Copyright  2002 by the Genetics Society of America

Interspecific Transfer of Wolbachia Between Two Lepidopteran Insects Expressing Cytoplasmic Incompatibility: A Wolbachia Variant Naturally Infecting Cadra cautella Causes Male Killing in Ephestia kuehniella

Tetsuhiko Sasaki,*,1 Takeo Kubo* and Hajime Ishikawa† *Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan and †University of the Air, Wakaba, Mihama-ku, Chiba 261-8586, Japan Manuscript received March 19, 2002 Accepted for publication August 19, 2002

ABSTRACT Wolbachia is known as the causative agent of various reproductive alterations in arthropods. The almond moth Cadra cautella is doubly infected with A- and B-group Wolbachia and expresses complete cytoplasmic incompatibility (CI). The Mediterranean flour moth Ephestia kuehniella carries A-group Wolbachia and ex- presses partial CI. In the present study, the Wolbachia in C. cautella was transferred to E. kuehniella from which the original Wolbachia had been removed. We obtained transfected lines of three different infection states: single infection with A, single infection with B, and double infection with A and B. The doubly transfected lines and those transfected with only A produced exclusively female progeny. Two lines of evidence suggested that the sex ratio distortion was due to male killing. First, reduced egg hatch rate was observed. Second, removal of the Wolbachia from the transfected lines resulted in the recovery of a normal The occurrence of male killing following transfection showed that host factors influence .1:1ف sex ratio of the determination of the reproductive phenotype caused by Wolbachia. The transfected E. kuehniella males carrying exclusively B-group Wolbachia expressed partial incompatibility when crossed with the uninfected females. In addition, the transfected lines were bidirectionally incompatible with the naturally infected strain, which was the first demonstration of bidirectional CI in a lepidopteran.

OLBACHIA is a group of rickettsia-like intracel- fected females, it can cause the rapid spread of this W lular bacteria found in many arthropods. These maternally inherited bacterium in the host population, maternally inherited bacteria are known as the causative as has been documented in Drosophila simulans (Turelli agents of various reproductive alterations such as cyto- and Hoffmann 1991) and the planthopper Laodelphax plasmic incompatibility (CI), thelytokous parthenogen- striatellus (Hoshizaki and Shimada 1995). esis, feminization of genetic males into functional females, Bidirectional incompatibility has also been reported and male killing (reviewed in Werren 1997; Bourtzis to occur in crosses between Wolbachia-infected males and Braig 1999; Stouthamer et al. 1999). and females of separate populations (Yen and Barr Of the various phenotypes associated with Wolbachia 1973; Breeuwer and Werren 1990; O’Neill and Karr infection, CI is the most commonly described. Typical 1990). This type of incompatibility has been associated CI is expressed unidirectionally: The cross between in- with the presence of different bacterial variants, sug- fected males and uninfected females is incompatible, gesting that some Wolbachia variants are unable to res- whereas the reciprocal cross is compatible. The incom- cue the sperm modified by another variant (Braig et patibility arises from defects in paternal chromatin al. 1994; Rousset and de Stordeur 1994; Hoffmann condensation during mitosis (O’Neill and Karr 1990; and Turelli 1997). Multiple infections within a single Reed and Werren 1995; Lassy and Karr 1996) and individual may also influence compatibility type. Unidi- results in embryonic mortality. In some haplodiploid rectional incompatibility has been reported in the crosses species, including wasps and mites, in which haploid between double-infected males and single-infected fe- embryos develop into males, CI causes male-biased sex males (Merc¸ot et al. 1995; Rousset and Solignac 1995; ratios (Breeuwer and Werren 1990; Breeuwer 1997). Sinkins et al. 1995; Perrot-Minnot et al. 1996). The mechanism of CI is generally explained by a dual Since Wolbachia and its host form a symbiotic system, action of Wolbachia: “modification” of sperm and “res- it would be important to investigate both bacterial and cue” in eggs. Since the unidirectional incompatibility host factors to understand the basis for the expression reduces the reproductive success of exclusively unin- of reproductive alterations. One method that can be used to examine roles played by Wolbachia and the host is to experimentally transfer the bacterium between 1Corresponding author: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo hosts. Wolbachia has been successfully transferred in 113-0033, Japan. E-mail: [email protected] several species of isopods and insects. When CI-inducing

Genetics 162: 1313–1319 (November 2002) 1314 T. Sasaki, T. Kubo and H. Ishikawa

Wolbachia was transferred intraspecifically, the recipi- containing tetracycline at a final concentration of 0.04% (w/w) ent expressed CI as the donor did (Chang and Wade for two generations (Sasaki and Ishikawa 1999). Microinjection: Injections were performed as previously de- 1994; Rousset and de Stordeur 1994; Sasaki and Ishi- scribed (Sasaki and Ishikawa 2000). Freshly laid eggs (Ͻ1 kawa 2000). On the other hand, several interspecific hr old) of the uninfected strain of E. kuehniella were placed transfers have shown that host factors can affect the on a piece of double-sided adhesive tape on a slide. The ovaries reproductive phenotype. The transfers between D. sim- of C. cautella collected by dissection from the adult females were ulans and D. melanogaster revealed the influence of the also placed on a slide. The ooplasm taken out of the ovary was injected into the eggs under a microscope equipped with a three- host on the intensity at which CI is expressed (Boyle dimensional micromanipulator and a microinjector. et al. 1993; Poinsot et al. 1998). Transfers of feminizing The injected eggs on the slide were kept at 25Њ in a plastic Wolbachia among isopods resulted in four situations: no dish (9 cm in diameter) containing a piece of moist filter reproductive effect, expression of feminization, death of paper. Five days after injection the sticky surface of the tape recipients, and failure of transfection (Bouchon et al. was covered with flour, and the slide was transferred onto the diet mixture in a plastic container. The eggs hatched on the 1998). Fujii et al. (2001) demonstrated that the Wol- sixth or seventh day after injection. The rate of egg hatching bachia causing feminization in the Asian corn borer, was checked by counting the cast-off shells left on the tape. Ostrinia scapulalis, induced male killing when trans- Adults emerged about 1 month after the eggs hatched. The ferred to the Mediterranean flour moth, Ephestia kueh- females were then individually transferred into plastic cups niella. Thus the importance of interactions between host (3 cm in diameter, 5 cm in height) where they were mated with males. The males used were either those developed from and Wolbachia has been established, highlighting the the injected eggs or those collected from the stock culture of need for further accumulation of data on interspecific the uninfected strain. A sample of eggs laid by each female transfers. was diagnosed for infection status by PCR. In this article, we report the transfer of Wolbachia PCR for detection of Wolbachia: The presence or absence from the almond moth Cadra cautella to E. kuehniella. of Wolbachia was tested by diagnostic PCR assays using Wol- bachia-specific primers for the ftsZ bacterial cell-cycle gene. C. cautella is doubly infected with wCauA and wCauB, The template DNA was prepared according to O’Neill et al. which belong to A- and B-group Wolbachia, respectively (1992). A total of 10–20 eggs were homogenized in 50 ␮lof (designated by Werren et al. 1995), and expresses com- STE (100 mm NaCl, 10 mm Tris-HCl, 1 mm EDTA, pH 8.0) plete CI (Kellen et al. 1981; Sasaki and Ishikawa containing 0.4 mg/ml of proteinase K and incubated for 90 Њ Њ 1999). While the double infection causes CI, the effect minat55 and then for 15 min at 95 . PCR was performed in a 20-␮l reaction mixture using Takara of each Wolbachia variant is not known because the EX Taq. Three primer sets were used for amplification of the two variants have not been separated in this host. E. ftsZ gene according to Werren et al. (1995). The general ftsZ kuehniella is infected with an A-group Wolbachia variant primers used were ftsZfl (5Ј-GTT GTC GCA AAT ACC GAT (wKue) and expresses partial CI (Sasaki and Ishikawa GC-3Ј) and ftsZrl (5Ј-CTT AAG TAA GCT GGT ATA TC-3Ј), Ј 1999). The present study was originally designed to ex- the A-group-specific ftsZ primers were ftsZAdf (5 -CTC AAG CAC TAG AAA AGT CG-3Ј) and ftsZAdr (5Ј-TTA GCT CCT amine whether the Wolbachia variants naturally in- TCG CTT ACC TG-3Ј), and the B-group-specific ftsZ primers fecting C. cautella could infect E. kuehniella and induce were ftsZBf (5Ј-CCG ATG CTC AAG CGT TAG AG-3Ј) and CI. Since both the donor and recipient insects express ftsZBr (5Ј-CCA CTT AAC TCT TTC GTT TG-3Ј). PCR cycling conditions were 94Њ for 3 min followed by 35 amplification CI in natural infections, we presumed that, once the Њ Њ Њ transfection was established, the transfected lines would cycles of 94 for30sec,55 for 30 sec, 72 for 1 min, and, finally, 72Њ for 5 min. also express CI and that the incompatibility level might Crossing experiments: The adults of E. kuehniella do not week. During this period, the female 1ف be affected by the hosts. Unexpectedly, however, wCauA feed and survive for caused male killing in E. kuehniella. Although the actual mates once or twice. Crossing experiments were performed phenotype caused by wCauA in C. cautella is obscure, it using single pairs of virgin individuals. Since the male larva is easily distinguished by a dark patch (the testes) on its back, is apparent that it does not cause male killing. The we separated the females and males at the late larval stage. A occurrence of male killing in the transfected E. kueh- female and a male, younger than 3 days after emergence, were niella clearly showed that the reproductive phenotype set in a plastic cup (3 cm in diameter, 5 cm in height) and changes are dependent on the combination of bacte- left there for 3 days. Most females deposited Ͼ100 eggs onto Ͻ rium and host. The E. kuehniella lines that were trans- the wall of the cup. Cups in which 50 eggs had been laid were discarded. From each pair, 50–100 eggs were collected fected with only wCauB expressed partial CI and were and placed onto 1% agarose in a plastic dish (35 mm in bidirectionally incompatible with the naturally infected diameter). After incubation for 7–8 days at 25Њ, the hatching strain. rate was scored for each single-pair cross. The data were ana- lyzed by Mann-Whitney U-tests.

MATERIALS AND METHODS RESULTS Insects: E. kuehniella and C. cautella, originally collected in Transfected lines: Of 208 E. kuehniella eggs injected Tsuchiura, Japan, were reared on a diet consisting of wheat bran, dried yeast, and glycerol (20:1:2 w/w) at 25Њ under a with the ooplasm of C. cautella, 130 eggs hatched. Insects 16 hr light:8 hr dark photoperiod. A Wolbachia-free strain of that developed from the injected eggs were designated E. kuehniella was established by rearing the insects on a diet as generation 0 (G0). G1 eggs were collected from each Transfection of Wolbachia in Ephestia 1315

TABLE 1 TABLE 2 Wolbachia infection in transfected E. kuehniella The survival rates of E. kuehniella lines at generation 2 % hatch % adult No. of No. of Line Female (mean Ϯ SD, n ϭ 5) emergence (mean Ϯ SD) Line broods Wolbachia detected broods established Line 3Aa 40.8 Ϯ 11.5 31.6 Ϯ 11.5 Line 1 39 wCauB 5 Line 1B Line 3ABa 37.2 Ϯ 9.0 30.8 Ϯ 4.6 Uninfected 34 Discarded Uninfected 87.6 Ϯ 6.7 61.8 Ϯ 17.3 Line 2 28 wCauB 8 Line 2B Uninfected 20 Discarded Females were crossed with uninfected males, and 50 eggs Line 3 36 wCauA 20 Line 3A were collected from the individual female. a Females at generation 4 postinjection were used. wCauA and wCauB 16 Line 3AB

and ZW in females (Traut and Rathjens 1973). If of 40 G0 females. A part (10–20 eggs) of each G1 brood feminization occurs, the transfected lines will be domi- was subjected to PCR assay, in which Wolbachia was nated by feminized individuals carrying ZZ chromo- detected in three broods. In two broods (lines 1 and somes because ZW females produce both ZZ and ZW 2), only wCauB was detected. Both wCauA and wCauB offspring, and the infected ZZ females (feminized indi- were detected in the other brood (line 3). G2 eggs were viduals) produce solely ZZ offspring. In this situation, diagnosed for infection as performed on the G1 eggs the removal of the feminizer should result in a male- (Table 1). Infected broods were pooled for each of line biased sex ratio (Kageyama et al. 1998). On the other 1 and line 2 and designated as line 1B and line 2B, hand, the removal of a male-killing agent should lead ف respectively. Two lines different in infection status were to the recovery of a normal sex ratio of 1:1. Eggs at G8 obtained from line 3: one infected with only wCauA were subjected to tetracycline treatment. The treatment (line 3A) and the other infected with both wCauA and had no effect on the sex ratio of the G8 adults, probably wCauB (line 3AB). At least 16 larvae were randomly because the manipulation by Wolbachia had been com- selected for each of the four infected lines and diag- pleted during the host’s embryonic development, be- nosed for infection by PCR. This assay showed that the fore the larvae started feeding on the antibiotic. After

G2 larvae were homogeneously infected within lines. treatment for two generations, the sex ratio reverted to When the G2 insects attained adulthood, only females unbiased levels in all of the 10 families examined (Table emerged in lines 3A and 3AB, whereas both females 3). Accordingly, it was concluded that the mechanism and males emerged in lines 1B and 2B. of the sex ratio distortion was male killing. Lines 3A and 3AB were maintained by crossing with It may be claimed that the male killing in lines 3A males from the uninfected strain. The all-female condi- tion has been consistently transmitted maternally to off- TABLE 3 spring for 22 generations. Female-biased sex ratio caused by wCauA: Emergence The effect of tetracycline treatment on the sex ratio of the of all-female populations in lines 3A and 3AB suggested transfected lines of E. kuehniella carrying wCauA that wCauA was the causative agent of the sex ratio Line No. of adults % female distortion. Wolbachia is known to cause a female-biased sex ratio by three distinct mechanisms, thelytokous par- Line 3A 36 39 thenogenesis, feminization of genetic males, and male 25 56 killing. The possibility that thelytokous parthenogenesis 37 36 was the mechanism was ruled out because no unfertil- 24 50 18 39 ized eggs collected from the virgin females hatched. To Line 3AB 30 47 distinguish feminization and male killing, the survival 29 62 rates of the transfected lines producing all-female off- 45 51 spring were examined (Table 2). The egg hatch rates 40 34 of the transfected lines were much lower than those of 27 55 Ͼ ف the uninfected strain, suggesting that the mechanism G8 eggs ( 100 eggs) were collected in bulk from 20 fe- was male killing rather than feminization. males for each line. The larvae were reared on food containing However, the data on hatching rates did not exclude tetracycline at 0.04% (w/w). When they attained adulthood, the possibility of feminization associated with a high only females emerged. Fifty eggs were collected from the indi- mortality caused by the possible virulence of wCauA in vidual female and reared on the food with tetracycline for one more generation. The number of males and females was E. kuehniella. Then we examined the effect of removal counted on emergence of G9 adults. No significant difference of Wolbachia from the transfected lines on the sex ratio. from 50% of females was observed in any of the 10 families In E. kuehniella, the sex chromosomes are ZZ in males examined (␹2-test, P Ͼ 0.05). 1316 T. Sasaki, T. Kubo and H. Ishikawa

TABLE 4 The second transfer of Wolbachia from C. cautella to E. kuehniella

Generation 2 Eggs at generation 1 No. of adults Line Wolbachia detected Infection status No. of broods (females:males) Line 4 wCauB wCauB 5 37:31 Line 5 wCauB wCauB 3 30:24 Line 6 wCauA and wCauB wCauA 2 66:0a wCauA and wCauB 3 41:0a Line 7 wCauA and wCauB Uninfected 2 79:57 wCauA 1 No adults wCauA and wCauB 2 63:0a Line 8 wCauB wCauB 2 31:38 Line 9 wCauB wCauB 3 27:38 Line 10 wCauA Uninfected 2 19:14 wCauA 2 40:0a Line 11 wCauB wCauB 2 49:43 From 523 injected eggs, we obtained eight Wolbachia-positive broods (designated as lines 4–11), of which three broods (lines 6, 7, and 10) were positive for wCauA. G2 eggs from each of the G1 females were diagnosed by PCR, and broods with the same infection status were pooled. The sex ratios were checked on emergence of adults. a Significantly different from 50% of females by ␹2-test (P Ͼ 0.01). and 3AB is not caused by wCauA but by another factor strated that wCauA caused male killing, while wCauB transferred from C. cautella. To confirm the causal rela- induced CI in E. kuehniella. tionship of transfection with wCauA with the appear- Effects of wCauA on C. cautella and E. kuehniella: ance of male killing in E. kuehniella, we performed the Since E. kuehniella naturally infected with wKue ex- transfer again. An all-female condition was found in E. presses CI, the occurrence of male killing in the wCauA- kuehniella lines transfected with wCauA, but not in lines transfected lines implies that the two Wolbachia vari- without wCauA (Table 4). ants, wKue and wCauA, differ in their ability to induce Cytoplasmic incompatibility induced by wCauB: The reproductive alterations. It was also shown that host infection in the wCauB-transfected lines was examined factors influence the determination of reproductive al- by testing 48 larvae selected at random for each of lines terations because wCauA does not cause male killing in 1B and 2B at generation 15. All 96 larvae tested were C. cautella. infected (data not shown), suggesting that the infection Prior to interpreting the occurrence of male killing was stable. The expression of CI then was examined in E. kuehniella, the effect of wCauA on its natural host at generations 16 and 17. The transfected males were should be considered. Although C. cautella expresses partially incompatible with uninfected females: The egg- complete CI, it is not clear whether or not wCauA is hatching rate in the cross between the uninfected fe- responsible for the induction of CI because the insect males and transfected males was significantly lower than is doubly infected. To examine the effect of wCauA, C. that within the uninfected strain (see Table 5; A vs. B and cautella infected with the single Wolbachia variant is A vs. C, both comparisons P Ͻ 0.001). The transfected needed, but such a strain has not been obtained. Three and naturally infected strains were bidirectionally in- situations can be assumed: Only wCauA induces CI, compatible: Egg-hatching rates were lower in the in- only wCauB induces CI, or both variants induce CI. We terstrain crosses than in the intrastrain crosses (F vs. H, assume that the third possibility is most likely because P Ͻ 0.001; J vs. L, P Ͻ 0.001; N vs. P, P Ͻ 0.001; O vs. if one or the other Wolbachia variant does not induce P, P Ͻ 0.05). In addition, a slight decrease of the egg- CI, it would be lost stochastically and not be maintained hatching rate was found in the crosses between trans- in the host population. Indeed in Nasonia vitripennis fected males and females as compared with the cross and D. simulans, it has been demonstrated that the two within the uninfected strain (A vs. F, J, and K, P Ͻ 0.01; Wolbachia variants forming double infection take part A vs. G, not significant). in CI (Perrot-Minnot et al. 1996 for N. vitripennis; Merc¸ot et al. 1995; Merc¸ot and Poinsot 1998 for D. simulans). In these insects, single-infected lines were DISCUSSION successfully segregated from the double-infected strain We established E. kuehniella lines infected with Wol- carrying A- and B-group Wolbachia. Lines carrying only bachia variants derived from C. cautella. It was demon- A or B Wolbachia were bidirectionally incompatible with Transfection of Wolbachia in Ephestia 1317

TABLE 5 Egg-hatching rates in crosses between E. kuehniella strains

Male Female Uninfected Line 1Ba Line 2Ba Naturally infected Uninfected A: 84.3 Ϯ 14.9 B: 36.7 Ϯ 28.3 C: 55.8 Ϯ 24.8 D: 36.8 Ϯ 26.7 26 30 17 23 Line 1Ba E: 81.4 Ϯ 17.9 F: 72.4 Ϯ 19.4 G: 76.9 Ϯ 16.5 H: 29.5 Ϯ 20.6 44 37 23 20 Line 2Ba I: 79.6 Ϯ 14.0 J: 68.2 Ϯ 19.7 K: 71.5 Ϯ 21.1 L: 31.6 Ϯ 18.8 19 17 31 17 Naturally infected M: 85.6 Ϯ 12.8 N: 31.2 Ϯ 32.3 O: 56.1 Ϯ 20.2 P: 78.3 Ϯ 13.2 13 23 16 20 Mean Ϯ SD (%) and numbers of pairs are given for each cross. a Insects at generations 16 and 17 postinjection were used. each other and unidirectional incompatibility occurred by a simple mutation. The first mechanism is possible, between single-infected females and double-infected considering that the genome of Wolbachia contains males. The mortality of single-infected eggs fertilized transposon-like sequences (Masui et al. 1999) and by sperm from double-infected males was pointed out phages (Masui et al. 2000, 2001). Genes may also move as the mechanism that maintains double infection. between Wolbachia variants through recombination of If wCauA induces CI in C. cautella, our data indicate the genome (Jiggins et al. 2001; Werren and Bartos that wCauA causes two distinct phenotypes in the two 2001). The second mechanism has not been supported different hosts. At least two explanations can be made because in various transfers more or less similar pheno- for the dual effect of wCauA. One is that the same types were found in the recipient and in the donor. bacterial gene causes CI and male killing: The bacterial In contrast, the observation that wCauA induces male action that induces CI in C. cautella may result in the killing depending on the host provides an example that mortality of males of E. kuehniella. Such differential host supports this mechanism. The involvement of host fac- responses could occur if the process of male develop- tors in the determination of the phenotype was also ment differs between the two insects. Alternatively, it is demonstrated by a transfer recently reported by Fujii possible that the genes responsible for the two effects et al. (2001), in which the feminizing Wolbachia of O. are different and that wCauA possesses both of them. scapulalis (wSca) caused male killing in E. kuehniella. In this case, C. cautella, but not E. kuehniella, may be The third mechanism is difficult to test until the genes resistant to the action of the male-killing gene, or the responsible for the phenotypes are identified. gene expression may be suppressed exclusively in C. Male killing and CI: The occurrence of male killing cautella because of differences in the intracellular envi- following the transfection with wSca (Fujii et al. 2001) ronment between the two insects. This explanation is and wCauA led us to suspect that male killing is the also plausible when wCauA does not participate in CI prototype of reproductive manipulation by Wolbachia induction in C. cautella and possesses the only male- in E. kuehniella, from which CI, or possibly another type killing gene. However, if wCauA does not induce CI of reproductive phenotype, evolves. It is generally be- and possesses only the male-killing gene that does not lieved that male killing evolves easily in insects (Hurst cause the phenotype in C. cautella, it is difficult to ex- et al. 1997, 1999) because male-killing bacteria have plain how wCauA is maintained in the host population. been found in several insects and because they belong hylogeny of Wolbachia and reproductive alterations: to diverse groups in the Eubacteria (Werren and Beu- Phylogenic analyses of Wolbachia have repeatedly shown a keboom 1998). lack of congruence between bacterial phylogeny and their The transition from male killing to CI at least theoreti- effects on hosts: Wolbachia that induce similar pheno- cally seems to be a possible evolutionary scenario be- types are not monophyletic, and closely related Wol- cause it could increase the fitness of both host and bachia can have different effects on their hosts (O’Neill Wolbachia. The host loses half of its offspring in male et al. 1992; Moran and Baumann 1994; Van Meer et killing but not in CI. For Wolbachia, CI would be a more al. 1999). Several mechanisms have been proposed for efficient mechanism than male killing in spreading and this incongruence (Van Meer et al. 1999): (i) Plasmids maintaining the infection in the host population. Male- or phages carry the genes responsible for the pheno- killing microbes are postulated to increase the number types; (ii) the host determines the phenotype induced of surviving female progeny by prevention of sibling by Wolbachia; or (iii) genes inducing different pheno- cannibalism, competition for food, or disadvantageous types are similar and can be derived from each other inbreeding (Skinner 1985; Hurst 1991). However, the 1318 T. Sasaki, T. Kubo and H. Ishikawa effects are indirect compared with the effect of CI that increases its potential as a tool for pest insect control. directly depresses the reproduction of uninfected fe- Wolbachia-mediated CI may be utilized as an alternative males. In addition, male killing produces selection on to sterile male release and also as a self-spreading mecha- the host against this trait. Male killing, as well as thelyto- nism by which useful genes are spread into insect popu- kous parthenogenesis and feminization, produces pop- lations (Sinkins et al. 1997). In both strategies, when ulations in which the sex ratio is female biased. In such the target pest population is already infected with Wol- populations, males have higher reproductive success bachia, a host strain that carries a different or additional than females, so selection on the host for genes prevents Wolbachia variant is needed. Interspecific transfers us- bacterial action and promotes the production of males ing various insects as Wolbachia sources will increase (Stouthamer et al. 1999). the chance to generate a desired strain of the pest insect CI induction by wCauB in E. kuehniella: E. kuehniella targeted. lines transfected with only wCauB were unidirectionally We thank Sugihiko Hoshizaki for comments on the manuscript. incompatible with the uninfected strain and were bidi- This work was supported by a grant-in-aid for scientific research (grant rectionally incompatible with the naturally infected no. 10740388) from the Ministry of Education, Science and Culture strain. To our knowledge, this is the first observation of of Japan. bidirectional CI in lepidopteran insects. Bidirectional CI has been described in the mosquito Culex pipiens (Yen and Barr 1973; Magnin et al. 1987), D. simulans (O’Neill LITERATURE CITED and Karr 1990), and Nasonia wasps (Breeuwer and Wer- Bouchon, D., T. Rigaud and P. Juchault, 1998 Evidence for wide- ren 1990). spread Wolbachia infection in isopod crustaceans: molecular In crosses between transfected males and females, the identification and host feminization. Proc. R. Soc. Lond. Ser. B 265: 1081–1090. egg-hatching rate was slightly lower than that in crosses Bourtzis, K., and H. R. Braig, 1999 The many faces of Wolbachia, within the uninfected strain (Table 5). This increase pp. 199–219 in Rickettsiae and Rickettsial Diseases at the Turn of the in mortality is not likely a consequence of inbreeding, Third Millenium, edited by D. Raoult and P. Brouqui. Elsevier, Paris. because it was observed in crosses between the two inde- Boyle, L., S. L. O’Neill, H. M. Robertson and T. L. Karr, 1993 pendently established transfected lines as well as within Interspecific and intraspecific horizontal transfer of Wolbachia each of them. It is also not likely due to a possible in Drosophila. Science 260: 1796–1799. Braig, H. R., H. Guzman, R. B. Tesh and S. L. O’Neill, 1994 Re- virulence of wCauB because the mortality decreased placement of the natural Wolbachia symbiont of Drosophila sim- when the transfected females were crossed with unin- ulans with a mosquito counterpart. Nature 367: 453–455. fected males (Table 5, E and I). The increased mortality Breeuwer, J. A. J., 1997 Wolbachia and cytoplasmic incompatibility in the spider mites Tetranychus urticae and T. turkestani. Heredity may be explained by an unequal transmission of wCauB. 79: 41–47. Some eggs of the transfected lines might harbor wCauB Breeuwer, J. A. J., and J. H. Werren, 1990 Microorganisms associ- at low density and therefore may have failed to rescue ated with chromosome destruction and reproductive isolation between two insect species. Nature 346: 558–560. the modified sperm from males with high bacterial den- Breeuwer, J. A. J., and J. H. Werren, 1993 Cytoplasmic incompati- sity. 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