Wolbachia is an intracellular microbe harbored by a wide variety of arthropods (including Drosophila) and filarial nematodes. Employing several different strategies including male killing, induced parthenogenesis, cytoplasmic incompatibility, and feminization, and acting by as-yet-unknown mechanisms, Wolbachia alters host reproduction to increase its representation within a population. Wolbachia is closely associated with gametic incompatibility but also interacts with Drosophila in other, little understood ways. We report here significant and widespread infection of Wolbachia within laboratory stocks and its real and potential impact on Drosophila research. We describe the results of a survey indicating that 30% of stocks currently housed at the Bloomington Drosophila Stock Center are infected with Wolbachia. Cells of both reproductive tissues and numerous somatic organs harbor Wolbachia and display considerable variation in infection levels within and between both tissue types. These results are discussed from the perspective of Wolbachia's potential confounding effects on both host fitness and phenotypic analyses. In addition to this cautionary message, the infection status of stock centers may provide further opportunities to study the genetic basis of host/symbiosis.

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WOLBACHIA is an intracellular microbe harbored by a wide variety of arthropod and filarial nematode hosts. During studies of reproductive isolation in Culex pipiens, a rickettsia-like microorganism, Wolbachia pipientis was determined to be the agent responsible for a form of inherited reproductive failure (LAVEN 1959; YEN and BARR 1973). This phenotype, termed cytoplasmic incompatibility (CI), is manifest when a Wolbachia-infected male mates with an uninfected female (Figure 1) or with a female infected with a different Wolbachia type. In addition to Diptera, CI has since been found to be a Wolbachia-induced trait in a wide diversity of arthropod orders, including Acarina (VELA et al. 2000), Coleoptera (WADE and STEVENS 1985), Homoptera (HOSHIZAKI and SHIMADA 1995), Hymenoptera (BREEUWER and WERREN 1990), Isoptera (BANDI et al. 1997), Lepidoptera (BROWER 1976), and Orthoptera (KAMODA et al. 2000). Since all Wolbachia are removed from spermatids prior to the completion of spermatogenesis (BRESSAC and ROUSSET 1993; SNOOK et al. 2000; CLARK et al. 2002), Wolbachia presumably modify sperm prior to the completion of spermatogenesis. The transfer and processing of at least two accessory gland proteins (Acp26Aa and Acp36De) is unaffected by Wolbachia (SNOOK et al. 2000), again suggesting that the primary effect is on sperm. The CI phenotype seen in incompatible embryos in Drosophila melanogaster (Figure 1) as well as in D. simulans (O'NEILL and KARR 1990; CALLAINI et al. 1996; LASSY and KARR 1996), C. pipiens (JOST 1970), Armadillidium vulgare (MORET et al. 2001), and Nasonia vitripennis (REED and WERREN 1995) is characterized by asynchronous mitotic divisions and chromatin bridges between nuclei, defects that accumulate with subsequent mitotic divisions, resulting in embryo lethality. The earliest CI defect, observed in both Nasonia and Drosophila, is delayed paternal pronuclear breakdown and entry into mitosis (REED and WERREN 1995; CALLAINI et al. 1997; TRAM and SULLIVAN 2002).

View larger version (62K): In this window In a new window Download PPT slide   FIGURE 1.— Cytoplasmic incompatibility. There are four different mating combinations between infected and uninfected males and females. Infected females (blue) produce infected offspring that develop normally regardless of paternal infection status. Uninfected males mate successfully with both infected and uninfected females. Infected males (yellow) with Wolbachia-modified sperm mated to uninfected females produce some embryos with early embryonic lethality, characterized by defects in early mitotic divisions (CI, lower left). In late telophase, nuclei (arrowheads) are abnormal and do not properly separate during anaphase/telophase. These defects are rescued when mated to infected females (rescue, lower right). Wolbachia are seen as small punctate dots with high concentrations associated with astral microtubules. Red, DNA; green, tubulin. Bar, 10 µm.

 
In addition to CI, Wolbachia has been found to manipulate host reproduction in other ways, including feminization, male killing, and parthenogenesis. Each of these Wolbachia-induced phenotypes serves the same purpose: to increase the prevalence of Wolbachia-infected individuals (STOUTHAMER et al. 1999). Although CI is by far the most prevalent effect of Wolbachia infection in Drosophila, male-killing Wolbachia have recently been described in Drosophila although their prevalence within this group has yet to be established (HURST et al. 2000; JAENIKE et al. 2003).

Wolbachia has also been found in a number of filarial nematodes, many of which are pathogenic to humans. Wolbachia's relationship with these filarial nematodes is obligate to a greater extent than in most arthropods and elimination of the microbe results in inhibited development or death of the host (RAO et al. 2002; CHIRGWIN et al. 2003; VOLKMANN et al. 2003). Wolbachia found in Onchocerca volvulus have been shown to play an inordinate role in the pathogenesis of river blindness (SAINT ANDRE et al. 2002) as well as in the pathogenicity of other filarial nematodes (TAYLOR et al. 2000; TAYLOR and HOERAUF 2001). Currently, Wolbachia is being considered as a target in the treatment of filariasis (CHIRGWIN et al. 2003; HOERAUF et al. 2003; VOLKMANN et al. 2003).

Wolbachia were probably first described in D. melanogaster by WOLSTENHOLME (1965), who described bacteriod particles in salivary glands and ovaries of two infected lines with reduced numbers of offspring of infected vs. uninfected males, consistent with CI. It was >20 years before Wolbachia was again reported in D. melanogaster (GLOVER et al. 1990; O'NEILL and KARR 1990) and was subsequently identified as W. pipientis by molecular methods (O'NEILL et al. 1992). Over this time period and continuing to the present, Wolbachia infection within D. melanogaster stocks commonly used in research has largely been ignored. The consequence of this infection, however, is not neutral. In addition to causing CI, Wolbachia in Drosophila have been shown to reduce sperm production (SNOOK et al. 2000), reduce egg production (HOFFMANN et al. 1990), and have an influence on longevity (FRY and RAND 2002). Wolbachia was recently identified as the causative agent in the seemingly non-Mendelian behavior of an allele of Sex-lethal (Sxl^f4). Oogenesis is normally disrupted in Sxl^f4/Sxl^f4 flies, but is restored when flies are infected with Wolbachia (STARR and CLINE 2002). The spread and persistence of CI-inducing Wolbachia in a population has been examined theoretically (CASPARI and WATSON 1959) and has been documented empirically in California (HOFFMANN et al. 1990; TURELLI and HOFFMANN 1991, 1995). However, fear of such spread in and among D. melanogaster is mitigated by the absence of significant levels of CI in this species, and a rapid male age-dependent decrease of CI (BOURTZIS et al. 1994, 1996, 1998; HOFFMANN et al. 1994, 1998; CLARK and KARR 2002; REYNOLDS and HOFFMANN 2002; WEEKS et al. 2002; CLARK et al. 2003). It is therefore more difficult to explain the widespread occurrence of Wolbachia in D. melanogaster in laboratory stocks and worldwide. Theoretical explanations for the spread and persistence of Wolbachia infections without CI invoke positive contributions of Wolbachia to other aspects of host fitness (TURELLI 1994; HOFFMANN et al. 1998), and other, unidentified host-symbiont interactions must be maintaining these infections. It is likely, then, that Wolbachia not only has played an as-yet-underappreciated role in the evolution of Drosophila (as well as of countless other arthropods and filarial nematodes), but also no doubt is an unrecognized yet important variable in Drosophila research.

The purpose of this report is to alert researchers to the frequency of Wolbachia infection in Drosophila stocks by determining the infection status of The Bloomington Drosophila Stock Center (BDSC) at Indiana University. The BDSC is the major repository for D. melanogaster stocks currently with 12,500 different stocks (http://flystocks.bio.indiana.edu), and as yet there has been no systematic information available regarding the Wolbachia infection status of this crucial resource. We also demonstrate Wolbachia proliferation in a variety of somatic organs and discuss one example of the confounding effects that Wolbachia can have on Drosophila research by highlighting the effect of Wolbachia on the recovery of homozygotes in two Drosophila stocks.

Wolbachia screen:

A pilot screen of 200 randomly chosen stocks were initially screened for Wolbachia. A total of 409 additional stocks were chosen to include adequate sampling of different subsets of stocks. These include most of the wild-type stocks, deficiency kits, as well as stocks from several different P-element mutagenesis screens. The infection status of stocks was determined using a PCR-based assay with Wolbachia-specific primers for the 16S rDNA gene (O'NEILL et al. 1992). Two females from each stock, 2–3 days old, were homogenized in 100 µl STE (400 mM NaCl/10 mM Tris Cl, pH 8.0/1 mM EDTA, pH 8.0) with 2 µl proteinase K (13.3 mg/ml) and incubated at 37° for 60 min, followed by 5 min at 95°, and then centrifuged briefly. Twenty-microliter PCR reactions were set up under standard conditions using the Wolbachia-specific 16s rRNA gene primers (994F and 99R) with 2.5 mM mgCl₂ and 1 µl of homogenate. Following 2 min denaturation at 95°, 40 cycles of amplification were performed with the following program: denaturation at 92° for 30 sec, annealing at 50° for 30 sec and extension for 30 sec at 72°, followed by further extension at 72° for 5 min. PCR products were run on 1% agarose gel.

Fly stocks:

A Wolbachia-infected wild-type line of D. melanogaster originally established from four females collected in Bermuda in 1954 was obtained from the Bloomington Drosophila Stock Center, Indiana University (no. 3839; designated DMB).

Confocal microscopy:

Wolbachia was visualized within various larval and pupal somatic organs from a wild-type D. melanogaster line (DMB) previously shown to exhibit CI (CLARK et al. 2002). An anti-wsp (Wolbachia specific protein) antibody (BRAIG et al. 1998; DOBSON et al. 1999), kindly provided by Scott O'Neill and Kostas Bourtzis, was used to label Wolbachia. Dissection, fixation, staining, and antibody labeling were done as previously described for Wolbachia visualization in spermatogenesis (CLARK et al. 2002, 2003). All internal organs within several third instar larvae were examined; selected organs of various different tissue types are shown. Pupal eyes and ovaries were similarly examined. Embryos from an incompatible cross (DMB Wolbachia-infected male x DMB Wolbachia-free female) and a compatible cross (DMB Wolbachia-infected male x DMB Wolbachia-infected female) were collected and fixed with heptane/methanol (KOSE and KARR 1995) and rehydrated in TBST (50 mM TRIS, 150 mM NaCl, 0.1% Tween, 0.05% NaN₃ pH 7.5) and then RNase A treated and stained with propidium iodide (5 µg/ml) for 1 hr. Images were obtained using a Zeiss LSM 510 confocal microscope with a Kr/Argon laser (488 nm) for detection of the Alexa Flour 488-labeled Wolbachia, and a He/Ne laser (543 nm) for detection of propidium iodide-nucleic acid staining. Images were composed of overlapping optical sections along the z-axis, projected onto a single image and superimposed over a single Nomarski image of the same area where indicated.

Cellular mechanism of CI in D. melanogaster:

Early embryonic defects associated with the expression of CI have been extensively described in D. simulans and N. vitripennis (CALLAINI et al. 1996; LASSY and KARR 1996; TRAM and SULLIVAN 2002). The penetrance of CI in both these species is high, usually approaching 100%. However, levels of CI in D. melanogaster have been typically much lower (<50%), although statistically significant (BOURTZIS et al. 1994, 1996; HOFFMANN et al. 1998; POINSOT et al. 1998; CLARK et al. 2002; REYNOLDS and HOFFMANN 2002). Thus, we first established that the modest reduction in egg viability in D. melanogaster was caused by a CI-like mechanism. We had previously measured CI in D. melanogaster (CLARK et al. 2002) and used this line (DMB) to examine cytological defects in crosses between infected males and either uninfected or infected females (Figure 1). The compatible cross showed normal development while the incompatible cross clearly displayed cytological defects in some embryos, identical to those observed in D. simulans, suggesting a similar mechanism of action.

Wolbachia infection of the BDSC:

Wolbachia infection of D. melanogaster stocks by a single Wolbachia strain has been reported a number of times (GLOVER et al. 1990; BOURTZIS et al. 1994, 1996; MIN and BENZER 1997; CLARK et al. 2002). To further describe the level and prevalence of infection in D. melanogaster, 609 stocks were surveyed for Wolbachia infection using a PCR assay and Wolbachia-specific primers. Our survey was designed to cover all major subdivisions of genetic lesions, all deficiency kits, and the full complement of wild-type stocks housed at the BDSC. Overall, 28.9% of the stocks surveyed tested positive for Wolbachia. In wild-type lines, 23.3% were infected, including lines from Bermuda; Bogota, Columbia; Koriba Dam, South Africa; Israel; Madeira, Portugal; Hikone, Japan; Oregon; Riverside, California; Maderia, Wisconsin; Manning, South Carolina; Monroe County, New York; Ceres, New York; Harwich, Massachusetts, and Amherst, Massachusetts (see supplementary material at http://www.genetics.org/supplemental/).

The BDSC infection data in Figure 2 illustrate the following important points. First, the overall frequency of infection of wild-type lines housed at the BDSC was similar to the overall infection rate of the entire BDSC (24% vs. 29%, respectively). Second, variation in infection rates was observed between classes of genetic lesions ranging from 10 to 45% (Figure 2A). Third, variation of infection status was observed both between and within chromosomes (Figure 2A). For example, 60% of X-linked lethal lines were found infected as compared to 16% on the second and third chromosomes, while X-linked lethals (Figure 2A), X-linked balancers and X-deficiency stocks showed variation within these classes (60% vs. 40% vs. 12%, respectively). Further work will be needed to establish the basis for these differences.

View larger version (25K): In this window In a new window Download PPT slide   FIGURE 2.— Wolbachia infection in D. melanogaster stocks. (A) Percentage of infected stocks categorized by general class of genotype. (B) Wolbachia infection in various P-element mutagenesis screens.

 
Similar levels of variation in infection were also found between P-collection lines (lines with a mapped P-insertion site) that have been deposited in the BDSC over the past 15 years. Overall this group showed a higher rate of infection compared to the overall rate (44.35% vs. 28.9%) (Figure 2B). Curiously, the original P-element screen from the Spradling laboratory was negative for infection (COOLEY et al. 1988). This may reflect the fact that this original screen used a P-element plasmid construct containing a neomycin resistance gene. Positive transformants were identified following growth on food vials containing the antibiotic neomycin, which may have removed Wolbachia infection if it had been present.

Wolbachia distribution in somatic tissues:

Wolbachia are consistently found in significant numbers within gonadal tissues of both sexes (DOBSON et al. 1999; CLARK and KARR 2002). Variation within and between Drosophila strains has also been observed (CLARK and KARR 2002) and this variation has been causally linked to the level of CI expression (BOYLE et al. 1993; CLARK and KARR 2002; CLARK et al. 2003; VENETI et al. 2003). Therefore, nearly all studies of Wolbachia in Drosophila have focused on the effects in the germline. For example, DOBSON et al. (1999) detected Wolbachia in the soma of various species via dot-blot assay, but suggest it is largely restricted to reproductive tissues in D. melanogaster. While searching for mutations that cause brain degeneration, MIN and BENZER (1997) discovered a variant Wolbachia strain in a D. melanogaster stock that causes lethality, presumably due to massive replication in adult brain tissues. To further examine this aspect of host/symbiont biology, we chose to examine third instar larval tissues in which Wolbachia were found in all tissues, both somatic and germline (Figure 3). Interestingly, we consistently observed a cell-by-cell mosaic pattern of infection with uninfected cells adjacent to infected cells. This is especially obvious in cells composing the fat bodies (Figure 3F).

View larger version (68K): In this window In a new window Download PPT slide   FIGURE 3.— Wolbachia in different organs from a wild-type (BDSC no. 3839) D. melanogaster third instar larva (A–G) and pupa (H and I). (A) Nerves, (B) Malpighian tubules, (C) salivary gland, (D) trachea, (E) haltare imaginal disc, (F) fat bodies, (G) proventriculus, (H) eye, (I) ovary. Red, DNA; green/yellow, Wolbachia. Bars, 10 µm.

 

Wolbachia distribution during spermatogenesis:

The growth and proliferation of Wolbachia during spermatogenesis has been previously described in wild-type D. melanogaster lines (CLARK and KARR 2002; CLARK et al. 2002). However, our survey found much greater variation of Wolbachia within developing spermatids in some mutant Drosophila stocks such as those shown in Figure 4. Three phenotypes were observed, including a typical wild-type distribution (Figure 4B) and two aberrant phenotypes (Figure 4, A and C) that apparently contain Wolbachia aggregations in higher numbers than those observed in wild-type lines.

View larger version (115K): In this window In a new window Download PPT slide   FIGURE 4.— Wolbachia in the nuclear end of sperm cysts. Elongated cysts removed from testes from three different Wolbachia-infected lines and stained with propidium iodide (staining both Drosophila and Wolbachia DNA). Variation is seen in Wolbachia (w) distribution within developing sperm cysts adjacent to spermatid nuclei (n). (A) BDSC no. 10030, w1118 P{EP}sd EP1088. (B) BDSC no. 97, Df(2L)JS32, dppd-ho/CyO, P{sevRas1.V12}FK1. (C) BDSC no. 1128, Df(2L)GpdhA/CyO.

 

Wolbachia infection in the BDSC:

Wolbachia infection in D. melanogaster differs in important ways from Wolbachia infection in its sister species, D. simulans. Two crucial aspects to these differences explain why Wolbachia infection in laboratory stocks and at the BDSC has remained undetected until recently. First, unless males are mated very young, Wolbachia usually do not cause high levels of CI in D. melanogaster (BOURTZIS et al. 1994, 1996; HOFFMANN et al. 1998; POINSOT et al. 1998; CLARK et al. 2002; REYNOLDS and HOFFMANN 2002), thereby mitigating any overt fitness effects on viability. Second, the density of Wolbachia in D. melanogaster can be quite low relative to D. simulans (BOYLE et al. 1993; CLARK and KARR 2002). However, in those cases where Wolbachia levels are sufficient, CI can be measured (BOURTZIS et al. 1996; POINSOT et al. 1998; CLARK et al. 2002; REYNOLDS and HOFFMANN 2002). We have shown (Figure 1) that the expression of CI in D. melanogaster is indistinguishable from cytological defects observed in N. vitripennis and D. simulans (REED 1995; CALLAINI et al. 1996; LASSY and KARR 1996; TRAM and SULLIVAN 2002). However, clear differences are seen during spermatogenesis, including differences in Wolbachia distribution within developing cysts as well as the total number of cysts infected (CLARK and KARR 2002; CLARK et al. 2002, 2003; VENETI et al. 2003). This supports the Wolbachia-infected spermatocyst/spermatocyte hypothesis recently put forward (CLARK et al. 2003). This hypothesis posits that the basic cellular unit for CI expression is the developing spermatocyst and further suggests that an "all-or-none" threshold of infection in individual cysts is essential for CI expression. Despite these important differences between the two species, mechanisms exist in D. melanogaster for CI expression and therefore have the potential to affect genetic studies.

In addition to causing CI in Drosophila, it is becoming clear that Wolbachia are insinuated into Drosophila biology in other important ways. The recent study showing an interaction between Sex-lethal (STARR and CLINE 2002) and Wolbachia and chico (see below) emphasizes the potential importance of Wolbachia on Drosophila research. However, awareness of Wolbachia is lacking among most Drosophila biologists. With a high proportion of Drosophila stocks infected with Wolbachia, it is likely that a significant number of laboratories working with Drosophila are also working with Wolbachia.

Variability of infection in the BDSC:

Certain classes of mutant stocks are infected at a higher rate than others, and significant variation within each class is apparent (Figure 2). What accounts for this difference? This question is difficult to answer because the precise history of stocks in the BDSC is limited in many cases and also because the original lines used for mutagenesis are not readily available. Presumably, a significant proportion of the variation observed is simply the result of a Wolbachia-infected line being used to establish or maintain the stock.

Wolbachia infection in P-element stocks:

P-element stocks offer a unique opportunity to study Wolbachia because (1) very large numbers of stocks have been generated by a small number of laboratories, (2) P-element mutagenesis does not utilize harsh chemical or ionization radiation used in other screens that might otherwise have unspecified effects on Wolbachia, and (3) the precise histories and parental stocks used in P-element screens are generally available (BERG and SPRADLING 1991).

Among the stocks generated in P-element mutagenesis screens, a majority of stocks were infected in two of the screens (Figure 2B) (RORTH et al. 1998; U. SCHAEFER, H. JACKLE, Y. HE, H. BELLEN and T. LAVERTY, personal communication). This high rate of infection suggests that these two screens were conducted in a Wolbachia-infected background. The uninfected stocks created in these screens may have lost the Wolbachia due to either host genetic, environmental, or other factors or the outcrossing to female flies (for their desired balancers) lacking Wolbachia. Likewise, in the screens with a low number of Wolbachia-infected stocks, infected stocks are likely the result of outcrossing to female flies infected with Wolbachia. In one screen, no infected stocks were observed (COOLEY et al. 1988). This is due to either the absence of Wolbachia from the initial flies or the use of an antibiotic in the P-element transposition selection.

The high rate of Wolbachia infection among stocks created in P-element mutagenesis screens may have a serendipitous explanation, or it may reflect previously unknown interactions between Wolbachia and P elements. Two of the wild-type stocks surveyed (Bloomington stocks no. 1, a Canton-S line, and no. 4265, the "Harwich" line) were used extensively in the early work on P-element-induced hybrid dysgenesis (KIDWELL and KIDWELL 1975; KIDWELL et al. 1977, 1983). Interestingly, Canton-S is negative for P elements as well as for Wolbachia, while Harwich contains both P elements and Wolbachia. One cross between these two lines (Canton-S female x Harwich male), potentially a cross exhibiting Wolbachia-induced CI, also results in offspring exhibiting complete gonadal dysgenesis. The reciprocal cross (Harwich female x Canton-S male) exhibits neither CI nor hybrid dysgenesis. Males resulting from the reciprocal cross, however, will be infected with Wolbachia and may exhibit CI when crossed to Wolbachia-free females. It is currently unknown what effect, if any, Wolbachia has on P-element activity or what effect P elements (or other mobile genetic elements) have on Wolbachia. There is, however, potential for both genomic conflict and cooperation between these two selfish genetic elements. For example, if cumulative effects of hybrid dysgenesis and/or CI were complete (resulting in no viable, fertile offspring from an M, Wolbachia– female x a P, Wolbachia+ male (as seen in the Canton-S female x Harwich male cross), then although the frequency of Wolbachia-infected and P-element-infected individuals would increase in such a population, new P-element transpositions would not spread. What effect, if any, mobile genetic elements have had on the spread of Wolbachia (or Wolbachia on the spread of mobile genetic elements) is unknown. Many of the wild-type lines (in addition to Canton-S and Harwich) from the BDSC tested in this survey have previously been characterized for their P-element status (KIDWELL et al. 1983). A total of 3/14 (21.4%) Wolbachia-infected lines were able to induce hybrid dysgenesis when mated to Canton-S females, compared to 8/37 (21.6%) of Wolbachia-free lines. Therefore, it seems that there is no evidence for long-term disequilibrium between P-element and Wolbachia infections.

Two previous studies in Drosophila suggested the presence of Wolbachia in nonreproductive tissues. Wolbachia was detected via dot-blot analysis in testes and ovaries, as well as in the male and female carcasses (DOBSON et al. 1999), and evidence for Wolbachia in neural tissues of the adult was found (MIN and BENZER 1997). This report extends these findings and shows extensive proliferation of Wolbachia throughout the soma of D. melanogaster (Figure 3). Thus, in addition to reproductive tissues, every organ system is potentially affected by the presence of Wolbachia. These findings are consistent with those found in other insect groups, including the adzuki bean beetle (IJICHI et al. 2002) and the tsetse fly (CHENG et al. 2000). Interestingly, in two testse fly species Wolbachia was limited to reproductive tissues whereas a third species exhibited Wolbachia infection of various somatic tissues (CHENG et al. 2000). These studies serve to underpin the complexities of interactions between host and microbe and serve as a reminder that such complexities may affect phenotypic and fitness studies in as-yet-unappreciated ways.

Wolbachia growth and proliferation during spermatogenesis has been described in wild-type D. melanogaster lines (CLARK and KARR 2002; CLARK et al. 2002). Wolbachia within testes of mutant stocks from the BDSC show much higher variation in bacterial distribution than those observed in wild-type lines. This includes variation in overall Wolbachia load, as well as the distribution of Wolbachia within developing spermatids. Most notable are lines with very large pockets of Wolbachia within cysts and developing spermatids (Figure 4). It is unclear if this variation is due to host factors, Wolbachia factors, or a combination of both. Also unclear is what effect these different Wolbachia distributions have on fertility or on other aspects of reproductive fitness.

Wolbachia infection in Drosophila has been studied over the past decade and, with few exceptions (MIN and BENZER 1997; SNOOK et al. 2000), infection causes negligible reduction of host fitness and may even enhance fitness in some host genotypes (FRY and RAND 2002). Thus Wolbachia is generally considered a commensal parasite, and recent indirect evidence suggests that mutualistic Wolbachia interactions occur in some groups of nematodes (BANDI et al. 1999; HOERAUF et al. 1999). Thus, host/Wolbachia interactions are expanding to include an ever-widening repertoire of important life-history traits. It is becoming increasingly clear that the entire range of such interactions is expressed in the Wolbachia/D. melanogaster symbiosis system. The nature and extent of such interactions is only just beginning to be explored and our results on the survey suggest that the BDSC should provide an excellent opportunity to explore these interactions further in this genetically tractable system.

Recent evidence has shown that Wolbachia can have dramatic effects on Drosophila mutant phenotypes (STARR and CLINE 2002), although the precise nature of these interactions are yet to be determined. In the course of our studies we also discovered an effect of Wolbachia on stocks that carry chico, a gene that encodes an insulin-receptor substrate involved in growth regulation. Two alleles, chi¹ and chi², produce adult progeny significantly smaller than their heterozygous siblings (BOHNI et al. 1999), and we determined that both the chi¹ and chi² lines were infected by the "A" strain of W. pipientis present in other D. melanogaster (supplementary Table S1 at http://www.genetics.org/supplemental/). Interestingly, removal of Wolbachia resulted in complete lethality of chi² homozygotes, while having little or no effect on chi¹ homozygotes (data not shown). However, subsequent introgression and deficiency analyses demonstrated that the Wolbachia effect was not directly linked to chico, but to some other unmapped loci. It will be of interest to map this interacting locus and determine what, if any, relationship it may have to chico.

Another recent study clearly illustrates the potential consequences of Wolbachia infection on Drosophila research. Isonuclear D. melanogaster lines differing in cytoplasmic genomes showed significant differences in longevity (DRIVER and TAWADROS 2000). These data were interpreted as support for a central role of mitochondria and mitochondrial damage in aging. When these same isonuclear lines were treated with tetracycline to remove Wolbachia, no difference in life span was detected (DRIVER et al. 2004). Wolbachia was clearly a confounding factor in the genetic analysis of longevity. It should be noted that chico has been the focus of much work on longevity (CLANCY et al. 2001). In light of our preliminary results showing an as-yet-understood interaction between Wolbachia and the chico mutant lines, we suggest that extra care be taken in such research to determine the infection status of fly lines.

Taken together, these results suggest that this commensal parasite can have profoundly different activities depending on genetic background or that independent variant strains of Wolbachia may arise in cultured laboratory strains. These results therefore may also have implications for the evolutionary and mechanistic dynamics between mutualism and parasitism. The question of whether parasitism inevitably evolves into stable mutualisms or whether a dynamic interaction between the two forms of symbiosis exists is a long-standing question in evolutionary biology (PRICE 1991; BRONSTEIN 1994; THOMPSON 1994; HERRE et al. 1999). Recent phylogenetic inference and theoretical analyses support the contention that mutualism and parasitism are dynamic evolutionary processes (HIBBERT et al. 2000; ROY and KIRCHNER 2000). The gathering evidence suggests that such dynamic processes can occur in the Wolbachia/Drosophila symbiosis system and perhaps for other obligate endocellular symbionts. For example, phylogenetic analyses place Wolbachia within the Rickettsiacaeae (O'NEILL et al. 1992; WERREN et al. 1995), suggesting that the Wolbachia-mediated rescue of chico stocks may have relevance for the evolution and mechanistic studies of these mammalian pathogens (ANDERSON and KARR 2001). The presence of a commensal parasite that can display mutualism within a clade of known pathogens suggests dynamic evolutionary interactions. Moreover, as a sister group to the Ehrlichia and Rickettsia whose last common ancestor was a parasite clearly demonstrates the lability of the evolutionary patterns (ANDERSON and KARR 2001).

The "sheltering" by an endosymbiont of deleterious mutations in the host as has apparently occurred in the chico mutant lines may be typical of the initial steps from commensal to obligate endosymbionts. Similar sheltering of deleterious mutations affecting male reproductive traits has been observed in parthenogenic-inducing Wolbachia in several parasitic wasps. Elimination of Wolbachia from several parthenogenic species results in the production of males, usually with the loss of various aspects of male sexual function and/or female receptivity. The degree to which total male sexual function is lost is likely due to the timing of the acquisition of parthogenesis-inducing Wolbachia (and consequently the duration of the sheltering of deleterious mutations affecting male sexual function) (reviewed in GOTTLIEB and ZCHORI-FEIN 2001). Further study of the unusual interactions so far documented, and others to be discovered, may provide further insight into the early stages of this dynamic evolutionary process.

This report demonstrates that an endocellular microbe has the capacity to indirectly affect phenotypic analyses. These observations did not arise from a concerted screen for genetic interactions but instead arose from our laboratory's general concern about the presence of Wolbachia (GLOVER et al. 1990; O'NEILL and KARR 1990; MIN and BENZER 1997). It therefore seems prudent to assume that other such interactions, even direct interactions as has been recently discovered in alleles of Sxl (STARR and CLINE 2002), will arise from a systematic examination of mutant lines.

As the purpose of this report is to highlight the potential importance of Wolbachia on Drosophila research, the message taken should not be the need for widespread antibiotic treatment of stocks to eliminate Wolbachia, but to be aware of the presence of Wolbachia. As Wolbachia is fully insinuated into Drosophila biology and likely has been a constant or recurrent selective factor throughout the evolutionary history of Drosophila, ignoring its contribution to Drosophila biology would be disadvantageous to further understanding the workings of this scientifically important organism. If stocks are to be treated with antibiotics, care should be taken to assess stock health and mutant phenotype expression before and after treatment. Probably other genes whose expression is influenced by Wolbachia infection status will be discovered. In fact, the Wolbachia-infected P-element mutagenesis screens may be of additional, unintentional value in the search for Wolbachia-Drosophila interactions.

We thank Kathy Matthews and the Bloomington Drosophila Stock Center for providing stocks used in this study. T.L.K. dedicates this article to Henning Chin for his many years of support, guidance, and inspiration.

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