Infection prevalence of Sodalis symbionts among stinkbugs

Diverse insects and other organisms are associated with microbial symbionts, which often significantly contribute to growth and survival of their hosts and/or drastically affect phenotypes of their hosts in a variety of ways. Sodalis glossinidius was first identified as a facultative bacterial symbiont of tsetse flies, and recent studies revealed that Sodalis-allied bacteria encompass diverse ecological niches ranging from free-living bacteria through facultative symbionts to obligate symbionts associated with a diverse array of insects. Despite potential ecological and evolutionary relevance of the Sodalis symbionts, their infection prevalence in natural insect populations has been poorly investigated. Here we surveyed diverse stinkbugs and allied terrestrial heteropteran bugs, which represented 17 families, 77 genera, 108 species, 310 populations and 960 individuals, for infection with the Sodalis symbionts. Diagnostic PCR detected relatively low infection frequencies of the Sodalis symbionts: 13.6% (14/103) of the species, 7.5% (22/295) of the populations, and 4.3% (35/822) of the individuals of the stinkbugs except for those belonging to the family Urostylididae. Among the urostylidid stinkbugs, strikingly, the Sodalis symbionts exhibited very high infection frequencies: 100% (5/5) of the species, 100% (15/15) of the populations, and 94.2% (130/138) of the individuals we examined. Molecular phylogenetic analysis based on bacterial 16S rRNA gene sequences revealed that all the symbionts were placed in the clade of Sodalis-allied bacteria while the symbiont phylogeny did not reflect the systematics of their stinkbug hosts. Notably, the Sodalis symbionts of the urostylidid stinkbugs were not clustered with the Sodalis symbionts of the other stinkbug groups on the phylogeny, suggesting their distinct evolutionary trajectories. The relatively low infection frequency and the overall host-symbiont phylogenetic incongruence suggest that the Sodalis symbionts are, in general, facultative symbiotic associates in the majority of the stinkbug groups. On the other hand, it is conceivable, although speculative, that the Sodalis symbionts may play some substantial biological roles for their host stinkbugs of the Urostylididae.


Introduction
Diverse insects are associated with symbiotic microorganisms [1]. Some symbionts are obligate companions essential for their hosts via, for example, provisioning of essential nutrients deficient in their host's diets, and often referred to as the primary symbionts [2,3]. Other symbionts are facultative associates not essential for their hosts, and often designated as the secondary symbionts [4,5].
Grasping infection prevalence of these symbionts is important for gaining insights into biological interactions with their hosts. The primary symbionts of obligate nature generally exhibit 100% infection frequencies in their host populations due to their indispensable roles. By contrast, the secondary symbionts of facultative nature exhibit variable infection frequencies ranging from near 0% to almost 100% depending on the symbiont species, the host species and populations, the environmental conditions, etc. For example, some Wolbachia strains attain 100% infection frequencies in their host populations by their selfish driving mechanisms such as cytoplasmic incompatibility and parthenogenesis induction [6,7]. The facultative symbionts Serratia, Regiella and Hamiltonella in natural aphid populations exhibit intermediate values between 0% to 100% [17][18][19][20], which probably reflect their context-dependent fitness consequences [8,9,11,13].
In this study, we surveyed diverse stinkbugs and allied terrestrial heteropteran bugs (order Hemiptera: suborder Heteroptera: infraorder Pentatomomorpha), which represent 17 families, 77 genera, 108 species, 310 populations and 960 individuals, for infection with Sodalis symbionts by diagnostic PCR and molecular phylogenetic approaches.

Insect samples
Additional file 1 lists the insect samples examined in this study. These insects were preserved in either acetone or ethanol [56], or freshly brought to the laboratory. For large specimens, dissected gonad was subjected to DNA extraction. For small specimens, dissected abdomen was subjected to DNA extraction. DNA extraction was performed using QIAamp DNA Mini kit (Qiagen).

Molecular phylogenetic analysis
A multiple alignment of the nucleotide sequences was generated by the program MAFFT version 7.127b [59]. The nucleotide substitution model, GTR + I + G, was selected using the program jModelTest 2 [60,61]. The phylogenetic analyses were conducted by Bayesian (BA) and maximum-likelihood (ML) methods with the programs MrBayes v3.2.2 [62] and RAxML version 7.2.6 [63], respectively. In the BA analysis, in total 37,500 trees were obtained for each analysis (ngen = 50,000,000, samplefreq = 1,000, burn in = 12,501, temp = 0.2) and multiple independent runs were conducted to ensure the stable results. Posterior probabilities were calculated for each node by statistical evaluation in BA, whereas bootstrap values were obtained with 1000 replications in ML.
In previous studies, 16S rRNA gene sequences of the Sodalis symbionts were determined for two scutellerid species Cantao ocellatus and Eucoryses grandis [38,39] and four urostylidid species Urostylis annulicornis, Urostylis striicornis, Urostylis westwoodii and Urochela quadrinotata [40]. In this study, we newly cloned and sequenced 16S rRNA gene of the Sodalis symbionts from the following heteropteran species: an acanthosomatid Elasmucha putoni; pentatomids Aelia fieberi (from two populations), Dolycoris baccarum, Glaucias subpunctatus, Lelia decempunctata, Nezara antennata (from three populations), Palomena angulosa, Picromerus lewisi and Piezodorus hybneri; a scutellerid Poecilocoris lewisi; and a rhopalid Rhopalus sapporensis (Figure 1). Molecular phylogenetic relationship of the Sodalis symbionts associated with the heteropteran bugs and other insects was inferred from the 16S rRNA gene sequences ( Figure 2). The phylogenetic pattern indicated that (i) all the symbiont sequences were placed in the clade of Sodalis-allied bacteria with high statistical supports, (ii) the symbiont sequences within the same host species tended to be closely related to each other, (iii) nonetheless, the overall phylogenetic relationship of the symbiont sequences did not reflect the systematics of the host stinkbugs, and (iv) notably, the Sodalis symbionts of the urostylidid stinkbugs were not clustered with the Sodalis symbionts of the other stinkbug groups on the phylogeny.
The relatively low infection frequencies and the overall host-symbiont phylogenetic incongruence favor the hypothesis that the Sodalis symbionts are, in general, facultative associates for the heteropteran bugs, as Wolbachia, Rickettsia, Spiroplasma, Lariskella, etc. [41,[64][65][66]. The majority of the plant-sucking heteropteran bugs harbor specific gut bacteria as the primary symbionts within the crypt cavities present in a posterior midgut region [1,67,68], which are important for normal growth, survival and reproduction of the host insects [69][70][71][72][73][74][75][76][77][78][79][80]. Probably, the majority of the Sodalis symbionts are, unlike the primary gut symbionts, not essential for their heteropteran hosts. On the other hand, it is conceivable, although speculative, that the Sodalis symbionts may play some substantial biological roles for their host stinkbugs in the Urostylididae. It deserves future studies what    biological roles, which are likely condition-dependent ones, the Sodalis symbionts play for their urostylidid hosts.

Conclusions
In conclusion, our results highlight that the Sodalis symbionts are facultative symbiotic bacteria commonly associated with diverse insects, as are Wolbachia, Rickettsia, Spiroplasma, Cardinium, Arsenophonus and other widespread facultative symbionts. In this study, we exhaustively surveyed diverse stinkbugs in Japan, but, considering the recent report on the infection prevalence of the Sodalis symbiont in African populations of the coffee bug Antestiopsis thunbergii (Pentatomidae) [41], the occurrence of the Sodalis symbionts seems widespread among world's stinkbugs and other insects. Future studies should focus on comprehensive survey of insect groups other than the heteropteran bugs, and also on effects and consequences of their infection to the host insects. Comparative studies on Sodalis-infected and uninfected host insects under the same genetic background combined with genomic and molecular biological analyses of the Sodalis symbionts will provide insights into ecological and evolutionary aspects of animal-microbe symbioses wherein the associations may range from free-living through facultative to obligate.

Additional file
Additional file 1: Stinkbug samples examined in this study and detection of Sodalis symbionts from the samples.

Competing interests
The authors declare that they have no competing interests.

Figure 2
Phylogenetic relationship between Sodalis symbionts of heteropteran bugs and other insects inferred from 16S rRNA gene sequences (1204 aligned nucleotide sites). A Bayesian phylogeny is shown with statistical support values (50% or higher) at the nodes: posterior probabilities of Bayesian analysis/bootstrap probabilities of maximum likelihood analysis. Asterisks indicate support values lower than 50%. Sequences obtained from stinkbugs are highlighted by boldface, wherein collection localities are indicated in parentheses and accession numbers in brackets.