Molecular Phylogenetic Characterization of Symbiotic Microbiota in the Intestinal Tracts of Wild Lepidopteran Stem Borer Eldana Saccharina Walker (Pyralidae)

Abstract

Intestinal tracts of plant feeding insects are potentially important sources of novel biocatalysts. However, the microbial populations in these guts, especially in the Lepidoptera, are not well characterized. In this study, thirty two (32) bacterial isolates from homogenized guts of late irtstar larvae of Eldana saccharina from papyrus rhizomes and infested sugarcane stalks were assayed for their ability to utilize alpha cellulose and sugarcane bagasse as sole carbon sources. All isolates were viable for 14 days in liquid fermentation cultures.

The microbial isolates were also characterized by a combination of microbiological and 16S rRNA gene sequence analysis. In addition, microbial communities in the gut homogenates were investigated by extracting total genomic DN A from eviscerated larval guts, followed by PCR amplification of the 16S rRNA gene. The diversity of the gut symbiotic bacteria was characterized by genetic profiling of the partial bacterial 16S rRNA gene by the Small Sub Unit PCR-Restriction Fragment Length polymorphism (SSU-RFLP) technique followed by clonal sequencing and phylogenetic analysis. A total of 48 cloned 16S rRNA gene sequences together with 16S rRNA gene sequences from 32 bacterial isolates obtained by two different culture methods were compared with sequences in the GenBank. database. Seventeen (\7) of the clonal sequences were closely affiliated to the alpha Proteobacteria subdivision of the Kingdom Bacteria, followed by·gamma proteobacteria (IS), Firmicutes (9), Actinobacteria (5) and Beta Proteobacteria (2).

In culture dependent studies, most of the isolates obtained were affiliated to the high G+C content, Gram positive bacteria in the Actinobacteria subdivision, represented by the Streptomyces operational taxonomic unit ( 18 isolates), Microbacterium (4 isolates)' and the Cellulosimicrobium (1 isolate). In addition, five (5) isolates clustered closely with the 1- Proteobacteria, three (3) with a.-Proteobacteria and one (1) isolate was affiliated to the Firmicutes group. Only three bacterial taxa were isolated from gut homogenates of laboratory reared control larvae.

Furthermore, only five bacterial taxa were detected in common by both culture dependent and independent methods. The results show a high microbial diversity in the guts of wild E. saccharina larvae and demonstrate that multiple approaches are necessary to characterize microbial diversity in a complex micro-biome like the insect gut. The results further suggest mutua1 benefits between microbial isolates and the host E. saccharina larvae and infer their potential applications in biotechnology. Moreover, these results provide the first comprehensive description of microbial diversity in the guts of the African Sugarcane stem borer and demonstrate that insect diet and habitat influence the composition of the gut microbial communities.