Novel Clade of Alphaproteobacterial Endosymbionts Associated with Stinkbugs and Other Arthropods

Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genusNysius (Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiontSchneideria nysicola within a pair of bacteriomes in the abdomen. Our survey ofNysius species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those ofWolbachia . Diagnostic PCR survey of 343 individuals representing 24 populations of fourNysius species revealed overall detection rates of the alphaproteobacteria at 77.6% inNysius plebeius , 87.7% inNysius sp. 1, 81.0% inNysius sp. 2, and 100% inNysius expressus . Further survey of diverse stinkbugs representing 24 families, 191 species, and 582 individuals detected the alphaproteobacteria from an additional 12 species representing six families. Molecular phylogenetic analysis showed that the alphaproteobacteria from the stinkbugs form a distinct and coherent monophyletic group in the orderRickettsiales together with several uncharacterized endosymbionts from fleas and ticks. The alphaproteobacterial symbiont clade was allied to bacterial clades such as the endosymbionts of acanthamoebae, the endosymbionts of cnidarians, andMidichloria spp., the mitochondrion-associated endosymbionts of ticks.In situ hybridization and electron microscopy identified small filamentous bacterial cells in various tissues ofN. plebeius , including the bacteriome and ovary. The concentrated localization of the symbiont cells at the anterior pole of oocytes indicated its vertical transmission route through host insect generations. The designation “Candidatus Lariskella arthropodarum” is proposed for the endosymbiont clade.