. In this study, we evaluated the spatial and temporal distribution of CyHV-3 strains in a small enzootic area, the lake of Cirata (West Java, Indonesia). Of the 365 samples analysed, from clinical or asymptomatic fish, 244 were found positive for CyHV-3, suggesting a high occurrence of the virus. Genotyping of these viral specimens with a range of molecular markers revealed the presence of numerous haplotypes in the host population, all related to the J lineage. In single individuals, mixed-genotype infections occurred at high frequency. The present results demonstrate that polymorphic molecular markers are suitable to monitor the genetic evolution of a viral population in an enzootic area.

First evidence of the activation of Cg-timp, an immune response component of Pacific oysters, through a damage-associated molecular pattern pathway.

In a previous work, we characterized a Crassostrea gigas cDNA (Cg-timp) encoding a protein which presents all the features of vertebrate tissue inhibitor of metalloproteinase (TIMP). The expression pattern of this gene led us to propose that Cg-timp is an important factor in oyster wound healing and defense mechanisms. Here we describe the analysis of Cg-timp expression in oysters challenged by live or dead bacteria as well as by bacterial secretory/excretory products and metalloproteinase. Surprisingly, bacterial secretory/excretory products activate Cg-timp gene expression whereas heat-inactivated ones do not. To address the question of the signal transduction pathway involved in Cg-timp gene activation, we isolated and sequenced Cg-timp promoter and upstream region. A 1-kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements and notably three NF-kappaB binding sites. The potential involvement of these motifs in Cg-timp gene regulation is discussed.

Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to alpha- and beta-Proteobacteria.

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.

Carotenoid and retinoid transport to fish oocytes and eggs: what is the role of retinol binding protein?

Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehydroretinol and retinyl-esters) that are utilized during embryonic development, after fertilization. The carotenoids (mainly astaxanthins) are transported in the plasma by the low density lipoproteins, high density lipoproteins, and very high density lipoproteins (VHDL) and were found to be associated also with serum albumin. Retinals were found to be associated vitellogenin (VTG), a component of the plasma VHDL fraction that is internalized by oocytes during vitellogenesis. However, the transport of retinols and retinyl-esters that were located in the oil droplet fraction of homogenized eggs, has yet to be elucidated. Retinols are more abundant in freshwater fish eggs than in eggs of marine fish species. Since retinol is transported in the plasma of vertebrates in association with retinol binding protein (RBP), recent studies on the molecular characterization and expression sites of RBP, could contribute to determining the involvement of RBP in transporting retinol to developing oocytes in vertebrates.Recently, results from our laboratory show that RBP mRNA levels in the liver and RBP plasma levels did not significantly change with the onset and during vitellogenesis in the Rainbow trout. These results were in contrast with a dramatic elevation in the mRNA levels of VTG in the liver and an increase in VTG plasma levels that was observed in the same females. Moreover, 17beta-estradiol treatment of immature fish, resulted in relatively lower mRNA levels of RBP in the liver, concomitantly with an increase in the level of VTG transcripts and the appearance of VTG in the plasma of treated fish. In addition, RBP was localized in the cytosol of ovulated oocytes. These results for Rainbow trout are similar to those reported for the chicken but differ from those of Xenopus, where an increase in RBP mRNA was reported in the liver and higher levels of retinal and retinol were found in the plasma of 17beta-estradiol treated animals. The results, reported here for the first time in Rainbow trout, showing RBP transcripts in the ovary, oviduct (the ovarian tissue adjacent to the gonopore) and oocytes, suggest a modulating role for RBP in follicular development, as has been suggested for the bovine ovary.

Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida.

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.

Inhibition of de novo synthesis of a jelly layer precursor protein by crustacean hyperglycemic hormone family peptides and posttranscriptional regulation by sinus gland extracts in Penaeus semisulcatus ovaries.

Mature penaeid oocytes possess extracellular cortical rods (CR) that contain precursor proteins of the jelly layer (JL) that forms a protective layer around eggs immediately after spawning and dissipates following the assembly of the hatching envelope. The temporal pattern of protein synthesis and mRNA expression of a jelly layer precursor protein in Penaeus semisulcatus ovaries was followed during vitellogenesis, and the regulation by sinus gland extracts (SGE) and crustacean hyperglycemic hormone (CHH) family peptides was evaluated. An approximately 33-kDa jelly layer precursor protein was previously identified in ovaries, CR, and JL and was named shrimp ovarian peritrophin-like protein (SOP), because its deduced amino acid sequence shows structural similarities to insect peritrophins. SOP was synthesized in ovarian explant fragments that were removed from vitellogenic ovaries and incubated in vitro, but synthesis was not detected in explants that were collected from previtellogenic ovaries. SOP transcripts were detected in all stages of ovarian development, but were more abundant in previtellogenic ovaries than in other stages. De novo synthesis of SOP was inhibited by P. semisulcatus SGE and by CHH family peptides that were purified from P. japonicus sinus glands. Sinus gland extracts, however, did not affect the steady state levels of SOP transcripts at any stage of ovarian development. These results suggest that SGE regulate SOP synthesis at the posttranscriptional level.

Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia.

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 10(4) CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the Chelex method could be used to detect fewer than 20 cultured Vibrio cells in sea-water or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.