Wolbachia is a group （genus） of cytoplasmic endosymbiotic bacteria found in arthropods and nematodes. Wolbachia is known to manipulate the reproduction of arthropod hosts to facilitate the spread of infection into the host population. From these reasons, Wolbachia is considered as a selfish genetic element in arthropods. The actions of Wolbachia infection on the arthropod hosts include feminization of genetic males, male−killing, cytoplasmic incompatibility （CI） and induction of parthenogenesis.

Ostrinia （Lepidoptera: Crambidae） is a group （genus） of small moths represented by the Asian corn borer moth O. furnacalis, the major pest of corn in the East Asia. Eight Ostrinia species found in Japan are divided into O. palustralis group, O. latipennis group and O. furnacalis group based on the morphological similarity. In the genus Ostrinia, Wolbachia-infection was found in O. furnacalis, O. scapulalis, O. orientalis, and O. zaguliaevi （these four species belong to O. furnacalis group）, and O. ovalipennis （this species belongs to O. latipennis group）. The sequences of a cell-cycle gene （ftsZ） as well as a Wolbachia surface protein gene （wsp） of Wolbachia that infect the four species of O. furnacalis group were identical, indicating that Wolbachia strain（s） in these host species are the same or very closely related. In the O. furnacalis group, Wolbachia infection was found only in females at a low frequency in the field. Wolbachia infecting O. furnacalis group causes production of all-female offspring. The all-female production was first interpreted as feminization of genetic males, since elimination of Wolbachia by antibiotic treatment resulted in the production of all-male offspring. However, a detailed analysis of the sexual genotype （male = ZZ, female = ZW） of individuals based on observations of the sex chromatin （W） and the comparative genomic hybridization of sex chromosomes led Kageyama & Traut （2004） to conclude that the all-female production in infected O. scapulalis is the consequence of male-specific death provoked by the feminizing effect of Wolbachia （wSca）. They also indicated that the all-male production after antibiotics treatment is caused by female-specific death, suggesting that wSca is indispensable for the survival of infected females. In contrast to O. furnacalis group, Wolbachia infection was found in both females and males of O. ovalipennis at a high frequency in the field. Because of the difficulty in rearing of O. ovalipennis in the laboratory, the action of Wolbachia in this species has not been clarified in detail.

In the present study, I first investigated the phylogenetic relationship of Wolbachia infecting O. ovalipennis and those infecting O. furnacalis group to infer the origin of Wolbachia strain that causes all-female production in Ostrinia. Secondly, I investigated the relative importance of Wolbachia and host genotypes in determining the type of reproductive alteration in the host. Thirdly, I tried to obtain a clue for the mechanism of all-female production by the examination of the sexual mosaics produced by antibiotics treatment of Wolbachia-infected adult females.

Characterization of Wolbachia strain infecting O. ovalipennis

I determined partial sequences of ftsZ and wsp gene of Wolbachia infecting O. ovalipennis. These sequences were the same as those of Wolbachia infecting O. furnacalis group. These results indicate that the same or a very close strain of Wolbachia infect O. ovalipennis and species of O. furnacalis group. Since the Wolbachia strain in O. ovalipennis does not cause all-female production in the hosts, these results may suggest that the ability to cause reproductive alteration was acquired by the Wolbachia infecting O. furnacalis group in the recent past in the evolutionary time scale.

Transinfection reveals the crucial importance of Wolbachia genotypes in determining the type of reproductive alteration in the host

wSca induced male-specific death, while another strain of Wolbachia （wKue） infecting the Mediterranean flour moth Ephestia kuehniella induces CI in the resident host. Transinfection of Wolbachia can be a powerful tool to elucidate the relative importance of Wolbachia and the host in determining the type of reproductive alterations. Recently, male-killing was shown to occur in E. kuehniella transinfected with wSca. Here, I transferred wKue to O. scapulalis by embryonic microinjection. In the O. scapulalis transinfected with wKue, not male killing, but CI occurred. Thus, in addition to wSca, wKue was shown to induce the same type of alteration in a foreign host as in its natural host. These results demonstrate the crucial role of Wolbachia genotype in determining the type of reproductive alteration. However, the present study also revealed the involvement of host factors. First, the degree of incompatibility induced by wKue in O. scapulalis was stronger than that in E, kuehniella, indicating the host factors can affect the level of CI. Second, the vertical transmission rate of wKue in O. scapulalis was generally low, suggesting that host affects the dynamics of Wolbachia transmission.

The mode of male-specific death in Wolbachia-infected O. furnacalis

To clarify the mode of sex-specific death, I observed sex chromatins of individuals derived from Wolbachia-infected and cured females of O. furnacalis through the larval development. Both genetic males and females were included in the hatched larvae in the broods derived from infected females. Likewise, both genetic males and females were found in the hatched larvae in most broods derived from cured females. In contrast, the last instar larvae derived from infected females were in the all-female condition, and those derived from cured females were in the all-male condition. These results clearly indicated that opposite sex specific lethality occurred during the larval development in the broods of Wolbachia-infected and cured females of O. furnacalis.

The interaction of Wolbachia and host occurs specifically during the embryonic stage

In O. scapulalis and O. furnacalis, the ability of Wolbachia to feminize genetic males was evidenced by the finding that antibiotics treatment of infected female adults led to the production of sexual mosaics, the genetic sex of which was male. Examination of organ（s） in the sexual mosaics feminized and not feminized by Wolbachia would allow us to estimate the mode and limitation of feminizing activity of Wolbachia. I observed sexual mosaics produced by tetracycline treatment of Wolbachia-infected O. furnacalis female adults. A small proportion of them had female wing pattern in the entire area. None of these mosaics had ovary in spite of their complete female-like wing pattern and having the bursa copulatrix. Surprisingly, none of the sexual mosaics were found to harbor Wolbachia. Then, I checked the presence of Wolbachia in the eggs and hatched larvae derived from tetracycline-treated Wolbachia-infected females. None of the larvae hatched from eggs laid after two days of tetracycline treatment harbored Wolbachia in spite of the occurrence of male-specific death. These results demonstrate that Wolbachia-infected genetic males are destined at the early embryonic stage to die later in the larval development, and that Wolbachia is indispensable for females only during the embryonic developmental stage.

W chromosome in Wolbachia-infected O. furnacalis is differentiated from that in normal females

In Wolbachia-infected insects, one of the Z chromosomes in the male （ZZ） may aggregate and appear like sex chromatin. To examine this possibility, I amplified microsatellite locus on W chromosome in O. furnacalis by PCR. The band found in normal females were not observed in any of the Wolbachia-infected females. Surprisingly, I found a band specific to females of Wolbachia-infected O. furnacalis, which is different from the band of normal females in size by 300 bp. The band was found in cured females, but not found in the all-male progeny produced by the cured females. The appearance of the band thus coincided with the presence of sex chromatin, indicating that sex chromatin in the Wolbachia-infected female is W chromosome. These results suggest that females in the Wolbachia-infected matrilines of O. furnacalis group have a W chromosome different from normal females.

In conclusion, this study clarified the uniqueness of reproductive alteration caused by Wolbachia infecting O. furnacalis group. I showed that the Wolbachia strain infecting O. furnacalis causes male-killing, and has the ability to feminize genetic males. Heretofore, male-killing and feminization of genetic males have been considered as entirely independent phenomena. However, the present study clearly demonstrated that feminization underlies the phenomenon of male-killing. This finding would help us to understand the mechanisms commonly underlying various types of Wolbachia-induced reproductive alterations in insects.

Wolbachia（以下，WOL）は昆虫などを宿主とする細胞内共生細菌で，遺伝的なオスのメス化，オス殺し，細胞質不和合など，自身および感染細胞質に有利になるように宿主の生殖を操作する．アワノメイガ属のWOL 感染は，ウスジロキノメイガ種群のマルバネキノメイガ（マルバネ）とアワノメイガ種群のアワノメイガ（アワ），アズキノメイガ（アズキ）などで知られている．アワノメイガ種群のWOL は同一か非常に近い系統で，感染率は低い．感染した寄主の子はメスに偏った性比になり，抗生物質で幼虫からWOLを除去するとその子は全オスになる．アズキではメスに偏る原因はWOL（wSca）のメス化作用によるオス致死，抗生物質処理によりオスに偏る原因はメス特異的致死であり，また，感染メスにはWOL が不可欠であることが示されている．一方，マルバネではWOLは雌雄ともに高い感染率を示すが，生殖操作の詳細はわかっていない．

本論文は，（1）マルバネに感染するWOLをアワノメイガ種群のWOLと比較して本属における感染の歴史を解明する，（2）生殖異常に対するWOLの系統と宿主の作用を調べる，（3）性モザイク個体を用いて性比がメスに偏る原因を考察すること，（4）感染系統と非感染系統のメスにおけるＷ染色体の構造の差異を比較することを目的とするものである．

マルバネに感染するWOLの特徴

マルバネとアワノメイガ種群でWOLのwsp遺伝子およびftsZ遺伝子の部分配列は一致した．しかし，マルバネではWOLは性比バイアスを起こさないと考えられ，アワノメイガ種群のWOLが比較的最近に性比異常を引き起こす能力を獲得したことが示された．

生殖異常の種類の決定に対するWOLの系統の重要性

wScaはアズキにオス殺しを起こすが，別科のスジコナマダラメイガ（スジコナ）のWOL （wKue）は宿主に細胞質不和合を起こす． wScaをスジコナに移植するとオス殺しを起こすとの報告があるので，アズキへのwKueの移植をしたところ細胞質不和合が起きた．つまりwScaと同様wKueも移植後，新寄主に元宿主と同じ生殖異常を起こした．これらはWOLの系統が生殖異常の種類決定に重要であることが示している．

アワにおけるWOLが引き起こすメスに偏った性比の原因

WOL感染メスおよび感染除去メスの子の幼虫期にメス特異的である性クロマチンの有無を観察した．感染，感染除去とも，孵化時には性比に有意な偏りは見られなかったが，終齢幼虫では感染メスの子は全てメス，感染除去メスの子は全てオスだった．以上から，本種でも感染メスの子でのオス致死，および，感染除去メスの子でのメス致死が示された．

アワの胚発生期におけるWOLと宿主の相互作用

感染メスの成虫に抗生物質を与えたところ，子の一部が性モザイク成虫になったが，遺伝的にはオスであった．一部の性モザイク個体は外見上完全なメスで，メス特有の交尾嚢も持っていたが卵巣は存在しなかった．モザイク個体からはWOLが検出されなかったので，抗生物質投与した感染メス成虫の産んだ卵と孵化幼虫でWOLの有無を調べたところ，WOLは投与3日後から孵化幼虫に観察されなくなったにもかかわらず，オス致死，性モザイクおよびメスの羽化はそれ以降も観察された．これらの結果は，WOLによるメス化とオス致死の決定時期が胚発生期より前であること，および，感染メスにとってWOLが必須なのは胚発生期までであることを示している．

アワ感染系統と非感染系統におけるＷ染色体の構造の差異

WOLによりオスのＺ染色体の一方が凝集して性染色体様になる可能性を調べた．Ｗ染色体上のマイクロサテライト領域特異的なプライマーによるPCRを行ったところ，感染メスでは非感染メスに特異的なバンドが見られず，サイズの異なる別のバンドが検出された．これは感染除去メスでも見られたが，感染除去メスの子であるオスには見られなかったので，Ｗ染色体上にあると推測された．バンドと性クロマチンの挙動は一致し，感染系統における性クロマチンがＷ染色体であることが示された．以上から，アワノメイガ種群ではメスのＷ染色体構造がWOL感染系統と非感染系統で異なっていることを示唆する．

以上，本研究はアワノメイガ属におけるWOL感染の歴史と感染による生殖操作の機構について多くの知見をもたらした．感染が除去されたメスの致死は通常のオス殺しよりもWOLに有利な新たな表現型として注目される．また，「オス殺し」と「メス化」は別の現象とされてきたが，「オス殺し」を起こすWOLがメス化能を持つことを示したことから，メス化によるオス殺しの可能性が示され，WOLによる生殖操作の機構に新たな理解をもたらした．これらの成果は学術的に高い価値を有するだけでなく，害虫管理など応用面にも有用である．よって，審査委員一同は本論文が博士（農学）を授与するに値することを認めた．