Primary Gut Symbiont and Secondary,Sodalis-Allied Symbiont of the Scutellerid Stinkbug Cantao ocellatus

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.Insects are among the largest animal groups on the earth, embracing 750,000 to several millions of species (37, 52). Diverse insects are symbiotically associated with microorganisms, especially bacteria (5-7). In some insects, symbiotic bacteria are harbored in specialized host cells called bacteriocytes (or mycetocytes), constituting obligate mutualistic associations. For example, Buchnera aphidicola is harbored within bacteriocytes in the abdominal body cavity of almost all aphids and provides essential amino acids that are lacking in the phloem sap diet of the insects (9, 47). Wigglesworthia glossinidia is localized in a midgut-associated bacteriome of tsetse flies and plays pivotal roles in biosynthesis of B vitamins that are deficient in the vertebrate blood diet of the insects (2, 34). These obligate endocellular symbionts are often collectively referred to as “primary symbionts.”In contrast, there are facultative endosymbiotic microorganisms not essential for their host insects, often collectively called “secondary symbionts.” For example, many aphids are known to harbor various facultative symbionts, which belong to distinct lineages in the Gamma- and Alphaproteobacteria (33, 43) and the Mollicutes (10). While the majority of those facultative bacteria are either parasitic or commensalistic for their hosts, some of them affect the host fitness beneficially in particular ecological contexts (29, 32, 36, 44, 51). In addition to the obligate primary symbiont Wigglesworthia, tsetse flies harbor the facultative secondary symbiont Sodalis glossinidius, whose biological function for the hosts is currently elusive (3, 8).Members of the suborder Heteroptera, known as true bugs and consisting of over 38,000 described species, are characterized by their sucking mouthparts, half-membranous forewings, and incomplete metamorphosis (46). In the Heteroptera, symbiotic associations with bacteria are mainly found in phytophagous groups, especially in stinkbugs of the infraorder Pentatomomorpha. These stinkbugs generally possess many sacs or tubular outgrowths, called crypts or ceca, in a posterior region of the midgut, whose lumen is densely populated by a specific bacterial symbiont (7, 16). In some cases, experimental elimination of the symbiotic bacteria resulted in retarded growth and high mortality of the host insects (1, 13, 21, 26, 27, 39), indicating that these gut symbionts play important biological roles. Most of the gut symbionts are vertically transmitted through host generations by such mechanisms as egg surface contamination in the families Pentatomidae and Acanthosomatidae (1, 27, 39, 40, 42), coprophagy in the Cydnidae and Coreidae (22, 45), and capsule transmission in the Plataspidae (20), whereas a case of environmental acquisition has been reported from the Alydidae (26). Thus far, gut symbiotic bacteria of some members of the Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae have been characterized using molecular techniques (21, 23, 25, 27, 38), while phylogenetic and biological aspects of gut symbiotic bacteria have been untouched in many other stinkbug groups.These gut symbiotic bacteria are, despite their extracellular localization, regarded as “primary symbionts” of the stinkbugs. On the other hand, some stinkbugs may, in addition to the gut symbiotic bacteria, also be associated with facultative “secondary symbionts.” For example, Wolbachia infections have been detected from diverse stinkbugs, most of which are probably of parasitic or commensalistic nature (24). Besides Wolbachia, there has been no report on facultative, secondary symbionts from stinkbugs.Members of the family Scutelleridae, often referred to as jewel bugs or shield-backed bugs, are stinkbugs characterized by their greatly enlarged convex scutellum that usually covers the entire abdomen. Some tropical species are also known for their vivid and beautiful body coloration (46). The family contains approximately 80 genera and 450 species, and in Japan, at least 7 genera and 9 species have been recorded (50). In the early 20th century, the presence of symbiotic bacteria was histologically described in midgut crypts of several scutellerid species (16, 31, 42). Since these pioneer works, however, no studies have been conducted on the symbiotic bacteria of scutellerid stinkbugs.Here we investigated the midgut symbiont of Cantao ocellatus, a scutellerid stinkbug widely distributed in Asian countries, including Japan, and known to guard their eggs and newborn nymphs (Fig. ​(Fig.1A)1A) (50). In addition to the gut symbiont, we also identified a Sodalis-allied facultative secondary symbiont from gonads of the insect.Open in a separate windowFIG. 1.(A) Adult female of Cantao ocellatus, guarding hatchlings under her body. (B) Dissected midgut from an adult female of C. ocellatus. 1st, midgut 1st section; 2nd, midgut 2nd section; 3rd, midgut 3rd section; 4th, midgut 4th section with crypts; hg, hindgut. (C) Enlarged image of the midgut 4th section with crypts. Arrowheads indicate three rows of crypts, while a fourth row is hidden behind. Glandular crypts (gc) are developed in adult females specifically, which may be involved in egg surface contamination with the symbiont. (D) An in situ hybridization image of the midgut 4th section, in which red and green signals indicate the gut symbiont and the host nucleus, respectively. Each arrow shows a crypt. (E) An enlarged image of the symbiotic bacteria in the crypts.