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

Insects.Collection data for the adult C. ocellatus insects used in this study are summarized in Table 1. Insect samples were collected from the Japanese mallotus tree, Mallotus japonicus, at nine localities in Japan in 2008 and 2009.

Dissection and DNA extraction.Ovaries, testes, and midgut 1st, 2nd, 3rd, and 4th sections were dissected from adult insects using a pair of fine forceps under a binocular microscope in a plastic petri dish filled with phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄ [pH 7.5]). These insect tissues were immediately subjected to DNA extraction using Nucleo Spin tissue kit (Macherey-Nagel).

DNA cloning and sequencing.A 1.5-kb segment of bacterial 16S rRNA gene was amplified with the primers 16SA1 (5′-AGAGTTTGATCMTGGCTCAG-3′) and 16SB1 (5′-TACGGYTACCTTGTTACGACTT-3′) (12). A 1.7-kb fragment of bacterial groEL gene was amplified with the primers Gro-F1 (5′-ATGGCAGCWAAAGACGTAAATTYGG-3′) and Gro-R1 (5′-TTACATCATKCCGCCCATGC-3′). PCR was conducted with Ampli Taq DNA polymerase (Applied Biosystems) and the supplemented buffer system, under the following temperature profile: 95°C for 10 min, followed by 35 cycles consisting of 95°C for 30 s, 55°C for 1 min, and 72°C for 2 min. The PCR products were subjected to the cloning using TA cloning vector pT7Blue (Takara Bio) and Escherichia coli DH5α competent cells (Takara Bio) with a blue-white selection system, using ampicillin and 5-bromo-4-chloro-3-indolyl-d-galactopyranoside (X-Gal). In order to check the length of the inserted DNA fragment, white colonies, which were expected to contain inserted plasmids, were directly subjected to PCR with the primers Univ19 (5′-GTTTTCCCAGTCACGACGT-3′) and Rev20 (5′-AGCTATGACCATGATTACGC-3′). When a PCR product of expected size (1.5 kb and 1.7 kb, respectively) was obtained, the product was subjected to restriction fragment length polymorphism (RFLP) genotyping. Three or more clones for each of the RFLP genotypes were cultured at 37°C in LB liquid medium overnight and subjected to plasmid extraction using a QIAprep-Spin miniprep kit (Qiagen). The purified plasmids were subjected to DNA sequencing with the primers Univ19 and Rev20 and the internal primers Eub925r (5′-CCGYCAATTCCTTTGAGTTT-3′) and Eub1405r (5′-GACGGGCGGTGTGTRCA-3′) for the 16S rRNA gene, groE-GS (5′-TAATGCTGATGAAAGCGT-3′) for the groEL gene of the midgut symbiont, and groE-SD (5′-ACTATCTCCGCCAACTC-3′) for the groEL genes of the Sodalis-allied symbiont, under a temperature profile of 96°C for 1 min, followed by 26 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, using a Genetic Analyzer (Applied Biosystems 3130x1).

Molecular phylogenetic analysis.Bacterial 16S rRNA and groEL gene sequences were subjected to molecular phylogenetic analysis together with those of gammaproteobacterial representatives. Multiple alignments were generated using the program ClustalW (48). Aligned nucleotide sites containing a gap were removed from the data set using the program SeaView (15), and the final alignment was corrected manually. Phylogenetic analyses were conducted by maximum likelihood (ML) and maximum parsimony (MP) methods. The best-fit substitution models were selected by using the program MrModeltest v2.1 (J. A. Nylander, 2004; http://www.ebc.uu.se/systzoo/staff/nylander.html ). ML phylogenies, with 100 bootstrap resamplings, were estimated using the program phyML 3.0 (18) with the GTR model for both 16S rRNA and groEL phylogenies. MP phylogenies, with 1,000 bootstrap resamplings, were estimated using the program phylo_win (15).

Relative rate test.A relative rate test, based on genetic distances estimated under the Kimura's two-parameter model (28), was performed using the program RRTree (41). For 16S rRNA gene sequences, 1,426 unambiguously aligned nucleotide sites were subjected to the analysis. For groEL gene sequences, 1,040 unambiguously aligned nucleotide sites at the first- and second-codon positions were analyzed, while nucleotide sites at the third-codon positions were omitted from the analysis due to saturated nucleotide substitutions.

Diagnostic PCR.The following specific primer sets were used for diagnostic PCR detection of 16S rRNA gene of the symbiotic bacteria: 16SA2 (5′-GTGCCAGCAGCCGCGGTAATAC-3′) and akagi16S780R (5′-GCTACGGAGGCTACTTATT-3′) for a 0.16-kb segment from the midgut symbiont; and sodalis370F (5′-CGRTRGCGTTAAYAGCGC-3′) and 16SB2 (5′-CGAGCTGACGACARCCATGC-3′) for a 0.62-kb segment from the Sodalis-allied symbiont. The temperature profile was 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min. To check the quality of the DNA samples, a 1.5-kb segment of the insect mitochondrial 16S rRNA gene was amplified with the primers MtrA1 (5′-AAWAAACTAGGATTAGATACCCTA-3′) and MtrB1 (5′-TCTTAATYCAACATCGAGGTCGCAA-3′) (11).

Whole-mount FISH.The Alexa 555-labeled oligonucleotide probe Al555-AKG447 (5′-GTGTATTATTTTTCATCACC-3′) was used for whole-mount fluorescent in situ hybridization (FISH) targeting 16S rRNA of the midgut symbiont. Midgut sections with crypts were dissected from adult females and fixed in Carnoy's solution (6:3:1 ethanol-chloroform-acetic acid) overnight. The fixed samples were incubated in ethanolic 6% H₂O₂ solution for a week to quench the autofluorescence of the tissues (30). The insects were thoroughly washed and equilibrated with a hybridization buffer (20 mM Tris HCl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide), to which the specific probe, SYTOX green and DAPI (4′,6-diamidino-2-phenylindole) were added at final concentrations of 0.1 μM, 0.5 μM, and 0.1 μg/ml, respectively. After an overnight incubation, the samples were thoroughly washed in PBSTx (PBS containing 0.3% Triton X-100) and observed under an epifluorescence microscope (Axiophoto; Carl Zeiss) and a laser confocal microscope (PASCAL5; Carl Zeiss). To confirm the specificity of the detection, the following control experiments were conducted: a no-probe control experiment, a no-SYTOX control experiment, and a competitive suppression control experiment with excess unlabeled probe.

Transmission electron microscopy.The midgut crypts were dissected from adult females in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde, prefixed in the fixative at 4°C overnight, and postfixed in the phosphate buffer containing 2% osmium tetroxide at 4°C for 60 min. After dehydration through an ethanol series, the materials were embedded in Spurr resin (Nisshin-EM). Ultrathin sections (thickness, 80 nm) were made on an ultramicrotome (Ultracat-N; Leichert-Nissei), mounted on collodion-coated copper meshes, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (model H-7000; Hitachi).

Egg sterilization experiments.In total, 17 egg masses laid by different females collected at Ishigaki Island in 2008 and 2009 were subjected to the experiments. Nine randomly selected egg masses were treated with 70% ethanol for 5 min and 4% formalin for 15 min, rinsed with 70% ethanol, and dried in air. For a control, five egg masses were treated with water for 20 min instead of the surface sterilization. The other three egg masses were kept untreated. Each of the 17 egg masses was separately placed in a plastic petri dish (5 cm in diameter and 1 cm in depth) with a wet cotton ball at 25°C under a long-day condition (16 h of light and 8 h of dark [16L/8D]) until hatching. The hatchlings were fed with raw peanuts, dry soybeans, and distilled water at 25°C (16L/8D) until they reached the 3rd instar. Ten to 20 nymphs were randomly collected from each of the egg masses and preserved in acetone and were subjected to DNA extraction and diagnostic PCR.

General observation of digestive tract.Digestive organs were dissected from adult females of C. ocellatus (Fig. 1B). The enlarged midgut 1st section was filled with liquid, followed by the elongated midgut 2nd and 3rd sections. The midgut 4th section bore a number of well-developed crypts, which were white, arranged in four rows, and fused to each other into a loose helical assemblage (Fig. 1C).

Bacterial gene sequences from midgut section with crypts.Insects collected at nine localities in Japan were examined. One to three insect samples from each of the localities were dissected, and their midgut 4th sections were subjected to DNA extraction and PCR amplification of the bacterial 16S rRNA gene. The PCR products were cloned, and three to eight clones per insect were sequenced. All of the bacterial sequences obtained were identical without geographic and populational variations. The bacterial groEL gene was also amplified, cloned, and sequenced from an insect collected at Onna, Okinawa Island.

Phylogenetic placement of the gut symbiont.Phylogenetic analyses of the 16S rRNA gene sequences revealed that the gut symbiont constituted a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Meanwhile, the lineage was placed in a cluster of diverse insect symbionts, including endocellular symbionts of aphids, sharpshooters, and tsetse flies, and also gut symbionts of acanthosomatids, plataspids, and pentatomids, although the phylogenetic assemblage was not conclusive, with relatively low bootstrap supports (Fig. 2). Phylogenetic analyses on the basis of groEL gene sequences yielded a similar phylogenetic relationship: the gut symbiont was placed in a cluster of insect symbionts consisting of endocellular symbionts of aphids, sharpshooters, and tsetse flies, and also gut symbionts of acanthosomatids and plataspids (Fig. 3).

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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.

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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.

Localization and morphology of the gut symbiont.Diagnostic PCR analysis of dissected tissues detected the symbiont DNA sequence preferentially in the midgut 4th section with crypts, while the midgut 2nd and 3rd sections of females also exhibited the symbiont infection (Fig. 4). In situ hybridization targeting 16S rRNA of the gut symbiont identified strong and specific signals in the lumen of the crypts in the midgut 4th section (Fig. 1D). In each of the crypts, tubular symbiont cells were distributed not uniformly but concentrated near the epithelial cell layer (Fig. 1E).

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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.

Fine structure of the midgut crypt and the gut symbiont.Electron microscopy revealed a peculiar histological configuration inside the midgut crypts. At the central region of the crypts, the symbiont cells were found in the extracellular space in a relatively dispersed manner (Fig. 5A). Meanwhile, at the peripheral zone of the crypts, the symbiont cells and the host epithelial cells were closely associated with each other in an intermingled manner (Fig. 5A and B). Some of the symbiont cells were seen in the epithelial cell layer, next to mitochondria and endoplasmic reticula, as if they were located in the host cytoplasm (Fig. 5C). However, close examination of a number of electron microscopic images, including those from serial sections, revealed that those symbiont cells were actually located in extracellular spaces. It was postulated that the symbiont cells were entrapped in narrow extracellular spaces constituted by convoluted projections from the epithelial cell layer. Of course, we cannot rule out the possibility that, while the majority of the symbiont cells were found extracellularly, a small population of the symbiont cells might be nearly or entirely engulfed by the gut epithelial cells.

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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.

Detection and phylogenetic placement of the Sodalis-allied symbiont.From ovarial DNA of an adult female collected at Ishigaki Island, bacterial 16S rRNA and groEL genes were detected. The PCR products were cloned, and eight clones were sequenced. All of the sequences were identical to each other. Phylogenetic analyses of the sequences revealed that the ovarial symbiont belongs to the Gammaproteobacteria, forming a well-supported clade with the secondary symbiont of tsetse flies Sodalis glossanidius, the bacteriome-associated symbiont of grain weevils, the symbiont of a louse fly, the bacteriocyte-associated symbiont of a pigeon louse, and the secondary symbiont of a Curculio weevil (Fig. 2 and 3).

AT richness of the gut symbiont genes.The 16S rRNA gene sequence of the gut symbiont exhibited an AT content of 48.4%, which was slightly higher than the AT contents of free-living gammaproteobacteria around 45% (Fig. 2). The groEL gene sequence of the gut symbiont was 60.1% AT, which was drastically higher than the values of free-living gammaproteobacteria of around 45% (Fig. 3). On the other hand, the 16S rRNA gene sequence and the groEL gene sequence of the Sodalis-allied symbiont were not AT biased: 44.4% and 43.2% AT, respectively (Fig. 2 and 3).

Accelerated molecular evolution of the gut symbiont genes.The evolutionary rates of the 16S rRNA and groEL gene sequences of the gut symbiont of C. ocellatus were significantly higher than those of free-living gammaproteobacteria, respectively. Meanwhile, the evolutionary rates in the Sodalis-allied symbiont of C. ocellatus were not significantly different from those of free-living gammaproteobacteria (Table 2).

Infection frequencies of the gut symbiont and the Sodalis-allied symbiont in natural host populations.Table 3 shows infection frequencies of the gut symbiont and the Sodalis-allied symbiont in natural populations of C. ocellatus. The gut symbiont was detected from all 116 insects representing 9 populations examined. Meanwhile, the Sodalis-allied symbiont was detected only from 6 of 144 insects examined. All of the infected insects were collected at Ishigaki Island, where the infection frequency with the Sodalis-allied symbiont was 11% (6/53).

Egg surface sterilization blocks vertical transmission of the gut symbiont.Table 4 summarizes the results of the egg surface sterilization experiments. The formalin and ethanol sterilization procedure did not affect the hatching rates of the treated eggs, indicating no detrimental effects of the treatment on the embryonic development. All of the nymphs from the sterilized eggs were free of the gut symbiont, whereas most of the nymphs from the water-treated eggs and untreated eggs were infected with the gut symbiont.