Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
ISSN: 0168-6496

hal-01682106
https://hal.univ-antilles.fr/hal-01682106
DOI : 10.1111/1574-6941.12366

Éditeur(s) : HAL CCSD Inter-Research
Résumé : International audience
Sphaeroma serratum is a marine isopod species that inhabits seashores from Europe to West Africa. The individuals live under stones in direct contact with reduced sediments and harbour a diverse bacterial community on the cuticle of their pleopods. We investigated the diversity of these epibiotic bacteria on male (pubescent and senescent) and female specimens with electron microscopic obser vations and molecular tools. The microbial community of S. serratum was shown to be composed of at least 5 bacterial morphotypes observed on the pleopodal cuticle in all male specimens. Using fluorescence in situ hybridization, we identified 5 major phylogenetic groups (α-, β-, γ- and δ-Proteobacteria and Archaea) whereas denaturing gradient gel electro - phoresis (DGGE) of PCR-amplified 16S rRNA gene fragments of epibiotic bacteria revealed 50 bands. The bacterial community associated with S. serratum seems more diverse than in other marine crustaceans, such as Rimicaris. The relative diversity of this bacterial community was also studied in relation to the molt cycle. The comparison of DGGE band patterns of several individuals from female, pubescent male and senescent male groups revealed that the bacterial community diversity was dependent on the sex and the age of the individuals and more generally on the molt status.
Marine Ecology Progress Series

hal-00743743
https://hal.univ-antilles.fr/hal-00743743
DOI : 10.3354/meps09711

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
The bivalve Codakia orbicularis, hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22- m-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.
ISSN: 0099-2240

hal-00750419
https://hal.univ-antilles.fr/hal-00750419
DOI : 10.1128/AEM.02659-08

Éditeur(s) : Wiley-blackwell Publishing, Inc
Résumé : Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 mu M Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. spiendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.
Environmental Microbiology (1462-2912) (Wiley-blackwell Publishing, Inc), 2010-04 , Vol. 12 , N. 4 , P. 951-963

Droits : 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
http://archimer.ifremer.fr/doc/00003/11421/8044.pdf
DOI:10.1111/j.1462-2920.2009.02138.x
http://archimer.ifremer.fr/doc/00003/11421/

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
Until now, the culture of sulphur-oxidizing bacterial symbionts associated with marine invertebrates remains impossible. Therefore, few studies focused on symbiont's physiology under stress conditions. In this study, we carried out a comparative experiment based on two different species of lucinid bivalves (Codakia orbiculata and Lucina pensylvanica) under comparable stress factors. The bivalves were starved for 6months in sulphide-free filtered seawater. For C.orbiculata only, starved individuals were then put back to the field, in natural sediment. We used in situ hybridization, flow cytometry and X-ray fluorescence to characterize the symbiont population hosted in the gills of both species. In L.pensylvanica, no decrease in symbiont abundance was observed throughout the starvation experiment, whereas elemental sulphur slowly decreased to zero after 3months of starvation. Conversely, in C.orbiculata, symbiont abundance within bacteriocytes decreased rapidly and sulphur from symbionts disappeared during the first weeks of the experiment. The modifications of the cellular characteristics (SSC - relative cell size and FL1 - genomic content) of the symbiotic populations along starvation were not comparable between species. Return to the sediment of starved C.orbiculata individuals led to a rapid (2-4weeks) recovery of symbiotic cellular characteristics, comparable with unstressed symbionts. These results suggest that endosymbiotic population regulation is host-species-dependent in lucinids.
ISSN: 0168-6496

hal-01545911
https://hal.archives-ouvertes.fr/hal-01545911
DOI : 10.1111/1574-6941.12366

Éditeur(s) : HAL CCSD Landes Bioscience
Résumé : International audience
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Autophagy

hal-00735751
https://hal.archives-ouvertes.fr/hal-00735751
PUBMED : 22966490

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
In marine invertebrates that acquire their symbionts from the environment, these are generally only taken up during early developmental stages. In the symbiosis between lucinid clams and their intracellular sulfur-oxidizing bacteria, it has been shown that the juveniles acquire their symbionts from an environmental stock of free-living symbiont forms, but it is not known if adult clams are still competent to take up symbiotic bacteria from the environment. In this study, we investigated symbiont acquisition in adult specimens of the lucinid clam Codakia orbiculata, using transmission electron microscopy, fluorescence in situ hybridization, immunohistochemistry and PCR. We show here that adults that had no detectable symbionts after starvation in aquaria for 6 months, rapidly reacquired symbionts within days after being returned to their natural environments in the field. Control specimens that were starved and then exposed to seawater aquaria with sulfide did not reacquire symbionts. This indicates that the reacquisition of symbionts in the starved clams returned to the field was not caused by high division rates of a small pool of remaining symbionts that we were not able to detect with the methods used here. Immunohistochemistry with an antibody against actin, a protein involved in the phagocytosis of intracellular bacteria, showed that actin was expressed at the apical ends of the gill cells that took up symbionts, providing further evidence that the symbionts were acquired from the environment. Interestingly, actin expression was also observed in symbiontcontaining cells of untreated lucinids freshly collected from the environment, indicating that symbiont acquisition from the environment occurs continuously in these clams throughout their lifetime.
ISSN: 1462-2912

hal-00743019
https://hal.univ-antilles.fr/hal-00743019
DOI : 10.1111/j.1462-2920.2012.02748.x

Éditeur(s) : HAL CCSD EDP Sciences
Résumé : Cette communication présente les principaux résultats du projet ExtraFlo sur l'estimation des pluies et crues extrêmes. A partir d'un jeu de données conséquent de plusieurs milliers de stations, une procédure d'inter-comparaison a permis d'évaluer les performances d'une trentaine d'implémentations différentes issues de différentes combinaisons (modèle probabiliste, méthode d'estimation, cadre local / régional / mixte local et régional). Le projet ExtraFlo a permis de mettre en évidence le manque de robustesse des approches basées sur le traitement statistique d'une série de quelques dizaines d'années d'observation. Des alternatives intéressantes existent, basées sur l'exploitation des records de pluie et de débit à l'échelle d'une région, de la collecte de données historiques quand cela est possible, de méthodes de simulation basées sur le couplage d'un générateur d'averses avec un modèle hydrologique pour la transformation des pluies en débit, ou des traces morphologiques laissées par les crues anciennes.
ISSN: 0018-6368

hal-01054313
https://hal.archives-ouvertes.fr/hal-01054313
DOI : 10.1051/1hb/2014010

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 _m in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host.
ISSN: 0099-2240

hal-00716079
https://hal.univ-antilles.fr/hal-00716079
DOI : 10.1128/AEM.01683-06