RNAi in the cereal weevil Sitophilus spp: Systemic gene knockdown in the bacteriome tissue

<p>Abstract</p> <p>Background</p> <p>The weevils <it>Sitophilus </it>spp. are among the most important cosmopolitan pests of stored cereal grains. However, their biology and physiology are poorly understood, mainly because the insect developmental stages take place within cereal grains and because of the lack of gene specific molecular manipulation.</p> <p>Results</p> <p>To gain access to the different insect developmental stages, weevil females were allowed to lay their eggs on starch pellets and hatched embryos were collected by dissolving starch with water. Embryos were transferred between two Glass Plates filled with packed Flour (GPF) to mimic compact texture of the cereal grain, and this system allowed us to recover specific developmental stages. To knockdown the gene expressed in the bacteria-bearing organ (the bacteriome), whole larvae were injected with dsRNA to target the <it>wpgrp1 </it>gene and they were then left to develop for a further 4 days period. Quantitative RT-PCR and Western blot analyses on the bacteriome of these animals revealed a down-regulation of the <it>wpgrp1 </it>expression, both at transcript and protein levels.</p> <p>Conclusion</p> <p>These results demonstrate that whole larval injection with dsRNA results in a high and systemic decrease of both mRNA and protein in the bacteriome tissue. This, along with the possibility of access to the insect developmental stages, opens up a new research avenue for exploring gene specific functions in the cereal weevils.</p

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri.

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species

Endosymbiosis allows Sitophilus oryzae to persist in dry conditions

Insects frequently associate with intracellular microbial symbionts (endosymbionts) that enhance their ability to cope with challenging environmental conditions. Endosymbioses with cuticle-enhancing microbes have been reported in several beetle families. However, the ecological relevance of these associations has seldom been demonstrated, particularly in the context of dry environments where high cuticle quality can reduce water loss. Thus, we investigated how cuticle-enhancing symbionts of the rice-weevil, Sitophilus oryzae contribute to desiccation resistance. We exposed symbiotic and symbiont-free (aposymbiotic) beetles to long-term stressful (47% RH) or relaxed (60% RH) humidity conditions and measured population growth. We found that symbiont presence benefits host fitness especially under dry conditions, enabling symbiotic beetles to increase their population size by over 33-fold within 3 months, while aposymbiotic beetles fail to increase in numbers beyond the starting population in the same conditions. To understand the mechanisms underlying this drastic effect, we compared beetle size and body water content and found that endosymbionts confer bigger body size and higher body water content. While chemical analyses revealed no significant differences in composition and quantity of cuticular hydrocarbons after long-term exposure to desiccation stress, symbiotic beetles lost water at a proportionally slower rate than did their aposymbiotic counterparts. We posit that the desiccation resistance and higher fitness observed in symbiotic beetles under dry conditions is due to their symbiont-enhanced thicker cuticle, which provides protection against cuticular transpiration. Thus, we demonstrate that the cuticle enhancing symbiosis of Sitophilus oryzae confers a fitness benefit under drought stress, an ecologically relevant condition for grain pest beetles. This benefit likely extends to many other systems where symbiont-mediated cuticle synthesis has been identified, including taxa spanning beetles and ants that occupy different ecological niches

Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae

Background: Insects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently. Results: To investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion an

Identification of the Weevil immune genes and their expression in the bacteriome tissue

<p>Abstract</p> <p>Background</p> <p>Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome).</p> <p>Results</p> <p>To study bacteriome immune specificity, we first identified immune-relevant genes of the weevil <it>Sitophilus zeamais </it>by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following <it>S. zeamais </it>primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the <it>coleoptericin </it>family and, intriguingly, homologs to genes described as immune modulators (that is, <it>PGRP-LB, Tollip</it>) were also shown to be highly expressed in the bacteriome.</p> <p>Conclusion</p> <p>The current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the <it>coleoptericin</it>, the <it>PGRP </it>and the <it>Tollip </it>genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.</p

Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae

Bacteria that establish an obligate intracellular relationship with eukaryotic hosts undergo an evolutionary genomic reductive process. Recent studies have shown an increase in the number of mobile elements in the first stage of the adaptive process towards intracellular life, although these elements are absent in ancient endosymbionts. Here, the genome of SOPE, the obligate mutualistic endosymbiont of rice weevils, was used as a model to analyze the initial events that occur after symbiotic integration. During the first phases of the SOPE genome project, four different types of insertion sequence (IS) elements, belonging to well-characterized IS families from γ-proteobacteria, were identified. In the present study, these elements, which may represent more than 20% of the complete genome, were completely characterized; their relevance as a source of gene inactivation, chromosomal rearrangements, and as participants in the genome reductive process are discussed herein. [Int Microbiol 2008; 11(1): 41-48

Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae

Bacteria that establish an obligate intracellular relationship with eukaryotic hosts undergo an evolutionary genomic reductive process. Recent studies have shown an increase in the number of mobile elements in the first stage of the adaptive process towards intracellular life, although these elements are absent in ancient endosymbionts. Here, the genome of SOPE, the obligate mutualistic endosymbiont of rice weevils, was used as a model to analyze the initial events that occur after symbiotic integration. During the first phases of the SOPE genome project, four different types of insertion sequence (IS) elements, belonging to well-characterized IS families from γ-proteobacteria, were identified. In the present study, these elements, which may represent more than 20% of the complete genome, were completely characterized; their relevance as a source of gene inactivation, chromosomal rearrangements, and as participants in the genome reductive process are discussed herein.Gil Garcia, Rosario, [email protected] ; Belda Cuesta, Eugenio, [email protected] ; Gosalbes Soler, Maria Jose, [email protected] ; Delaye, Luis, [email protected] ; Silva Moreno, Francisco J., [email protected] ; Moya Simarro, Andres, [email protected] ; Latorre Castillo, Amparo, [email protected]

Transcriptomic-based selection of reference genes for quantitative real-time PCR in an insect endosymbiotic model

Reference genes are a fundamental tool for analyses of gene expression by real-time quantitative PCR (qRT-PCR), in that they ensure the correct comparison between conditions, stages, or treatments. Because of this, selection of appropriate genes to use as references is crucial for proper application of the technique. Nevertheless, efforts to find appropriate, stably expressed transcripts are still lacking, in particular in the field of insect science. Here, we took advantage of a massive transcriptomic high-throughput analysis of various developmental stages of the gut and associated-bacteriomes of the cereal weevil Sitophilus oryzae and identified a subset of stably expressed genes with the potential to be used as housekeeping genes from the larva to the adult stage. We employed several normalization techniques to select the most suitable genes among our subset. Our final selection includes two genes–TAO, and YTH3–which can also be used to compare transcript abundance at various developmental stages in symbiotic insects, and in insects devoid of endosymbionts (aposymbiotic). Since they are well conserved, these genes have the potential to be useful for many other insect species. This work confirms the interest in using large-scale, unbiased methods for reference gene selection

Bacteriocyte cell death in the pea aphid/ Buchnera symbiotic system

International audienceSymbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning