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. 2009 Jan 15;7:2.
doi: 10.1186/1741-7007-7-2.

Host-symbiont Co-Speciation and Reductive Genome Evolution in Gut Symbiotic Bacteria of Acanthosomatid Stinkbugs

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Free PMC article

Host-symbiont Co-Speciation and Reductive Genome Evolution in Gut Symbiotic Bacteria of Acanthosomatid Stinkbugs

Yoshitomo Kikuchi et al. BMC Biol. .
Free PMC article

Abstract

Background: Host-symbiont co-speciation and reductive genome evolution have been commonly observed among obligate endocellular insect symbionts, while such examples have rarely been identified among extracellular ones, the only case reported being from gut symbiotic bacteria of stinkbugs of the family Plataspidae. Considering that gut symbiotic communities are vulnerable to invasion of foreign microbes, gut symbiotic associations have been thought to be evolutionarily not stable. Stinkbugs of the family Acanthosomatidae harbor a bacterial symbiont in the midgut crypts, the lumen of which is completely sealed off from the midgut main tract, thereby retaining the symbiont in the isolated cryptic cavities. We investigated histological, ecological, phylogenetic, and genomic aspects of the unique gut symbiosis of the acanthosomatid stinkbugs.

Results: Phylogenetic analyses showed that the acanthosomatid symbionts constitute a distinct clade in the gamma-Proteobacteria, whose sister groups are the obligate endocellular symbionts of aphids Buchnera and the obligate gut symbionts of plataspid stinkbugs Ishikawaella. In addition to the midgut crypts, the symbionts were located in a pair of peculiar lubricating organs associated with the female ovipositor, by which the symbionts are vertically transmitted via egg surface contamination. The symbionts were detected not from ovaries but from deposited eggs, and surface sterilization of eggs resulted in symbiont-free hatchlings. The symbiont-free insects suffered retarded growth, high mortality, and abnormal morphology, suggesting important biological roles of the symbiont for the host insects. The symbiont phylogeny was generally concordant with the host phylogeny, indicating host-symbiont co-speciation over evolutionary time despite the extracellular association. Meanwhile, some local host-symbiont phylogenetic discrepancies were found, suggesting occasional horizontal symbiont transfers across the host lineages. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts.

Conclusion: Comprehensive studies of the acanthosomatid bacterial symbiosis provide new insights into the genomic evolution of extracellular symbiotic bacteria: host-symbiont co-speciation and drastic genome reduction can occur not only in endocellular symbiotic associations but also in extracellular ones. We suggest that many more such cases might be discovered in future surveys.

Figures

Figure 1
Figure 1
Specialized organs of Elasmostethus humeralis for harboring the symbiotic bacteria. (A) An adult female. (B) A dissected midgut: m1, midgut first section; m2, midgut second section; m3, midgut third section; m4, midgut fourth section with crypts; h, hindgut. (C) An enlarged image of the midgut fourth section, with butterfly-shaped symbiotic organ consisting of a number of crypts fused two-dimensionally. (D) A sectioned image of the symbiotic organ, stained with hematoxylin and eosin: c, midgut crypt; g, midgut main tract. (E) An in situ hybridization image of the symbiotic organ, wherein the symbiotic bacteria (red) and the insect nuclei (blue) are visualized. (F) A dissected ventral abdomen of a female insect, on which a pair of lubricating organs is seen (arrows). (G) An enlarged image of the lubricating organs (arrows) in the posterior tip of the abdomen. (H) A dissected lubricating organ, with the yellow membranous tissue surrounding the organ removed: r, chitinous ridge region; s, sac-like region. (I) An in situ hybridization image of the dissected lubricating organ, in which the symbiotic bacteria (red) are specifically detected in the sac-like region. Inset is a confocal image, showing tubulet-like structures harboring the symbiont. Bars, 2 mm in (A), 0.5 mm in (B), 0.2 mm in (C), 100 μm in (D) and (E), 1 mm in (F), 0.5 mm in (G), 0.25 mm in (H) and (I), and 100 μm in (I, inset).
Figure 2
Figure 2
Transmission electron microscopy of the symbiotic organ of acanthosomatid stinkbugs. (A) Midgut crypts of Elasmostethus humeralis. (B) Midgut crypts of Elasmostethus nubilus. (C) Symbiotic bacteria of E. humeralis. (D) Symbiotic bacteria of E. nubilus. Bars, 1 μm in (A) and (B); 0.3 μm in (C) and (D). Abbreviations: M, mitochondrion; N, nucleus; S, symbiont.
Figure 3
Figure 3
Phylogenetic placement of the symbiotic bacteria from the acanthosomatid stinkbugs in the γ-Proteobacteria on the basis of 16S rRNA gene sequences. A total of 1271 aligned nucleotide sites were subjected to the analysis. A Bayesian phylogeny is shown. On the nodes, posterior probabilities in the Bayesian analysis are shown above, and bootstrap probabilities (maximum parsimony (MP) analysis/maximum likelihood (ML) analysis) are shown below. Branches supported by less than 50% posterior probabilities were collapsed into polytomies. Asterisks indicate support values lower than 50%. Sequence accession numbers are in brackets. Percent AT contents of the sequences are in parentheses.
Figure 4
Figure 4
Phylogenetic placement of the symbiotic bacteria from the acanthosomatid stinkbugs in the γ-Proteobacteria on the basis of groEL gene sequences. A total of 1040 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 Bayesian tree is shown. Support values for the nodes are indicated as in Figure 3. Asterisks indicate support values lower than 50%. Sequence accession numbers are in brackets. Percent AT contents of the sequences are in parentheses. Note that the AT-content values are based on the data of all codon positions.
Figure 5
Figure 5
Diagnostic PCR detection of the symbiotic bacteria from dissected tissues of acanthosomatid stinkbugs. The results of Elasmostethus humeralis (A and B), Elasmostethus nubilus (C and D), and Elasmucha putoni (E and F) are shown. (A, C, and E) Detection of mitochondrial COI gene of the host insects. (B, D, and F) Detection of 16S rRNA gene of the symbiotic bacteria. Lane 1, head; lane 2, flight muscle; lane 3, foregut; lane 4, midgut first section; lane 5, midgut second section; lane 6, midgut third section; lane 7, midgut fourth section with crypts; lane 8, hindgut; lane 9, abdominal tip containing the lubricating organ; lane 10, ovary; lane 11, egg before hatching; lane 12, no template control; lane 13, positive control (template DNA from midgut crypts of E. humeralis TK used for phylogenetic analyses); lane M, DNA size markers (1500, 1000, 900, 800, 700, 600, 500, 400, 300, and 200 bp from top to bottom).
Figure 6
Figure 6
Effects of symbiont elimination on fitness parameters and phenotype of Elasmostethus humeralis. (A) Adult emergence rate (%). Emerged insects per total insects and P values of Fisher's exact probability test are indicated. (B) Developmental time to adulthood (days). Means and standard deviations are shown. Sample sizes and P values of Mann-Whitney U test are indicated. (C) Body size in terms of thorax width (mm). Means and standard deviations are shown. Sample sizes and P values of Mann-Whitney U test are indicated. (D) A normal adult female from a control egg mass. (E) A symbiont-free adult female from a surface-sterilized egg mass, exhibiting abnormal coloration. Bars, 2.5 mm.
Figure 7
Figure 7
Phylogenetic relationship of the acanthosomatid stinkbugs on the basis of mitochondrial COI gene sequences. A total of 611 aligned nucleotide sites were subjected to the analysis. A Bayesian phylogeny is shown. Support values for the nodes are indicated as in Figure 3. The phylogeny includes an additional four stinkbug species as outgroup taxa, whose family names are shown in parentheses. Sequence accession numbers are in brackets.
Figure 8
Figure 8
Phylogenetic congruence between the acanthosomatid stinkbugs and their symbiotic bacteria. Phylogenetic tree of the host insects (left; cf. Figure 7) and that of the symbiotic bacteria (right; cf. Figure 3) are contrasted. Each symbiont is connected to its host by a dashed line. Dots indicate the 10 co-divergence events inferred by TreeMap.
Figure 9
Figure 9
Pulsed field gel electrophoresis of the symbiont genomic DNA prepared from dissected midgut crypts of the acanthosomatid stinkbugs. (A) Elasmostethus humeralis. (B) Elasmostethus nubilus. (C) Sastragala esakii. Lane M1, size marker (Saccharomyces cerevisiae chromosomes); lane M2, size marker (lambda ladder); lane S1, I-CeuI digestion for E. humeralis; lane S2, I-CeuI digestion for E. nubilus; lane S3, AscI digestion for E. nubilus; lane S4,ApaI digestion for S. esakii; lane S5, SmaI digestion for S. esakii. Sizes of genomic DNA fragments are depicted on the gel images. Estimated genome sizes are shown under the gel images.

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