The shutting down of the insulin pathway: a developmental window for Wolbachia load and feminization.

Using the isopod Armadillidium vulgare as a case study, we review the significance of the "bacterial dosage model", which connects the expression of the extended phenotype to the rise of the Wolbachia load. In isopods, the Insulin-like Androgenic Gland hormone (IAG) induces male differentiation: Wolbachia feminizes males through insulin resistance, presumably through defunct insulin receptors. This should prevent an autocrine development of the androgenic glands so that females differentiate instead: feminization should translate as IAG silencing and increased Wolbachia load in the same developmental window. In line with the autocrine model, uninfected males expressed IAG from the first larval stage on, long before the androgenic gland primordia begin to differentiate, and exponentially throughout development. In contrast in infected males, expression fully stopped at stage 4 (juvenile), when male differentiation begins. This co-occurred with the only significant rise in the Wolbachia load throughout the life-stages. Concurrently, the raw expression of the bacterial Secretion Systems co-increased, but they were not over-expressed relative to the number of bacteria. The isopod model leads to formulate the "bacterial dosage model" throughout extended phenotypes as the conjunction between bacterial load as the mode of action, timing of multiplication (pre/post-zygotic), and site of action (soma vs. germen).

Hygroregulation, a key ability for eusocial insects: Native Western European honeybees as a case study.

Sociality has brought many advantages to various hymenoptera species, including their ability of regulating physical factors in their nest (e.g., temperature). Although less studied, humidity is known to be important for egg, larval and pupal development, and also for nectar concentration. Two subspecies of Apis mellifera of the M evolutionary lineage were used as models to test the ability of a superorganism (i.e. honeybee colony) to regulate the humidity in its nest (i.e. "hygroregulation hypothesis") in four conservation centers: two in France (A. m. mellifera) and two in Portugal (A. m. iberiensis). We investigated the ability of both subspecies to regulate the humidity in hives daily, but also during the seasons for one complete year. Our data and statistical analysis demonstrated the capacity of the bees to regulate humidity in their hive, regardless of the day, season or subspecies. Furthermore, the study showed that humidity in beehives is stable even during winter, when brood is absent, and when temperature is known to be less stable in the beehives. These results suggest that humidity is important for honeybees at every life stage, maybe because of the 'imprint' of the evolutionary history of this hymenopteran lineage.

Occurrence and diversity of both bacterial and fungal communities in dental unit waterlines subjected to disinfectants.

Chemical disinfectants are widely advocated to reduce the microbial contamination in dental unit waterlines (DUWL). However, until now their efficacy has been poorly examined after long-term application. In this study, through quantitative PCR and high-throughput sequencing, both bacterial and fungal communities were profiled from 8- to 12-year-old DUWL treated with disinfectants commonly used by European dentists. Water was collected from the tap water supplying units to the output exposure point of the turbine handpiece following a stagnation period and dental care activity. Results showed that (i) the unit itself is the principal source of microbial contamination and (ii) water stagnation, DU maintenance practices and quality of water supplying DU appeared as parameters driving the water quality. Despite disinfecting treatment combined to flushing process, the microbial contamination remained relevant in the studied output water, in association with a high bacterial and fungal diversity. The occurrence of potentially pathogenic microorganisms in these treated DUWL demonstrated a potential infectious risk for both patients and dental staff. A disinfectant shock before a prolonged stagnation period could limit the microbial proliferation inside DUWL. Necessity to proceed to regular water quality control of DUWL was highlighted.

Host origin and tissue microhabitat shaping the microbiota of the terrestrial isopod Armadillidium vulgare.

We present the first in-depth investigation of the host-associated microbiota of the terrestrial isopod crustacean Armadillidium vulgare. This species is an important decomposer of organic matter in terrestrial ecosystems and a major model organism for arthropod-Wolbachia symbioses due to its well-characterized association with feminizing Wolbachia 16S rRNA gene pyrotags were used to characterize its bacterial microbiota at multiple levels: (i) in individuals from laboratory lineages and field populations and (ii) in various host tissues. This integrative approach allowed us to reveal an unexpectedly high bacterial diversity, placing this species in the same league as termites in terms of symbiotic diversity. Interestingly, both animal groups belong to the same ecological guild in terrestrial ecosystems. While Wolbachia represented the predominant taxon in infected individuals, it was not the only major player. Together, the most abundant taxa represented a large scope of symbiotic interactions, including bacterial pathogens, a reproductive parasite (Wolbachia) and potential nutritional symbionts. Furthermore, we demonstrate that individuals from different populations harboured distinct bacterial communities, indicating a strong link between the host-associated microbiota and environmental bacteria, possibly due to terrestrial isopod nutritional ecology. Overall, this work highlights the need for more studies of host-microbiota interactions and bacterial diversity in non-insect arthropods.

Characterization of biofilm formation in natural water subjected to low-frequency electromagnetic fields.

Electromagnetic field (EMF) treatment has proven to be effective against mineral scaling in water systems. Therefore, it should be assessed for the treatment of other deposits such as biofilms. In this study, a commercial device producing low-frequency EMF (1-10 kHz) was applied to a reactor fed with natural water for 45 days. The treatment promoted the concentration of microorganisms in suspension and limited the amount of sessile microorganisms in the biofilm, as determined by the measurement of total DNA, qPCR and microscopy. The structure of the bacterial community was assessed by t-RFLP and pyrosequencing analysis. The results showed that EMF treatment affected both planktonic and sessile community composition. EMFs were responsible for a shift in classes of Proteobacteria during development of the biofilm. It may be speculated that the EMF treatment affected particle solubility and/or microorganism hydration. This study indicated that EMFs modulated biofilm formation in natural water.

Molecular tools to detect anatoxin-a genes in aquatic ecosystems: Toward a new nested PCR-based method.

Over the last few decades, cyanobacterial mass occurrence has become a recurrent feature of aquatic ecosystems. This has led to ecosystem exposure and health hazards associated with cyanotoxin production. The neurotoxin anatoxin-a and its homologs can be synthesized by benthic cyanobacterial species in lotic systems, but also by planktonic lacustrine species such as Dolichospermum (also known as Anabaena). However, only a few studies have focused on anatoxin-a occurrence and its biosynthesis genes in freshwater lakes. The initial aim of this study was to evaluate the molecular tools available in the literature to detect anatoxin-a biosynthesis genes in lacustrine environments. Having tested different sets of PCR primers, we found that that some sets of primers, such as anxC, were too specific and did not amplify anatoxin-a biosynthesis genes in all producing strains. On the other hand, some sets of primers, such as atxoa, seemed not to be specific enough, amplifying numerous non-specific bands in environmental samples, especially those from sediments. Furthermore, anaC and anaF amplification exhibited different band intensities during electrophoresis, suggesting a high variation in number of gene copies between samples. As a result, we proposed a new nested PCR-based method which considerably improved the amplification of the anaC gene in our environmental samples, eliminating non-specific bands and weak detections. Using this tool, our study also highlighted that anatoxin-a genes are widely distributed throughout freshwater lakes. This suggests the need for further ecological investigations into anatoxin-a in these ecosystems.

Pyrosequencing analysis of bacterial diversity in dental unit waterlines.

Some infections cases due to exposure to output water from dental unit waterlines (DUWL) have been reported in the literature. However, this type of healthcare-associated risk has remained unclear and up until now the overall bacterial composition of DUWL has been poorly documented. In this study, 454 high-throughput pyrosequencing was used to investigate the bacterial community in seven dental offices (N = 7) and to identify potential bacterial pathogenic sequences. Dental unit waters (DUW) were collected from the tap water supplying units (Incoming Water; IW) to the output exposure point of the turbine handpiece (Output water; OW) following a stagnation period (OWS), and immediately after the last patient of the sampling day (OWA). A high bacterial diversity was revealed in DUW with 394 operational taxonomic units detected at the genus level. In addition to the inter-unit variability observed, results showed increased total bacterial cell concentration and shifts in bacterial community composition and abundance at the genus level, mainly within the Gamma- and Alpha-Proteobacteria class, as water circulated in the dental unit (DU). Results showed that 96.7%, 96.8% and 97.4% of the total sequences from IW, OWS and OWA respectively were common to the 3 defined water groups, thereby highlighting a common core microbiome. Results also suggested that stagnation and DU maintenance practices were critical to composition of the bacterial community. The presence of potentially pathogenic genera was detected, including Pseudomonas and Legionella spp. Emerging and opportunistic pathogenic genera such as Mycobacterium, Propionibacterium and Stenotrophomonas were likewise recovered in DUW. For the first time, an exhaustive evaluation of the bacterial communities present in DUW was performed taking into account the circulation of water within the DU. This study highlights an ignored diversity of the DUWL bacterial community. Our findings also contribute to a better appreciation of the potential infectious risk associated with dental care and suggest the importance of better managing microbial quality in DUW.

Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses.

Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.

Influence of changing plant food sources on the gut microbiota of saltmarsh detritivores.

Changes in agricultural land-use of saltmarshes along the German North Sea coast have favoured the succession of the marsh grass Elytrigia atherica over the long-established Spartina anglica. Consequently, E. atherica represents a potential food source of increasing importance for plant-feeding soil detritivores. Considering the importance of this ecological guild for decomposition processes and nutrient cycling, we focussed on two sympatric saltmarsh soil macrodetritivores and their associated gut microbiota to investigate how the digestive processes of these species may be affected by changing plant food sources. Using genetic fingerprints of partial 16S rRNA gene sequences, we analysed composition and diversity of the bacterial gut community in a diplopod and an amphipod crustacean in relation to different feeding regimes representing the natural vegetation changes. Effects of syntopy on the host-specific gut microbiota were also taken into account by feeding the two detritivore species either independently or on the same plant sample. Bacterial community composition was influenced by both the host species and the available plant food sources, but the latter had a stronger effect on microbial community structure. Furthermore, bacterial diversity was highest after feeding on a mixture of both plant species, regardless of the host species. The gut microbiota of these two detritivores can thus be expected to change along with the on-going succession at the plant community level in this environment. Cloning and sequencing of bacterial 16S rRNA gene fragments further indicated a host-related effect since the two detritivores differed in terms of predominant bacterial taxa: diplopods harboured mainly representatives of the phyla Bacteroidetes and Gammaproteobacteria. In contrast, the genus Vibrio was found for the amphipod host across all feeding conditions.

Viral cystatin evolution and three-dimensional structure modelling: a case of directional selection acting on a viral protein involved in a host-parasitoid interaction.

BACKGROUND: In pathogens, certain genes encoding proteins that directly interact with host defences coevolve with their host and are subject to positive selection. In the lepidopteran host-wasp parasitoid system, one of the most original strategies developed by the wasps to defeat host defences is the injection of a symbiotic polydnavirus at the same time as the wasp eggs. The virus is essential for wasp parasitism success since viral gene expression alters the immune system and development of the host. As a wasp mutualist symbiont, the virus is expected to exhibit a reduction in genome complexity and evolve under wasp phyletic constraints. However, as a lepidopteran host pathogenic symbiont, the virus is likely undergoing strong selective pressures for the acquisition of new functions by gene acquisition or duplication. To understand the constraints imposed by this particular system on virus evolution, we studied a polydnavirus gene family encoding cyteine protease inhibitors of the cystatin superfamily. RESULTS: We show that cystatins are the first bracovirus genes proven to be subject to strong positive selection within a host-parasitoid system. A generated three-dimensional model of Cotesia congregata bracovirus cystatin 1 provides a powerful framework to position positively selected residues and reveal that they are concentrated in the vicinity of actives sites which interact with cysteine proteases directly. In addition, phylogenetic analyses reveal two different cystatin forms which evolved under different selective constraints and are characterized by independent adaptive duplication events. CONCLUSION: Positive selection acts to maintain cystatin gene duplications and induces directional divergence presumably to ensure the presence of efficient and adapted cystatin forms. Directional selection has acted on key cystatin active sites, suggesting that cystatins coevolve with their host target. We can strongly suggest that cystatins constitute major virulence factors, as was already proposed in previous functional studies.

Bracovirus gene products are highly divergent from insect proteins.

Recently, several polydnavirus (PDV) genomes have been completely sequenced. The dsDNA circles enclosed in virus particles and injected by wasps into caterpillars appear to mainly encode virulence factors potentially involved in altering host immunity and/or development, thereby allowing the survival of the parasitoid larvae within the host tissues. Parasitoid wasps generally inject virulence factors produced in the venom gland. As PDV genomes are inherited vertically by wasps through a proviral form, wasp virulence genes may have been transferred to this chromosomal form, leading to their incorporation into virus particles. Indeed, many gene products from Cotesia congregata bracovirus (CcBV), such as PTPs, IkappaB-like, and cystatins, contain protein domains conserved in metazoans. Surprisingly however, CcBV virulence gene products are not more closely related to insect proteins than to human proteins. To determine whether the distance between CcBV and insect proteins is a specific feature of BV proteins or simply reflects a general high divergence of parasitoid wasp products, which might be due to parasitic lifestyle, we have analyzed the sequences of wasp genes obtained from a cDNA library. Wasp sequences having a high similarity with Apis mellifera genes involved in a variety of biological functions could be identified indicating that the high level of divergence observed for BV products is a hallmark of these viral proteins. We discuss how this divergence might be explained in the context of the current hypotheses on the origin and evolution of wasp-bracovirus associations.