ABSTRACT
Here we report a novel clade of secondary endosymbionts associated with insects and other arthropods. Seed bugs of the genus
Nysius
(Hemiptera: Lygaeidae) harbor the primary gammaproteobacterial symbiont
Schneideria nysicola
within a pair of bacteriomes in the abdomen. Our survey of
Nysius
species for their facultative bacterial associates consistently yielded a novel type of alphaproteobacterial 16S rRNA gene sequence in addition to those of
Wolbachia
. Diagnostic PCR survey of 343 individuals representing 24 populations of four
Nysius
species revealed overall detection rates of the alphaproteobacteria at 77.6% in
Nysius plebeius
, 87.7% in
Nysius
sp. 1, 81.0% in
Nysius
sp. 2, and 100% in
Nysius 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 order
Rickettsiales
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, and
Midichloria
spp., the mitochondrion-associated endosymbionts of ticks.
In situ
hybridization and electron microscopy identified small filamentous bacterial cells in various tissues of
N. 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.