Primary gut symbiont and secondary, Sodalis-allied symbiont of the Scutellerid stinkbug Cantao ocellatus

Abstract

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.

FIG. 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.

FIG. 2.

Phylogenetic placement of the symbiotic bacteria of C. ocellatus on the basis of 16S rRNA gene sequences. A maximum likelihood (ML) tree inferred from a total of 1,243 aligned nucleotide sites is shown, whereas a maximum parsimony (MP) analysis gave substantially the same topology (data not shown). Bootstrap values higher than 50% are indicated at the nodes in the order of ML/MP. Asterisks indicate support values lower than 50%. Sequence accession numbers and AT contents of the nucleotide sequences are in brackets and parentheses, respectively. As for insect endosymbionts, the name of the host insect is also indicated in parentheses. P-symbiont, primary symbiont; S-symbiont, secondary symbiont.

FIG. 3.

Phylogenetic placement of the symbiotic bacteria from C. ocellatus on the basis of groEL gene sequences. A total of 1,104 aligned nucleotide sites at first and second codon positions were subjected to the analysis. The third codon positions were not used because of saturated nucleotide substitutions. Analysis of deduced amino acid sequences gave substantially the same results (data not shown). A maximum likelihood tree is shown. Support values for the nodes are indicated as in Fig. 2. Sequence accession numbers and AT contents of the nucleotide sequences are in brackets and parentheses, respectively. Note that the AT content values are based on the data of all codon positions.

FIG. 4.

Diagnostic PCR detection of the midgut symbiont in dissected tissues of adult C. ocellatus. Negative control, distilled water; positive control, DNA from midgut crypts of C. ocellatus; lane M, DNA size markers (from bottom to top) from 100 bp to 1,000 bp in 100-bp increments and 1,500 bp.

FIG. 5.

Transmission electron microscopy of the midgut crypts in adult females of C. ocellatus. (A) Image of the interface between the crypt cavity and the epithelial cell layer; (B) enlarged image of the transitional zone; (C) enlarged image of the symbiont cells indicating an extracellular, rather than endocellular location. Asterisks and arrows indicate the symbiont cells and mitochondria, respectively. N, nucleus of the epithelial cell of the midgut crypt.