Raul Bettencourt

Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos

There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso‐ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria‐inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi‐females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality.

Hemolymph-dependent and -independent responses in Drosophila immune tissue.

Insects possess an antimicrobial defense response that is similar to the mammalian innate immune response. The innate immune system is designed to recognize conserved components of microorganisms called pathogen‐associated molecular patterns (PAMPs). How host receptors detect PAMPs and transmit the signals to mount the immune response is being elucidated. Using GFP‐Dorsal, ‐Dif, and ‐Relish reporter proteins in ex vivo assays, we demonstrate that Drosophila fat bodies, a major immune tissue, have both hemolymph‐dependent and ‐independent responses. Microbial preparations such as lipoteichoic acid (LTA) and peptidoglycan (PGN) can stimulate some responses from dissected and rinsed larval fat bodies. Therefore, at least some aspects of recognition can occur on fat body cell surfaces, bypassing the requirement of hemolymph. Our results also show that supernatants from bacterial cultures can stimulate the nuclear translocation of Dorsal in dissected fat bodies, but this stimulation is strictly hemolymph‐dependent. Various biochemical assays suggest that the factors from bacterial supernatants that stimulate the hemolymph‐dependent nuclear translocation are likely made up of proteins. We further show that Dorsal mutant larvae have much lower phenoloxidase activity, consistent with a more important role of Dorsal in innate immunity than previously shown.

The insect immune protein hemolin is expressed during oogenesis and embryogenesis

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca(2+) indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components.

Dispersal of Steinernema glaseri (Nematoda: Steinernematidae) in adult Japanese beetles, Popillia japonica (Coleoptera: Scarabaeidae)

Japanese beetle adults, Popillia japonica, can become infected with and disperse the entomopathogenic nematode, Steinernema glaseri, under laboratory and field conditions. After a 24-h exposure to 10 000 infective juveniles/20 adult beetles, 45% of the beetles died within 4 days post-treatment, but only 59% of these were infected with the nematode. Corresponding control mortality was 6.5%. An average of 238 infective juveniles were produced/beetle. Beetles exposed to 4000 and 10 000 infectives/10 adults carried with them an average of 17 and 59 infectives/adult on external body surfaces respectively. When beetles that had been exposed to 4000 infectives/20 adults were transferred to, and held in, cages containing soil for 2 weeks, up to 89% of the adults died, as did 74% of the P. japonica larvae that were subsequently placed in the cages. When adults that had been exposed to 50 000 infectives/250 beetles in moist sand for 16 h were released into screened cages in the field at soil temperatures of over 25...

Toll and Toll-9 in Drosophila innate immune response.

In both insects and mammals, members of the Toll receptor family play important roles in the initial events leading to the activation of immunity genes. The prototypic Toll in Drosophila appears to be activated by a host protein ligand after microbial stimulation. The cellular events and the biological response after Toll activation, however, require further investigation. We used transgenic Drosophila strains expressing NF-κB and Toll proteins to investigate innate immune response in whole larvae and dissected larval fat bodies. Substantial activation of antimicrobial peptide genes was observed after septic injury. To circumvent the contribution of injury-induced response, we used dissected larval fat bodies to show that commercially available microbial compounds were able to alter the cellular distribution of Toll. The results also demonstrate that complex cellular events, including receptor trafficking, likely take place after stimulation of the larval immune tissue. By genome-wide expression analysis, we further show that Toll and Toll-9 may utilize the same signaling pathway in activating many immunity genes. Thus, the innate immune response in Drosophila is regulated by complex mechanisms, which involve Toll and other Toll-related proteins.

Changes of gill and hemocyte-related bio-indicators during long term maintenance of the vent mussel Bathymodiolus azoricus held in aquaria at atmospheric pressure

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus has been the subject of several studies aimed at understanding the physiological adaptations that vent animals have developed in order to cope with the particular physical and chemical conditions of hydrothermal environments. In spite of reports describing successful procedures to maintain vent mussels under laboratory conditions at atmospheric pressure, few studies have described the mussels physiological state after a long period in aquaria. In the present study, we investigate changes in mucocytes and hemocytes in B. azoricus over the course of several months after deep-sea retrieval. The visualization of granules of mucopolysaccharide or glycoprotein was made possible through their inherent auto-fluorescent property and the Alcian blue-Periodic Acid Schiff staining method. The density and distribution of droplets of mucus-like granules was observed at the ventral end of lamellae during acclimatization period. The mucus-like granules were greatly reduced after 3 months and nearly absent after 6 months of aquarium conditions. Additionally, we examined the depletion of endosymbiont bacteria from gill tissues, which typically occurs within a few weeks in sea water under laboratory conditions. The physiological state of B. azoricus after 6 months of acclimatization was also examined by means of phagocytosis assays using hemocytes. Hemocytes from mussels held in aquaria up to 6 months were still capable of phagocytosis but to a lesser extent when compared to the number of ingested yeast particles per phagocytic hemocytes from freshly collected vent mussels. We suggest that the changes in gill mucopolysaccharides and hemocyte glycoproteins, the endosymbiont abundance in gill tissues and phagocytosis are useful health criteria to assess long term maintenance of B. azoricus in aquaria. Furthermore, the laboratory set up to which vent mussels were acclimatized is an applicable system to study physiological reactions such as hemocyte immunocompetence even in the absence of the high hydrostatic pressure found at deep-sea vent sites.

The Transcriptome of Bathymodiolus azoricus Gill Reveals Expression of Genes from Endosymbionts and Free-Living Deep-Sea Bacteria

Deep-sea environments are largely unexplored habitats where a surprising number of species may be found in large communities, thriving regardless of the darkness, extreme cold, and high pressure. Their unique geochemical features result in reducing environments rich in methane and sulfides, sustaining complex chemosynthetic ecosystems that represent one of the most surprising findings in oceans in the last 40 years. The deep-sea Lucky Strike hydrothermal vent field, located in the Mid Atlantic Ridge, is home to large vent mussel communities where Bathymodiolus azoricus represents the dominant faunal biomass, owing its survival to symbiotic associations with methylotrophic or methanotrophic and thiotrophic bacteria. The recent transcriptome sequencing and analysis of gill tissues from B. azoricus revealed a number of genes of bacterial origin, hereby analyzed to provide a functional insight into the gill microbial community. The transcripts supported a metabolically active microbiome and a variety of mechanisms and pathways, evidencing also the sulfur and methane metabolisms. Taxonomic affiliation of transcripts and 16S rRNA community profiling revealed a microbial community dominated by thiotrophic and methanotrophic endosymbionts of B. azoricus and the presence of a Sulfurovum-like epsilonbacterium.

Microbial diversity in deep-sea sediments from the Menez Gwen hydrothermal vent system of the Mid-Atlantic Ridge.

Deep-sea hydrothermal sediments are known to support remarkably diverse microbial consortia. Cultureindependent sequence-based technologies have extensively been used to disclose the associated microbial diversity as most of the microorganisms inhabiting these ecosystems remain uncultured. Here we provide the first description of the microbial community diversity found on sediments from Menez Gwen vent system. We compared hydrothermally influenced sediments, retrieved from an active vent chimney at 812 m depth, with non-hydrothermally influenced sediments, from a 1400 m depth bathyal plain. Considering the enriched methane and sulfur composition of Menez Gwen vent fluids, and the sediment physicochemical properties in each sampled area, we hypothesized that the site-associated microbes would be different. To address this question, taxonomic profiles of bacterial, archaeal and micro-eukaryotic representatives were studied by rRNA gene tag pyrosequencing. Communities were shown to be significantly different and segregated by sediment geographical area. Specific mesophilic, thermophilic and hyperthermophilic archaeal (e.g., Archaeoglobus, ANME-1) and bacterial (e.g., Caldithrix, Thermodesulfobacteria) taxa were highly abundant near the vent chimney. In contrast, bathyal-associated members affiliated to more ubiquitous phylogroups from deep-ocean sediments (e.g., Thaumarchaeota MGI, Gamma- and Alphaproteobacteria). This study provides a broader picture of the biological diversity and microbial biogeography, and represents a preliminary approach to the microbial ecology associated with the deep-sea sediments from the Menez Gwen hydrothermal vent field.

Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria

The deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the continental European coast Mytilus galloprovincialis are two bivalves species living in highly distinct marine habitats. Mussels are filter-feeding animals that may accumulate rapidly bacteria from the environment. Contact with microorganism is thus inevitable during feeding processes where gill tissues assume a strategic importance at the interface between the external milieu and the internal body cavities promoting interactions with potential pathogens during normal filtration and a constant challenge to their immune system. In the present study B. azoricus and M. galloprovincialis were exposed to Vibrio alginolyticus, Vibrio anguillarum and Vibrio splendidus suspensions and to a mixture of these Vibrio suspensions, in order to ascertain the expression level of immune genes in gill samples, from both mussel species. The immune gene expressions were analyzed by means of quantitative-Polymerase Chain Reaction (qPCR). The gene expression results revealed that these bivalve species exhibit significant expression differences between 12 h and 24 h post-challenge times, and between the Vibrio strains used. V. splendidus induced the strongest gene expression level in the two bivalve species whereas the NF-κB and Aggrecan were the most significantly differentially expressed between the two mussel species. When comparing exposure times, both B. azoricus and M. galloprovincialis showed similar percentage of up-regulated genes at 12 h while a marked increased of gene expression was observed at 24 h for the majority of the immune genes in M. galloprovincialis. This contrasts with B. azoricus where the majority of the immune genes were down-regulated at 24 h. The 24 h post-challenge gene expression results clearly bring new evidence supporting time-dependent transcriptional activities resembling acute phase-like responses and different immune responses build-up in these two mussel species when challenged with Vibrio bacteria. High Pressure Liquid Chromatography (HPLC)-Electrospray ionization mass spectrometry (ESI-MS/MS) analyses resulted in different peptide sequences from B. azoricus and M. galloprovincialis gill tissues suggesting that naïve animals present differences, at the protein synthesis level, in their natural environment. B. azoricus proteins sequences, mostly of endosymbiont origin, were related to metabolic, energy production, protein synthesis processes and nutritional demands whereas in M. galloprovincialis putative protein functions were assumed to be related to structural and cellular integrity and signaling functions.