Strict host-symbiont cospeciation and reductive genome evolution in insect gut bacteria

Abstract

Host-symbiont cospeciation and reductive genome evolution have been identified in obligate endocellular insect symbionts, but no such example has been identified from extracellular ones. Here we first report such a case in stinkbugs of the family Plataspidae, wherein a specific gut bacterium is vertically transmitted via "symbiont capsule." In all of the plataspid species, females produced symbiont capsules upon oviposition and their gut exhibited specialized traits for capsule production. Phylogenetic analysis showed that the plataspid symbionts constituted a distinct group in the gamma-Proteobacteria, whose sister group was the aphid obligate endocellular symbionts Buchnera. Removal of the symbionts resulted in retarded growth, mortality, and sterility of the insects. The host phylogeny perfectly agreed with the symbiont phylogeny, indicating strict host-symbiont cospeciation despite the extracellular association. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts. These findings suggest that not the endocellular conditions themselves but the population genetic attributes of the vertically transmitted symbionts are probably responsible for the peculiar genetic traits of these insect symbionts. We proposed the designation "Candidatus Ishikawaella capsulata" for the plataspid symbionts. The plataspid stinkbugs, wherein the host-symbiont associations can be easily manipulated, provide a novel system that enables experimental approaches to previously untouched aspects of the insect-microbe mutualism. Furthermore, comparative analyses of the sister groups, the endocellular Buchnera and the extracellular Ishikawaella, would lead to insights into how the different symbiotic lifestyles have affected their genomic evolution.

Figure 1. Adult Females of the Plataspid Stinkbugs and Their Posterior Midgut

(A and B) M. cribraria; (C and D) B. subaeneus; (E and F) C. parvipictum. Bars, 1 mm. AEM, anterior enlarged midgut section; BEM, brownish enlarged midgut end section; REC, rectum; SCM, swollen crypt-bearing midgut section [28].

Figure 2. Phylogenetic Placement of the Symbiotic Bacteria from the Plataspid Stinkbugs in the γ-Subclass of the Proteobacteria on the Basis of 16S rRNA Gene Sequences

A total of 1,213 aligned nucleotide sites were subjected to the analysis. A Bayesian tree is shown, while MP tree and ML tree exhibited substantially the same topologies. On each of the nodes, posterior probabilities in the Bayesian analysis are shown above, and bootstrap probabilities (MP analysis/ML analysis) are shown below. Asterisks indicate bootstrap values lower than 50%. In brackets are sequence accession numbers. In parentheses are collection localities for the stinkbug symbionts and host insect names for the other symbionts, respectively.

Figure 3. Effects of Symbiont Elimination on the Adult Emergence Rate of the Plataspid Stinkbugs

(A) M. punctatissima; (B) M. cribraria; (C) B. subaeneus; (D) C. parvipictum. Means and standard deviations are shown. Open columns, with capsules; filled columns, without capsules. Sample sizes and p values of Wilcoxon test are indicated.

Figure 4. Effects of Symbiont Elimination on Adult Body Size and Phenotype of the Plataspid Stinkbugs

(A) Adult females of M. punctatissima (left) and M. cribraria (right) emerged from the control egg masses with capsules (top) and those from the treated egg masses without capsules (bottom). (B) Thorax width of M. punctatissima and (C) thorax width of M. cribraria. Means and standard deviations are shown. Open columns, with capsules; filled columns, without capsules. Sample sizes are labeled on the columns. Asterisks indicate statistically significant differences (median test; *** p < 0.001 after Bonferroni correction).

Figure 5. Phylogenetic Congruence between the Plataspid Stinkbugs and Their Symbiotic Bacteria

(A) A Bayesian phylogeny of the host insects on the basis of mitochondrial 16S rRNA gene sequences (847 aligned nucleotide sites). In addition to the plataspid stinkbugs, four pentatomomorphan species were examined as out-group taxa. In parentheses are taxonomic affiliations and in brackets are sequence accession numbers. (B) A Bayesian phylogeny of the plataspid symbionts on the basis of the same data of Figure 2 (16S rRNA gene, 1,213 aligned nucleotide sites). Dots indicate codivergence events inferred by the jungles algorithm [34]. Posterior probabilities in the Bayesian analysis and bootstrap values in the MP/ML analyses are presented as shown in Figure 2.

Figure 6. PFGE of the Symbiont Genomic DNA Prepared from the Posterior Midgut of a Female Adult of the Plataspid Stinkbugs

Lane 1, M. punctatissima; lane 2, M. cribraria; lane 3, C. parvipictum; lane 4, yeast PFGE marker; lane 5, lambda PFGE marker. Marker sizes are shown on the right side.