Evolutionarily conserved Wolbachia-encoded factors control pattern of stem-cell niche tropism in Drosophila ovaries and favor infection

Abstract

Wolbachia are intracellular bacteria that infect invertebrates at pandemic levels, including insect vectors of devastating infectious diseases. Although Wolbachia are providing novel strategies for the control of several human pathogens, the processes underlying Wolbachia’s successful propagation within and across species remain elusive. Wolbachia are mainly vertically transmitted; however, there is also evidence of extensive horizontal transmission. Here, we provide several lines of evidence supporting Wolbachia’s targeting of ovarian stem cell niches—referred to as “niche tropism”—as a previously overlooked strategy for Wolbachia thriving in nature. Niche tropism is pervasive in Wolbachia infecting the Drosophila genus, and different patterns of niche tropism are evolutionarily conserved. Phylogenetic analysis, confirmed by hybrid introgression and transinfection experiments, demonstrates that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germ-line cells passing through infected niches, supporting previous suggestions of a contribution of Wolbachia from stem-cell niches toward vertical transmission. These results support the role of stem-cell niches as a key component for the spreading of Wolbachia in the Drosophila genus and provide mechanistic insights into this unique tissue tropism.

The most common maternally transmitted bacteria in invertebrates are alphaproteobacteria belonging to the genus Wolbachia, representing the largest pandemic on the planet (reviewed by ref. 1). These Rickettsia-like bacteria are estimated to infect a great number of invertebrate species, including insect vectors of infectious diseases and pathogenic filarial worms. Recently, it has been shown that Wolbachia strains derived from Drosophila melanogaster, when introduced into mosquito vectors, can invade and sustain themselves in mosquito populations (2). Several phenotypes observed in Drosophila are also maintained in the mosquito nonnative hosts: reduction of adult lifespan, reproductive manipulation, and resistance against several pathogens, including Dengue, Chikungunya, West Nile Virus, and both chicken and human Plasmodium (3–6).

Because Wolbachia are maternally transmitted, their presence in the germ line is essential for their vertical propagation to the next generation. However, Wolbachia are often found in several somatic tissues as well, and this distribution varies among different Wolbachia–host associations (7–11). The role of these bacteria in somatic cells is not clear.

Wolbachia can also move horizontally within and between species (12–16). The mechanism by which horizontal transmission occurs in nature is poorly understood. Regardless of how Wolbachia reach a new host, after the initial infection event, reaching the germ line is an essential requirement for successful transmission to the next generation (1). It has been previously reported in D. melanogaster that, upon recent infection through microinjection, Wolbachia enter the region of the ovary containing the germarium. Several germaria reside at the anterior tip of each ovary and house all of the stem cells necessary to make an egg (Fig. 1A). Within the germarium, the major route for Wolbachia to enter the germ line in this artificial infection model is through the somatic stem-cell niche (SSCN; Fig. 1A, light blue cells) (17). The SSCN is the microenvironment that harbors the somatic stem cell (Fig. 1A, dark blue cells), which in turn generates the somatically derived follicle cells that envelope the germ line and secrete the eggshell. This observation in D. melanogaster raised the possibility of tropism for stem-cell niches as a mechanism to facilitate reaching the germ line during horizontal infection.

Fig. 1.

Wolbachia tropism for stem-cell niches is present across the Drosophila genus, with specific patterns of distribution. (A) Representative diagram of a Drosophila germarium with the regions and cell types indicated: GSCN, in green [formed by TF cells (light green) and CCs (dark green)]; GSC in yellow; escort cells in gray; SSCN in light blue; SSC in dark blue; and germ line in red. (B–L) Wolbachia distribution in germaria of different Drosophila species. DNA is in blue, germ-line marker (Vasa) is in red, and Wolbachia is in green. Wolbachia highly infect the SSCN in all species and also infect the GSCN in several species (G–L). (Scale bar: 10 µm.) (M) Frequency of SSCN tropism. (N) Frequency of GSCN tropism. Brackets indicate groups with statistically similar frequencies. Groups are statistically significantly different from each other. N ∼ 100 germaria each. For details see SI Appendix, Table S2. Error bars represent SEM. D. ana, D. ananassae; D. inn, D. innubila; D. mau, D. mauritiana; D. mel, D. melanogaster; D. sech, D. sechellia; D. sim, D. simulans; D. tei, D. teisseri; D. trop, D. tropicalis; D. yak, D. yakuba.

The same work also showed that Wolbachia accumulate at the SSCN in maternally infected flies. Additionally, in another fruit fly, Drosophila mauritiana, Wolbachia also target the germ-line stem-cell niche (GSCN; Fig. 1A, green cells) in long-term maternally infected flies (18). The GSCN is a somatic structure at the anterior tip of the germarium, composed of terminal filament (TF) and cap cells (CC) (Fig. 1A; TF, light green; CC, dark green) that support the germ-line stem cells (GSC; Fig. 1A, yellow cells). The GSCs are the source of the germ-line cells that develop into the eggs. These observations and subsequent work in other invertebrates (19–21) suggest that stem-cell niche tropism plays a widespread role in germ-line infection during long-term maternal transmission of Wolbachia, in addition to the potential role during horizontal transmission.

Here, using cell biological, phylogenetic, genetic, and transinfection tools, we provide evidence that stem-cell niche tropism is an evolutionarily conserved mechanism for Wolbachia hereditary and nonhereditary transmission. We show that this tropism is a widespread occurrence across the Drosophila genus. Phylogenetic analyses reveal selective pressures promoting strong conservation of the same pattern of niche tropism among closely related Wolbachia strains. Furthermore, quantification of bacterial densities across different regions of the germarium shows an increase of Wolbachia loads in the germ line during or immediately after interaction with infected stem-cell niches. Finally, through hybrid crosses and transinfection experiments, we show that Wolbachia-encoded factors, rather than the host genetic background, are the major determinants of different patterns of stem-cell niche tropism.

Results

Wolbachia Tropism to the Somatic Stem-Cell Niche Is Pervasive Across the Drosophila Genus in All Species Tested.

To determine whether niche targeting is an evolutionarily conserved occurrence across the Drosophila genus, we conducted a survey of 11 different Wolbachia strains that naturally infect nine different Drosophila species (SI Appendix, Supplemental Materials and Methods and Table S1). Using immunohistochemistry, we quantified the frequency of Wolbachia’s niche tropism in the germaria of all 11 Wolbachia strain–Drosophila species pairs. In every ovary analyzed, we found that Wolbachia preferentially infect the border region (BR) between regions 2a and 2b of the germarium (Fig. 1A; for controls, see SI Appendix, Fig. S1). This region contains the SSCN, and preferential Wolbachia infection at the BR characterizes SSCN tropism (SI Appendix, Supplemental Materials and Methods). By comparing Wolbachia levels at the BR to the neighboring somatic regions 2a and 2b, we found that Wolbachia was enriched in the SSCN in 100% of individuals for each species (n = 119 flies; Fig. 1 B–L). Visual assessment of confocal imaging of ∼10 randomly sampled germaria from each ovary showed a frequency of SSCN tropism of greater than 80% (n = 1,194 total germaria; Fig. 1M; P = 0.0012). To quantify levels of Wolbachia enrichment at the SSCN, representative confocal Z stacks were subjected to image analysis of Wolbachia voxel density in the soma of the different germarial regions (SI Appendix, Supplemental Materials and Methods, Fig. S2A). In every species analyzed, there was an increase in Wolbachia load in the soma of the SSCN region normalized to the somatic cells in adjacent region 2b ranging from 2- to 59-fold (SI Appendix, Fig. S2B; t test between BR and 2b statistically significant, P < 0.01 for all species). This analysis indicates a strong selective pressure for an evolutionarily conserved Wolbachia tropism to the SSCN.

Wolbachia Target the Germ-Line Stem-Cell Niche in a Subset of Species.

In addition to Wolbachia tropism to the SSCN, we observed Wolbachia infection in the GSCN (Fig. 1A, green TF and CC), characterized as GSCN tropism (SI Appendix, Supplemental Materials and Methods). Six of eleven Drosophila–Wolbachia pairs analyzed showed GSCN tropism (Fig. 1 G–L). Occurrence of GSCN tropism is more variable than SSCN tropism, with frequencies ranging from 37% to 99% of GSCNs targeted (Fig. 1N and SI Appendix, Table S2; n = 647 total germaria). ANOVA analyses defined three distinct groups: high frequency (HF) of GSCN targeting (Fig. 1 J–L and N; P = 0.80), moderate frequency (MF) of GSCN targeting (Fig.1 G–I and N; P = 0.087), and low/no frequency (LF) of GSCN targeting (Fig. 1 B–F and N; P = 0.44). Voxel intensity measurements showed that Wolbachia density is from 2.5- to 26.5-fold enriched in the GSCN normalized to region 2b soma (SI Appendix, Supplemental Materials and Methods and Fig. S2C). In addition, Wolbachia infection of the escort cells was also noted in some species (SI Appendix, Supplemental Results and Fig. S3, and Movie S1). Escort cells are a stable, nondividing, stromal population of cells that are attached to the basement membrane of the germarium and support the progression of early germ-line cysts in region 1 and 2A of the germarium (see gray cells in Fig. 1A). Relative to SSCN tropism, targeting of the GSCN occurred at a lower frequency and density. These observations show that, although targeting of stem-cell niches in the Drosophila ovary is a widespread occurrence, the patterns of distribution are not the same in all Drosophila host–Wolbachia strain pairs.

Phylogenetic Analyses Suggest That Differential Niche Tropisms Are Mediated by Wolbachia-Encoded Factors.

In broad terms, we see two different patterns of stem-cell niche tropism in the Drosophila ovary: (i) targeting of only the SSCN (herein referred to as SSCN pattern) or (ii) targeting of both the SSCN and the GSCN (herein referred to as GSCN pattern). This observation of differential patterning of stem-cell niches led us to investigate the relative contributions of host factors and bacterial factors toward the distinct Wolbachia tropism patterns. We reconstructed the evolution of niche tropism on phylogenetic trees of both Wolbachia and Drosophila (Fig. 2A) (22, 23) to determine whether patterns of niche tropism were primarily determined by factors derived from the Wolbachia strains or derived from the Drosophila host species. To quantify the correlation of niche tropism pattern to the two different phylogenies, we used a computer simulation model of randomized character distributions to compare with the distribution of niche tropism pattern on each of the phylogenies (SI Appendix, Supplemental Results and Fig. S4) (24). This analysis indicated that there is an ∼10-fold lower probability that the association of niche tropism with the Wolbachia phylogeny is due to random chance than the association with the Drosophila phylogeny. Therefore, closely related Wolbachia strains are more likely to display similar patterns of tropism compared with the tropism patterns observed in closely related Drosophila species. Furthermore, the phylogenetic analysis suggests that the different patterns of niche tropism evolved in Wolbachia and that the pattern of shared Wolbachia niche tropism in Drosophila results from characteristics of the infecting Wolbachia strain rather than characteristics of the host Drosophila species.

Fig. 2.

Wolbachia strain determines differential targeting of the germ-line stem-cell niche. (A) Different patterns of niche targeting are correlated with Drosophila and Wolbachia phylogenies (22, 23). MYA, million years ago. Green, blue, and red lines indicate high, moderate, and low frequency of GSCN tropism, respectively. (B) Diagram showing experimental design of the hybrid cross to introgress Wolbachia A into species B genetic background. (C) Wolbachia strains wMau and wSh were introgressed into D. sechellia and D. mauritiana, respectively. Representative images of Wolbachia niche targeting in the parental (Upper) and F₅ hybrid (Lower) host germaria. The red and green arrows represent the direction of Wolbachia transfer. The male genital arch is shown to confirm successful introgression of the male genetic background. (Scale bar: 10 µm.) (D) Quantification of GSCN targeting in parental (solid bars) and hybrid (striped bars) species (Log reg, P_wolb = 4.7 × 10⁻²² and P_host = 0.18). N_{D.sech wSh} = 120, N_{D.mau wSh} = 140, N_{D.mau wMau} = 100, N_{D.sech wMau} = 109 (N = number of germaria). Error bars represent SEM.

Hybrid Crosses Confirm That Bacterial Factors Mediate Stem-Cell Niche Tropism.

The phylogenetic analyses suggest that Wolbachia factors mediate differential stem-cell niche tropism patterns. To experimentally evaluate this hypothesis, we generated hybrid flies between Drosophila species harboring two different Wolbachia strains that display the two different Wolbachia tropism patterns, using genetic introgression (SI Appendix, Fig. S5). The rationale for this experiment is as follows: if the pattern of tropism is mediated by the Wolbachia strain, the Wolbachia patterning in the germaria in the hybrid host will be the same as the original maternal host, regardless of the introgressed male host genetic background (Fig. 2B).

Hybrid fly lines were created by crossing D. mauritiana flies infected with Wolbachia wMau, which display a GSCN tropism pattern, and Drosophila sechellia flies infected with Wolbachia wSh, which display a SSCN tropism pattern. Wolbachia wMau, infecting both the parental D. mauritiana and hybrid D. sechellia, display a high frequency of GSCN tropism pattern (greater than 85%; Fig. 2 C and D; n = 209 total germaria). In contrast, Wolbachia wSh, infecting both the parental D. sechellia and hybrid D. mauritiana, display high frequencies of the SSCN tropism pattern, with greater than 90% of germaria analyzed only infecting the SSCN (Fig. 2 C and D; n = 260 total germaria). Regardless of genetic background, both Wolbachia strains maintain the maternal niche tropism pattern in the hybrid host. Logistic regression analysis was performed to evaluate the relative contributions of the Wolbachia strain and the host genetic background to the differential patterns of stem-cell niche tropism. We found no evidence of host influence on niche tropism pattern (P = 0.18); however, the Wolbachia strain does have a highly statistically significant effect (P = 4.7 × 10⁻²²). Image analysis of representative images confirms GSCN tropism in wMau-infected flies and SSCN tropism in wSh-infected flies (SI Appendix, Fig. S6).

During the hybrid crosses, together with the Wolbachia strain, other maternally inherited components, such as the mitochondria, are also transmitted. To eliminate the possibility that maternally transmitted organelles and other factors have a role in determining Wolbachia niche tropism pattern, we analyzed a fly line whose Wolbachia infection was established via microinjection (Drosophila simulans artificially infected with wMel) (25). The results indicate that the Wolbachia strain is necessary and sufficient to determine the pattern of niche tropism in a nonnative host. wMel-infected flies always display the SSCN tropism pattern only, regardless of genetic background and maternally inherited components (SI Appendix, Supplemental Results and Fig. S7; n = 246 total germaria).

These results are in agreement with our phylogenetic analysis and support the hypothesis that stem-cell niche tropism is largely mediated by Wolbachia factors rather than the host genetic background.

Wolbachia Factors also Direct Qualitative Differences Within Niche Tropism Pattern.

We also observed variability in the pattern of Wolbachia distribution in the TF cells. Some TFs were fully infected, with all cells densely infected with Wolbachia; others had a discontinuous pattern of infection, with only some TF cells densely infected, interspersed with noninfected TF cells. Interestingly, two Wolbachia strains that naturally infect D. simulans had this noticeable difference, which was most evident in young flies. Wolbachia wRi displays a discontinuous TF pattern of infection (Figs. 1H and 3A); Wolbachia wNo fully infects the TF (Figs. 1J and 3A).

Fig. 3.

Wolbachia strain directs patterning within the GSCN. (A) Wolbachia distribution in GSCN of wRi and wNo infected D. simulans 198,169 (Upper) and F₅ backcrossed strains (Lower). (Scale bar: 10 µm.) (B) Quantification of parental F₀ (solid bars) and F₅ (striped bars) strains. (Log reg, P_wolb = 6.5 × 10⁻¹¹ and P_host = 0.54). N_{D.sim198 wNo} = 120, N_{D.sim169 wNo} = 122, N_{D.sim169 wRi} = 100, N_{D.sim198 wRi} = 130 (N = number of germaria). Error bars represent SEM. Lamin C labels TF and CCs.

Because we have shown that Wolbachia factors are mediating the overall patterns of niche tropism, we investigated whether they also influence qualitative differences within the same pattern. After backcrossing to introgress the host genetic backgrounds (Fig. 2B and SI Appendix, Fig. S5), we observed that wRi-infected flies, regardless of host strain genetic background, display a high frequency of discontinuous terminal filament infection, with ∼80% of highly infected niches having a discontinuous pattern (Fig. 3; n = 230 total germaria). Wolbachia wNo-infected flies display a low frequency of discontinuous terminal filament infection, with ∼20% of infected niches having a discontinuous pattern, regardless of host strain genetic background (Fig. 3; n = 242 total germaria). Logistic regression analysis confirms that the Wolbachia strain plays a more significant role in the discontinuous GSCN pattern than the fly genetic background (P = 6.5 × 10⁻¹¹ and P = 0.54, respectively). These results demonstrate that Wolbachia-encoded factors also direct specific differences in the distribution of bacteria within the GSCN.

Wolbachia Levels in the Germ Line Increase with Proximity to Infected Niches.

To assess the contribution of stem-cell niche tropism toward Wolbachia enrichment in the germ line, we quantified the Wolbachia density in the germ line in the different germarial regions of each of the Drosophila–Wolbachia pairs (SI Appendix, Fig. S2C). For contribution from the SSCN, we compared the density of Wolbachia in germ-line cysts in region 2a to the density of Wolbachia in germ-line cysts in region 2b (SI Appendix, Supplemental Materials and Methods). These two regions contain germ-line cells before (2a) and after (2b) developing cysts pass through the niche (Fig. 1A). In all species, except Drosophila tropicalis, we observed a similar trend: after passage through the border region containing the highly infected SSCNs, the levels of Wolbachia in germ-line cysts in region 2b are higher than the levels of Wolbachia in region 2a, with fold-changes (2b/2a) ranging from 1.3 to 25 (SI Appendix, Fig. S8M). Although there is high variability in Wolbachia load from germ-line cyst to germ-line cyst, 7 of 11 species, have a statistically significant increase of Wolbachia load from 2a to 2b [see white arrows in SI Appendix, Fig. S8 B–F, J, K, and M (quantification); t test, P < 0.05].

For contribution from the GSCN, we compared the relative fraction of Wolbachia in region 1 of the germ line across species with GSCN tropism and without GSCN tropism (SI Appendix, Fig. S8N). Species with GSCN tropism had a higher relative density of Wolbachia in region 1 (compared with the whole germarium) than species with only SSCN [green asterisks in SI Appendix, Fig. S8 G–L and N (quantification)].

In the majority of Drosophila species analyzed, Wolbachia tropism to the stem-cell niches correlates with higher densities of Wolbachia in the adjacent germ line. These results agree with previous work (17, 19–21) supporting a passage of Wolbachia from the niche into the germ line.

Increase of Wolbachia Density from Regions 2a to 2b Is Contributed to by Wolbachia Proliferation in the Niche and Germ Line.

For the niche to be a source for Wolbachia into the germ line, we expect Wolbachia to be dividing in the niche. Using an antibody against the conserved bacterial cell division protein FtsZ (named after filamenting temperature sensitive mutant Z), we observed substantial Wolbachia division within the SSCN (SI Appendix, Supplemental Materials and Methods and Fig. S9A) (20, 26). In addition to passage from the SSCN, Wolbachia are actively dividing in the germ line, which also contributes to the increase in Wolbachia’s density in region 2b. Region-specific differences in the rate of Wolbachia division could play a major role in the increase of Wolbachia in region 2b. However, our analysis indicates that the fraction of Wolbachia dividing in both regions 2a and 2b of the germarium is the same (SI Appendix, Fig. S9 B and C). Even with the same division rate of Wolbachia in these regions, differences in cyst development timing could also play a role in the increase of Wolbachia density in region 2b. However, studies in D. melanogaster demonstrate that the developmental time that germ-line cysts remain in region 2b is not significantly different from the time the germ-line cysts are present in the surrounding regions 2a and 3, ruling out this possibility in at least D. melanogaster (27). These data suggest that Wolbachia division within the germ line, in combination with Wolbachia passage from the niche, contributes to the increase of Wolbachia density in region 2b.

Discussion

To understand the spread of Wolbachia in nature, it is important to elucidate the mechanisms of horizontal and vertical transmission. Because the majority of transmission events are maternal, to effectively infect a population, Wolbachia must infect the female’s germ line during both long-term stable vertical transmission and recent horizontal introduction into a new host. Here, we provide evolutionary, cytological, genetic, and developmental evidence for a mechanism in which stem-cell niche tropism promotes germ-line colonization across the Drosophila genus. We also demonstrate that factors encoded by the Wolbachia strain, rather than the host species, are the major determinants of the type of stem-cell niche that is infected.

In a survey of niche tropism, we show that Wolbachia display tropism for two different stem cell niches in the Drosophila ovary: the SSCN and the GSCN. Several studies have described Wolbachia preferential infection of different tissues, host cells, and subcellular locations in the Drosophila genus, including adult brain, embryonic neuroblasts, specific regions of the oocyte during oogenesis, and posterior or anterior areas of the early embryo (9, 28–30). Considering Wolbachia’s transmission across generations, a site in the host of particular interest is the germplasm, which is a highly specialized, maternally synthesized cytoplasm that is deposited in the posterior pole of the egg and induces the formation of the germ line in the embryo (ref. 31 and reviewed by ref. 32). During late oogenesis and early embryonic development, Wolbachia efficiently colonize the germplasm in D. melanogaster, giving rise to a highly infected germ line, ensuring Wolbachia transmission to the subsequent generation (28, 33). However, germplasm infection is not observed in several other Drosophila species (28, 29). Surprisingly, targeting of the SSCN is more prevalent in the Drosophila genus than targeting of the germplasm. To our knowledge, with the exception of infection of the adult oocyte, the preferential infection of the SSCN reported here is the most conserved Wolbachia tropism reported in the Drosophila genus.

Given that Wolbachia does not colonize the germplasm of the embryo in every Drosophila species, there must be an alternative mechanism to ensure its vertical transmission. The strong phylogenetic conservation of patterns and the pervasive presence of tropism for stem-cell niches in the Drosophila germarium are suggestive of a significant role for niche tropism in transmission. Previous work has implicated stem-cell niche tropism as a mechanism facilitating horizontal transmission of Wolbachia in D. melanogaster (17). Our confocal imaging analysis suggests that stem-cell niches in the Drosophila germarium also play a role in vertical transmission of Wolbachia. Similar to our findings, there is a surprising observation from the Wolbachia strains infecting filarial nematodes. In the filarial worm, Wolbachia are excluded from the precursor of the germ-cell lineage; infection of the gonad happens later in development, through the invasion via the distal tip cell, the nematode equivalent to the stem-cell niche (20). Furthermore, studies on a bedbug and a leafhopper suggest that Wolbachia are transmitted to the germ line via a putative stem-cell niche (19, 21). These observations support a hypothesis of stem-cell niche tropism as a mechanism for Wolbachia dissemination shared during both horizontal and vertical transmission.

Our data clearly show that the SSCN prevails over the GSCN in terms of occurrence and evolutionary conservation. To provide an explanation for these observations, we propose a model that considers Wolbachia transmission to the germ line during development from the stem-cell niches. The differences in the anatomic features between niches and associated cells, as well as the developmental time periods in which Wolbachia can be transmitted from each niche, suggest that the SSCN is better suited for Wolbachia transmission to the germ line.

The model presented in Fig. 4 displays potential routes of Wolbachia entry into the germ line from the surrounding niches and other somatic cells during Drosophila oogenesis. The GSCN contacts the germ-line stem cell, providing a potential route for the Wolbachia present in this niche to enter the germ line (Fig. 4C, dark blue arrows). In addition, when escort cells are highly infected, it is possible to have transmission from these somatic cells into the germ line until the developing cyst reaches the BR (Fig. 4C, light blue arrow; see also SI Appendix, Fig. S3 and Movie S1). Therefore, transmission into the germ line could occur for a total of ∼2.5 d, the estimated time for germ-line transit from the germ-line stem-cell niche to the BR (Fig. 4B, see blue line in timeline) (27, 34).

Fig. 4.

Model for Wolbachia transmission from the stem-cell niches into the germ line. Wolbachia originating from the SSCN, rather than from the GSCN, are more likely to invade the germ line. (A) Diagram of egg formation with developmental stages and timeline in days (27, 36; diagram adapted from ref. 18). Developmental timeline is colored according to potential for Wolbachia transmission from the GSCN and escort cells (blue, days 0–2.5) or from the SSCN, either directly (red, day 2.5) or indirectly (orange, days 2.5–7.3). (B) Diagram of potential sources of Wolbachia transmission into the germ cells from somatic cells present in the germarium and representative egg chambers. (C) Magnification of Wolbachia transfer from the GSCN (dark blue arrows) or the escort cells (light blue arrows). (D) Magnification of Wolbachia transmission directly from the SSCN (red arrows). (E–G) The somatic tissue infected with Wolbachia originating from the SSC can indirectly transmit Wolbachia into the germ line for the rest of egg development (orange arrows).

In comparison, the SSCN provides several routes for Wolbachia transmission into the germ line (Fig. 4 D–G), both direct and indirect. Because the SSCN contacts all developing germ-line cysts, it can transmit Wolbachia directly into the germ-line cells that must pass through the border region (Fig. 4 B and D, red arrows). The possibility of Wolbachia passage into the germ line was initially suggested for D. melanogaster by confocal analysis (see supplementary table 1 in ref. 17), further corroborated by EM studies (21). The data presented here suggest that the SSCN can deliver Wolbachia directly into the germ line in all species of Drosophila analyzed in this study.

The SSCN can also transmit Wolbachia indirectly. The infected niche is a constant source of Wolbachia into the SSC, which, in turn, divides and transmits Wolbachia into the developing follicle cells (Fig. 4D, orange arrows) (see also supplementary figure 2 b–d and supplementary movie in ref. 17). The follicle cells can transmit Wolbachia into the germ line of developing egg chambers through the remaining stages of germ-line development, providing an extended period of developmental time for transmission (Fig. 4B, developmental stages indicated by orange line; Fig. 4 E–G, orange arrows). Furthermore, several yolk proteins produced by the follicle cells are actively transported into the oocyte during the final stages of oogenesis (35). This process may provide a facilitated mechanism for Wolbachia present in the follicle cells to transfer into the oocyte (Fig. 4G and SI Appendix, Fig. S10). From the border region, it takes approximately 5 d for the completion of oogenesis (36). Compared with the previous 2.5 d of cyst development in regions 1 and 2A, where there is the potential for Wolbachia transmission from the GSCN and escort cells, the developmental time available for transmission of Wolbachia derived from the SSCN is about twice as long (Fig. 4 A and B, blue line vs. red/orange line in timeline). Ultimately, it is easier for Wolbachia to reach the germ line through the SSCN (rather than the GSCN) during vertical transmission and probably during horizontal transmission as well. These developmental and anatomical features of the niches provide an explanation to the phylogenetic, genetic, and cytological data presented here.

This work highlights bacterial localization as a fundamental aspect of Wolbachia–host interactions being maintained during Wolbachia evolution. Our current understanding of the mechanisms involved in Wolbachia localization is limited (36). Toward dissecting the mechanistic basis of stem-cell niche tropism, we investigated the relative role of bacterial versus host factors in the different patterns of niche tropism. Through hybrid crosses and transinfection experiments, we showed that bacterial intrinsic factors are the major determinant of the pattern of niche tropism and also determine differences within the same pattern.

There are extensive comparative genomic analyses of different Wolbachia strains used in this study (37–39). At this point, we cannot attribute differences in the targeting of stem-cell niches to specific genes or proteins due to a large number of genomic differences across the Wolbachia strains analyzed (38, 39). Indeed, it has been suggested that Wolbachia is one of the most highly recombining intracellular bacterial genomes known to date (37). Nevertheless, the data presented here provide the foundation for future approaches toward the identification of genetic pathways mediating Wolbachia’s stem-cell niche tropism in hosts.

Wolbachia-based technologies are emerging as a promising tool for the control of vectors of deadly human diseases, including Dengue fever, West Nile virus, and malaria (3–6, 41, 42). Understanding the basis of Wolbachia targeting of specific tissues in the host and its consequences toward bacterial transmission will provide further mechanistic insight into their extremely successful propagation and is also relevant for developing new Wolbachia-based vector control approaches.

Materials and Methods

SSCN tropism was defined as Wolbachia accumulation in the somatic cells residing at the border between regions 2a and 2b, as previously described (17). GSCN tropism was defined as Wolbachia accumulation in the TF and CCs, as previously described (18). Fly stocks utilized in this study, husbandry, immunohistochemistry, FISH, introgression crosses, phylogenetic analyses, image analysis, FtsZ analysis, and statistical analysis are provided in SI Appendix, Supplemental Materials and Methods.