Genome fragment of Wolbachia endosymbiont transferred to X chromosome of host

Natsuko Kondo*, Naruo Nikoh†‡, Nobuyuki Ijichi†§, Masakazu Shimada*, and Takema Fukatsu†¶

*Department of Systems Science (Biology), and §Department of Biological Science at the Graduate School of Arts and Sciences, University of Tokyo, Meguro, Tokyo 153-8902, Japan; †Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan; and ‡Bio-Oriented Technology Research Advancement Institution, Saitama 331-8537, Japan

Edited by Fotis C. Kafatos, European Molecular Biology Laboratory, Heidelberg, Germany, and approved September 5, 2002 (received for review April 16, 2002) The adzuki bean , chinensis, is triple-infected tact between eukaryotic host cells and inhabiting microbial with distinct lineages of Wolbachia endosymbiont, wBruCon, associates, may apply to these cases. In fact, mitochondria and wBruOri, and wBruAus, which were identified by their wsp (Wol- chloroplasts have experienced gene transfer to the nucleus bachia surface protein) gene sequences. Whereas wBruCon and accompanied by drastic reduction in their genome size at early wBruOri caused cytoplasmic incompatibility of the host insect, stages of their endosymbiotic (6, 7). In plants and wBruAus did not. Although wBruCon and wBruOri were easily , transfer of mitochondrial genes to the nuclear genome eliminated by antibiotic treatments, wBruAus persisted over five has been shown to be a currently ongoing process (15, 16). It treated generations and could not be eliminated. The inheritance appears meaningful that obligate endosymbiotic bacteria tend to pattern of wBruAus was, surprisingly, explained by sex-linked exhibit a remarkably reduced genome size in comparison with inheritance in male-heterozygotic organisms, which agreed with their free-living relatives (17–20). However, no case of horizon- XY). Quantitative PCR tal gene transfer from prokaryotic endosymbiont to eukaryotic ,20 ؍ the karyotype of C. chinensis (2n analysis demonstrated that females contain around twice as much host has been described to date. wsp titer as males, which is concordant with an X chromosome Members of the genus Wolbachia are rickettsia-like endocel- linkage. Specific PCR and Southern blot analyses indicated that the lular bacteria associated with and other invertebrates. wBruAus-bearing strain of C. chinensis contains only a fraction of Infection with Wolbachia often causes a wide range of effects on the Wolbachia gene repertoire. Several genome fragments of the reproduction and physiology of hosts such as wBruAus were isolated using an inverse PCR technique. The frag- cytoplasmic incompatibility, parthenogenesis, feminization, ments exhibited a bacterial genome structure containing a number male killing, etc. Through host generations, Wolbachia are of ORFs typical of the ␣-proteobacteria, although some of the ORFs inherited solely in the maternal lineage by transovarial trans- contained disruptive mutations. In the flanking region of ftsZ gene, mission (21–23). Therefore, the life cycle of Wolbachia has an a non-long terminal repeat (non-LTR) retrotransposon sequence, intimate association with germ line cells of the host. Here we which is typical of insects but not found from bacteria, was present. report an unprecedented case of prokaryote–eukaryote hori- These results strongly suggest that wBruAus has no microbial zontal gene transfer: a genome fragment from the Wolbachia entity but is a genome fragment of Wolbachia endosymbiont endosymbiont has been transferred to the X chromosome of a transferred to the X chromosome of the host insect. beetle. Materials and Methods or a long time, horizontal gene transfers between genomes of Materials. Three strains of Callosobruchus chinensis (Coleoptera: different organisms have been believed to be rather excep- F Bruchidae) were used in this study. Strain jC was triple-infected tional and idiosyncratic phenomena. However, recent accumu- with wBruCon, wBruOri, and wBruAus (infection status, COA; lation of microbial genome data has revealed an exciting view of ref. 24). Strains k10 and r13 were two isofemale lines derived evolution where horizontal gene transfers have commonly taken from two wild populations, kkC98 and mrC98, respectively (24). place between unrelated prokaryotic lineages in a dynamic These strains were double-infected with wBruCon and wBruOri manner (1–3). Now it is widely accepted that horizontal gene (infection status, CO). These insects were maintained on adzuki transfer is an important and universal pathway for bacteria to seeds at 30°C and 70% relative humidity in a long-day regimen reorganize their genome and to quickly acquire novel features of 16 h light͞8 h dark. such as drug resistance, pathogenicity, metabolic properties, and others (3–5). On the other hand, horizontal gene transfers Antibiotic Treatment. Antibiotic treatment was conducted by between prokaryote and eukaryote are still regarded as unusual, feeding the insects on artificial beans containing tetracycline or except for those derived from mitochondria and chloroplasts (6, rifampicin. A mixture of milled adzuki bean powder and either 7), organelles of prokaryotic endosymbiont origin (8, 9). tetracycline or rifampicin [0.03% (wt͞wt)] was kneaded with an Mechanisms of horizontal gene transfer between prokaryotes, aliquot of water, shaped into small balls that mimicked bean such as transformation, transduction and conjugation, are rela- grains, freeze-dried, and coated with collodion to imitate the tively well understood (1, 3). In contrast, mechanisms underlying peel of beans. The insects were allowed to oviposit and main- prokaryote–eukaryote gene transfer are unknown, although tained on the artificial beans. several hypothetical models have been considered. One model is the ‘‘food hypothesis,’’ which applies to prokaryotic genes iden- Examination of Inheritance. Offspring of mating experiments were tified in phagocytic unicellular eukaryotes like Trichomonas and subjected to specific PCR detection of wBruCon, wBruOri, and Entamoeba (10–12). Unicellular eukaryotes often live close to prokaryotes and frequently use them as food, which means that they have a constant exposure to prokaryotic DNA (13, 14). This paper was submitted directly (Track II) to the PNAS office. However, this model appears not applicable to multicellular Abbreviations: non-LTR retrotransposon, non-long terminal repeat retrotransposon; wsp, eukaryotes like animals, as foreign prokaryotes are not easily Wolbachia surface protein. accessible to the germ line cells because of germ–soma separa- Data deposition: The DNA sequences reported in this paper have been deposited in the tion and absence of active phagocytosis. An alternative model, GenBank database (accession nos. AB080664, AB080665, and AB081842). the ‘‘endosymbiont hypothesis’’ that rests on a permanent con- ¶To whom correspondence should be addressed. E-mail: [email protected].

14280–14285 ͉ PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.222228199 Downloaded by guest on October 2, 2021 wBruAus by using specific reverse PCR primers wspConR, wspOriR, and wspAusR, in combination with universal forward primer wspF, as described (24).

Quantitative PCR. Real-time fluorescence detection quantitative PCR was performed using TaqMan PCR and an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems) as de- scribed (24, 25). To estimate the titer of the respective three Wolbachia, copy number of the wsp gene was quantified. A Fig. 1. Specific PCR detection of Wolbachia before and after tetracycline double-fluorescence-labeled probe for detection, TQwspPRB, treatment. wBruCon, wBruOri, and wBruAus were detected by PCR using was targeted to a conserved region of the three types of wsp specific primers. 1, untreated; 2, after treatment for 1 generation; 3, after treatment for 5 generations; 4, 10 generations after treatment for 1 genera- sequences. Highly specific amplifying primers were designed to tion. C, wBruCon; O, wBruOri; A, wBruAus; M, DNA size markers (1,000, 700, variable flanking regions between the three sequences: 500, 400, 300, 200, and 100 bp from top to bottom). Each sample contains TQwspCF and TQwspCR; TQwspOF and TQwspOR; and whole DNA from an adult female. Although data from only one insect for each TQwspAF and TQwspAR (24, 25). For standardized compari- treatment were shown, reproducibility of the results was confirmed for more son between males and females of different body size and DNA than 10 individuals. content, titer of wBruAus in each total DNA sample (wsp copies per microliter) was normalized by DNA concentration (nano- Ϸ 8 grams per microliter) in the same sample. of magnitude than that of wBruCon and wBruOri ( 10 wsp copies equivalent per adult female). Secondly, infection with Detection of Wolbachia Genes. PCR detection of Wolbachia genes wBruAus showed no detectable reproductive symptoms of the was conducted using the following primers as described: wspF host insect, although infections with wBruCon and wBruOri and wspR for wsp (24); ftsF and ftsR for ftsZ (26); groEfl and caused significant levels of cytoplasmic incompatibility. Thirdly, groErl for groE (27); and wp16SF and wp16SR for 16S rDNA although the titer of wBruAus in oocytes and unfertilized eggs (28). Southern blot detection of Wolbachia genes was conducted was extremely low (Ͻ102 wsp copies equivalent per egg), the using the probes for atpD, cytB, ftsZ, gltA, glyA, gyrA, Isw1, lipA, Wolbachia was stably inherited through host generations in the polA, rpoB, sdhA, sucD, thrA, tufA, and wsp, which were kindly laboratory, and the infection rate in natural populations was provided by S. Masui (RIKEN Center for Developmental Biol- Ͼ97% on average. In this study, further unusual properties of ogy, Kobe, Japan). wBruAus atypical of endosymbiotic bacteria emerged.

Cloning of Wolbachia Genome Fragments. Total DNA of the insects Antibiotic Resistance of wBruAus. In an attempt to obtain was digested with BamHI or HindIII, and self-ligated using T4 Wolbachia-free C. chinensis, a triple-infected strain, jC, was DNA ligase. The circularized DNA products were subjected reared with artificial beans containing tetracycline or rifamp- to nested long-inverted PCR by using outward specific primers icin. Fig. 1 shows the results of specific PCR detection of

for Wolbachia genes. PCR was conducted using LA TaqDNA wBruCon, wBruOri, and wBruAus after tetracycline treatments. EVOLUTION polymerase (TaKaRa, Otsu, Japan) under a temperature profile Tetracycline treatment of only one generation was sufficient to of 94°C for 2 min followed by 30 cycles of 94°C for 1 min, 60°C eliminate wBruCon and wBruOri. On the other hand, wBruAus for 1 min, and 68°C for 5 min, and a final extension step at 60°C persisted throughout five generations of tetracycline treatment for 5 min. For flanking sequence of wsp gene, primers wsp3FINV and could not be eliminated. After the treatment, the insects (5Ј-TACAACCAAACGCACCACCGCCAGCTATAA-3Ј) and were transferred to and maintained on normal beans. The TQwspAF (24) were used for the first PCR, and primers insects, named a strain jCAus, contained wBruAus but were wsp3FINV and wsp5RINV (5Ј-ACACCTTTGGCAACTGCT- completely free of wBruCon and wBruOri. The same patterns GTGAGTAGTCAA-3Ј) were used for the second PCR. For 5Ј of tetracycline sensitivity, resistant wBruAus and sensitive flanking sequence of ftsZ gene, primers fts3F (5Ј-GAGAAATT- wBruCon and wBruOri, were found in other strains of C. TAAGTGGCCCTATAGTCAC-3Ј) and fts5R (5Ј-AAACA- chinensis (data not shown). The same results were obtained from GAGTCATATCTCCGCCACCAGT-3Ј) were used for the first rifampicin treatments (data not shown). Thus, it was shown that PCR, and primers fts3F and fts5R2 (5Ј-AAGCCCTGGCAT- wBruAus is exceptionally resistant to tetracycline and rifampicin, GACCACCAAGTCAGT-3Ј) were for the second PCR. The although Wolbachia and other endosymbiotic bacteria are, in sizes of these PCR products were consistent with the results of general, susceptible to these antibiotics. Southern blot analysis. To obtain 3Ј flanking sequence of ftsZ gene, standard PCR was conducted using primers fts3F and X-Linked Inheritance of wBruAus. In mating experiments between wCZC (5Ј-CCAAGCCATTGTAAAGATGATTTAAGAA- C. chinensis strains of different infection status, we observed the CAT-3Ј). These amplified products were cloned into pT7Blue inheritance of the three Wolbachia. As expected, wBruCon and vector (Novagen), and purified plasmids containing the clones wBruOri were maternally inherited to the offspring. Unexpect- were subjected to DNA sequencing. edly, however, we found that wBruAus was passed to the offspring not only maternally but also paternally. Fig. 2 shows the Source of the Genome Data of Wolbachia sp. from Drosophila mela- inheritance of wBruAus in reciprocal crosses between COA nogaster. Preliminary sequence data of Wolbachia sp. from D. insects and CO insects. The crosses between COA fathers and melanogaster were obtained from the Institute for Genomic CO mothers produced all CO males and, surprisingly, all COA Research through the web site at www.tigr.org. females. When these COA females were mated with CO males, wBruAus exhibited 1:1 segregation in both males and females Results and Discussion (Fig. 2A). The crosses between CO fathers and COA mothers Peculiar Properties of wBruAus. In recent studies (24, 25), it has produced all COA offspring, superficially showing typical ma- been demonstrated that the adzuki bean beetle, C. chinensis, ternal inheritance. However, when the COA female offspring is triple-infected with distinct lineages of Wolbachia, called were mated with CO males, wBruAus did not exhibit maternal wBruCon, wBruOri, and wBruAus. Among them, wBruAus inheritance but 1:1 segregation in both males and females (Fig. showed peculiar properties. First, the titer of wBruAus (Ϸ107 2B). Notably, all these patterns were perfectly explained by wsp copies equivalent per adult female) was smaller by an order sex-linked inheritance in male-heterozygotic organisms (Fig. 2),

Kondo et al. PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 ͉ 14281 Downloaded by guest on October 2, 2021 Fig. 4. Specific PCR detection of Wolbachia genes from the original triple- infected strain jC and the tetracycline-treated, wBruAus-bearing strain jCAus. Lanes 1, jC; lanes 2, jCAus; lanes 3, no template control; lanes M, DNA size markers (1,500, 1,000, 700, 500, 400, 300, and 200 bp from top to bottom). A Fig. 2. Inheritance of wBruAus. (A) Cross between wBruAus-bearing (COA) faint band in lane 2 of the 16S rDNA panel is due to a nonspecific PCR product. males and wBruAus-free (CO) females. (B) Cross between wBruAus-free (CO) males and wBruAus-bearing (COA) females. Shade indicates presence of wBruAus. The inheritance patterns are in agreement with X-linked inheri- Structure of the Wolbachia Genome Fragment in the Strain jCAus. We tance. Sex chromosome types deduced are shown in rectangles. XA means the cloned and sequenced the amplified fragments of wsp, ftsZ, and X chromosome carrying wBruAus. Numbers beneath the rectangles are the gyrA genes from the total DNA of the strain jCAus. In addition, number of offspring obtained. we successfully isolated large DNA fragments flanking wsp and ftsZ genes by using an inverse PCR technique. A 4,141-bp fragment containing wsp (Fig. 5A), an 11,400-bp fragment ϭ which agreed with the karyotype of C. chinensis (2n 20, XY; containing ftsZ (Fig. 5B), and a 373-bp fragment of gyrA were ref. 29; T.F., unpublished data). obtained. These DNA fragments encoded bacterial genes that showed high sequence similarity to those of the ␣-proteobacte- Sex-Linked Difference in Titer of wBruAus. From the antibiotic ria, such as Wolbachia, Rickettsia, and Brucella. In the 4.1-kb insensitivity and the X-linked inheritance, a possibility was fragment, four bacterial ORFs were identified: dnaX, wsp, and suggested that wBruAus has no microbial entity but is a bacterial two ORFs homologous to RP866 and RP416 in the genome of genome fragment associated with X chromosome of the host Rickettsia prowazekii (17). In the 11.4-kb fragment, seven bac- insect. If so, female insects should contain twice as much titer of terial ORFs were detected: ftsZ, sdhB, pgpA, g3pdh, czcR,an wBruAus as male insects. Fig. 3 shows the titer of wBruAus in ORF homologous to RP741 in the genome of R. prowazekii, and adult males and females of C. chinensis in terms of wsp gene an ORF homologous to BMEI0172 in the genome of Brucella copies per nanogram of total insect DNA. As expected, females melitensis (30). One ORF showed a partial weak homology to contained about twice as much wBruAus titer as males, which acetyltransferase genes from various bacteria, archea, and confirmed the idea of the X chromosome linkage. plants.

Specific PCR Detection of Wolbachia Genes from the Strain jCAus. In Non-LTR Retrotransposon-Like Sequence in the Wolbachia Genome addition to wsp, we attempted PCR detection of several Wolba- Fragment. Notably, a non-LTR retrotransposon-like sequence, chia genes, ftsZ, groE, and 16S rDNA, in the strain jCAus, which which showed a significant similarity to I and You elements from Drosophila species (31) and MosquI elements from Aedes aegypti contains only wBruAus (Fig. 4). Whereas wsp and ftsZ were (32), was identified in the upstream region of ftsZ. The sequence amplified by PCR, groE and 16S rDNA were not detected. contained two ORFs typical of non-LTR retrotransposons: ORF1, encoding a protein of unknown function, and ORF2, Southern Blot Detection of Wolbachia Genes from the Strain jCAus. encoding a protein with endonuclease and reverse transcriptase Furthermore, we attempted to detect Wolbachia genes in the Aus activities (31). Deduced amino acid sequence similarities be- strain jC by using Southern blotting. The following 15 genes tween the retrotransposon-like sequence and a You element were targeted: atpD, cytB, ftsZ, gltA, glyA, gyrA, Isw1, lipA, polA, (accession no. AJ302712) were 41% in ORF1 and 47% in ORF2, rpoB, sdhA, sucD, thrA, tufA, and wsp. Whereas all of the genes respectively. I element is known to be involved in the I–R system were detected from the strain jC, only three genes, ftsZ, gyrA, and of hybrid dysgenesis in D. melanogaster (33). A number of I and wsp, were detected from the strain jCAus (data not shown). These You elements, which constitute a distinct clade in the non-LTR results indicated that the strain jCAus contains only a fraction of retrotransposon phylogeny (31), have been identified in the the Wolbachia gene repertoire, supporting the idea that the genomes of D. melanogaster and other species. Because non-LTR wBruAus strain in fact is a genome fragment of Wolbachia retrotransposons of this family have been found in insect ge- located on the host chromosome. nomes but not in bacterial genomes, this finding may favor the idea that the Wolbachia genome fragment is located on a chromosome of the host insect.

Comparison with the Wolbachia Genome from D. melanogaster. The genome fragments of wBruAus were compared with the pre- liminary genome sequences of wMel, a strain of Wolbachia from D. melanogaster, determined by the Institute for Genomic Re- search (Fig. 5). Gene arrangements on the genome fragments were, in general, conserved between wBruAus and wMel, al- though several differences were identified. In wBruAus wsp was next to RP416, whereas in wMel these genes were not on the Fig. 3. Sex-linked difference in titer of wBruAus. Titers of wBruAus in adult same genome fragment. Only in wMel was a histidine kinase-like males and females were quantified in terms of wsp gene copies per nanogram sequence found between sdhB and acetyltransferase-like se- of total insect DNA. The difference was statistically significant (Mann– quence, and was an unidentified ORF located between Whitney U test, P Ͻ 0.001). BMEI0172 and RP741. In the upstream region of ftsZ, where

14282 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.222228199 Kondo et al. Downloaded by guest on October 2, 2021 Fig. 5. Structure of the genome fragments of wBruAus obtained by inverse PCR, aligned with genome sequences of wMel, a strain of Wolbachia from D. melanogaster.(A) A 4.1-kb fragment containing wsp gene. (B) An 11.4-kb fragment containing ftsZ gene. Arrows indicate the position of intermittent stop codons. Arrowheads show the position of frame-shift substitutions. Filled ORFs contain either stop codons or frame-shift substitutions, whereas shaded ORFs are structurally intact. A non-LTR retrotransposon-like sequence was located upstream of the ftsZ gene. All ORFs on wMel genome fragments are structurally intact. Question marks indicate unidentified ORFs. EVOLUTION

non-LTR retrotransposon-like sequence was identified in and (v) the location of the transferred genome fragment is wBruAus, comM and an unidentified ORF were found in identified to be X chromosome. wMel. These results indicated that the genome fragments of wBruAus certainly retain the Wolbachia genome structure. Are Wolbachia Genes on Host Chromosome Functional? Among 12 ORFs identified on the Wolbachia genome fragment (excluding Horizontal Gene Transfer from Wolbachia Endosymbiont to X Chro- the non-LTR retrotransposon region), more than half, 7 ORFs, mosome of Host Insect. From all these results taken together, it is contained stop codons or frame-shift substitutions, although concluded that wBruAus, a strain of Wolbachia identified from these ORFs in the wMel genome were structurally intact (Fig. 5). Preliminary RT-PCR analyses showed that wsp and ftsZ are not C. chinensis, has no bacterial entity but is a genome fragment of transcribed in the strain jCAus (data not shown). These results Wolbachia horizontally transferred to the genome of the host indicate that most of the Wolbachia genes became pseudogenes insect. The Wolbachia genome fragment is at least 11 kb on or after the horizontal transfer and were no longer functional. (excluding the non-LTR retrotransposon region), probably However, it is unknown whether all of the transferred Wolbachia much larger, in size, containing a number of bacterial ORFs, and genes are inactivated, or whether some of them survive on the located on the X chromosome of the host. Because the genome host chromosome. It will be necessary to clone the whole size of Wolbachia is established to be 0.95–1.66 Mb (34), the Wolbachia genome fragment, to determine its structure, and to transferred gene fragment may account for Ͼ1% of the whole examine the expression of all ORFs on the fragment. genome of the original bacterium. Thus far, there have been several reports of putative prokaryote–eukaryote horizontal Process of Symbiont–Host Horizontal Gene Transfer? At present, gene transfers in which a gene highly homologous to a prokary- neither mechanism nor process involved in the Wolbachia–insect otic one was detected in the nuclear genome of eukaryotes horizontal gene transfer is understood. Through generations of (10–12). However, evolutionary processes underlying these pu- host insects, Wolbachia are maternally inherited via infection of tative gene transfers were difficult to infer because the events are developing oocytes. In eggs, Wolbachia cells localize at the posterior pole where germ cells develop (35), and are closely likely to have occurred quite anciently and the original structures associated with astral microtubules of the host cells during of the transferred genes are no longer retained. Therefore, our mitosis (36). Therefore, although speculative, it is conceivable finding provides unprecedented evidence of horizontal gene that these intimate associations of Wolbachia with proliferating transfer from an endosymbiotic prokaryote to a host multicel- germ cells have provided a favorable condition for the observed lular eukaryote in that (i) the transfer must be a recent event, (ii) horizontal gene transfer. the donor bacterium is unequivocally identified to be Wolbachia, (iii) a large fragment of bacterial genome is transferred, (iv) the Evolutionary Origin? Absence of Wolbachia in other Callosobru- structure of the transferred bacterial genome is highly preserved, chus species (37) suggests that wBruAus was acquired by the

Kondo et al. PNAS ͉ October 29, 2002 ͉ vol. 99 ͉ no. 22 ͉ 14283 Downloaded by guest on October 2, 2021 ancestor of C. chinensis through horizontal transmission from an extensive survey of infection and diversity of Wolbachia (44–46), unrelated host. In the DNA databases, a wsp sequence from the only PCR detection was conducted without examining the tephritid fruit fly Dacus destillatoria (accession no. AF295344) inheritance pattern of the genes. Considering that insects are the shows a very high similarity (98.9%) to the wsp gene of wBruAus, most diverse eukaryotic group in the terrestrial ecosystem (47) although a biological connection between the fruit fly and the and that infection frequency of Wolbachia in natural insect bruchid beetle is obscure. Probably, the acquisition of bacterial populations reaches Ϸ20–70% worldwide (45, 46), it would not wBruAus preceded the transfer of its genome fragment to the be surprising if future careful studies revealed other cases of host chromosome. If so, a worldwide survey of C. chinensis Wolbachia–host gene transfer. Other endosymbiotic systems populations might lead to the discovery of relict bacterial such as Buchnera in aphids, in which the obligate symbiont wBruAus, which would provide further insights into the evolu- genome exhibits remarkable degeneration and reduction (18, tionary origin and process of the horizontal gene transfer. 20), might conceal similar horizontal gene transfer events. Genome sequencing of Arabidopsis thaliana revealed a contin- Mechanism of Maintenance in Host Populations? In all local popula- uous stretch of nearly 75% of mitochondrial genome located tions of C. chinensis examined in Japan, the frequency of wBruAus, on chromosome 2 of the plant (48). Similarly, other cases of which can be considered to be ‘‘chromosomal Wolbachia,’’ was Ͼ symbiont–host gene transfer might come from genome sequenc- consistently 90% (24). At present, the mechanism whereby the ing projects of various eukaryotic organisms now in progress. Wolbachia genome fragment on the host chromosome prevails in populations is a mystery. Fixation by chance through drift cannot be Evolutionary Implications. It has been pointed out that Wolbachia- ruled out but appears unlikely because of the consistent prevalence induced cytoplasmic incompatibility can promote reproductive in many local populations. The chromosomal Wolbachia might be isolation of host insects (49, 50). If genes of Wolbachia respon- able to increase its frequency by hitchhiking with coexisting bac- terial Wolbachia that cause cytoplasmic incompatibility. The chro- sible for cytoplasmic incompatibility are transferred to the host mosomal Wolbachia might be tightly linked to genes that confer a genome in a functional form, this would also reinforce repro- positive fitness effect to the insect, or genes that enhance its own ductive isolation and ultimately lead to speciation. Endosymbi- transmission in a selfish manner like meiotic drive genes (38). otic associations with microorganisms have been suggested to act Alternatively, the chromosomal Wolbachia itself might behave as a as a source of evolutionary innovations for their hosts (51). selfish genetic element like Medea, a maternal-effect chromosomal Implications of the ongoing genome transfer between the sym- factor known from flour (39). The last possibility is intrigu- biont and host can be far-reaching in this context. ing in that the Medea phenotype and Wolbachia-induced cytoplas- mic incompatibility can be explained by the same ‘‘poison– We thank A. Sugimura, S. Kumagai, and K. Sato for technical and antidote’’ or ‘‘modification–rescue’’ mechanism (40, 41). secretarial assistance; S. Masui for Wolbachia gene probes; and S. L. O’Neill for reading the manuscript. Preliminary sequence data of Wolbachia sp. from D. melanogaster, which was accomplished with Other Symbiont–Host Horizontal Gene Transfers to Be Found? Previ- support from the National Institutes of Health, were obtained from the ous studies have reported that genetic recombination can occur Institute for Genomic Research through the web site www.tigr.org. This between co-infecting Wolbachia strains (42, 43). In this study, we research was supported by the Program for Promotion of Basic Research first demonstrated that genetic materials can be exchanged Activities for Innovation Biosciences (ProBRAIN) of the Bio-Oriented between Wolbachia and host insect. It is unknown whether the Technology Research Advancement Institution. N.K. was supported by Wolbachia–host gene transfer is an orphan exception, or whether the Research Fellowship of the Japan Society for the Promotion of other cases are to be found. It should be noted that, in previous Science for Young Scientists.

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