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.