Insect-Microbe Mutualism without Vertical Transmission: a Stinkbug Acquires a Beneficial Gut Symbiont from the Environment Every Generation

Abstract

The broad-headed bug Riptortus clavatus (Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus Burkholderia. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous hostsymbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the Burkholderia symbiont in R. clavatus. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The Burkholderia symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that R. clavatus postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-Burkholderia relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-Rhizobium and alder-Frankia relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

“…culturable on standard microbiological media, and orally acquired by young nymphs of R. pedestris from the environment without vertical transmission (Kikuchi et al, 2007). The discovery of environmental acquisition of beneficial symbiont without vertical transmission in R. pedestris was unprecedented among insects.…”

“…In these stinkbug groups, notably, Burkholderia infection was coincident with development of the midgut crypts: all the 39 species with the midgut crypts were Burkholderia-positive (Table 1; Figure 2), whereas the other 22 species exhibited neither the crypts nor the Burkholderia infection (Table 1; Supplememtary Figure S1). (Kikuchi et al, , 2007.…”

“…Considering that the Burkholderia symbiont of R. pedestris is culturable on standard bacterial media (Kikuchi et al, 2007), we attempted to isolate Burkholderia symbionts using YG medium agar plates from the following lygaeoid/coreoid stinkbugs: Y. exilis (Berytidae); D. pallipes (Blissidae); P. exiguus and T. hemipterus (Rhyparochromidae); P. antennata (Pachygronthidae); A. sordidus, H. lativentris and C. puntiger (Coreidae); and additionally R. pedestris and L. chinensis (Alydidae). Within 3 days after plating of homogenized midgut crypts, a number of white, circular and convex colonies appeared for all the stinkbug samples (Figure 4g-l).…”

“…Similar to the pentatomoid stinkbugs, R. pedestris possesses numerous crypts in the posterior midgut, but the bacterium in the symbiotic organ is quite different, belonging to the genus Burkholderia in the b-Proteobacteria . Aposymbiotic insects suffered marked reduction in body size and weight, indicating mutualistic nature of the symbiosis (Kikuchi et al, 2007). However, distinct from the pentatomoid symbionts thus far studied, the Burkholderia symbiont was present in the rhizosphere of the food plants of R. pedestris, easily Figure 1 Phylogenetic relationship of the pentatomomorphan stinkbug families.…”

“…It should be noted that in these cases, the symbionts outside the host organisms are also protected from the above-mentioned environmental stresses in the soil. In this context, it is remarkable as well as understandable that environmental symbiont transmission was recently discovered in an insect-microbe mutualism, wherein the symbiont was soil bacteria of the genus Burkholderia (Kikuchi et al, 2007).…”