Re-opening of the symbiont sorting organ with aging in Riptortus pedestris

Because environments are full of diverse microorganisms including parasites and pathogens, how to select and maintain a beneficial microbial partner is a critical issue for host organisms. The bean bug Riptortus pedestris (Heteroptera: Alydidae) acquires a specific gut symbiont, Burkholderia insecticola, from environmental soil in the second instar stage and houses it in a crypt-bearing midgut region called M4. To sort the Burkholderia symbiont from a wide variety of soil microbes, R. pedestris develops a specialized organ named constricted region (CR). The CR, located in front of the crypt-bearing symbiotic region, is immediately closed after colonization of M4 by the Burkholderia symbiont to block any contamination of microbes ingested with food. By using a food coloring and a red fluorescent protein (RFP)-expressing Burkholderia symbiont, we here revealed that the closed CR is reopened at a later developmental stage of R. pedestris. Although the CR was re-opened at the late phase of the fifth instar, oral administration of food coloring and green fluorescent protein (GFP)-expressing symbiont demonstrated that ingested food and bacteria were stopped at the M4B despite the opened CR. Observations using confocal microscopy revealed reverse flow of gut content from M4 to M3 through the opened CR, the flow pressure of which seemed to prevent any contamination of the symbiotic M4 region. The morphological change of the CR with aging may cause a risk of contamination, but another mechanism, the reverse flow, plausibly maintains the specificity of gut symbiont in R. pedestris.