dMany stinkbugs (Insecta: Hemiptera: Heteroptera) are associated with bacterial symbionts in a posterior region of the midgut. In these stinkbugs, adult females excrete symbiont-containing materials from the anus for transmission of the beneficial symbionts to their offspring. For ensuring the vertical symbiont transmission, a variety of female-specific elaborate traits at the cellular, morphological, developmental, and behavioral levels have been reported from diverse stinkbugs of the families Plataspidae, Urostylididae, Parastrachiidae, etc. Meanwhile, such elaborate female-specific traits for vertical symbiont transmission have been poorly characterized for the largest and economically important stinkbug family Pentatomidae. Here, we investigated the midgut symbiotic system of a pentatomid stinkbug, Plautia splendens. A specific gammaproteobacterial symbiont was consistently present extracellularly in the cavity of numerous crypts arranged in four rows on the midgut fourth section. The symbiont was smeared on the egg surface upon oviposition by adult females, orally acquired by newborn nymphs, and thereby transmitted vertically to the next generation and important for growth and survival of the host insects. We found that, specifically in adult females, several rows of crypts at the posterior end region of the symbiotic midgut were morphologically differentiated and conspicuously enlarged, often discharging the symbiotic bacteria from the crypt cavity to the main tract of the symbiotic midgut. The female-specific enlarged end crypts were also found in other pentatomid stinkbugs Plautia stali and Carbula crassiventris. These results suggest that the enlarged end crypts represent a female-specific specialized morphological trait for vertical symbiont transmission commonly found among stinkbugs of the family Pentatomidae. Many insects are associated with microbial mutualists in the gut lumen, inside the body cavity, or within the cells (1, 2). For ecological and evolutionary persistence of such mutualistic associations, vertical symbiont transmission through host generations is among the pivotal processes, although nonvertical (i.e., horizontal or environmental) symbiont transmission may also be prevalent in marine invertebrates, plants, and some insects (3-5).In intracellular associations such as Buchnera in aphids and Wigglesworthia in tsetse flies, vertical symbiont transmission usually occurs prenatally to developing eggs or embryos within maternal bodies (1,(6)(7)(8). In extracellular associations such as gut microbes in termites and stinkbugs, by contrast, vertical symbiont transmission tends to occur postnatally outside the maternal bodies via nymphal feeding of symbiont-containing excrements (1, 9, 10).The majority of plant-sucking stinkbugs (order Hemiptera: suborder Heteroptera: infraorder Pentatomomorpha: superfamily Pentatomoidea) are associated with bacterial symbionts of a beneficial nature in a posterior region of the midgut, where numerous crypts develop and harbor specific symbiotic bacteria the...

BackgroundThe lesser grain borer Rhyzopertha dominica (Coleoptera: Bostrichidae) is a stored-product pest beetle. Early histological studies dating back to 1930s have reported that R. dominica and other bostrichid species possess a pair of oval symbiotic organs, called the bacteriomes, in which the cytoplasm is densely populated by pleomorphic symbiotic bacteria of peculiar rosette-like shape. However, the microbiological nature of the symbiont has remained elusive.ResultsHere we investigated the bacterial symbiont of R. dominica using modern molecular, histological, and microscopic techniques. Whole-mount fluorescence in situ hybridization specifically targeting symbiotic bacteria consistently detected paired bacteriomes, in which the cytoplasm was full of pleomorphic bacterial cells, in the abdomen of adults, pupae and larvae, confirming previous histological descriptions. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont constituted a distinct bacterial lineage allied to a variety of insect-associated endosymbiont clades, including Uzinura of diaspidid scales, Walczuchella of giant scales, Brownia of root mealybugs, Sulcia of diverse hemipterans, and Blattabacterium of roaches. The symbiont gene exhibited markedly AT-biased nucleotide composition and significantly accelerated molecular evolution, suggesting degenerative evolution of the symbiont genome. The symbiotic bacteria were detected in oocytes and embryos, confirming continuous host–symbiont association and vertical symbiont transmission in the host life cycle.ConclusionsWe demonstrate that the symbiont of R. dominica constitutes a novel bacterial lineage in the Bacteroidetes. We propose that reductive evolution of the symbiont genome may be relevant to the amorphous morphology of the bacterial cells via disruption of genes involved in cell wall synthesis and cell division. Genomic and functional aspects of the host-symbiont relationship deserve future studies.

Diverse insects are obligatorily associated with microbial symbionts, wherein the host often develops special symbiotic organs and vertically transmits the symbiont to the next generation. What molecular factors underpin the host-symbiont relationship is of great interest but poorly understood. Here we report a novel protein preferentially produced in a female-specific symbiotic organ of the stinkbug Plautia stali, whose posterior midgut develops numerous crypts to host a Pantoea-allied bacterial mutualist. In adult females, several posteriormost crypts are conspicuously enlarged, presumably specialized for vertical symbiont transmission. We detected conspicuous protein bands specific to the female’s swollen crypts by gel electrophoresis, and identified them as representing a novel mucin-like glycoprotein. Histological inspections confirmed that the mucin protein is localized to the female’s swollen crypts, coexisting with a substantial population of the symbiotic bacteria, and excreted from the swollen crypts to the midgut main tract together with the symbiotic bacteria. Using RNA interference, we successfully suppressed production of the mucin protein in adult females of P. stali. However, although the mucin protein was depleted, the symbiont population persisted in the swollen crypts, and vertical symbiont transmission to the next generation occurred. Possible biological roles and evolutionary trajectory of the symbiosis-related mucin protein are discussed.