Symbiotic bacteria associated with gut symbiotic organs and female genital accessory organs of the leaf beetle Bromius obscurus (Coleoptera: Chrysomelidae)

Leaf beetles(Coleoptera: Chrysomelidae) constitute one of the most species-rich insect families, and live exclusively on leaves or other plant parts. Early histological works described the presence of symbiotic bacteriain gut-associated symbiotic organs of some chrysomelid species, but their microbiological nature has been poorly characterized except for those associated with reed beetles of the subfamily Donaciinae. Here we investigated symbiotic bacteriaof the leaf beetle Bromius obscurus(L.) belonging to the subfamily Eumolpinae. Specific bacterial 16S ribosomal RNAand gyrB gene sequences were consistently obtained from the symbiotic organs, which radially surround the foregut-midgut junction, of all adult males and females examined. In adult females, the same sequences were also obtained from a pair of genital accessory organs, which are presumably for vertical symbiont transmission. Whole mount in situ hybridization specifically detectedthe symbiont in the gut symbiotic organs endocellularly and also in the female genitalaccessory organs extracellularly. In the gut symbiotic organs, the endocellular symbiont cells were small and rosette-like or aggregated and granule-like, whereas in the female genital organsthe extracellular symbiont cells were of a condensed form. Molecular phylogeneticanalysis showed that the symbiont of B. obscurus constitutes a distinct lineage in the Gammaproteobacteria. Molecular evolutionary analysis has identified significantly accelerated molecular evolutionand a highly adenine–thymine-biased nucleotide composition of the symbiont genes, presumably reflecting reductive evolution of the symbiont genome. These results suggest an intimate and stable host-symbiont association in B. obscurus, in which the symbiont may play some important, though hitherto unknown, biological roles in its herbivorous insect host.