Members of the family Bruchidae (Insecta, Coleoptera) were examined for Wolbachia infection. Out of seven species investigated, which represented three subfamilies and five genera, amplified bands were detected only from Callosobruchus chinensis by diagnostic PCR with Wolbachia-specific primers. Bacterial 16S rDNA was amplified by PCR, cloned and sequenced from the total DNA of C. chinensis. Molecular phylogenetic analysis demonstrated that the sequence belongs to a monophyletic group of the genus Wolbachia in the α-Proteobacteria. The Wolbachia was detected from all tissues and body parts such as ovary, gut, fat body, muscle, wing, leg, head and antenna. In six geographic populations from central Japan, all the individuals examined, more than 400 in total, possessed the Wolbachia, indicating high prevalence of Wolbachia among natural populations of C. chinensis.
Endosymbiotic associations with microorganisms are quite ubiquitous in many groups of insects and other arthropods (Buchner, 1965; Baumann and Moran, 1997). There is an enormous variety of endosymbiotic relationships in which the host and symbiont interact with various degrees of interdependency. Some endosymbionts are obligate and essential endosymbiotic companions for the host, whereas others are regarded as facultative guest microbes of a commensal or rather parasitic nature. Members of the genus Wolbachia, which are widely distributed among various groups of insects, crustaceans and nematodes (Werren et al., 1995a; Werren, 1997; Werren and O'Neill, 1997), are the most intensively studied endosymbiont of the latter type. It has been shown that Wolbachia infection causes various sex-related aberrations in their arthropod hosts such as cytoplasmic incompatibility (Hoffmann et al., 1990; Hoffmann and Turelli, 1997), parthenogenesis (Stouthamer et al., 1990; Stouthamer, 1997) and feminization of genetic males (Rigaud et al., 1991; Rigaud, 1997; Kageyama et al., 1998). Because Wolbachia is inherited solely through maternal lineage of the host by vertical transmission, these reproductive manipulations can be interpreted as the strategy by which Wolbachia increases the frequency of infected females in populations of the host, often at the expense of host's fitness. In this context, Wolbachia is regarded as a selfish genetic element (Werren et al., 1988), is expected to conflict with the host in an evolutionary sense, and, therefore, is an attractive subject in evolutionary biology.
The azuki bean beetle, Callosobruchus chinensis (Coleoptera, Bruchidae), is known as a pest of stored azuki bean, Vigna angularis. In addition to the importance as a pest insect, this beetle has been widely used as a model organism in population biology because various parameters related to its population dynamics can easily be measured, quantified and controlled under laboratory conditions (Utida, 1941; Hassell et al., 1989; Shimada and Tuda, 1996). Thus, if C. chinensis harbors an endosymbiotic microorganism, it will provide us with a good system to investigate the dynamics of host-symbiont interactions.
In this paper, we report the discovery of Wolbachia in C. chinensis based on molecular phylogenetic and PCR analyses. We also report high prevalence of Wolbachia among natural populations in Japan and widespread distribution in host's entire body.
MATERIALS AND METHODS
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).
Species, strain codes, original places of collection and their year in bruchids examined.
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.
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 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.
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.
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.
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.
Among seven species of Bruchidae examined in this study, only C. chinensis exhibited the amplified bands by diagnostic PCR with two Wolbachia-specific primer sets, ftsZ and wsp (Fig. 1). Molecular phylogenetic analysis confirmed that C. chinensis possesses a bacterium of the genus Wolbachia (Fig. 2). So far as we know, there has been no report on the endosymbiont, including Wolbachia, from the family Bruchidae except for Rickettsia sp. in Kytorhinus sharpianus (Fukatsu and Shimada, 1999), although various types of endosymbiotic microorganisms have been found from related coleopteran groups such as Chrysomelidae, Curculionidae and Rhynchophoridae (Buchner, 1965; Campbell et al., 1992; Heddi et al., 1998).
Although our sampling of bruchid species was far from complete, the result that Wolbachia was detected only in C. chinensis suggests that the infection with Wolbachia may not be common in the family Bruchidae. Even in the genus Callosobruchus, Wolbachia was not detected in C. maculatus nor C. rhodesianus but found only in C. chinensis. On the other hand, in the species C. chinensis, the Wolbachia was universally distributed among natural populations in central Japan. From these results, it is conceivable that the Wolbachia was acquired by a common ancestor of C. chinensis, possibly from either an unexamined bruchid carrier or a phylogenetically distant arthropod. In fact, several studies have claimed that interspecific transmissions must have occurred throughout the evolutionary history of Wolbachia (O'Neill et al., 1992; Werren et al., 1995b).
Since Wolbachia is passed to the next host generation by vertical transmission and causes sex-related aberrations in the host, it has been widely assumed that Wolbachia specifically infect reproductive tissues. Recently, however, it was reported that Wolbachia are much more widely distributed among host tissues in insects than previously appreciated (Dobson et al. 1999), which is concordant with our finding that Wolbachia was detected by PCR not only from an ovary but from all the other tissues and body parts of C. chinensis.
So far, several works have been conducted on Wolbachia infection rates in natural populations, Drosophila simulans in California (Turelli and Hoffman 1995), D. melanogaster in Australia (Hoffmann et al. 1994), and Laodelphax striatellus in northeastern Japan (Noda 1984; Hoshizaki and Shimada 1995), in which most of local populations were not infected perfectly. Compared with these reports, perfect infection of C. chinensis by Wolbachia (Fig. 4) appears quite remarkable, because complete infection rate has not been reported except the population of L. striatellus in southwestern Japan (Noda 1984; Hoshizaki and Shimada 1995).
In theoretical models, factors determining an infection rate of Wolbachia in a host population are, first, fidelity of vertical transmission of Wolbachia, secondly, fitness cost of an infected host such as decrease in viability and fecundity, thirdly, the degree of cytoplasmic incompatibility, and finally, initial frequency of invasion (Caspari and Watson, 1959; Fine 1978; Turelli and Hoffmann, 1995). These factors in C. chinensis populations should be investigated in detail to identify the determinants of the remarkable complete infection. In addition, we plan to analyse the wsp sequence and phylogenetic differences of the Wolbachia in C. chinensis from different geographical populations.
The azuki bean beetle C. chinensis is a useful material for population biological studies because it is easy to establish and maintain experimental populations in order to examine its life history parameters. Therefore, this species will provide us with a model system to understand the interaction and dynamics between Wolbachia and its host insect.
We thank Dr. T. Sasaki for kindly providing PCR primers. We are also grateful to Y. Harimoto, A. Sugimura, S. Kumagai and K. Sato for technical and secretarial assistance. In addition, we thank two anonymous referees for giving many valuable comments on an earlier version of the ms. This research was supported in part by Grants-in-Aids for Scientific Research (08304043) to M. S. and T. F., and International Scientific Research (09044202) to M. S. from the Ministry of Education, Science and Culture of Japan, and also supported by Industrial Science and Technology Frontier Program “Technological Development of Biological Resources in Bioconsortia” to T. F. from the Ministry of International Trade and Industry of Japan.