The Immune Cellular Effectors of Terrestrial Isopod Armadillidium vulgare: Meeting with Their Invaders, Wolbachia

Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10(3)Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001).The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals

Détection et caractérisation des bactéries intracellulaires associées au champignon ectomycorhizien Laccaria bicolor S238N

Texte intégral accessible uniquement aux membres de l'Université de LorraineLaccaria bicolor S238N is an ectomycorrhizal fungus used commercially in France for controlled mycorrhization. During the production of the inoculum in fermentor, bacterial contaminations occur recurrently. Fluorescent in situ hybridization associated with confocal microscopy has allowed to detect several types of intracellular bacteria in the pure cultures of the fungus. Most of these endobacteria belong to the Firmicutes. Some have been identified as Paenibacilli, and belong to the same species as bacteria isolated from a L. bicolor S238N culture in fermentor. In contrast, in non axenic mycelium samples such as mycorrhizas, endobacteria affiliated to the a-proteobacteria were detected. Since some observations suggest that the endobacteria sometimes colonize live fungal cells, it could be a case of bacterial endosymbiosis.Laccaria bicolor S238N est un champignon ectomycorhizien utilisé commercialement en France dans le cadre de la mycorhization contrôlée. Lors de la production de l'inoculum en fermenteur, des contaminations bactériennes apparaissent régulièrement. La technique d'hybridation in situ associée à la microscopie confocale a permis de détecter plusieurs types de bactéries intracellulaires dans les cultures pures du champignon. La majorité des endobactéries était affiliée aux Firmicutes. Certaines ont été identifiées comme des Paenibacilli appartenant à la même espèce que des bactéries isolées à partir d'une culture en fermenteur de L. bicolor S238N. En revanche, dans des échantillons de mycélium non axéniques comme les mycorhizes, les endobactéries détectées étaient majoritairement des a-protéobactéries. Comme les observations suggèrent que certaines endobactéries colonisent des cellules fongiques vivantes, il pourrait s'agir d'un cas d'endosymbiose bactérienne

Détection et caractérisation des bactéries intracellulaires associées au champignon ectomycorhizien Laccaria bicolor S238N

Laccaria bicolor S238N est un champignon ectomycorhizien utilisé commercialement en France dans le cadre de la mycorhization contrôlée. Lors de la production de l'inoculum en fermenteur, des contaminations bactériennes apparaissent régulièrement. La technique d'hybridation in situ associée à la microscopie confocale a permis de détecter plusieurs types de bactéries intracellulaires dans les cultures pures du champignon. La majorité des endobactéries était affiliée aux Firmicutes. Certaines ont été identifiées comme des Paenibacilli appartenant à la même espèce que des bactéries isolées à partir d'une culture en fermenteur de L. bicolor S238N. En revanche, dans des échantillons de mycélium non axéniques comme les mycorhizes, les endobactéries détectées étaient majoritairement des a-protéobactéries. Comme les observations suggèrent que certaines endobactéries colonisent des cellules fongiques vivantes, il pourrait s'agir d'un cas d'endosymbiose bactérienne.Laccaria bicolor S238N is an ectomycorrhizal fungus used commercially in France for controlled mycorrhization. During the production of the inoculum in fermentor, bacterial contaminations occur recurrently. Fluorescent in situ hybridization associated with confocal microscopy has allowed to detect several types of intracellular bacteria in the pure cultures of the fungus. Most of these endobacteria belong to the Firmicutes. Some have been identified as Paenibacilli, and belong to the same species as bacteria isolated from a L. bicolor S238N culture in fermentor. In contrast, in non axenic mycelium samples such as mycorrhizas, endobacteria affiliated to the a-proteobacteria were detected. Since some observations suggest that the endobacteria sometimes colonize live fungal cells, it could be a case of bacterial endosymbiosis.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Divergent evolution and clade-specific duplications of the Insulin-like Receptor in malacostracan crustaceans

International audienceThe Insulin-like Receptors (IRs) are an important protein family, represented by three members in vertebrates, two of which are well-known for their implication in metabolism (Insulin Receptor) and growth (IGF Receptor). In contrast, little is known about these receptors in invertebrates, in which a single gene generally exists except for a part of insects and other occasional species-specific duplications. In this study, we used publicly available sequences as well as de novo assembled transcriptomes to investigate the IR evolution in malacostracan crustaceans, animals in which the Insulin/IGF pathway is known to be implicated in sexual development through the androgenic gland hormone. We described the evolutionary divergence of malacostracan IRs compared to all the other metazoan sequences, including other pancrustaceans. We also demonstrated two well conserved duplications of IRs: one specific to the whole malacostracan class, another one specific to the decapod order. The potential implications for malacostracan biology are discussed

Iron Availability Modulates the Persistence of Legionella pneumophila in Complex Biofilms.

International audienceLegionella pneumophila is a pathogenic bacteria found in biofilms in freshwater. Iron is an essential nutrient for L. pneumophila growth. In this study, complex biofilms were developed using river water spiked with L. pneumophila, and the persistence of L. pneumophila in these complex biofilms was evaluated. In order to study the role of iron in the persistence of L. pneumophila, river water was supplied with either iron pyrophosphate or iron chelators (deferoxamine mesylate, DFX for ferric iron and dipyridyl, DIP for ferrous iron) to modulate iron availability. The addition of iron pyrophosphate and DFX did not markedly affect the persistence of L. pneumophila in the biofilms, whereas that of DIP had a beneficial effect. Since DIP specifically chelates ferrous iron, we hypothesized that DIP may protect L. pneumophila from the deleterious effects of ferrous iron. In conclusion, ferrous iron appears to be important for the persistence of L. pneumophila in complex biofilms. However, further studies are needed in order to obtain a better understanding of the role of ferrous iron in the behavior of this bacterium in the environment

Intra-amoeba multiplication induces chemotaxis and biofilm colonization and formation for Legionella.

Legionella pneumophila, a facultative intracellular bacterium, is the causative agent of legionellosis. In the environment this pathogenic bacterium colonizes the biofilms as well as amoebae, which provide a rich environment for the replication of Legionella. When seeded on pre-formed biofilms, L. pneumophila was able to establish and survive and was only found at the surface of the biofilms. Different phenotypes were observed when the L. pneumophila, used to implement pre-formed biofilms or to form mono-species biofilms, were cultivated in a laboratory culture broth or had grown intracellulary within the amoeba. Indeed, the bacteria, which developed within the amoeba, formed clusters when deposited on a solid surface. Moreover, our results demonstrate that multiplication inside the amoeba increased the capacity of L. pneumophila to produce polysaccharides and therefore enhanced its capacity to establish biofilms. Finally, it was shown that the clusters formed by L. pneumophila were probably related to the secretion of a chemotaxis molecular agent

The "soil microbial loop" is not always needed to explain protozoan stimulation of plants

Protozoa stimulate plant growth, but we do not completely understand the underlying mechanisms, and different hypotheses seek to explain this phenomenon. To test these hypotheses, we grew the grass Yorkshire fog (Holcus lanatus) in pots with soil, which contained either (1) no organisms but bacteria – or (2) bacteria and protozoa. Half of the pots received a glucose treatment so as to mimic an additional root exudation.We measured plant growth and plant nitrogen uptake, along with various microbial pools and processes that support plant growth. Protozoan presence significantly enhanced soil nitrogen mineralization, plant nitrogen uptake from organic nitrogen sources, plant nitrogen content, and plant growth. By contrast, we found no evidence that glucose addition, mimicking root exudation, increased soil nitrogen availability and plant nitrogen uptake. Moreover, although protozoan presence affected bacterial community structure, it did not affect the proportion of IAA-producing bacteria in the community or plant root morphology. These results refute the ''soil microbial loop'' hypotheses, which suggest that protozoan stimulation of plant growth results from complex interactions between plant roots, bacteria and protozoa. Our experiment thus favours the simple explanation that increased nitrogen availability is the key factor behind the positive protozoan effect on plant growth. To exploit natural resources in an efficient and environmentally friendly way, we need to understand in detail the functioning of ecosystems. This study stresses that to achieve this, it is still urgent, besides investigating intricate food-web and signal compound interactions, also to focus on the basic stoichiometric and energetic aspects of organisms. (Résumé d'auteur

Efficacy of dental unit waterlines disinfectants on a polymicrobial biofilm.

International audienceDue to their high surface-volume ratio, their laminar flow and frequent stagnation periods, dental unit waterlines (DUWL) foster the attachment of microorganisms and the development of biofilm, resulting in the continuous contamination of the outlet water from dental units; this contamination may be responsible for a potential risk of infection due to the exposure of patients and medical staff to droplet inhalation or splashed water. In this study, the anti-biofilm activity of three disinfectants recommended by dental unit manufacturers -Calbenium(©), Oxygenal 6(©) and Sterispray(©) - was evaluated. A dynamic model simulating DUWL conditions was developed and polymicrobial biofilms containing bacteria (Pseudomonas aeruginosa), fungi (Candida albicans) and Free Living Amoeba (FLA: Vermamoeba vermiformis) were allowed to form. The ability of disinfectants to reduce biofilm formation or to eradicate an already formed biofilm was evaluated. Results showed the various effects of the tested disinfectants according to their composition, concentration and the targeted species. V. vermiformis was resistant to disinfectants, regardless of the tested concentrations and the concentrations recommended by manufacturers were not the most appropriate. Results also showed that Calbenium(©) was the most effective disinfectant to reduce already formed biofilms; its maximum efficiency was observed from 0.5% on both P. aeruginosa and C. albicans compared to 2 and 3% respectively for Sterispray(©). The maximum efficiency of Oxygenal(©) was observed from 3% on P. aeruginosa but Oxygenal(©) was unable to totally eliminate C. albicans in the tested conditions, contrary to other disinfectants. Calbenium(©) was able to prevent biofilm formation efficiently even if it displayed no prophylactic activity against V. vermiformis. Overall, the FLA survival may contribute to maintaining other species. Finally the tested disinfectants were partially active against sessile microorganisms and more suitable concentrations could be used to increase their efficacy. Their use in a prophylactic rather than curative way should be recommended

Isopod ovary organisation (A) and inferred infection dynamics (B).

<p><b>A</b>: The germarium is composed of oogonia that mature and increase in diameter as they move away from the germarium in the course of ovary maturation The oocytes are encased within two sheets of follicle cells and a layer of connective tissue (dark gray). At spawning muscles compress the ovary to expel the oocytes through the oviduct, which is on the germarium side. <b>B</b>: We propose two non exclusive hypotheses could explain <i>Wolbachia</i> enrichment in the course of oocyte and ovary maturation (Immature, Mature I, Mature II stages): oocyte selection (oocytes uninfected by <i>Wolbachia</i> are preferentially destroyed by oosorption) and/or maturing oocytes acquire <i>Wolbachia</i> secondarily from somatic tissues through follicle cells.</p