The burrower bug Macroscytus japonensis (Hemiptera: Cydnidae) acquires obligate symbiotic bacteria from the environment

Many plant-feeding stinkbugs belonging to the infraorder Pentatomomorpha possess a specialized symbiotic organ at the posterior end of the midgut, in which mutualistic bacterial symbionts are harbored extracellularly. In species of the superfamily Pentatomoidea, these symbionts typically are verticallytransmitted from host mothers to offspring, whereas in species of the superfamilies Coreoidea and Lygaeoidea they are acquired from the environment. In the pentatomoid family Cydnidae, vertical symbiont transmission has been reported in several species. Here, we report the first case of environmental symbiont acquisition in Cydnidae, observed in the burrower bug Macroscytus japonensis. A comprehensive survey of 72 insect samples from 23 sites across the Japanese archipelago revealed that (1) symbionts exhibit remarkably high diversity, forming six distinct phylogenetic groups within the Enterobacteriaceae of the γ-Proteobacteria, (2) most symbionts are cultivable and closely related to free-living Pantoea-allied bacteria, and (3) symbiont phylogenetic groups do not reflect the host phylogeny. Microbial inspection of eggs revealed the absence of bacteria on the egg surface. These results strongly suggest that symbionts are acquired from the environment, not vertical transmission. Rearing experiments confirmed environmental symbiont acquisition. When environmental symbiont sources were experimentally withheld, nymphs became aposymbiotic and died before molting to the second instar, indicating that nymphs environmentally acquire symbionts during the first-instar stage and that symbionts are essential for nymphal growth and survival. This study highlights Cydnidae as the only pentatomoid family that includes species that environmentally acquire symbionts and those that vertically transmit symbionts, providing an ideal platform for comparative studies of the ecological and environmental factors that influence the evolution of symbiont transmission modes.