Publications by authors named "Saulnier Denis"

32 Publications

Description of the unusual digestive tract of and the potential impact of infection.

PeerJ 2020 24;8:e9966. Epub 2020 Sep 24.

MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, Montpellier, France.

Background: Ephippidae fish are characterized by a discoid shape with a very small visceral cavity. Among them has a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due to infection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ in is still scarce. Therefore, the aims of this study are to characterize the GIT in non-infected juveniles to then investigate the impact of on this multifunctional organ.

Methods: In the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged by were analysed and compared to the previously established GIT reference.

Results: The overlapped shape of the GIT of is probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na/K ATPase pump. An identification key allowing the characterization of the GIT sections using histology is given. Secondly, individuals challenged with exhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.
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http://dx.doi.org/10.7717/peerj.9966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520087PMC
September 2020

Quorum Sensing Inhibitory and Antifouling Activities of New Bromotyrosine Metabolites from the Polynesian Sponge n. sp.

Mar Drugs 2020 May 21;18(5). Epub 2020 May 21.

IRD, Université de la Polynésie française, Ifremer, ILM, EIO, Papeete F-98713, French Polynesia.

Four new brominated tyrosine metabolites, aplyzanzines C-F (-), were isolated from the French Polynesian sponge n. sp., along with the two known 2-aminoimidazolic derivatives, purealidin A () and previously isolated, respectively, from the sponges and Verongula sp. Their structures were assigned based on the interpretation of their NMR and HRMS data. The compounds exhibited quorum sensing inhibition (QSi) and antifouling activities against several strains of bacteria and microalgae. To our knowledge, the QSi activity of this type of bromotyrosine metabolite is described here for the first time.
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http://dx.doi.org/10.3390/md18050272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281015PMC
May 2020

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing.

Vet Res 2020 May 7;51(1):60. Epub 2020 May 7.

Université Paris-Saclay, INRAE, UVSQ, VIM, 78350, Jouy-En-Josas, France.

Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys.
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http://dx.doi.org/10.1186/s13567-020-00782-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204230PMC
May 2020

Influence of temperature and pearl rotation on biomineralization in the pearl oyster, .

J Exp Biol 2018 09 21;221(Pt 18). Epub 2018 Sep 21.

Ifremer, UMR EIO 241, Labex Corail, Centre du Pacifique, BP 49, 98719 Taravao, Tahiti, French Polynesia.

The objective of this study was to observe the impact of temperature on pearl formation using an integrative approach describing the rotation of the pearls, the rate of nacre deposition, the thickness of the aragonite tablets and the biomineralizing potential of the pearl sac tissue though the expression level of some key genes. Fifty pearl oysters were grafted with magnetized nuclei to allow the rotation of the pearls to be described. Four months later, 32 of these pearl oysters were exposed to four temperatures (22, 26, 30 and 34°C) for 2 weeks. Results showed that the rotation speed differed according to the movement direction: pearls with axial movement had a significantly higher rotation speed than those with random movement. Pearl growth rate was influenced by temperature, with a maximum between 26 and 30°C but almost no growth at 34°C. Lastly, among the nine genes implicated in the biomineralization process, only expression was significantly modified by temperature. These results showed that the rotation speed of the pearls was not linked to pearl growth or to the expression profiles of biomineralizing genes targeted in this study. On the basis of our results, we consider that pearl rotation is a more complex process than formerly thought. Mechanisms involved could include a strong environmental forcing in immediate proximity to the pearl. Another implication of our findings is that, in the context of ocean warming, pearl growth and quality can be expected to decrease in pearl oysters exposed to temperatures above 30°C.
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http://dx.doi.org/10.1242/jeb.186858DOI Listing
September 2018

Influence of water temperature and food on the last stages of cultured pearl mineralization from the black-lip pearl oyster Pinctada margaritifera.

PLoS One 2018 5;13(3):e0193863. Epub 2018 Mar 5.

Ifremer, UMR 241 « Ecosystèmes Insulaires Océaniens », Labex Corail, Centre du Pacifique, Tahiti, French Polynesia.

Environmental parameters, such as food level and water temperature, have been shown to be major factors influencing pearl oyster shell growth and molecular mechanisms involved in this biomineralization process. The present study investigates the effect of food level (i.e., microalgal concentration) and water temperature, in laboratory controlled conditions, on the last stages of pearl mineralization in order to assess their impact on pearl quality. To this end, grafted pearl oysters were fed at different levels of food and subjected to different water temperatures one month prior to harvest to evaluate the effect of these factors on 1) pearl and shell deposition rate, 2) expression of genes involved in biomineralization in pearl sacs, 3) nacre ultrastructure (tablet thickness and number of tablets deposited per day) and 4) pearl quality traits. Our results revealed that high water temperature stimulates both shell and pearl deposition rates. However, low water temperature led to thinner nacre tablets, a lower number of tablets deposited per day and impacted pearl quality with better luster and fewer defects. Conversely, the two tested food level had no significant effects on shell and pearl growth, pearl nacre ultrastructure or pearl quality. However, one gene, Aspein, was significantly downregulated in high food levels. These results will be helpful for the pearl industry. A wise strategy to increase pearl quality would be to rear pearl oysters at a high water temperature to increase pearl growth and consequently pearl size; and to harvest pearls after a period of low water temperature to enhance luster and to reduce the number of defects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837120PMC
June 2018

Development of a duplex Taqman real-time PCR assay for rapid identification of Vibrio splendidus-related and V. aestuarianus strains from bacterial cultures.

J Microbiol Methods 2017 09 12;140:67-69. Epub 2017 Jul 12.

Laboratoire de Génétique et Pathologie des Mollusques Marins, SG2M-RBE Ifremer, av du Mus de Loup, 17390 La Tremblade, French Polynesia.

To enable the rapid and accurate identification of Vibrio splendidus-related and V. aestuarianus strains associated with Pacific cupped oyster Crassostrea gigas mortality, we developed a duplex Taqman real-time PCR assay and evaluated its efficacy. This technique proved to be rapid, sensitive, and specific and will be particularly valuable for epidemiologic studies.
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http://dx.doi.org/10.1016/j.mimet.2017.07.002DOI Listing
September 2017

Response of the pearl oyster Pinctada margaritifera to cadmium and chromium: Identification of molecular biomarkers.

Mar Pollut Bull 2017 May 18;118(1-2):420-426. Epub 2017 Mar 18.

Ifremer, UMR 241 EIO, UPF-ILM-IRD, Labex Corail, BP 49, 98719 Taravao, Tahiti, French Polynesia. Electronic address:

This study was designed to identify in the pearl oyster Pinctada margaritifera, used as a bio-accumulator, molecular biomarkers for the presence of heavy metals in the lagoon environment. Pearl oysters were exposed to 2 concentrations (1 and 10μgL) of cadmium (Cd) and chromium (Cr) compared to a control. Twelve target genes encoding proteins potentially involved in the response to heavy metal contamination with antioxidant, detoxification or apoptosis activities were selected. P. margaritifera accumulated Cd but not Cr, and mortality was related to the amount of Cd accumulated in tissues. In response to Cd-Cr contamination, metallothionein (MT) was significantly up-regulated by Cd-Cr at both concentrations, while 7 others (SOD, CAT, GPX, GSTO, GSTM, CASP, MDR) were down-regulated. Based on the development of these molecular tools, we propose that the pearl oyster, P. margaritifera, could be used as a sentinel species for heavy metal contamination in the lagoons of tropical ecosystems.
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http://dx.doi.org/10.1016/j.marpolbul.2017.03.012DOI Listing
May 2017

An updated assessment of spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B.

PeerJ 2017 5;5:e2856. Epub 2017 Jan 5.

PSL CRIOBE USR3278 CNRS-EPHE-UPVD, Labex CORAIL, Papetoai, Moorea, French Polynesia; Current affiliation:  UMR250/9220 ENTROPIE IRD-CNRS-UR, Labex CORAIL, Promenade Roger-Laroque, Noumea cedex, New Caledonia, France.

The adaptative bleaching hypothesis (ABH) states that, depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to "switch" or "shuffle" their algal symbionts), coral bleaching can lead to a change in the composition of their associated community and, thus, contribute to the coral's overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations and to detect low levels of coral-associated . Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional molecular methods, to assess the presence of low-abundance (background) spp. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional clade, among clades A-D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral- associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or "shuffle" different clades. Altogether our data suggest that corals from French Polynesia may favor a trade-off between optimizing symbioses with a specific clade(s), maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.
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http://dx.doi.org/10.7717/peerj.2856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289445PMC
January 2017

Effects of local Polynesian plants and algae on growth and expression of two immune-related genes in orbicular batfish (Platax orbicularis).

Fish Shellfish Immunol 2016 Nov 12;58:82-88. Epub 2016 Sep 12.

CRIOBE, USR3278-CNRS/EPHE/UPVD/PSL, BP1013 Papetoai, 98729 Moorea, French Polynesia; Laboratoire d'Excellence "CORAIL", 98729 Moorea, French Polynesia.

The emerging orbicular batfish (Platax orbicularis) aquaculture is the most important fish aquaculture industry in French Polynesia. However, bacterial infections are causing severe mortality episodes. Therefore, there is an urgent need to find an effective management solution. Besides the supplying difficulty and high costs of veterinary drugs in French Polynesia, batfish aquaculture takes place close to the coral reef, where use of synthetic persistent drugs should be restricted. Medicinal plants and bioactive algae are emerging as a cheaper and more sustainable alternative to chemical drugs. We have studied the effect of local Polynesian plants and the local opportunistic algae Asparagopsis taxiformis on batfish when orally administered. Weight gain and expression of two immune-related genes (lysozyme g - Lys G and transforming growth factor beta - TGF-β1) were studied to analyze immunostimulant activity of plants on P. orbicularis. Results showed that several plants increased Lys G and TGF-β1 expression on orbicular batfish after 2 and 3 weeks of oral administration. A. taxiformis was the plant displaying the most promising results, promoting a weight gain of 24% after 3 weeks of oral administration and significantly increasing the relative amount of both Lys G and TGF-β1 transcripts in kidney and spleen of P. orbicularis.
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http://dx.doi.org/10.1016/j.fsi.2016.09.011DOI Listing
November 2016

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system.

BMC Microbiol 2016 07 19;16(1):157. Epub 2016 Jul 19.

Ifremer, UMR 241 EIO, UPF-ILM-IRD, Labex Corail, B.P. 7004, 98719, Taravao, Tahiti, French Polynesia.

Background: Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology.

Results: Analyses of the bacterial community identified in water from BFT and "clear seawater" (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments.

Conclusion: This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.
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http://dx.doi.org/10.1186/s12866-016-0770-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4952143PMC
July 2016

Symbiodinium clades A and D differentially predispose Acropora cytherea to disease and Vibrio spp. colonization.

Ecol Evol 2016 01 9;6(2):560-72. Epub 2016 Jan 9.

USR3278 CRIOBE CNRS-EPHE-UPVDBP 1013 Papetoai Moorea 98729 Polynésie française; Laboratoire d'Excellence "CORAIL" 58 Avenue Paul Alduy Perpignan Cedex 66860 France.

Coral disease outbreaks have increased over the last three decades, but their causal agents remain mostly unclear (e.g., bacteria, viruses, fungi, protists). This study details a 14-month-long survey of coral colonies in which observations of the development of disease was observed in nearly half of the sampled colonies. A bimonthly qPCR method was used to quantitatively and qualitatively evaluate Symbiodinium assemblages of tagged colonies, and to detect the presence of Vibrio spp. Firstly, our data showed that predisposition to disease development in general, and, more specifically, infection by Vibrio spp. in Acropora cytherea depended on which clades of Symbiodinium were harbored. In both cases, harboring clade D rather than A was beneficial to the coral host. Secondly, the detection of Vibrio spp. in only colonies that developed disease strongly suggests opportunistic traits of the bacteria. Finally, even if sporadic cases of switching and probably shuffling were observed, this long-term survey does not suggest specific-clade recruitment in response to stressors. Altogether, our results demonstrate that the fitness of the coral holobiont depends on its initial consortium of Symbiodinium, which is distinct among colonies, rather than a temporary adaptation achieved through acquiring different Symbiodinium clades.
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http://dx.doi.org/10.1002/ece3.1895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729262PMC
January 2016

Identification of genes associated with shell color in the black-lipped pearl oyster, Pinctada margaritifera.

BMC Genomics 2015 Aug 1;16:568. Epub 2015 Aug 1.

Laboratoire d'Excellence "CORAIL", USR 3278 CNRS-CRIOBE- EPHE, Perpignan, France, Papetoai, Moorea, French Polynesia.

Background: Color polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.

Results: Out of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.

Conclusions: Our results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.
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http://dx.doi.org/10.1186/s12864-015-1776-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521380PMC
August 2015

Use of Natural Antimicrobial Peptides and Bacterial Biopolymers for Cultured Pearl Production.

Mar Drugs 2015 Jun 11;13(6):3732-44. Epub 2015 Jun 11.

AiMB. 17 Rue d'Ouessant, 29280 Plouzané, France.

Cultured pearls are the product of grafting and rearing of Pinctada margaritifera pearl oysters in their natural environment. Nucleus rejections and oyster mortality appear to result from bacterial infections or from an inappropriate grafting practice. To reduce the impact of bacterial infections, synthetic antibiotics have been applied during the grafting practice. However, the use of such antibiotics presents a number of problems associated with their incomplete biodegradability, limited efficacy in some cases, and an increased risk of selecting for antimicrobial resistant bacteria. We investigated the application of a marine antimicrobial peptide, tachyplesin, which is present in the Japanese horseshoe crab Tachypleus tridentatus, in combination with two marine bacterial exopolymers as alternative treatment agents. In field studies, the combination treatment resulted in a significant reduction in graft failures vs. untreated controls. The combination of tachyplesin (73 mg/L) with two bacterial exopolysaccharides (0.5% w/w) acting as filming agents, reduces graft-associated bacterial contamination. The survival data were similar to that reported for antibiotic treatments. These data suggest that non-antibiotic treatments of pearl oysters may provide an effective means of improving oyster survival following grafting procedures.
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http://dx.doi.org/10.3390/md13063732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483653PMC
June 2015

Rearing effect of biofloc on antioxidant and antimicrobial transcriptional response in Litopenaeus stylirostris shrimp facing an experimental sub-lethal hydrogen peroxide stress.

Fish Shellfish Immunol 2015 Aug 4;45(2):933-9. Epub 2015 Jun 4.

Ifremer, Centre Océanologique du Pacifique, Unité de recherche Resources Marines, B.P 7004, 98719, Taravao, French Polynesia; Ifremer, UMR 5244 IHPE, UPVD, CNRS, Université de Montpellier, F-34095, Montpellier, France.

This study compares the antioxidant and antimicrobial transcriptional expression of blue shrimps reared according to two different systems, BioFloc Technology (BFT) and Clear sea Water (CW) and their differential responses when facing an experimental sublethal hydrogen peroxide stress. After 30 days of rearing, juvenile shrimps were exposed to H2O2 stress at a concentration of 30 ppm during 6 h. The oxidative stress caused by H2O2 was examined in the digestive glands of the shrimp, in which antioxidant enzyme (AOE) and antimicrobial peptide (AMP) gene expression were analysed by quantitative real-time PCR. Results showed that rearing conditions did not affect the expression of genes encoding AOEs or AMPs. However, H2O2 stress induced a differential response in expression between shrimps from the two rearing treatments (BFT and CW). Comparative analysis of the expression profiles indicates that catalase transcripts were significantly upregulated by H2O2 stress for BFT shrimps while no change was observed for CW shrimps. In contrast, H2O2 caused down-regulation of superoxide dismutase and glutathione transferase transcripts and of the three AMP transcripts studied (penaeidin 2 and 3, and crustin) for CW shrimps, while no effect was observed on BFT shrimp transcript levels. These results suggested that BFT shrimps maintained antioxidant and AMP responses after stress and therefore can effectively protect their cells against oxidative stress, while CW shrimp immune competence seems to decrease after stress.
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http://dx.doi.org/10.1016/j.fsi.2015.05.041DOI Listing
August 2015

Factors other than metalloprotease are required for full virulence of French Vibrio tubiashii isolates in oyster larvae.

Microbiology (Reading) 2015 May 20;161(Pt 5):997-1007. Epub 2015 Feb 20.

IFREMER, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins Avenue de Mus de Loup, 17390 La Tremblade, France.

Vibrio tubiashii is a marine pathogen isolated from larval and juvenile bivalve molluscs that causes bacillary necrosis. Recent studies demonstrated the isolation of this species in a French experimental hatchery/nursery affecting Crassostrea gigas spat in 2007. Here, using larvae of C. gigas as an interaction model, we showed that the French V. tubiashii is virulent to larvae and can cause bacillary necrosis symptoms with an LD50 of about 2.3 × 10(3) c.f.u. ml(-1) after 24 h. Moreover, complete or gel permeation HPLC fractionated extracellular products (ECPs) of this strain appeared toxic to larvae. MS-MS analysis of the different ECP fractions revealed the existence of an extracellular metalloprotease and other suspected virulence factors. This observation is also supported by the expression level of some potential virulence factors. The overall results suggest that the pathology caused by the French V. tubiashii in C. gigas oysters is caused by a group of toxic factors and not only the metalloprotease.
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http://dx.doi.org/10.1099/mic.0.000058DOI Listing
May 2015

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

J Invertebr Pathol 2014 Nov 22;123:49-59. Epub 2014 Sep 22.

UMR 7266 CNRS-Université de La Rochelle, LIENSs, Equipe Approches Moléculaires, Environnement-Santé, Avenue Michel Crépeau, 17000 La Rochelle, France; Fédération de Recherche en Environnement et Développement Durable, FR CNRS 3097, Université de La Rochelle, France. Electronic address:

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.
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http://dx.doi.org/10.1016/j.jip.2014.09.006DOI Listing
November 2014

Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera.

PLoS One 2014 14;9(8):e103944. Epub 2014 Aug 14.

Ifremer, UMR 241 « Ecosystèmes Insulaires Océaniens », Labex Corail, Centre du Pacifique, Taravao, Tahiti, Polynésie Française.

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103944PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133174PMC
April 2015

First description of French V. tubiashii strains pathogenic to mollusk: I. Characterization of isolates and detection during mortality events.

J Invertebr Pathol 2014 Nov 9;123:38-48. Epub 2014 May 9.

Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities). Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities. These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas.
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http://dx.doi.org/10.1016/j.jip.2014.04.009DOI Listing
November 2014

First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes.

Fish Shellfish Immunol 2011 Dec 23;31(6):795-800. Epub 2011 Jul 23.

Littoral Environnement et Sociétés (LIENSs), UMR 6250, CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, F-17042 La Rochelle Cedex 01, France.

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine (L-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of L-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.
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http://dx.doi.org/10.1016/j.fsi.2011.07.016DOI Listing
December 2011

Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility.

Vet Res 2011 Feb 7;42:27. Epub 2011 Feb 7.

Ifremer (Institut Français de Recherche pour l'Exploitation de la Mer), Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France.

In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 μVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection.
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http://dx.doi.org/10.1186/1297-9716-42-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042938PMC
February 2011

Vibriosis induced by experimental cohabitation in Crassostrea gigas: evidence of early infection and down-expression of immune-related genes.

Fish Shellfish Immunol 2011 Feb 31;30(2):691-9. Epub 2010 Dec 31.

Laboratoire de Génétique et Pathologie, Ifremer, Av du Mus de Loup, 17390 La Tremblade, France.

The understanding of reciprocal interactions between Crassostrea gigas and Vibrio sp., whether these be virulent or avirulent, is vital for the development of methods to improve the health status of cultured oysters. We describe an original non-invasive experimental infection technique using cohabitation, designed to explore these interactions. Using real-time PCR techniques we examined the dynamics of virulent and avirulent Vibrio sp. in oyster hemolymph and tank seawater, and made a parallel study of the expression of four genes involved in oyster immune defense: Cg-BPI, Cg-EcSOD, Cg-IκB, Cg-TIMP. No mortality occurred in control animals, but oysters put in cohabitation for 2-48 h with animals previously infected by two Vibrio pathogens suffered mortalities from 2 to 16 days post-cohabitation. Our results show that virulent Vibrio infect healthy individuals after only 2 h of cohabitation, with values ranging from 4.5 x 10² to 2 x 10⁴ cells ml⁻¹ hemolymph. Simultaneously, an approximate ten-fold increase of the total Vibrio population was observed in control animals, with a 6.6-78.5-fold up-expression of targeted genes. In contrast, oysters exposed to harmful bacteria had mean expression levels strongly down-regulated by a factor of 9.2-29 (depending on the gene) compared with control animals. Although oysters were still found to be infected by virulent Vibrio after 6-48 h of cohabitation, no significant differences were noted when comparing levels of each transcript in control and infected oysters at the same sampling times during this period: the important differences were noted before 6 h cohabitation. Taken together, our data support (1) the hypothesis that virulent Vibrio disturbs the immune response of this invertebrate host both rapidly and significantly, although this occurs specifically during an early and transient period during the first 6 h of cohabitation challenge, and that (2) expression of targeted genes is not correlated with vibriosis resistance.
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http://dx.doi.org/10.1016/j.fsi.2010.12.017DOI Listing
February 2011

A large-scale epidemiological study to identify bacteria pathogenic to Pacific oyster Crassostrea gigas and correlation between virulence and metalloprotease-like activity.

Microb Ecol 2010 May 11;59(4):787-98. Epub 2009 Dec 11.

Laboratoire de Génétique et Pathologie, IFREMER, BP 33, av. du Mus de Loup, 17390, La Tremblade, France.

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.
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http://dx.doi.org/10.1007/s00248-009-9620-yDOI Listing
May 2010

Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity.

Environ Microbiol 2009 Aug 1;11(8):1959-70. Epub 2009 Apr 1.

Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, F-75015, Paris, France.

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.
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http://dx.doi.org/10.1111/j.1462-2920.2009.01918.xDOI Listing
August 2009

Real-time PCR assay for rapid detection and quantification of Vibrio aestuarianus in oyster and seawater: a useful tool for epidemiologic studies.

J Microbiol Methods 2009 May 2;77(2):191-7. Epub 2009 Feb 2.

Laboratoire de Génétique et Pathologie, IFREMER, av. du Mus de Loup, 17390 La Tremblade, France.

Because Vibrio aestuarianus is known to cause serious infections in Pacific oyster Crassostrea gigas, a real-time PCR assay was developed targeting the dnaJ gene of this bacterium. Only V. aestuarianus strains isolated from C. gigas mortality events in different geographic areas and the reference strain tested positive, whereas no amplification products was obtained with type strains belonging to 23 other species of Vibrio. Sensitivity and reproducibility of the method were assessed using either seawater or oyster homogenate samples spiked with one V. aestuarianus strain. All these samples were stored at -20 degrees C in order to mimic retrospective or grouped natural sample analysis without quantification bias due to prolonged freezing. Analysis of standard curves revealed excellent correlation values between light microscopy cell enumerations and PCR Threshold Cycle (Ct) values, and acceptable PCR reaction efficiencies for all type of samples. Quantification curves of both sample types were equivalent, with a detection level as low as 1.6 V. aestuarianus cells in the PCR reaction tube, corresponding to 1.6 x 10(2) cells ml(-1) and 1.6 x 10(2) cells mg(-1) in seawater and entire oyster samples, respectively, taking into account the dilution factor used for appropriate template DNA preparation. Comparison of PCR assay reproducibility according to the complexity of samples revealed that seawater samples gave more reproducible quantification measures than samples from oyster homogenate, with precision of measured Ct values inferior to 0.4 and 0.6 respectively at 99% confidence. Use of the real-time PCR assay allowed us to monitor V. aestuarianus load in oysters naturally infected with this pathogen. Furthermore, we were able to detect V. aestuarianus in samples of seawater in which oysters had been reared and in algal cultures used for feeding oysters. Because of the rapidity and reliability of the real-time PCR assay method used in this study, just a few hours are needed compared with the two days required using the classic culture method, this technique will be particularly valuable in mollusc pathology laboratories, for monitoring the source and course of infections by V. aestuarianus in pathogenesis and epidemiologic studies, as well as for designing appropriate prophylactic control measures.
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http://dx.doi.org/10.1016/j.mimet.2009.01.021DOI Listing
May 2009

Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus.

Appl Environ Microbiol 2008 Dec 3;74(23):7108-17. Epub 2008 Oct 3.

Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, Paris, France.

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Deltavsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Deltavsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Deltavsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.
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http://dx.doi.org/10.1128/AEM.01261-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592927PMC
December 2008

Correlation between detection of a plasmid and high-level virulence of Vibrio nigripulchritudo, a pathogen of the shrimp Litopenaeus stylirostris.

Appl Environ Microbiol 2008 May 21;74(10):3038-47. Epub 2008 Mar 21.

Laboratoire de Génétique et Pathologie, Ifremer, 17390 La Tremblade, France.

Vibrio nigripulchritudo, the etiological agent of Litopenaeus stylirostris summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (n = 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (n = 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in Vibrio shilonii, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed.
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http://dx.doi.org/10.1128/AEM.02680-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394918PMC
May 2008

Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg).

Aquat Toxicol 2007 Aug 12;84(1):92-102. Epub 2007 Jun 12.

Ifremer La Tremblade, Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France.

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.
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http://dx.doi.org/10.1016/j.aquatox.2007.06.002DOI Listing
August 2007

Combination of a pesticide exposure and a bacterial challenge: in vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg).

Aquat Toxicol 2007 Aug 12;84(1):92-102. Epub 2007 Jun 12.

Ifremer La Tremblade, Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France.

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.
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http://dx.doi.org/10.1016/j.aquatox.2007.06.002DOI Listing
August 2007

Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp (Litopenaeus stylirostris) in New Caledonia.

Syst Appl Microbiol 2006 Nov 18;29(7):570-80. Epub 2006 Jan 18.

IFREMER, Département Aquaculture en Nouvelle-Calédonie, BP 2059, 98846 Nouméa cedex, Nouvelle-Calédonie.

A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the "Summer Syndrome" from strains isolated from other contexts, especially the other shrimp vibriosis "Syndrome 93". These results highlight that the "Summer Syndrome" is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose.
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http://dx.doi.org/10.1016/j.syapm.2005.12.005DOI Listing
November 2006

Pathogenicity of Vibrio penaeicida for white shrimp Litopenaeus vannamei: a cysteine protease-like exotoxin as a virulence factor.

Dis Aquat Organ 2005 Nov;67(3):201-7

Centro de Investigaciones Biológicas del Noroeste, Apdo. Postal 128, Unidad de Patología Marina, La Paz, Baja California Sur, Mexico.

The pathogenicity of Vibrio penaeicida Strains KH-1 and AM101, their culture-free supernatant (CFS), and their protein fraction obtained by 40% of ammonium sulfate precipitation (PFs40) were assessed in experimental challenges against juvenile Litopenaeus vannamei. Live Vibrio cells, CFS, and PFs40 from the AM101 strain produced a significantly higher mortality (p < 0.05) compared to the KH-1 strain. Toxicity and median lethal doses (LD50) of Fast Protein Liquid Chromatography (FPLC) products were evaluated on L. vannamei. The first FPLC fraction sample (A) from PFs40 of the AM101 strain displayed LD50 values of 1.68 and 5.61 microg protein ind.(-1), respectively. The second FPLC process from Fraction A showed a peak (A1) also with toxic effects to shrimp. PFs40, Fraction A, and Peak A1 showed a 38.5 kDa molecular band (SDS-PAGE), with activity on a gelatin protease zymogram. The lethal effect of PFs40 and Fraction A was inhibited by Proteinase K, CuCl2, E-64, and heat (60 and 100 degrees C) treatments, but was not inhibited by EDTA-Na2, aprotinin, and soy trypsin treatments. These results and the zymogram inhibition test suggest the presence of a cysteine protease-like proteinaceous exotoxin as a dominant protease, secreted by V. penaeicida Strain AM101.
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http://dx.doi.org/10.3354/dao067201DOI Listing
November 2005