The Bacterial Diversity In Rice Water Weevil And Its Relationship With Host Reproduction

Symbiotic bacteria play important roles in a number of biological processes of hosts including growth, reproduction, resistance to pathogens and harmful substances. The rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera, Curculionidae), is an important invasive pest in China and can cause serious damage to rice plants. Previous studies showed that Wolbachia, one of major symbiotic bacteria in this weevil, is essential for the reproduction of its host. However, it is not clear whether there exists other symbiotic bacteria in this weevil, and whether they also have any importance for the host reproduction. In this study, based on the amplification and analysis of bacterial 16S rRNA sequences, bacterial species in guts and ovaries of L. oiyzophilus adults collected from different locations was identified and the diversity of bacterial community analyzed. Relationships between major bacteria and their reproduction as well as the underlying molecular mechanism were also explored. The results were summarized as follows.From five geographic populations of rice water weevil (Beaumont of Texas, Colt of Arkansas and Biggs of California in the USA; Tanghai of Hebei and Yueqing of Zhejiang in China), a total of 19 bacteria were identified from guts, which belong to Alpha-, Beta-, Gamma-proteobacteria, Bacilli, Sphingobacteria and Actinobacteria. The dominant symbiotic bacteria are Pantoea and Wolbachia. Except the Beaumont population, bacteria diversity in each of the investigated weevil populations is relatively low. In the ovary, only five bacteria were identified which belong to Alpha-, Gamma-proteobacteria and Bacilli, with Wolbachia and Nardonella being the dominant species.The quantitative changes of major symbiotic bacteria were detected in weevil adults treated with antibiotics, and, by combining results regarding change of reproductive phenotype in treated weevils, the relative importance of various bacteria for the reproduction of this weevil was evaluated. The results showed that, as expected, Wolbachia in the adults treated with tetracycline (leading to abnormal reproduction) was significantly lower than in the control adults (treated with water), however, the treatment using gentamicin (incapable of killing Wolbachia and not negatively impacting weevil reproduction) did not reduce the Wolbachia density significantly. In adults separately treated with tetracycline and gentamycin, Nardonella were maintained at a high level, irrespective of the status of weevil reproduction. In the adults treated with gentamicin, which can reproduce healthily, Rickettsia significantly reduced. These results indicated that changes in Nardonella and Rickettsia density has no evident relations with the reproduction status of rice water weevil, suggesting both of these two bacteria are not essential for the reproduction of this weevil. These findings strengthen our previous conclusion that Wolbachia is the essential symbiotic bacterium for the reproduction of rice water weevil.By using the adults which were separately treated with tetracycline and gentamicin and thus in two substantially different Wolbachia infection status, i.e., Wolbachia-removed and Wolbachia-infected, two cDNA libraries were constructed using the suppressive subtraction hybridization (SSH). Sequences from each library were cloned, and analyzed using by GO (Gene Ontology) annotation. Moreover,35 genes were quantitatively detected for their expression in the adults treated separately with tetracycline and gentamicin, i.e., in the adults with Wolbachia removal and those harboring Wolbachia. It was found that Wolbachia can affect the expression of multiple genes of this weevil, including the genes related to metabolic process, cellular process, catalytic activity, reproduction, immunity and death. Three genes changed their expression significantly due to the deletion of Wolbachia, namely transcript antisense to ribosomal RNA protein 1 (TAR1), a hypothetical protein, and juvenile hormone epoxide hydrolase-like protein 5 precursor.In sum, in this study the composition and diversity of bacterial community in L. oryzophilus were determined, and importance of main symbiotic bacteria for the reproduction of this weevil and some underlying molecular mechanism were investigated. The results would enrich our knowledge on the relationship between weevils and symbiotic bacteria, also provide important information as to the evolution of symbiotic bacteria, reproductive mechanism in weevils and the interaction mechanism between symbiotic bacteria and their host.