Cytoplasmic Incompatibility and Sperm Cyst Infection in Different Drosophila-Wolbachia Associations

Corresponding author: Kostas Bourtzis, 2 Seferi St., University of Ioannina, Aginio 30100, Greece., kbourtz{at}cc.uoi.gr (E-mail)

Communicating editor: D. CHARLESWORTH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Wolbachia are a group of maternally transmitted obligatory intracellular -proteobacteria that infect a wide range of arthropod and nematode species. Wolbachia infection in Drosophila in most cases is associated with the induction of cytoplasmic incompatibility (CI), manifested as embryonic lethality of offspring in a cross between infected males and uninfected females. While the molecular basis of CI is still unknown, it has been suggested that two bacterial functions are involved: mod (for modification) modifies the sperm during spermatogenesis and resc (for rescue) acts in the female germline and/or in early embryos, neutralizing the modification. There is considerable variation in the level of incompatibility in different Wolbachia/host interactions. We examine the relationship between the levels of CI in a number of naturally infected and transinfected Drosophila hosts and the percentage of Wolbachia-infected sperm cysts. Our results indicate the presence of two main groups of Drosophila-Wolbachia associations: group I, which exhibits a positive correlation between CI levels and the percentage of infected sperm cysts (mod⁺ phenotype), and group II, which does not express CI (mod^- phenotype) irrespective of the infection status of the sperm cysts. Group II can be further divided into two subgroups: The first one contains associations with high numbers of heavily Wolbachia-infected sperm cysts while in the second one, Wolbachia is rarely detected in sperm cysts, being mostly present in somatic cells. We conclude that there are three requirements for the expression of CI in a host-Wolbachia association: (a) Wolbachia has to be able to modify sperm (mod⁺ genotype), (b) Wolbachia has to infect sperm cysts, and (c) Wolbachia has to be harbored by a permissive host.

WOLBACHIA pipientis is an intracellular maternally transmitted bacterium found in arthropods and nematodes. Wolbachia manipulates host reproduction by inducing parthenogenesis, feminization, male killing, and cytoplasmic incompatibility, all of which enhance transmission of the bacterium (WERREN 1997 ; STOUTHAMER et al. 1999 ; STEVENS et al. 2001 ). On the other hand, Wolbachia has evolved intimate relationships with nematodes (SIRONI et al. 1995 ), a parasitic wasp (DEDEINE et al. 2001 ), and mosquitoes (DOBSON et al. 2002A ). Wolbachia-induced cytoplasmic incompatibility (CI) was first described in mosquitoes (YEN and BARR 1971 ) as embryonic lethality in crosses between infected males and uninfected females. Since then, CI has been described in crustaceans (MORET et al. 2001 ), arachnids (BREEUWER 1997 ), and many insects including wasps, planthoppers, moths, beetles, and Drosophila, making it the most common and widespread Wolbachia-induced phenotype (O'NEILL et al. 1997 ; CHARLAT et al. 2002 ).

Although the molecular mechanisms of CI are not known, Wolbachia apparently modify developing sperm of infected males. Mature sperm does not contain Wolbachia. When sperm from an infected male fertilizes an egg, bacteria of the same CI type must be present in the egg to rescue this modification. Otherwise, abnormal mitosis occurs, which typically results in zygotic death (LASSY and KARR 1996 ; CALLAINI et al. 1997 ; TRAM and SULLIVAN 2002 ). Thus, the strategy employed by CI-inducing Wolbachia in diploid hosts is to reduce the fertility of uninfected females to spread into uninfected populations.

The mod resc model provides a general framework for the investigation of CI (WERREN 1997 ). It assumes the existence of two bacterial functions, modification (mod) that occurs during sperm development and rescue (resc), which takes place in the egg. According to this model, four different CI types could exist: mod⁺ resc⁺, mod ⁺ resc^-, mod^- resc⁺, and mod^- resc^-. The mod⁺ resc⁺ type corresponds to most strains described so far, which induce CI and rescue their own modification. A mod^- resc^- Wolbachia that neither induces CI nor rescues CI induced by other mod⁺ strains has been described for Drosophila simulans (HOFFMANN et al. 1996 ). A mod^- resc⁺ strain does not induce but can rescue CI induced by closely related strains (BOURTZIS et al. 1998 ; MERCOT and POINSOT 1998 ). mod⁺ resc^- strains have not been found yet, possibly because such strains may not be able to maintain themselves in a population (but see CHARLAT et al. 2001 for an alternative view).

A number of factors have been proposed to affect expression of CI, including bacterial genotype, host genotype, and bacterial density (REYNOLDS and HOFFMANN 2002 ; WEEKS et al. 2002 ). These factors can interact in complex ways to affect strength and type of CI. Several studies have suggested that Wolbachia strains might act independently of the host genome (MONTCHAMP-MOREAU et al. 1991 ; GIORDANO et al. 1995 ; HOFFMANN et al. 1996 ). However, the host background may affect CI levels. The contribution of host genotype to the expression of CI has been studied in Nasonia by genome introgression experiments (BREEUWER and WERREN 1993 ; BORDENSTEIN and WERREN 1998 ) and in Drosophila by transinfection experiments. Transfer of Wolbachia from D. simulans into D. melanogaster leads to a shift from high to low CI levels (BOYLE et al. 1993 ). Conversely, the reciprocal transfer of Wolbachia from D. melanogaster to D. simulans results in high CI (POINSOT et al. 1998 ).

BREEUWER and WERREN 1993 (pp. 571–572) proposed a bacterial dosage model according to which "unidirectional incompatibility is strongly influenced by the dose of bacteria in the male (e.g., in the spermatocytes) relative to the dose in the egg. Sperm is incompatible with an egg when the number of bacteria is greater in the male strain than in the female strain. Conversely, a cross is compatible when the paternal strain harbors equal (e.g., intrastrain crosses) or lower numbers of cytoplasmic bacteria." Several studies have demonstrated that the degree of CI correlates with bacterial numbers in eggs and the proportion of infected cysts in testes of Drosophila species (BOYLE et al. 1993 ; ROUSSET and DE STORDEUR 1994 ; SOLIGNAC et al. 1994 ; GIORDANO et al. 1995 ; BOURTZIS et al. 1996 ; POINSOT et al. 1998 ; MCGRAW et al. 2001 ). Although a positive correlation was observed when comparisons involved a single Wolbachia strain within a single host, no such correlation could be established when different Wolbachia strains and/or different hosts were compared.

CLARK et al. 2002 , CLARK et al. 2003 described the distribution of Wolbachia during spermatogenesis of different Drosophila-Wolbachia associations and discussed the possible implications on the mechanism of CI. Building on these studies, we determined and compared the percentage of Wolbachia-infected sperm cysts in the testes of naturally infected and transinfected Drosophila lines. Using these quantitative data, we investigated the relationship between the percentage of Wolbachia-infected sperm cysts and the levels of CI. By comparing the same Wolbachia strain in different hosts and different Wolbachia strains in the same host background, we investigated the relative contribution of bacterial and host factors to bacterial distribution, density, and levels of CI. Finally, we discuss the modification rescue and bacterial dosage models in light of these results.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila lines:
Drosophila lines, Wolbachia strains, and their sources are listed in Table 1. Flies were grown at 25° on cornmeal-agar-sugar-yeast medium under uncrowded conditions. Wolbachia strains are named according to ZHOU et al. 1998 .

DNA extraction, PCR, and sequencing:
Total DNA was extracted from whole individuals as described (O'NEILL et al. 1992 ). Presence of Wolbachia was confirmed by PCR using primers 99F and 994R as described in the same article. For cloning, PCR products amplified by primers 81F and 691R (BRAIG et al. 1998 ) were purified using the QIAquick gel extraction kit (QIAGEN, Chatsworth, CA) and ligated into pGEM-T Easy (Promega, Madison, WI). Three independent clones were sequenced for each strain to eliminate polymerase errors and generate consensus sequences. Alternatively, PCR products from at least three individuals were directly sequenced using the same primers.

Cytoplasmic incompatibility assay:
Tests were performed at 25°. Thirty single-pair matings were performed on apple juice/agar plates. Two-day-old virgin females and 1-day-old virgin males were used. Plates were replaced daily for 2–3 days. Hatching rates were scored 36 hr after egg collection on at least 50 eggs per cross. Flies were frozen immediately after egg collection for DNA extraction and verification of their infection status with PCR.

Confocal microscopy:
Testes were removed from up to 1-day-old virgin males in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween, 0.05% NaN₃, pH 7.5) and further dissected on glass slides to expose germline cysts. Tissue samples were flattened under a coverslip and frozen in liquid nitrogen. Coverslips were removed and slides placed in ice-cold ethanol for 3 min and fixed in 4% paraformaldeyde for 12 min. Slides were then washed three times with TBST for 15 min each, then blocked in 1% BSA in TBST, and incubated with Wolbachia surface protein (WSP) antibody (DOBSON et al. 1999 ) at a 1:500 dilution overnight at 4°. After three washes with TBST, slides were incubated for 1 hr at room temperature with a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG-labeled antibody (Molecular Probes, Eugene, OR) and 2 mg/ml Rnase A (Sigma, St. Louis) in TBST. After three washes in TBST, slides were stained for 3 min with 1 µg/ml 4',6-diamidino-2-phenylindole (Molecular Probes), rinsed, stained with 5 µg/ml propidium iodide (Molecular Probes) for 20 min, rinsed, and mounted with ProLong antifade medium (Molecular Probes).

Optical sections were taken by using a confocal laser-scanning microscope (Leica TCS-NT) and projected onto single images. Images were further processed using Photoshop 6.0 (Adobe).

Wolbachia load in testes:
The percentages of infected cysts were determined by scoring the mature (elongated) cysts of every male examined.

Statistical analysis:
All statistical tests were performed with SPSS (version 10).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Wolbachia infection and Drosophila spermatogenesis:
For details about distribution and proliferation of Wolbachia during spermatogenesis in Drosophila see CLARK et al. 2002 , CLARK et al. 2003 . One of the main goals of the present study was to determine distribution and density of Wolbachia during spermatogenesis of naturally infected and transinfected Drosophila hosts. The infection pattern of Wolbachia-infected sperm cysts was determined following the principles established by CLARK et al. 2002 . Cysts were counted as infected when Wolbachia infection was detected in spermatids and had the pattern of D. simulans infected with wRi (Fig 1A) or the patchy pattern of D. melanogaster infected with wMel (Fig 1B). Cysts were counted as uninfected either when there were no Wolbachia at all or when only the somatic cyst cells that surround the spermatids contained (usually very few) bacteria (Fig 1C). In testes that harbored strains other than wRi, immature infected cysts were very rarely detected. Seven different Wolbachia strains from three subgroups of both A and B groups were studied (see Table 1). In particular, different Drosophila species infected with the same Wolbachia variant as well as a single Drosophila species, D. simulans, infected with different Wolbachia strains are included. Such an analysis should allow us to determine the contribution of bacterial and/or host factors to the different degree of sperm cyst infection and different CI levels. We observed that all these Drosophila strains contained Wolbachia of somatic origin under the sheath of their testes as well as in other somatic tissue. However, they differed in the infection pattern of sperm cysts (Fig 1). Percentages of infected sperm cysts are given in Table 1 (hereafter, "infected sperm cyst" will refer to a cyst with Wolbachia within spermatids), together with CI levels estimated in this and other studies. For every Drosophila-Wolbachia association studied, the CI type of the infected host as well as the partial wsp gene sequences of all the Wolbachia strains were confirmed. All CI assays of the present study were performed using males no more than 1 day old because it was recently reported that male age is an important factor for the detection of associations expressing low levels of CI (REYNOLDS and HOFFMANN 2002 ; WEEKS et al. 2002 ).

View larger version (69K): In this window In a new window Download PPT slide   Figure 1. Wolbachia distribution close to sperm nuclei of mature cysts in (A) D. simulans Riverside, (B) D. melanogaster CS, and (C) D. simulans transinfected with wMa. D–F are transmission images of A–C, respectively. Bacteria are stained green-yellow and nuclei are stained red. Bar, 10 µm.

Mel subgroup Wolbachia strains and cytoplasmic incompatibility:
D. melanogaster yw^67C23 (wMel, group A), D. melanogaster Canton-S (wMelCS, group A), and D. melanogaster popcorn (wMelPop, group A) associations showed few infected cysts, 10% or less, and a patchy distribution of bacteria (Fig 1B). The D. melanogaster yw^67C23 (wMel) association expresses low levels of CI while the other two associations do not express detectable CI (HOLDEN et al. 1993 ; BOURTZIS et al. 1996 ; MIN and BENZER 1997 ). On the basis of these data, wMel was considered as a mod⁺ variant while wMelCS and wMelPop were considered mod^-. However, recent studies clearly show that both latter strains are also mod⁺ (MCGRAW et al. 2001 ; REYNOLDS and HOFFMANN 2002 ; WEEKS et al. 2002 ). While D. simulans transinfected with wMel exhibits high numbers of heavily infected cysts and is known to express high levels of CI (POINSOT et al. 1998 ), the D. simulans Coffs (wCof, group A) strain has high numbers of heavily infected cysts, while it is not capable of inducing CI (HOFFMANN et al. 1996 ; present study). In a parallel study, we showed that D. yakuba, D. teissieri, and D. santomea, naturally infected with Wolbachia, were not able to express CI while carrying wCof-like (group A) Wolbachia, as judged by partial wsp gene sequences (S. ZABALOU, S. CHARLAT, A. NIRGIANAKI, D. LACHAISE, H. MERÇOT and K. BOURTZIS, unpublished results). The vast majority of the cysts of these Drosophila species were uninfected. Only a small number contained a few bacteria in the somatic cyst cells (Fig 1C).

Ri subgroup Wolbachia strains and cytoplasmic incompatibility:
Analysis of the D. simulans Riverside (wRi, group A) association shows that most mature sperm cysts from newly eclosed males were heavily infected with bacteria (Fig 1A; see also CLARK et al. 2002 ). This association is known to express high levels of CI (HOFFMANN et al. 1986 ). Interestingly, transinfected lines of D. yakuba (wRi), D. teissieri (wRi), and D. santomea (wRi) contained, although variable (40–70%), high numbers of heavily infected cysts showing a wRi-infected D. simulans-like pattern (Fig 1A). The transinfected lines also express high levels of CI (S. ZABALOU, S. CHARLAT, A. NIRGIANAKI, D. LACHAISE, H. MERÇOT and K. BOURTZIS, unpublished data).

Pip subgroup Wolbachia strains and cytoplasmic incompatibility:
Analysis of the D. simulans Noumea (wNo, group B), D. simulans Kilimanjaro (wKi, group B), as well as the transinfected D. simulans Watsonville (wMa, group B) associations indicate that 20–30% of the mature sperm cysts from newly eclosed males are heavily infected (Fig 1A), while the rest contain a few scattered bacteria, probably in the cyst cells (Fig 1C). The D. simulans wNo association has been shown to induce moderate levels of CI (MERCOT et al. 1995 ) while the D. simulans wKi and the transinfected D. simulans wMa do not express CI at all (GIORDANO et al. 1995 ; BOURTZIS et al. 1998 ; MERCOT and POINSOT 1998 ). On the other hand, the D. mauritiana (wMa, group B) association has >70% heavily infected cysts, while it is not able to induce CI.

Is there any correlation between the percentage of Wolbachia-infected sperm cysts and/or tropism in Drosophila sperm cysts and expression of cytoplasmic incompatibility?
Fig 2 clearly shows two main groups of Drosophila-Wolbachia associations. Group I consists of all wRi-infected Drosophila hosts, D. simulans (wMel), D. simulans (wNo), and D. melanogaster (wMel). This group of associations has variable numbers of Wolbachia-infected sperm cysts and expresses variable degrees of CI. Group II consists of D. simulans Coffs (wCof), D. mauritiana (wMa), D. melanogaster (wMelCS), D. melanogaster (wMelPop), D. simulans (wMa), D. simulans (wKi), and wCof-like infected D. yakuba, D. teissieri, and D. santomea. This group of associations does not express CI, irrespective of the infection status of the sperm cysts. Group II can be further divided into two subgroups: The first one contains D. simulans Coffs (wCof) and D. mauritiana (wMa) associations with high numbers of heavily Wolbachia-infected sperm cysts while the second one contains D. melanogaster (wMelCS), D. melanogaster (wMelPop), D. simulans (wMa), D. simulans (wKi), and wCof-like infected D. yakuba, D. teissieri, and D. santomea, where Wolbachia are rarely detected in sperm cysts, mostly being present in somatic cells.

View larger version (7K): In this window In a new window Download PPT slide   Figure 2. Two main groups of Drosophila-Wolbachia associations: group I of associations shows variable numbers of Wolbachia-infected sperm cysts and expresses variable levels of CI (mod+ phenotype) while group II does not express CI (mod- phenotype) irrespective of the infection status of the sperm cysts.

Is there any correlation between the percentage of Wolbachia-infected sperm cysts in the Drosophila-bacterial associations and the respective levels of CI? Pearson correlation analysis indicates an overall positive correlation (r = 0.618, N = 16, P = 0.011) that becomes even stronger if the analysis is restricted to the CI-expressing Drosophila-bacterial associations of group I (r = 0.923, N = 7, P = 0.003).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We used a Wolbachia specific antibody and confocal microscopy to study Wolbachia distribution and density in mature sperm cysts of 16 naturally infected and transinfected Drosophila-Wolbachia associations. This antibody had been raised against the major surface protein of Wolbachia (WSP protein) and has been used in previous studies to determine the Wolbachia tissue tropism in different host species by Western blot analyses (BRAIG et al. 1998 ; DOBSON et al. 1999 ). Recently, the same antibody was used to detect Wolbachia cells in transinfected insect cell lines by confocal microscopy (DOBSON et al. 2002B ) and also to study, in more detail, Wolbachia distribution during spermatogenesis of different Drosophila lines (CLARK et al. 2002 , CLARK et al. 2003 ). CLARK and KARR 2002 also used a quantitative PCR method and a general nucleic acid stain to quantify Wolbachia numbers in relation to CI levels in one D. melanogaster line and two D. simulans lines, all of them naturally infected. Their data did not allow any conclusion on the relative role of host or Wolbachia factors.

Our study suggests two main groups of Drosophila-Wolbachia associations. Group I— consisting of all wRi-infected (natural and transinfected) Drosophila hosts, the transinfected D. simulans (wMel), and the naturally infected D. simulans (wNo) and D. melanogaster (wMel)—shows variable numbers of Wolbachia-infected sperm cysts and expresses variable degrees of CI. Group II—consisting of the naturally infected D. simulans Coffs (wCof), D. mauritiana (wMa), D. melanogaster (wMelCS), and D. melanogaster (wMelPop), the transinfected D. simulans (wMa), the naturally infected D. simulans (wKi), and wCof-like infected D. yakuba, D. teissieri, and D. santomea—does not express CI irrespective of the infection status of the sperm cysts. On the basis of this evidence, we suggest that there are two distinct requirements for a Wolbachia strain to modify sperm: Wolbachia has to infect sperm cysts and Wolbachia has to be able to modify the sperm (mod⁺ genotype).

Group I of Drosophila-bacterial associations contains known mod⁺ Wolbachia strains (HOFFMANN et al. 1986 ; BOURTZIS et al. 1994 , BOURTZIS et al. 1996 ; MERCOT et al. 1995 ). Interestingly, this group has a strong positive correlation between CI expression levels and percentage of infected sperm cysts. BREEUWER and WERREN 1993 introduced the bacterial dosage model according to which bacterial density influences the degree of CI. Several studies have suggested that variation in bacterial density is an important determinant of CI levels (BOYLE et al. 1993 ; ROUSSET and DE STORDEUR 1994 ; SOLIGNAC et al. 1994 ; GIORDANO et al. 1995 ; BOURTZIS et al. 1996 ; POINSOT et al. 1998 ). However, it has to be pointed out that differential densities of Wolbachia within the testes may not be primarily responsible for different rates of CI. The total number of Wolbachia within testes contributes to the modification of sperm only when that Wolbachia is mod⁺ and is located within the spermatocytes and/or spermatids. Dosage seems important inasmuch as it is responsible for the total number of Wolbachia-infected sperm cysts. The total number of Wolbachia within the spermatogonial stem cells is likely to determine the total number of Wolbachia-infected sperm cysts, but Wolbachia at this stage may not be imprinting sperm. Thus, the Wolbachia dosage within the germline component of a cyst may be important in determining the modification of sperm. Such differences exist within, for example, the D. simulans-Wolbachia associations that we have examined for the naturally infected D. simulans (wRi) vs. D. simulans (wNo) and could be an explanation for lines with different rates of CI. Different Wolbachia densities present within cysts from different species (for example, D. simulans vs. D. melanogaster) may explain differences in the CI expression levels between these species. Therefore, there are two possible aspects of the bacterial dosage model, as applied to our data. First, overall density of bacteria in the testes may be correlated with percentage of sperm cells infected, thus affecting the CI levels. Second, the number of bacteria in infected cells may influence the probability and therefore the level of modification. BREEUWER and WERREN 1993 found that males from partially cured mothers produced sperm that induced mortality in CI crosses, whereas typically fully infected males produced complete paternal genome loss and therefore conversion to males. This indicates that CI may not be an all-or-none phenomenon.

Group II of Drosophila-Wolbachia associations do not express CI. Does this group include mod⁺ or mod^- Wolbachia strains? Recent studies suggested that the age of the male host plays a significant role in the expression of the modification component of CI (REYNOLDS and HOFFMANN 2002 ; WEEKS et al. 2002 ). Therefore, we performed crosses to confirm the CI status of all 16 host-bacterial associations studied using young males up to 1 day old (data not shown). Our results show that the first subgroup associations of naturally infected D. simulans (wCof) and D. mauritiana (wMa) indeed do not express CI. Our observations are similar to prior reports (GIORDANO et al. 1995 ; HOFFMANN et al. 1996 ) suggesting that Wolbachia strains wCof and wMa may not be able to cause modification, although they are able to infect the sperm cysts of their respective hosts at high percentage levels. It should be noted that the wMa strain naturally present in D. mauritiana was also unable to induce CI after transfer to an uninfected strain of D. simulans (DSW strain), a host known to express high levels of CI, suggesting that the inability to induce CI is due to the bacterial genetic background (mod^- genotype) (GIORDANO et al. 1995 ). However, some of the second subgroup of associations certainly includes mod⁺ strains. For example, wMelPop infects the vast majority of the sperm cysts and induces high levels of CI when transferred to D. simulans, although it is unable to infect sperm cysts or induce detectable CI levels in its natural host D. melanogaster (MIN and BENZER 1997 ; MCGRAW et al. 2001 ; this study). Similarly, wMelCS is able to induce CI in its natural host D. melanogaster, as reported recently (REYNOLDS and HOFFMANN 2002 ), although in the present study we were unable to detect significant levels of CI. This contradiction may be explained by the use of D. melanogaster Canton-S strains of different origin and genotype, which may significantly affect the expression levels of CI (SOLIGNAC et al. 1994 ; BOURTZIS et al. 1996 ; S. ZABALOU and K. BOURTZIS, unpublished data). These and previous data (POINSOT et al. 1998 ) suggest that the inter- and intraspecies differences observed in the number of infected sperm cysts may be under host control. This could explain the fact that wMa heavily infects the sperm cysts of its natural host D. mauritiana, but only weakly infects the cysts in D. simulans, and also that wCof heavily infects sperm cysts of D. simulans but wCof-like strains only weakly infect the cysts in other Drosophila species such as D. yakuba, D. teissieri, and D. santomea. It has to be noted that D. yakuba, D. teissieri, and D. santomea carry Wolbachia strains that have the same partial wsp gene sequences as the wCof strain of D. simulans (LACHAISE et al. 2000 ; A. NIRGIANAKI and K. BOURTZIS, unpublished data). It should also be noted at this point that D. yakuba, D. teissieri, and D. santomea allow the wRi strain to heavily infect their sperm cysts, suggesting that this strain is capable of replicating irrespective of the host genetic background or the fact that the ability of these species to suppress their native bacteria is strain specific (S. ZABALOU and K. BOURTZIS, unpublished data). Until these wCof-like bacteria are transferred to permissive hosts, it remains to be seen whether they are truly mod^- strains.

How does the host control the Wolbachia infection of the sperm cysts and consequently the apparent levels of CI? Does the host remove Wolbachia from sperm cysts at some point during spermatogenesis? Does the host prevent sperm cysts from being infected? If so, how early does this happen in development? Do different hosts regulate the replication rate of different Wolbachia strains during spermatogenesis in a different way? The results of the present study clearly suggest that host factors determine whether and at what level the sperm cysts will become infected and consequently may also determine CI levels, if the cysts are infected with a mod⁺ strain. Indeed, it has been suggested (TURELLI 1994 ) that there is selection on only the host to reduce the CI level, which would imply that host factors are crucial for control of the percentage of Wolbachia-infected sperm cysts. It has to be noted that upon transfer of wMa from its natural host D. mauritiana to the new host D. simulans, no CI could be detected. However, this transfer at the same time is accompanied by a significant reduction in the number of infected sperm cysts, most likely impairing induction of CI. On the other hand, there have been reports that naturally infected D. simulans (wMa) associations exhibit from none to moderate CI levels (JAMES and BALLARD 2000 ). Thus, both wCof and wMa may turn out to be mod⁺ strains. In that case three requirements for the expression of CI in a host-Wolbachia association have been identified: (a) Wolbachia has to be able to modify sperm (mod⁺ genotype), (b) Wolbachia has to infect sperm cysts and, (c) Wolbachia has to be harbored by a permissive host.

The present study is based on a wsp-gene-based classification system for Wolbachia strains (ZHOU et al. 1998 ). However, several lines of evidence suggest that this classification system may be of limited biological relevance. Our results do not allow us to correlate Wolbachia distribution and percentage of Wolbachia-infected sperm cysts with the wsp gene sequence. BOURTZIS et al. 1998 had suggested that this system could be used to infer whether or not two Wolbachia strains are compatible, but POINSOT et al. 1998 clearly showed that such predictions could not be made. SCHULENBURG et al. 2000 indicated that the presence of a significant substitution rate heterogeneity between lineages in the wsp gene prevents a reliable estimation of divergence dates and also limits the applicability of a simple sequence-based classification system such as that proposed for the wsp gene. In addition, recent reports about the recombination between Wolbachia strains and particularly within the wsp gene further complicate phylogenetic interpretations (JIGGINS et al. 2001 ; WERREN and BARTOS 2001 ). Several projects are currently in progress to determine the full genome sequence of a variety of Wolbachia strains (SLATKO et al. 1999 ; OEHLER and BOURTZIS 2000 ). We hope this will lead to the development of additional molecular markers for both phylogenetic studies and an accurate prediction of the Wolbachia-induced reproductive phenotypes.

	ACKNOWLEDGMENTS

We thank George Markakis for help with statistics, Androniki Nirgianaki, Stefanos Siozos, and Harris Pavlikaki for their help at early stages of this study and Sylvain Charlat, Steve Dobson, Greg Hurst, Stefan Oehler, and Fabrice Vavre for critical reading of the manuscript. We also thank two anonymous reviewers for their comments that helped to significantly improve the manuscript. This research was supported in part by a grant from the European Union (QLK3-CT2000-01079) to K.B.

Manuscript received September 16, 2002; Accepted for publication February 25, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BORDENSTEIN, S. R. and J. H. WERREN, 1998  Effects of A and B Wolbachia and host genotype on interspecies cytoplasmic incompatibility in Nasonia. Genetics 148:1833-1844.[Abstract/Free Full Text]

BOURTZIS, K., A. NIRGIANAKI, P. ONYANGO, and C. SAVAKIS, 1994  A prokaryotic dnaA sequence in Drosophila melanogaster: Wolbachia infection and cytoplasmic incompatibility among laboratory strains. Insect Mol. Biol. 3:131-142.[Medline]

BOURTZIS, K., A. NIRGIANAKI, G. MARKAKIS, and C. SAVAKIS, 1996  Wolbachia infection and cytoplasmic incompatibility in Drosophila species. Genetics 144:1063-1073.[Abstract]

BOURTZIS, K., S. L. DOBSON, H. R. BRAIG, and S. L. O'NEILL, 1998  Rescuing Wolbachia have been overlooked. Nature 391:852-853.[Medline]

BOYLE, L., S. L. O'NEILL, H. M. ROBERTSON, and T. L. KARR, 1993  Interspecific and intraspecific horizontal transfer of Wolbachia in Drosophila. Science 260:1796-1799.[Abstract/Free Full Text]

BRAIG, H. R., W. ZHOU, S. L. DOBSON, and S. L. O'NEILL, 1998  Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis.. J. Bacteriol. 180:2373-2378.[Abstract/Free Full Text]

BREEUWER, J. A., 1997  Wolbachia and cytoplasmic incompatibility in the spider mites Tetranychus urticae and T. turkestani.. Heredity 79:41-47.

BREEUWER, J. A. and J. H. WERREN, 1993  Cytoplasmic incompatibility and bacterial density in Nasonia vitripennis.. Genetics 135:565-574.[Abstract]

CALLAINI, G., R. DALLAI, and M. G. RIPARBELLI, 1997  Wolbachia-induced delay of paternal chromatin condensation does not prevent maternal chromosomes from entering anaphase in incompatible crosses of Drosophila simulans.. J. Cell Sci. 110:271-280.[Abstract]

CHARLAT, S., C. CALMET, and H. MERÇOT, 2001  On the mod resc model and the evolution of Wolbachia compatibility types. Genetics 159:1415-1422.[Abstract/Free Full Text]

CHARLAT, S., K. BOURTZIS and H. MERÇOT, 2002 Wolbachia-induced cytoplasmic incompatibility, pp. 623–644 in Symbiosis, Mechanisms and Model Systems (Cellular Origin and Life in Extreme Habitats, Vol. 4), edited by J. SECKBACH. Kluwer, Dordrecht, The Netherlands.

CLARK, M. E. and T. L. KARR, 2002  Distribution of Wolbachia within Drosophila reproductive tissue: implications for the expression of cytoplasmic incompatibility. Integr. Comp. Biol. 42:332-339.[Abstract/Free Full Text]

CLARK, M. E., Z. VENETI, K. BOURTZIS, and T. L. KARR, 2002  The distribution and proliferation of the intracellular bacteria Wolbachia during spermatogenesis in Drosophila. Mech. Dev. 111:3-15.[Medline]

CLARK, M. E., Z. VENETI, K. BOURTZIS, and T. L. KARR, 2003  Wolbachia distribution and cytoplasmic incompatibility in Drosophila: the cyst as the basic cellular unit of CI expression. Mech. Dev. 120:85-98.

DEDEINE, F., F. VAVRE, F. FLEURY, B. LOPPIN, and M. E. HOCHBERG et al., 2001  Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp. Proc. Natl. Acad. Sci. USA 98:6247-6252.[Abstract/Free Full Text]

DOBSON, S. L., K. BOURTZIS, H. R. BRAIG, B. F. JONES, and W. ZHOU et al., 1999  Wolbachia infections are distributed throughout insect somatic and germ line tissues. Insect Biochem. Mol. Biol. 29:153-160.[Medline]

DOBSON, S. L., E. J. MARSLAND, and W. RATTANADECHAKUL, 2002a  Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive. Genetics 160:1087-1094.[Abstract/Free Full Text]

DOBSON, S. L., E. J. MARSLAND, Z. VENETI, K. BOURTZIS, and S. L. O'NEILL, 2002b  Characterization of Wolbachia host cell range via the in vitro establishment of infections. Appl. Environ. Microbiol. 68:656-660.[Abstract/Free Full Text]

GIORDANO, R., S. L. O'NEILL, and H. M. ROBERTSON, 1995  Wolbachia infections and the expression of cytoplasmic incompatibility in Drosophila sechellia and D. mauritiana.. Genetics 140:1307-1317.[Abstract]

HOFFMANN, A. A., M. TURELLI, and G. M. SIMMONS, 1986  Unidirectional incompatibility between populations of Drosophila simulans.. Evolution 40:692-701.

HOFFMANN, A. A., D. CLANCY, and J. DUNCAN, 1996  Naturally-occurring Wolbachia infection in Drosophila simulans that does not cause cytoplasmic incompatibility. Heredity 76:1-8.

HOLDEN, P. R., P. JONES, and J. F. BROOKFIELD, 1993  Evidence for a Wolbachia symbiont in Drosophila melanogaster.. Genet. Res. 62:23-29.[Medline]

JAMES, A. C. and J. W. BALLARD, 2000  Expression of cytoplasmic incompatibility in Drosophila simulans and its impact on infection frequencies and distribution of Wolbachia pipientis.. Evolution 54:1661-1672.[Medline]

JIGGINS, F. M., J. H. VON DER SCHULENBURG, G. D. HURST, and M. E. MAJERUS, 2001  Recombination confounds interpretations of Wolbachia evolution. Proc. R. Soc. Lond. B Biol. Sci. 268:1423-1427.[Medline]

LACHAISE, D., M. HARRY, M. SOLIGNAC, F. LEMEUNIER, and V. BENASSI et al., 2000  Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from Sao Tome. Proc. R. Soc. Lond. B Biol. Sci. 267:1487-1495.[Medline]

LASSY, C. W. and T. L. KARR, 1996  Cytological analysis of fertilization and early embryonic development in incompatible crosses of Drosophila simulans.. Mech. Dev. 57:47-58.[Medline]

MCGRAW, E. A., D. J. MERRITT, J. N. DROLLER, and S. L. O'NEILL, 2001  Wolbachia-mediated sperm modification is dependent on the host genotype in Drosophila. Proc. R. Soc. Lond. B Biol. Sci. 268:2565-2570.[Medline]

MERÇOT, H. and D. POINSOT, 1998  and discovered on Mount Kilimanjaro. Nature 391:853.[Medline]

MERÇOT, H., B. LLORENTE, M. JACQUES, A. ATLAN, and C. MONTCHAMP-MOREAU, 1995  Variability within the Seychelles cytoplasmic incompatibility system in Drosophila simulans.. Genetics 141:1015-1023.[Abstract]

MIN, K. T. and S. BENZER, 1997  Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death. Proc. Natl. Acad. Sci. USA 94:10792-10796.[Abstract/Free Full Text]

MONTCHAMP-MOREAU, C., J. F. FERVEUR, and M. JACQUES, 1991  Geographic distribution and inheritance of three cytoplasmic incompatibility types in Drosophila simulans.. Genetics 129:399-407.[Abstract]

MORET, Y., P. JUCHAULT, and T. RIGAUD, 2001  Wolbachia endosymbiont responsible for cytoplasmic incompatibility in a terrestrial crustacean: effects in natural and foreign hosts. Heredity 86:325-332.[Medline]

OEHLER, S., and K. BOURTZIS, 2000 The first International Wolbachia Conference: a minireview. Symbiosis 29: 151–161.

O'NEILL, S. L., R. GIORDANO, A. M. COLBERT, T. L. KARR, and H. M. ROBERTSON, 1992  16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc. Natl. Acad. Sci. USA 89:2699-2702.[Abstract/Free Full Text]

O'NEILL, S. L., A. A. HOFFMANN and J. H. WERREN, 1997 Influential Passengers: Inherited Microorganisms and Arthropod Reproduction. Oxford University Press, Oxford.

POINSOT, D., K. BOURTZIS, G. MARKAKIS, C. SAVAKIS, and H. MERÇOT, 1998  Wolbachia transfer from Drosophila melanogaster into D. simulans: host effect and cytoplasmic incompatibility relationships. Genetics 150:227-237.[Abstract/Free Full Text]

REYNOLDS, K. T. and A. A. HOFFMANN, 2002  Male age, host effects and the weak expression or non-expression of cytoplasmic incompatibility in Drosophila strains infected by maternally-transmitted Wolbachia. Genet. Res. 80:79-87.[Medline]

ROUSSET, F. and E. DE STORDEUR, 1994  Properties of Drosophila simulans strains experimentally infected by different clones of the bacterium Wolbachia. Heredity 72:325-331.

SCHULENBURG, J. H., G. D. HURST, T. M. HUIGENS, M. M. VAN MEER, and F. M. JIGGINS et al., 2000  Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing. Mol. Biol. Evol. 17:584-600.[Abstract/Free Full Text]

SIRONI, M., C. BANDI, L. SACCHI, B. DI SACCO, and G. DAMIANI et al., 1995  Molecular evidence for a close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Mol. Biochem. Parasitol. 74:223-227.[Medline]

SLATKO, B. E., S. L. O'NEILL, A. L. SCOTT, J. L. WERREN, and M. L. BLAXTER, 1999  The Wolbachia Genome Consortium. Microb. Comp. Genomics 4:161-165.[Medline]

SOLIGNAC, M., D. VAUTRIN, and F. ROUSSET, 1994  Widespread occurrence of the proteobacteria Wolbachia and partial cytoplasmic incompatibility in Drosophila melanogaster.. C. R. Acad. Sci. Paris 317:461-470.

STEVENS, L., L. GIORDANO, and R. FIALHO, 2001  Male-killing, nematode infections, bacteriophage infection, and virulence of cytoplasmic bacteria in the genus Wolbachia. Annu. Rev. Ecol. Syst. 32:519-545.

STOUTHAMER, R., J. A. BREEUWER, and G. D. HURST, 1999  Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53:71-102.[Medline]

TRAM, U. and W. SULLIVAN, 2002  Role of delayed nuclear envelope breakdown and mitosis in Wolbachia-induced cytoplasmic incompatibility. Science 296:1124-1126.[Abstract/Free Full Text]

TURELLI, M., 1994  Evolution of incompatibility-inducing microbes and their hosts. Evolution 48:1500-1513.

WEEKS, A. R., K. T. REYNOLDS, and A. A. HOFFMANN, 2002  Wolbachia dynamics and host effects: What has (and has not) been demonstrated? Trends Ecol. Evol. 17:257-262.

WERREN, J. H., 1997  Biology of Wolbachia. Annu. Rev. Entomol. 42:587-609.[Medline]

WERREN, J. H. and J. D. BARTOS, 2001  Recombination in Wolbachia. Curr. Biol. 11:431-435.[Medline]

YEN, J. H. and A. R. BARR, 1971  New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens.. Nature 232:657-658.[Medline]

ZHOU, W., F. ROUSSET, and S. O'NEILL, 1998  Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc. R. Soc. Lond. B Biol. Sci. 265:509-515.[Medline]

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