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

Corresponding author: Tetsuhiko Sasaki, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan., sasaki{at}biol.s.u-tokyo.ac.jp (E-mail)

Communicating editor: M. J. SIMMONS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 expresses 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 sex ratio of 1:1. The occurrence of male killing following transfection showed that host factors influence 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.

WOLBACHIA is a group of rickettsia-like intracellular bacteria found in many arthropods. These maternally inherited bacteria are known as the causative agents of various reproductive alterations such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males into functional females, and male killing (reviewed in WERREN 1997 ; BOURTZIS and BRAIG 1999 ; STOUTHAMER et al. 1999 ).

Of the various phenotypes associated with Wolbachia infection, CI is the most commonly described. Typical CI is expressed unidirectionally: The cross between infected males and uninfected females is incompatible, whereas the reciprocal cross is compatible. The incompatibility arises from defects in paternal chromatin condensation during mitosis (O'NEILL and KARR 1990 ; REED and WERREN 1995 ; LASSY and KARR 1996 ) and results in embryonic mortality. In some haplodiploid species, including wasps and mites, in which haploid embryos develop into males, CI causes male-biased sex ratios (BREEUWER and WERREN 1990 ; BREEUWER 1997 ). The mechanism of CI is generally explained by a dual action of Wolbachia: "modification" of sperm and "rescue" in eggs. Since the unidirectional incompatibility reduces the reproductive success of exclusively uninfected females, it can cause the rapid spread of this maternally inherited bacterium in the host population, as has been documented in Drosophila simulans (TURELLI and HOFFMANN 1991 ) and the planthopper Laodelphax striatellus (HOSHIZAKI and SHIMADA 1995 ).

Bidirectional incompatibility has also been reported to occur in crosses between Wolbachia-infected males and females of separate populations (YEN and BARR 1973 ; BREEUWER and WERREN 1990 ; O'NEILL and KARR 1990 ). This type of incompatibility has been associated with the presence of different bacterial variants, suggesting that some Wolbachia variants are unable to rescue the sperm modified by another variant (BRAIG et al. 1994 ; ROUSSET and DE STORDEUR 1994 ; HOFFMANN and TURELLI 1997 ). Multiple infections within a single individual may also influence compatibility type. Unidirectional incompatibility has been reported in the crosses between double-infected males and single-infected females (MERCOT et al. 1995 ; ROUSSET and SOLIGNAC 1995 ; SINKINS et al. 1995 ; PERROT-MINNOT et al. 1996 ).

Since Wolbachia and its host form a symbiotic system, it would be important to investigate both bacterial and host factors to understand the basis for the expression 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 hosts. Wolbachia has been successfully transferred in several species of isopods and insects. When CI-inducing Wolbachia was transferred intraspecifically, the recipient expressed CI as the donor did (CHANG and WADE 1994 ; ROUSSET and DE STORDEUR 1994 ; SASAKI and ISHIKAWA 2000 ). On the other hand, several interspecific transfers have shown that host factors can affect the reproductive phenotype. The transfers between D. simulans and D. melanogaster revealed the influence of the host on the intensity at which CI is expressed (BOYLE et al. 1993 ; POINSOT et al. 1998 ). Transfers of feminizing Wolbachia among isopods resulted in four situations: no reproductive effect, expression of feminization, death of recipients, and failure of transfection (BOUCHON et al. 1998 ). FUJII et al. 2001 demonstrated that the Wolbachia causing feminization in the Asian corn borer, Ostrinia scapulalis, induced male killing when transferred to the Mediterranean flour moth, Ephestia kuehniella. Thus the importance of interactions between host and Wolbachia has been established, highlighting the need for further accumulation of data on interspecific transfers.

In this article, we report the transfer of Wolbachia from the almond moth Cadra cautella to E. kuehniella. C. cautella is doubly infected with wCauA and wCauB, which belong to A- and B-group Wolbachia, respectively (designated by WERREN et al. 1995 ), and expresses complete CI (KELLEN et al. 1981 ; SASAKI and ISHIKAWA 1999 ). While the double infection causes CI, the effect of each Wolbachia variant is not known because the two variants have not been separated in this host. E. kuehniella is infected with an A-group Wolbachia variant (wKue) and expresses partial CI (SASAKI and ISHIKAWA 1999 ). The present study was originally designed to examine whether the Wolbachia variants naturally infecting C. cautella could infect E. kuehniella and induce CI. Since both the donor and recipient insects express CI in natural infections, we presumed that, once the transfection was established, the transfected lines would also express CI and that the incompatibility level might be affected by the hosts. Unexpectedly, however, wCauA caused male killing in E. kuehniella. Although the actual phenotype caused by wCauA in C. cautella is obscure, it is apparent that it does not cause male killing. The occurrence of male killing in the transfected E. kuehniella clearly showed that the reproductive phenotype changes are dependent on the combination of bacterium and host. The E. kuehniella lines that were transfected with only wCauB expressed partial CI and were bidirectionally incompatible with the naturally infected strain.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Insects:
E. kuehniella and C. cautella, originally collected in Tsuchiura, Japan, were reared on a diet consisting of wheat bran, dried yeast, and glycerol (20:1:2 w/w) at 25° under a 16 hr light:8 hr dark photoperiod. A Wolbachia-free strain of E. kuehniella was established by rearing the insects on a diet containing tetracycline at a final concentration of 0.04% (w/w) for two generations (SASAKI and ISHIKAWA 1999 ).

Microinjection:
Injections were performed as previously described (SASAKI and ISHIKAWA 2000 ). Freshly laid eggs (<1 hr old) of the uninfected strain of E. kuehniella were placed on a piece of double-sided adhesive tape on a slide. The ovaries of C. cautella collected by dissection from the adult females were also placed on a slide. The ooplasm taken out of the ovary was injected into the eggs under a microscope equipped with a three-dimensional micromanipulator and a microinjector.

The injected eggs on the slide were kept at 25° in a plastic dish (9 cm in diameter) containing a piece of moist filter paper. Five days after injection the sticky surface of the tape was covered with flour, and the slide was transferred onto the diet mixture in a plastic container. The eggs hatched on the sixth or seventh day after injection. The rate of egg hatching was checked by counting the cast-off shells left on the tape.

Adults emerged about 1 month after the eggs hatched. The females were then individually transferred into plastic cups (3 cm in diameter, 5 cm in height) where they were mated with males. The males used were either those developed from the injected eggs or those collected from the stock culture of the uninfected strain. A sample of eggs laid by each female was diagnosed for infection status by PCR.

PCR for detection of Wolbachia:
The presence or absence of Wolbachia was tested by diagnostic PCR assays using Wolbachia-specific primers for the ftsZ bacterial cell-cycle gene. The template DNA was prepared according to O'NEILL et al. 1992 . A total of 10–20 eggs were homogenized in 50 µl of STE (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 0.4 mg/ml of proteinase K and incubated for 90 min at 55° and then for 15 min at 95°.

PCR was performed in a 20-µl reaction mixture using Takara EX Taq. Three primer sets were used for amplification of the ftsZ gene according to WERREN et al. 1995 . The general ftsZ primers used were ftsZfl (5'-GTT GTC GCA AAT ACC GAT GC-3') and ftsZrl (5'-CTT AAG TAA GCT GGT ATA TC-3'), the A-group-specific ftsZ primers were ftsZAdf (5'-CTC AAG CAC TAG AAA AGT CG-3') and ftsZAdr (5'-TTA GCT CCT TCG CTT ACC TG-3'), and the B-group-specific ftsZ primers were ftsZBf (5'-CCG ATG CTC AAG CGT TAG AG-3') and ftsZBr (5'-CCA CTT AAC TCT TTC GTT TG-3'). PCR cycling conditions were 94° for 3 min followed by 35 amplification cycles of 94° for 30 sec, 55° for 30 sec, 72° for 1 min, and, finally, 72° for 5 min.

Crossing experiments:
The adults of E. kuehniella do not feed and survive for 1 week. During this period, the female mates once or twice. Crossing experiments were performed using single pairs of virgin individuals. Since the male larva is easily distinguished by a dark patch (the testes) on its back, we separated the females and males at the late larval stage. A female and a male, younger than 3 days after emergence, were set in a plastic cup (3 cm in diameter, 5 cm in height) and left there for 3 days. Most females deposited >100 eggs onto the wall of the cup. Cups in which <50 eggs had been laid were discarded. From each pair, 50–100 eggs were collected and placed onto 1% agarose in a plastic dish (35 mm in diameter). After incubation for 7–8 days at 25°, the hatching rate was scored for each single-pair cross. The data were analyzed by Mann-Whitney U-tests.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transfected lines:
Of 208 E. kuehniella eggs injected with the ooplasm of C. cautella, 130 eggs hatched. Insects that developed from the injected eggs were designated as generation 0 (G₀). G₁ eggs were collected from each of 40 G₀ females. A part (10–20 eggs) of each G₁ brood was subjected to PCR assay, in which Wolbachia was detected in three broods. In two broods (lines 1 and 2), only wCauB was detected. Both wCauA and wCauB were detected in the other brood (line 3). G₂ eggs were diagnosed for infection as performed on the G₁ eggs (Table 1). Infected broods were pooled for each of line 1 and line 2 and designated as line 1B and line 2B, respectively. Two lines different in infection status were obtained from line 3: one infected with only wCauA (line 3A) and the other infected with both wCauA and wCauB (line 3AB). At least 16 larvae were randomly selected for each of the four infected lines and diagnosed for infection by PCR. This assay showed that the G₂ larvae were homogeneously infected within lines. When the G₂ insects attained adulthood, only females emerged in lines 3A and 3AB, whereas both females and males emerged in lines 1B and 2B.

Lines 3A and 3AB were maintained by crossing with males from the uninfected strain. The all-female condition has been consistently transmitted maternally to offspring for 22 generations.

Female-biased sex ratio caused by wCauA:
Emergence of all-female populations in lines 3A and 3AB suggested that wCauA was the causative agent of the sex ratio distortion. Wolbachia is known to cause a female-biased sex ratio by three distinct mechanisms, thelytokous parthenogenesis, feminization of genetic males, and male killing. The possibility that thelytokous parthenogenesis was the mechanism was ruled out because no unfertilized eggs collected from the virgin females hatched. To distinguish feminization and male killing, the survival rates of the transfected lines producing all-female offspring were examined (Table 2). The egg hatch rates of the transfected lines were much lower than those of the uninfected strain, suggesting that the mechanism was male killing rather than feminization.

However, the data on hatching rates did not exclude the possibility of feminization associated with a high mortality caused by the possible virulence of wCauA in E. kuehniella. Then we examined the effect of removal of Wolbachia from the transfected lines on the sex ratio. In E. kuehniella, the sex chromosomes are ZZ in males and ZW in females (TRAUT and RATHJENS 1973 ). If feminization occurs, the transfected lines will be dominated by feminized individuals carrying ZZ chromosomes because ZW females produce both ZZ and ZW offspring, and the infected ZZ females (feminized individuals) produce solely ZZ offspring. In this situation, the removal of the feminizer should result in a male-biased sex ratio (KAGEYAMA et al. 1998 ). On the other hand, the removal of a male-killing agent should lead to the recovery of a normal sex ratio of 1:1. Eggs at G₈ were subjected to tetracycline treatment. The treatment had no effect on the sex ratio of the G₈ adults, probably because the manipulation by Wolbachia had been completed during the host's embryonic development, before the larvae started feeding on the antibiotic. After treatment for two generations, the sex ratio reverted to unbiased levels in all of the 10 families examined (Table 3). Accordingly, it was concluded that the mechanism of the sex ratio distortion was male killing.

It may be claimed that the male killing in lines 3A and 3AB is not caused by wCauA but by another factor transferred from C. cautella. To confirm the causal relationship of transfection with wCauA with the appearance of male killing in E. kuehniella, we performed the transfer again. An all-female condition was found in E. kuehniella lines transfected with wCauA, but not in lines without wCauA (Table 4).

Cytoplasmic incompatibility induced by wCauB:
The infection in the wCauB-transfected lines was examined by testing 48 larvae selected at random for each of lines 1B and 2B at generation 15. All 96 larvae tested were infected (data not shown), suggesting that the infection was stable. The expression of CI then was examined at generations 16 and 17. The transfected males were partially incompatible with uninfected females: The egg-hatching rate in the cross between the uninfected females and transfected males was significantly lower than that within the uninfected strain (see Table 5; A vs. B and A vs. C, both comparisons P < 0.001). The transfected and naturally infected strains were bidirectionally incompatible: Egg-hatching rates were lower in the interstrain crosses than in the intrastrain crosses (F vs. H, P < 0.001; J vs. L, P < 0.001; N vs. P, P < 0.001; O vs. P, P < 0.05). In addition, a slight decrease of the egg-hatching rate was found in the crosses between transfected males and females as compared with the cross within the uninfected strain (A vs. F, J, and K, P < 0.01; A vs. G, not significant).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We established E. kuehniella lines infected with Wolbachia variants derived from C. cautella. It was demonstrated that wCauA caused male killing, while wCauB induced CI in E. kuehniella.

Effects of wCauA on C. cautella and E. kuehniella:
Since E. kuehniella naturally infected with wKue expresses CI, the occurrence of male killing in the wCauA-transfected lines implies that the two Wolbachia variants, wKue and wCauA, differ in their ability to induce reproductive alterations. It was also shown that host factors influence the determination of reproductive alterations because wCauA does not cause male killing in C. cautella.

Prior to interpreting the occurrence of male killing in E. kuehniella, the effect of wCauA on its natural host should be considered. Although C. cautella expresses complete CI, it is not clear whether or not wCauA is responsible for the induction of CI because the insect is doubly infected. To examine the effect of wCauA, C. cautella infected with the single Wolbachia variant is needed, but such a strain has not been obtained. Three situations can be assumed: Only wCauA induces CI, only wCauB induces CI, or both variants induce CI. We assume that the third possibility is most likely because if one or the other Wolbachia variant does not induce CI, it would be lost stochastically and not be maintained in the host population. Indeed in Nasonia vitripennis and D. simulans, it has been demonstrated that the two Wolbachia variants forming double infection take part in CI (PERROT-MINNOT et al. 1996 for N. vitripennis; MERCOT et al. 1995 ; MERCOT and POINSOT 1998 for D. simulans). In these insects, single-infected lines were successfully segregated from the double-infected strain carrying A- and B-group Wolbachia. Lines carrying only A or B Wolbachia were bidirectionally incompatible with each other and unidirectional incompatibility occurred between single-infected females and double-infected males. The mortality of single-infected eggs fertilized by sperm from double-infected males was pointed out as the mechanism that maintains double infection.

If wCauA induces CI in C. cautella, our data indicate that wCauA causes two distinct phenotypes in the two different hosts. At least two explanations can be made for the dual effect of wCauA. One is that the same bacterial gene causes CI and male killing: The bacterial action that induces CI in C. cautella may result in the mortality of males of E. kuehniella. Such differential host responses could occur if the process of male development differs between the two insects. Alternatively, it is possible that the genes responsible for the two effects are different and that wCauA possesses both of them. In this case, C. cautella, but not E. kuehniella, may be resistant to the action of the male-killing gene, or the gene expression may be suppressed exclusively in C. cautella because of differences in the intracellular environment between the two insects. This explanation is also plausible when wCauA does not participate in CI induction in C. cautella and possesses the only male-killing gene. However, if wCauA does not induce CI and possesses only the male-killing gene that does not cause the phenotype in C. cautella, it is difficult to explain how wCauA is maintained in the host population.

hylogeny of Wolbachia and reproductive alterations:
Phylogenic analyses of Wolbachia have repeatedly shown a lack of congruence between bacterial phylogeny and their effects on hosts: Wolbachia that induce similar phenotypes are not monophyletic, and closely related Wolbachia can have different effects on their hosts (O'NEILL et al. 1992 ; MORAN and BAUMANN 1994 ; VAN MEER et al. 1999 ). Several mechanisms have been proposed for this incongruence (VAN MEER et al. 1999 ): (i) Plasmids or phages carry the genes responsible for the phenotypes; (ii) the host determines the phenotype induced by Wolbachia; or (iii) genes inducing different phenotypes are similar and can be derived from each other by a simple mutation. The first mechanism is possible, considering that the genome of Wolbachia contains transposon-like sequences (MASUI et al. 1999 ) and phages (MASUI et al. 2000 , MASUI et al. 2001 ). Genes may also move between Wolbachia variants through recombination of the genome (JIGGINS et al. 2001 ; WERREN and BARTOS 2001 ). The second mechanism has not been supported because in various transfers more or less similar phenotypes were found in the recipient and in the donor. In contrast, the observation that wCauA induces male killing depending on the host provides an example that supports this mechanism. The involvement of host factors in the determination of the phenotype was also demonstrated by a transfer recently reported by FUJII et al. 2001 , in which the feminizing Wolbachia of O. scapulalis (wSca) caused male killing in E. kuehniella. The third mechanism is difficult to test until the genes responsible for the phenotypes are identified.

Male killing and CI:
The occurrence of male killing following the transfection with wSca (FUJII et al. 2001 ) and wCauA led us to suspect that male killing is the prototype of reproductive manipulation by Wolbachia in E. kuehniella, from which CI, or possibly another type of reproductive phenotype, evolves. It is generally believed that male killing evolves easily in insects (HURST et al. 1997 , HURST et al. 1999 ) because male-killing bacteria have been found in several insects and because they belong to diverse groups in the Eubacteria (WERREN and BEUKEBOOM 1998 ).

The transition from male killing to CI at least theoretically seems to be a possible evolutionary scenario because it could increase the fitness of both host and Wolbachia. The host loses half of its offspring in male killing but not in CI. For Wolbachia, CI would be a more efficient mechanism than male killing in spreading and maintaining the infection in the host population. Male-killing microbes are postulated to increase the number of surviving female progeny by prevention of sibling cannibalism, competition for food, or disadvantageous inbreeding (SKINNER 1985 ; HURST 1991 ). However, the effects are indirect compared with the effect of CI that directly depresses the reproduction of uninfected females. In addition, male killing produces selection on the host against this trait. Male killing, as well as thelytokous parthenogenesis and feminization, produces populations in which the sex ratio is female biased. In such populations, males have higher reproductive success than females, so selection on the host for genes prevents bacterial action and promotes the production of males (STOUTHAMER et al. 1999 ).

CI induction by wCauB in E. kuehniella:
E. kuehniella lines transfected with only wCauB were unidirectionally incompatible with the uninfected strain and were bidirectionally incompatible with the naturally infected strain. To our knowledge, this is the first observation of 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 and KARR 1990 ), and Nasonia wasps (BREEUWER and WERREN 1990 ).

In crosses between transfected males and females, the egg-hatching rate was slightly lower than that in crosses within the uninfected strain (Table 5). This increase in mortality is not likely a consequence of inbreeding, because it was observed in crosses between the two independently established transfected lines as well as within each of them. It is also not likely due to a possible virulence of wCauB because the mortality decreased when the transfected females were crossed with uninfected males (Table 5E and Table 5I). The increased mortality may be explained by an unequal transmission of wCauB. Some eggs of the transfected lines might harbor wCauB at low density and therefore may have failed to rescue the modified sperm from males with high bacterial density. Such a dosage effect, proposed by BREEUWER and WERREN 1993 , also explains the observation that the egg-hatching rate in the cross between 2B females and 1B males was the lowest among the four crosses within transfected lines. The density of wCauB might be higher in line 1B than in line 2B because the 1B males caused a significantly (P < 0.05) stronger CI than the 2B males did when crossed with the uninfected and naturally infected females.

BRAIG et al. (1990) conducted a Wolbachia transfer from the mosquito Aedes albopictus into D. simulans and reported that the transfected Drosophila line was bidirectionally incompatible with all other naturally infected strains. Our observation of bidirectional CI in E. kuehniella provides an additional example in which a new incompatibility property can be introduced by interspecific transfer. If wCauB is transferred into naturally infected E. kuehniella, the transfected strain possibly would express unidirectional incompatibility against the original strain, as documented in D. simulans (SINKINS et al. 1995 ; ROUSSET et al. 1999 ).

From applied perspectives, the ability of Wolbachia to cause bidirectional incompatibility in a new host species increases its potential as a tool for pest insect control. Wolbachia-mediated CI may be utilized as an alternative to sterile male release and also as a self-spreading mechanism by which useful genes are spread into insect populations (SINKINS et al. 1997 ). In both strategies, when the target pest population is already infected with Wolbachia, a host strain that carries a different or additional Wolbachia variant is needed. Interspecific transfers using various insects as Wolbachia sources will increase the chance to generate a desired strain of the pest insect targeted.

	ACKNOWLEDGMENTS

We thank Sugihiko Hoshizaki for comments on the manuscript. This work was supported by a grant-in-aid for scientific research (grant no. 10740388) from the Ministry of Education, Science and Culture of Japan.

Manuscript received March 19, 2002; Accepted for publication August 19, 2002.

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