Materials

Laboratory strains of bruchid beetles used in this study are listed in Table 1(a). They were maintained on their host seeds at 30°C and 70% r.h. in a long-day regimen 16 hr light-8 hr dark. Natural populations of C. chinensis examined in this study are shown in Table 1(b). We collected adult beetles in fields of the azuki bean (Vigna angularis) and/or the sasage bean (V. sinensis) at six localities in central Japan (see Table 1(b) and later Fig. 4 for a map).

Table 1

Species, strain codes, original places of collection and their year in bruchids examined.

Sample preparation

For standard DNA analyses, adult insects were preserved in 100% acetone until DNA extraction. In order to examine the distribution of Wolbachia among tissues, live female adults of C. chinensis, jC strain were subjected to the following dissection procedure. The body of C. chinensis was divided into eleven parts: antennae, head, fore and hind wings, legs (fore-, mid-, and hind-), prothorax, muscle (from thorax), meso- and metathorax, gut, ovarioles with eggs, fat body, and abdomen. The dissection of body parts/tissues was carefully conducted in 100% ethanol with clean forceps. To minimize the contamination from other tissues, after a body part was carefully isolated from an insect, it was transferred to and rinsed with fresh ethanol.

DNA extraction

For standard DNA analyses, a single whole adult beetle preserved in acetone was subjected to DNA extraction. To examine the distribution of Wolbachia in the insect body, DNA was extracted from the isolated body part/tissue from a single insect. QIAamp Tissue Kit (QIAGEN) was used for DNA extraction essentially according to manufacturer's instruction. The material was put into a 1.5 ml plastic tube, and the preservative was removed well with a micropipette. Then, the material was homogenized with a plastic pestle in 200 μl of lysis buffer containing Proteinase K. After 3 hr or longer incubation at 55°C, DNA was purified from the lysate using QIAamp micro-spin columns.

PCR

PCR was conducted using Takara Taq polymerase (Takara) and its supplemented buffer system, under temperature profiles of 94°C for 2 min followed by 30 amplification cycles. Following primer sets and conditions were used. Around 1.6 kb of insect mitochondrial small subunit ribosomal RNA gene fragment (MtrDNA) was amplified with primers MtrA1 [5′-AAWAAACTAGGATTAGATACCCTA-3′] and MtrB1 [5′-TCTTAATYCAACATCGAGGTCGCAA-3′] (Fukatsu and Shimada, 1999) with cycles of 1 min at 94°C, 1 min at 48°C and 3 min at 68°C. About 1.5 kb of eubacterial 16S ribosomal RNA gene fragment (16S rDNA) was produced with primers 16SA1 [5′-AGAGTTTGATCMTGGCTCAG-3′] and 16SB1 [5′-TACGGYTACCTTGTTACGACTT-3′] (Fukatsu and Nikoh, 1998) with cycles of 1 min at 94°C, 1 min at 50°C and 2 min at 70°C. A 0.75 kb fragment of Wolbachia homolog of ftsZ, a cell-division related gene of bacteria, was specifically amplified with primers fts1 [5′-GTATGCCGATTGCAGAGCTTG-3′] and fts2 [5′ GCCATGAGTATTCACTTGGCT-3′], which were designed according to a previous report (Holden et al., 1993), with cycles of 1 min at 94°C, 1 min at 50°C and 1 min at 70°C. About 0.6 kb of Wolbachia surface protein gene fragment (wsp) was specifically produced with primers wsp1 [5′-GGATCCGGGTCCAATAAGTGATGAAGAAAC-3′] and wsp2 [5′-GGATCCTTAAAACGCTACTCCAGCTTCTGC-3′], which were provided by Dr. T. Sasaki, with cycles of 1min at 94°C, 1 min at 50°C and 1 min at 70°C. Although the wsp primers contained an additional BamHI site at the 5′ end for cloning, it did not affect specificity of the PCR reaction. The PCR products were electrophoresed in TAE-agarose gels, stained with ethidium bromide, and observed under an UV transilluminator.

DNA cloning and sequencing

Amplified product of 16S rDNA from C. chinensis, jC strain was subjected to cloning and sequencing essentially as described previously (Fukatsu and Nikoh, 1998). The product was directly cloned with TA-cloning vector pT7Blue (Novagen) and E. coli JM109 competent cell (Takara) using ampicillin and X-gal blue-white selection system. White colonies that were expected to contain inserted plasmid were directly subjected to PCR using primers on the flanking region of the vector's cloning site, U-19 [5′-GTTTTCCCAGTCACGACGT-3′] and BT7 [5′-TAATACGACTCACTATAGGG-3′], in order to check the length of the inserted DNA fragment (1.5 kb). The colonies identified to contain the gene fragment were isolated and cultured in 1.5 ml LB medium with ampicillin overnight, and subjected to plasmid extraction using QIAprep-Spin Miniprep Kit (QIAGEN). The purified plasmids, eluted with 30 μl TE buffer, were used for sequencing template DNA. Dye terminator labelled cycle sequencing reaction was conducted with BigDye DNA Sequencing Kit (PE Applied Biosystems). Six primers, 16SA1, 16SA2 [5′-GTGCCAGCAGCCGCGGTAATAC-3′], 16SA3 [5′ TGCATGGYTGTCGTCAGCTCG-3′], 16SB1, 16SB2 [5′-CGAGCTGACGACARCCATGCA-3′] and 16SB3 [5′-GTATTA-CCGCGGCTGCTGGCAC-3′] were used for sequencing. The tem-perature profile was 2 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 50°C and 2 min at 70°C. Reaction products were analyzed by ABI PRISM 377 DNA sequencer (PE Applied Biosystems). Determined partial sequences were edited and connected using DNASIS V. 3.0 (Hitachi Software Engineering Co., Ltd.).

Nucleotide sequence accession number

The sequence of 16S rDNA of Wolbachia sp. from C. chinensis, jC strain has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB025965.

Molecular phylogenetic analysis

The sequence data used for molecular phylogenetic analyses were searched for and retrieved from the DDBJ sequence database. Multiple alignment was performed by Clustal W (Thompson et al., 1994). The final alignment was inspected and corrected manually. Ambiguously aligned regions were excluded from the phylogenetic analysis. Nucleotide sites that included alignment gap(s) were also omitted from the aligned data set. Neighbor-joining tree (Saitou and Nei, 1987) was constructed with Kimura's two parameter distance (Kimura, 1980) using the program package Clustal W (Thompson et al., 1994), and bootstrap test (Felsenstein, 1981) was conducted with 1,000 resamplings.

Detection of Wolbachia in the Bruchidae

Twelve laboratory strains of bruchid beetles, which represented seven species, five genera and three subfamilies of Bruchidae, were subjected to DNA extraction to conduct diagnostic PCR experiments with Wolbachia-specific primers. DNA of a large molecular size was extracted from these insects (data not shown). Quality of the extracted DNA was confirmed by successful PCR amplification of 1.6 kb segment of insect MtrDNA (data not shown). When PCR was conducted with the specific primers to amplify ftsZ homolog of Wolbachia, an amplified product of expected size, approximately 0.75kb, was detected only from C. chinensis, strains jC and mrC97 (Fig. 1a). With another specific primer set to detect wsp gene fragment (0.6 kb) of Wolbachia, the same result was obtained (Fig. 1b). Therefore, it was strongly suggested that only C. chinensis was infected by Wolbachia among the bruchid species examined.

Fig. 1

Diagnostic PCR detection of Wolbachia with specific primer sets from laboratory strains of bruchid beetles. (a) ftsZ homolog specific to Wolbachia. (b) wsp gene specific to Wolbachia. Lane 1, Z. subfasciatus, C100 strain; lane 2, Z. subfasciatus, US strain; lane 3, C. chinensis, jC strain; lane 4, C. chinensis, mrC97 strain; lane 5, C. maculatus, hQ strain; lane 6, C. maculatus, iQ strain; lane 7, C. rhodesianus; lane 8, A. obtectus; lane 9, B. dorsalis, Harataima strain; lane 10, B. dorsalis, Tatsuno strain; lane 11, K. sharpianus, Mitsuma strain; lane 12, K. sharpianus, Yoneyama strain; lane 13, Ephestia kuehniella with Wolbachia sp. for a positive control; lane 14, no template control; lane M, DNA size markers (2000, 1500, 1000, 700, 525/500, 400, 300, 200 and 100 bps from the top to the bottom). Arrow indicates the specific PCR product. Other faint bands are due to non-specific amplification.

Molecular phylogeny of Wolbachia of C. chinensis based on 16S rDNA

To confirm the diagnostic PCR result, we determined the nucleotide sequence of bacterial 16S rDNA fragment amplified from C. chinensis, jC strain. The sequence showed high similarity to the 16S rDNA sequences of Wolbachia spp. deposited in the DNA databases. In the molecular phylogenetic tree (Fig. 2), the sequence constituted a compact monophyletic group in the α-Proteobacteria, supported by 100% bootstrap value, with Wolbachia sequences derived from other insects.

Fig. 2

Phylogenetic position of the bacterial 16S rDNA sequence (1,592 bp) amplified and cloned from C. chinensis, jC strain. The 16S rDNA sequence was analyzed by neighbor-joining method with sequences of representatives of the α-Proteobacteria and E. coli as an outgroup. A total of 1,271 unambiguously aligned nucleotide sites were subjected to the analysis. Bootstrap values obtained with 1,000 resamplings are shown at nodes. Numbers in brackets are the accession numbers of the sequence data.

Wolbachia infection in the body of C. chinensis

The distribution of the Wolbachia in the body of C. chinensis was checked by PCR experiments with Wolbachiaspecific primers. Using ftsZ primers (Fig. 3a), the amplified product was detected from all the body parts/tissues examined; antennae, head, wings, legs, prothorax, muscle, meso- and metathorax, gut, ovarioles with eggs, fat body, and abdomen. Using wsp primers (Fig. 3b), the same result was obtained.

Fig. 3

Distribution of the Wolbachia in the body of C. chinensis, examined by PCR with Wolbachia specific primers. (a) ftsZ, and (b) wsp. Lane 1, antennae; lane 2, head; lane 3, fore and hind wings; lane 4, fore-, midand hindlegs; lane 5, prothorax; lane 6, muscle from thorax; lane 7, mesothorax and metathorax; lane 8, gut; lane 9, ovarioles with eggs; lane 10, fat body; lane 11, abdomen; lane 12, Ephestia kuehniella with Wolbachia sp. for a positive control; lane 13, no template control; lane M, DNA size markers (2000, 1500, 1000, 700, 525/500, 400, 300, 200 and 100 bps from the top to the bottom). Arrow indicates the specific PCR product. Other faint bands are due to non-specific amplification.

Fig. 4

Locations of six collection sites with the infection rates of Wolbachia in C. chinensis in central Japan. Solid regions in pie diagrams represent the infection rates. The number of samples and the infection rates (%) in males (left) and females (right) are shown above and beneath each pie diagram, respectively. See Table 1(b) for detailed information on localities of the populations.

Wolbachia infection among natural populations of C. chinensis

In our field survey, occurrence of Wolbachia in natural populations of C. chinensis in Japan was investigated by PCR experiments (Fig. 4). All the individuals, 136 males and 273 females in total, collected at the six localities were Wolbachia-positive with ftsZ primers.