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

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

Host PGRP Gene Expression and Bacterial Release in Endosymbiosis of the Weevil Sitophilus zeamais

Intracellular symbiosis (endosymbiosis) with gram-negative bacteria is common in insects, yet little is known about how the host immune system perceives the endosymbionts and controls their growth and invasion without complete bacterial clearance. In this study, we have explored the expression of a peptidoglycan recognition protein gene of the weevil Sitophilus zeamais (wPGRP); an ortholog in Drosophila (i.e., PGRP-LB) was recently shown to downregulate the Imd pathway (A. Zaidman-Remy, M. Herve, M. Poidevin, S. Pili-Floury, M. S. Kim, D. Blanot, B. H. Oh, R. Ueda, D. Mengin-Lecreulx, and B. Lemaitre, Immunity 24:463-473, 2006). Insect challenges with bacteria have demonstrated that wPGRP is induced by gram-negative bacteria and that the level of induction depends on bacterial growth. Real-time reverse transcription-PCR quantification of the wPGRP gene transcript performed at different points in insect development has shown a high steady-state level in the bacteria-bearing organ (the bacteriome) of larvae and a high level of wPGRP up-regulation in the symbiotic nymphal phase. Concomitantly, during this stage fluorescence in situ hybridization has revealed an endosymbiont release from the host bacteriocytes. Together with the previously described high induction level of endosymbiont virulence genes at the nymphal phase (C. Dale, G. R. Plague, B. Wang, H. Ochman, and N. A. Moran, Proc. Natl. Acad. Sci. USA 99:12397-12402, 2002), these findings indicate that insect mutualistic relationships evolve through an interplay between bacterial virulence and host immune defense and that the host immunity engages the PGRP gene family in that interplay