Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host

Shelbi L Russell; Jennie Ruelas Castillo; William T Sullivan

doi:10.1371/journal.pbio.3002335

. 2023 Oct 24;21(10):e3002335. doi: 10.1371/journal.pbio.3002335

Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host

Shelbi L Russell ^1,^*, Jennie Ruelas Castillo ², William T Sullivan ³

Editor: Nancy A Moran⁴

PMCID: PMC10597519 PMID: 37874788

Abstract

The alphaproteobacterium Wolbachia pipientis infects arthropod and nematode species worldwide, making it a key target for host biological control. Wolbachia-driven host reproductive manipulations, such as cytoplasmic incompatibility (CI), are credited for catapulting these intracellular bacteria to high frequencies in host populations. Positive, perhaps mutualistic, reproductive manipulations also increase infection frequencies, but are not well understood. Here, we identify molecular and cellular mechanisms by which Wolbachia influences the molecularly distinct processes of germline stem cell (GSC) self-renewal and differentiation. We demonstrate that wMel infection rescues the fertility of flies lacking the translational regulator mei-P26 and is sufficient to sustain infertile homozygous mei-P26-knockdown stocks indefinitely. Cytology revealed that wMel mitigates the impact of mei-P26 loss through restoring proper pMad, Bam, Sxl, and Orb expression. In Oregon R files with wild-type fertility, wMel infection elevates lifetime egg hatch rates. Exploring these phenotypes through dual-RNAseq quantification of eukaryotic and bacterial transcripts revealed that wMel infection rescues and offsets many gene expression changes induced by mei-P26 loss at the mRNA level. Overall, we show that wMel infection beneficially reinforces host fertility at mRNA, protein, and phenotypic levels, and these mechanisms may promote the emergence of mutualism and the breakdown of host reproductive manipulations.

Wolbachia bacterial symbionts are being harnessed as biological control agents by exploiting their manipulation of host fertility. This study reveals that the wMel strain can have a beneficial impact on fruit fly development in both mutant and wild-type flies; this phenotype is essential to the long-term use of these bacteria for host population control.

Introduction

Endosymbiotic bacteria have evolved diverse strategies for infecting and manipulating host populations [1,2], which are now being leveraged for biological control applications [3]. Many of these bacteria reside within host cells and navigate female host development to colonize offspring, thus linking their fitness to that of their hosts through vertical transmission [4]. Inherited endosymbionts with reproductive manipulator capabilities go a step further by altering host development in ways that rapidly increase the frequency of infected, reproductive females in the host population. Reproductive manipulator strategies include cytoplasmic incompatibility (CI), where infected sperm require rescue by infected eggs, male-killing, feminization, and parthenogenesis [5,6]. These manipulations can be highly effective at driving bacterial symbionts into host populations, regardless of costs to the host individual or population [7].

Despite the success of parasitic reproductive manipulation, natural selection favors symbionts that increase the fertility of infected mothers, even if these variants reduce the efficacy of the parasitic mechanisms that initially drove the infection to high frequency [8]. In associations between arthropods and strains of the alphaproteobacterium Wolbachia pipientis, this scenario may be common. For example, measured fecundity in populations of Drosophila simulans infected with the wRi strain of Wolbachia swung from −20% to +10% across a 20-year span following wRi’s CI-mediated sweep across California in the 1980s [9]. This transition from fitness cost to benefit coincided with weakening of CI strength over the same time frame [10]. Fertility-enhancing mechanisms may be at work in other strains of Wolbachia that have reached high infection frequencies in their native hosts, yet do not currently exhibit evidence of parasitic reproductive manipulation [11]. Importantly, these fitness benefits may have also played a role in the early stages of population infection, when infected hosts are at too low of frequencies for CI to be effective [12].

Currently, the wMel strain of Wolbachia and its encoded CI mechanism are successfully being used to biologically control non-native hosts [3]. In Aedes aegypti mosquitoes, CI causes nearly 100% mortality of offspring born to uninfected mothers [13]. However, in its native host, the fruit fly Drosophila melanogaster, wMel exhibits CI that rarely exceeds 50% mortality and is extremely sensitive to paternal age [14–16], as well as grandmother age because titer increases with age [17,18]. Despite weak CI in its native host, wMel is found at moderate to high infection frequencies in populations worldwide [19,20]. Other data suggest that these frequencies may be explained by some emergent beneficial function that increases host fitness [21–25]. The molecular basis for these beneficial functions could be related to the loss of CI efficacy in D. melanogaster. Given that wMel’s use in non-native hosts relies on strong and efficient CI, it is essential that we learn the basis for its beneficial functions that could ultimately undermine CI function.

Host germline stem cell (GSC) maintenance and differentiation pathways are powerful targets for Wolbachia-mediated reproductive manipulation. Wolbachia strains have strong affinities for host germline tissues [26,27], positioning them at the right place to manipulate and enhance host fertility. In the strains that form obligate associations with Brugia filarial nematodes [28] and Asobara wasps [29], Wolbachia is required in the germline to prevent premature differentiation and achieve successful oogenesis (reviewed in [30]). In the facultative wMel-D. melanogaster association, the wMel strain can partially rescue select loss of function alleles of the essential germline maintenance and differentiation genes sex lethal (sxl) and bag-of-marbles (bam) in female flies [31–33]. It is known that wMel encodes its own factor, toxic manipulator of oogenesis (TomO), that partially recapitulates Sxl function in the GSC through derepression and overexpression of the translational repressor Nanos (Nos) [34,35]. However, Nos expression is negatively correlated with Bam expression in the early germarium [36]. Therefore, Bam’s function in cyst patterning and differentiation in wMel-infected mutant flies cannot be explained by shared mechanisms with sxl rescue or TomO’s known functions.

In a previous screen, our lab identified the essential fertility gene meiotic-P26 (mei-P26) as a host factor that influences wMel infection intensity in D. melanogaster cell culture, potentially through modulating protein ubiquitination [37]. Mei-P26 is a Trim-NHL protein that confers a wide range of functions through its multiple domains: its protein-binding NHL and B-box domains allow it to act as an adapter for multiple translational repressor complexes (e.g., Sxl, Nanos (Nos), Argonaute-1 (AGO1), see S1 to S2A Figs and S1 to S2 Tables). Its E3-ubiquitin ligase domain likely enables it to modulate proteolysis [38]. Given mei-P26’s in vitro role in infection [37] and its in vivo role in GSC maintenance [38], differentiation [39,40], and meiosis [40,41], we selected it as a candidate gene for modulating wMel–host interactions.

We report that D. melanogaster flies infected with the wMel strain of Wolbachia partially rescue mei-P26 mutations and exhibit reinforced fertility. In flies homozygous for hypomorphic mei-P26, infection with wMel elevates fertility in both males and females to a level sufficient to maintain a stable stock, whereas the uninfected stock is unsustainable. Infection rescues GSC maintenance and cyst differentiation by mitigating the downstream effects of perturbed mei-P26 function on other protein and mRNA expression. Specifically, wMel infection restores a wild-type-like expression profile for phosphorylated Mothers against decapentaplegic (pMad), Sxl, Bam, and Oo18 RNA-binding (Orb) proteins, as well as tumorous testis (tut) and benign gonadal cell neoplasm (bgcn) mRNAs. We find evidence of wMel’s beneficial reproductive manipulator abilities in Oregon R (OreR) wild-type flies, illustrating how bacterially mediated developmental resilience may be selected for in nature. These results are essential to understanding how wMel reaches high frequencies in natural D. melanogaster populations and these beneficial functions may be able to be harnessed for biological control applications.

Results

Here, we explore the effects of wMel Wolbachia infection on D. melanogaster’s essential fertility gene mei-P26 through dosage knockdown with an RNAi construct, disrupted function with a hypomorphic allele, mei-P26[1], and full knockdown with a null allele, mei-P26[mfs1]. Using fly fecundity assays, immunocytochemistry, and dual-RNAseq of both host and bacterial transcripts, we show that wMel can compensate for the loss of mei-P26 to significantly rescue host fertility.

Infection with wMel rescues female D. melanogaster fertility in mei-P26 deficient flies

Infection with wMel significantly rescued fertility defects induced by the loss of mei-P26 function relative to the OreR wild-type strain, as measured by offspring produced per female D. melanogaster per day (Figs 1A–1E and S3; p< = 2.2e-16 to 2.9e-2, Wilcoxon rank sum test; see S2–S6 Figs). Offspring production requires successful maintenance of the GSC, differentiation of a GSC daughter cell into a fully developed and fertilized egg, embryogenesis, and hatching into a first-instar larva. Breaking offspring production down into egg lay and egg hatch components (Fig 1B and 1C) revealed that as allelic strength increased, from nos-driven RNAi to hypomorphic and null alleles, fertility impacts shifted from those that impacted differentiation/development (egg hatch) to those that also impacted egg production (egg lay).

Fig 1 — (A–C) Beeswarm boxplots of (A) overall offspring production, broken into (B) eggs laid and (C) eggs hatched, from single female RNAi, hypomorphic, and null *mei-P26* knockdown female flies crossed to single OreR males of the same infection state and age (3–7 days old). The null allele *mei-P26[mfs1]* reduced egg lay rates in both mutant heterozygotes and homozygotes below an average of 20 for both infected and uninfected females, necessitating an analysis of females with and without offspring (see D). Wilcoxon rank sum test *p< = 0.05, **< = 0.01, ***< = 0.001, ****< = 1e-4. Fisher’s exact test [**]p< = 0.01, [****]< = 1e-4. (D–E) (D) Barplots of the proportion of female samples of the indicated genotypes with offspring and without offspring, infected and uninfected with *Wolbachia*. (E) Table listing the percentage of wild-type fecundity demonstrated by wMel-infected (left) or uninfected (right) *mei-P26* mutants in order of increasing severity, from top to bottom (see also S4–S6 Tables). Cell values from low to high proportions are colored from light to dark blue, respectively. (F, G) Knockdown *mei-P26 D*. *melanogaster* fecundity versus female age, fit with a local polynomial regression (dark gray bounds = 95% confidence intervals). Infection with wMel increases offspring production over the first 25 days of the (F) *mei-P26* RNAi and (G) *mei-P26[1]* hypomorphic female lifespan. (H–O) Confocal mean projections of D. *melanogaster* ovarioles and germaria (3–7 days old). (H–I’) Intracellular wMel FtsZ localizes to germline (yellow, Vas+, inner dotted outline) and somatic (Vas-, space between inner dotted and outer dashed outlines) cells at high titers, with low background in uninfected flies. Bacteria are continuously located in the *mei-P26[1]* germline, starting in the GSC (arrowhead near Hts-bound spectrosome). Somatic cell regions are indicated with empty arrowheads. (J–O) Cytology reveals that wMel-infection rescues (J, K) *mei-P26* RNAi, (L, M) *mei-P26[1]* (see also S5 Fig), and (N, O) *mei-P26[1/mfs1]*-induced oogenesis defects. Fluorescence channels were sampled serially and overlaid as indicated on each set of images as follows: cyan = anti-Hu Li Tai Shao (Hts) ring canal isoform (-RC) or fusome isoform (-FUS), yellow = anti-Vasa (Vas), blue = wMel anti-Filamenting temperature-sensitive mutant Z (FtsZ), and red = PI DNA staining. Scale bars: H–I’ = 10 μm, L–O = 50 μm, J, K = 100 μm. (P) Diagram of a wild-type D. *melanogaster* germarium, highlighting key events and cell types by region as in [42]. Somatic cells in beige, GSC-derived cells are brightly colored gradients leading to differentiated cyst cells in yellow, with specified oocytes in blue. The black structure that originates in the GSC is the spectrosome, which becomes a branching fusome as the CB is formed, moves away from the niche, and divides into CCs. The data underlying this figure can be found in S3 Table and on Dryad at doi.org/10.7291/D1DT2C. CB, cystoblast; CC, cystocyte; GSC, germline stem cell; OreR, Oregon R; PI, propidium iodide.

Infection with wMel mediated a significant degree of fertility rescue for all allelic strengths and nos:GAL4>UAS-driven RNAi knockdowns, suggesting a robust bacterial rescue mechanism that compensates for loss of protein dosage and function. Rescue was more than complete for nos-driven mei-P26 RNAi depletion (from 26% deficient offspring production in uninfected flies): wMel-infected mei-P26 RNAi females produced 44% more offspring than infected OreR wild-type females (37 versus 25 larvae/female/day, p< = 1.15E-02 Wilcoxon rank sum test, S4 Table and Fig 1A and 1E) due to a higher rate of egg production (p< = 6.99E-05 Wilcoxon rank sum test, S5 Table and Fig 1B and 1E), opposed to a higher hatch rate (Fig 1C and 1E and S6 Table). This may suggest a synergistic function between wMel and low-dose mei-P26 in the GSC. In uninfected and wMel-infected mei-P26[1] hypomorphic females, offspring production decreased 94% and 73%, respectively, compared to OreR wild-type of the matching infection status (p< = 2.26E-14 and 3.45E-12 Wilcoxon rank sum test, respectively, S4 Table and Fig 1A and 1E). Comparing uninfected and wMel-infected female mei-P26[1] fecundity revealed that wMel infection increases the number of offspring produced per day (1.6 versus 7 larvae/female/day, p< = 9.37E-05 Wilcoxon rank sum test, S4 Table and Fig 1A) through increasing both the rate of egg lay (p< = 2.82E-02 Wilcoxon rank sum test, S5 Table and Fig 1B) and egg hatch (p< = 2.55E-03 Wilcoxon rank sum test, S6 Table and Fig 1C). In uninfected and wMel-infected mei-P26[1]/mei-P26[mfs1] trans-heterozygous females, offspring production decreased 99% and 82%, respectively, relative to OreR wild-type (p< = 3.40E-04 and 5.01E-10 Wilcoxon rank sum test, S4 Table and Fig 1A and 1D). Comparing uninfected and wMel-infected trans-heterozygous flies showed that infection elevates offspring production (0.2 versus 4.6 larvae/female/day, p< = 1.03E-05 Fisher’s exact test, S4 Table and Fig 1A) through increasing the rate females lay eggs (p< = 4.71E-05 Wilcoxon rank sum test; Fig 1B) and the rate those eggs hatch (p< = 4.20E-03 Wilcoxon rank sum test; S6 Table and Fig 1C). In females homozygous for the most severe allele, mei-P26[mfs1], offspring production decreased 100% and 97% in uninfected and wMel-infected flies relative to wild-type, respectively (p< = 5.14E-06 Fisher’s exact test, S4 Table and Fig 1A). Offspring production was marginally rescued in mei-P25[mfs1] flies by wMel infection (0 (uninfected) versus 0.76 (infected) larvae/female/day, p< = 5.14E-06 Fisher’s exact test, S4 Table, Fig 1A), in part due to infected flies laying eggs at a higher rate than uninfected flies (p< = 3.04E-05 Wilcoxon rank sum test, Fig 1B). However, no female laid 20 or more eggs in one day, precluding an estimation hatch rate rescue (Fig 1C). See S2–S6 Tables and S3 Fig for a full description of the fecundity assays and data.

wMel-mediated mei-P26 rescue is robust and sufficient to rescue this gene in a stable stock. Infection with wMel elevates the number of offspring produced per day across the fly lifespan in mei-P26[1] hypomorphic and nos-driven RNAi-knockdown females (p< = 1.5e-11 to 3.4e-2 Kolmogorov–Smirnov test, S7 Table and Fig 1F and 1G). However, the underlying number of eggs laid per day and the hatch rate do vary considerably with age for both infection states and genotypes (S4A–S4D Fig). The wMel rescue mechanism is not specific to females, as the weaker impacts of mei-P26 loss on male fertility are mitigated by wMel-infection (S4E–S4G Fig; p< = 6.1e-6 to 2.9e-2 Wilcoxon rank sum test, S3–S5 Tables). Thus, we were able to establish a homozygous stock of mei-P26[1] flies infected with wMel Wolbachia. In contrast, the uninfected stock only lasted a few generations without balancer chromosomes (S4H and S4I Fig). Given the severe, yet significantly rescuable nature of the mei-P26[1] allele (Figs 1A–1C, S2B and S2D), we proceeded with this genotype for many of our subsequent assays for specific immunocytological rescue phenotypes.

Female germline morphology is rescued by wMel infection in mei-P26-deficient flies

In the D. melanogaster germarium, wMel is continuously present at high titers in both germline and somatic cells (Fig 1H–1H’, compared to control in Fig 1I–1I’), consistent with previous reports [26,27,43]. The bacteria localize more strongly to the germline-derived cells than the somatic cells (co-localization of FtsZ and Vas in Fig 1H–1H’). Intracellular wMel can be identified in the GSC, the cystoblast (CB), the cystocytes (CCs), and the developed cyst. This positioning puts wMel in all of the critical cell types and stages that mei-P26 is active, enabling the bacterium to compensate for mei-P26’s developmental functions in GSC maintenance and differentiation.

Comparing the cytology of mei-P26 knockdown oocytes infected and uninfected with Wolbachia confirmed that wMel-infected ovarioles exhibit far fewer developmental defects than uninfected, more closely resembling OreR wild-type (Figs 1J–1O and S5 versus Fig 1P; Vasa (Vas) = germline [44], Hu Li Tai Shao (Hts) = cytoskeletal spectrosome/fusome [45], propidium iodide (PI) = DNA). Specifically, wMel-infected mei-P26-knockdown ovarioles exhibited more normally formed cysts, consisting of somatically derived follicle cells surrounding germline-derived nurse cells and an oocyte, than uninfected ovarioles. Aberrant phenotypes included cysts lacking the follicle cell exterior (Fig 1K and 1M), the germline-derived interior (Fig 1M and 1N), or a normal number of differentiated nurse cells (Fig 1O).

These comparative data for a range of mei-P26 alleles, RNAi-knockdowns, and host ages indicate that wMel rescues mei-P26’s developmental functions in early host oogenesis. In contrast, wMel does not rescue mei-P26’s role in meiosis. Segregation defects were not rescued by wMel infection, as indicated by elevated X-chromosome nondisjunction (NDJ) rates in both infected and uninfected mei-P26[1] homozygous hypomorphs (NDJ = 7.7% and 5.6% by experiment, respectively, S6A and S6B Fig). In the following sections, we analyze wMel’s ability to rescue mei-P26 function at each of the critical time points in early oogenesis.

Host GSCs are maintained at higher rates in wMel-infected than uninfected mei-P26-deficient flies

GSCs were quantified by immunofluorescence staining of the essential GSC markers pMad and Hts in “young” 4- to 7-day-old (Fig 2) and “aged” 10- to 13-day-old females (S7A–S7C Fig). Positive pMad staining indicates the cell is responding to quiescent signals from the surrounding somatic GSC niche cells. Positive Hts staining requires localization to a single punctate spectrosome, opposed to a branching fusome [46].

Staining revealed an increase in the average number of GSCs per mei-P26[1] germarium with wMel infection, relative to uninfected germaria in young, 4- to 7-day-old flies (infected/hypomorph: 1.0 versus uninfected/hypomorph: 0.58, p< = 2.9e-4 Wilcoxon rank sum test, S8 Table), but GSC maintenance did not reach OreR wild-type rates (infected/WT: 2.3 and uninfected/WT: 1.9, p< = 1.2e-4 to 2.8e-4 Wilcoxon rank sum test, Fig 2A–2E and S8 Table). wMel-infected mei-P26[1] germaria also had more cells manifesting other GSC properties, such as a large cytoplasm and physical attachment to the cap cells of the somatic niche [46], than uninfected germaria. In contrast to the mei-P26[1] allele, RNAi knockdown of mei-P26 did not significantly affect the numbers of GSCs per germarium (infected/RNAi: 2.1 and uninfected/RNAi: 1.9, S7A–S7C Fig and S8 Table), suggesting that the modest reductions in mei-P26 dosage have a stronger impact on differentiation than GSC maintenance. OreR wild-type germaria did not exhibit different numbers of GSCs due to wMel infection (Fig 2C–2E and S8 Table).

The number of GSCs per germarium in wMel-infected mei-P26[1] females converges on OreR wild-type values in aged 10- to 13-day-old germaria (S7C Fig). As wild-type flies age, the number of GSCs per germarium declines naturally [47], even in wMel-infected flies (aged/infected/WT: 1.6 versus young/infected/WT: 2.3, p< = 0.019 Wilcoxon rank sum test, Figs 2C–2E and S7C and S8 Table). Both infected and uninfected mei-P26[1] females appear to retain some of their GSCs as they age (aged/infected/hypomorph: 1.5 and aged/uninfected/hypomorph: 0.94, p< = 0.013–0.014 Wilcoxon rank sum test, S7C Fig and S8 Table). While OreR wild-type infected and uninfected flies lose from 3% to 31% of their GSCs in the first 2 weeks, infected and uninfected mei-P26[1] flies increase their GSC abundance by 43% and 63% in this time (S7C Fig), potentially through recruiting differentiated GSCs [48]. Taking both age-dependent trajectories in GSC retention into account, by the time wMel-infected mei-P26[1] germaria are 10 to 13 days old, they contain the same average number of GSCs per germarium as wild-type flies and significantly more GSCs than uninfected mei-P26[1] flies (p< = 0.015 Wilcoxon rank sum test, S7C Fig and S8 Table). Therefore, Wolbachia enhances stem cell maintenance in both young and old mei-P26[1] females.

Infected mei-P26[1] GSCs are functional, as indicated by their ability to divide by mitosis (Fig 2F–2J and S9 Table). We identified nuclei in mitosis by positive anti-phospho-histone H3 serine 10 (pHH3) staining, a specific marker of Cdk1 activation and mitotic entry [49]. Significantly fewer GSCs were in mitosis in uninfected mei-P26[1] germaria than infected mei-P26[1] and uninfected OreR wild-type germaria (0.09% versus 6%, p = 4.7e-2 and 4.6e-2 Fisher’s exact test, Fig 2F–2J and S9 Table). There was no difference in the frequency of GSCs in mitosis between uninfected and infected wild-type germaria (5% to 6%, Fig 2H–2J and S9 Table). Mitotic cystoblasts and cystocytes were only significantly enriched in infected mei-P26[1] germaria compared to infected wild-type germaria (p< = 1.3e-2 Wilcoxon rank sum test, S7D Fig and S10 Table), despite over-replication during transit-amplifying (TA) mitosis being a common phenotype in uninfected mei-P26[1] germaria (discussed below and [50]).

Regulation of host Sxl expression is rescued in wMel-infected germaria

Proper Sxl expression is required for GSC maintenance and differentiation [51]. High levels of Sxl in the GSC maintains stem cell quiescence [52]. When the GSC divides and the CB moves away from the niche, Sxl cooperates with Bam and Mei-P26 to bind to the nos 3′ untranslated region (UTR) and down-regulate Nos protein levels to promote differentiation [50,53].

We quantified Sxl localization patterns in whole ovaries stained with anti-Sxl antibodies by confocal microscopy of whole oocytes, revealing that Sxl dysregulation due to mei-P26 loss is mitigated by wMel infection (Fig 3). Less Sxl is expressed in uninfected mei-P26[1] germarium region 1 and more Sxl is expressed across regions 2a and 2b, relative to OreR wild-type (p< = 5.7e-10 to 5.3e-5 Wilcoxon rank sum test, Fig 3A–3B’ versus 3F–3H’ and 3I, S7E Fig, and S11 Table). Infection with wMel increased Sxl expression in germarium region 1, had no impact on region 2a, and suppressed expression in region 2b relative to uninfected mei-P26[1], replicating an expression pattern similar to that seen in wild-type germaria (p< = 5.7e-10 to 8.4e-3 Wilcoxon rank sum test, Figs 3A–3B’ versus 3C–3E’ and 3I and S7E and S11 Table). Interestingly, wMel infection may impact Sxl expression in OreR germaria, as the protein is significantly elevated in region 2a of wMel-infected relative to uninfected germaria (Fig 3I). Thus, Sxl dysregulation due to mei-P26 loss can be partially rescued by the presence of wMel, suggesting that wMel’s mechanism for mei-P26 rescue may underlie how Nos regulation in wMel-infected Sxl mutants [31,34].

Fig 3 — (A–H’) Confocal mean projections of D. *melanogaster* germaria stained with antibodies against Sxl and pMad. Three phenotypes for each *mei-P26[1]* infection state are shown, (left) one as similar to wild-type as possible, (middle) one representative of GSC loss, pMad-negative, and (right) one representative of a developmentally altered phenotype retaining pMad-positive GSCs. We sampled the fluorescence channels serially and overlaid them as indicated on each set of images: cyan = anti-Sxl, red = PI, green = anti-pMad. Scale bars = 25 μm. (I) Bar-scatter plots of relative Sxl fluorescence expression levels across the germarium, by region (yellow outlines over Sxl-channel images in A’–H’). Colors as labeled in Figs 1A–1D and 2E, from left to right: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected *mei-P26[1]*, light pink = uninfected *mei-P26[1]*. Wilcoxon rank sum test * = p<0.05, ** = 0.01, *** = 0.001, **** = 1e-4. (J) Model of wMel Sxl expression rescue in *mei-P26[1]* germaria. Green dashed line represents wMel’s up-regulation of pMad expression in the GSC (Fig 2A–2E). Purple dashed line represents wMel’s up-regulation of Sxl in the GSC/CB and down-regulation of Sxl by germarium region 2b. Arrows with + symbols indicate wMel’s action on gene expression. The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C. CB, cystoblast; GSC, germline stem cell; OreR, Oregon R; PI, propidium iodide.

Bam expression is partially restored in wMel-infected mei-P26 mutant germaria

In wild-type female flies, Bam expression begins immediately after the cystoblast daughter cell moves away from its undifferentiated sister, which remains bound to the GSC niche ([50,54] and illustrated in Fig 1P). Bam binds to the fusome and stabilizes CyclinA to promote TA mitosis, producing 16 cyst cells from a single CB [55]. Immediately following these 4 mitoses, Bam expression is down-regulated in wild-type ovaries to limit germline cysts to 16 germline-derived cells.

Through anti-Bam immunofluorescence confocal imaging and quantification, we determined that wMel-infection mitigates aspects of Bam dysregulation in mei-P26[1] germaria (Fig 4). Loss of Mei-P26 deregulates and extends Bam expression past germarium region 1 (Fig 4A–4B” versus 4C–4F” and 4G; S7F Fig and S12 Table), producing excess nurse-like cells [50]. Infection with wMel enables Bam up-regulation at the CB-to-16-cell stage in mei-P26[1] germaria relative to uninfected germaria (p< = 0.016, Fig 4G). Bam may also be down-regulated in stage 2a and 2b wMel-infected mei-P26[1] germaria because uninfected germaria express significantly more Bam than wild-type (p< = 0.019 Wilcoxon rank sum test, Fig 4G and S12 Table), whereas wMel-infected germaria do not (Fig 4G and S12 Table). Unfortunately, variance in mei-P26[1] Bam fluorescence intensity among samples is too large to resolve whether uninfected and infected germaria differ in Bam expression after the 16-cell stage with this dataset.

Fig 4 — (A–F”) Confocal mean projections of D. *melanogaster* germaria stained with antibodies against Bam and pMad. Two phenotypes for each *mei-P26[1]* infection state are shown, (left) one as similar to wild-type as possible and (right) one representative of developmentally altered phenotypes. (G) Bar-scatter plots of relative Bam fluorescence expression levels across the germarium, by region (yellow outlines over Bam-channel images in A’–F’). We subdivided region 1 into GSC and CB/cystocyte subsections because Bam expression ramps up in the CB. (H) Bar-scatter plot of the ratio of relative Bam to pMad fluorescence. As pMad and Bam expression are mutually exclusive in the GSC and CB/cystocytes, respectively, OreR wild-type values are near zero. Values significantly higher than this reflect dysregulation of the stem cell state. (I) Model of wMel rescue of Bam expression in *mei-P26[1]* germaria. Colors as labeled in Figs 1A–1C, 2E and 3I, from left to right: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected *mei-P26[1]*, light pink = uninfected *mei-P26[1]*. Wilcoxon rank sum test * = p<0.05, ** = 0.01, **** = 1e-4. Scale bars = 25 μm. The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C. CB, cystoblast; GSC, germline stem cell; OreR, Oregon R.

Infection with wMel alters Bam expression in the GSC and CB to achieve an expression profile similar to and more extreme than wild-type germaria, respectively (Fig 4H and 4I). Both infected and uninfected mei-P26[1] germaria exhibit lower Bam expression than wild-type in the GSC niche (Fig 4G). This contrasts with expectations from the literature: bone morphogenic protein (BMP) signaling from the somatic niche induces pMad expression in the GSC, which directly represses Bam transcription, preventing differentiation [56]. Loss of mei-P26 function in the GSC should derepress Brat, resulting in pMad repression and inappropriate Bam expression [38]. However, we see lower Bam expression in both wMel-infected and uninfected mei-P26[1] GSCs relative to OreR wild-type (p< = 1.3e-6 to 1.4e-3 Wilcoxon rank sum test, Fig 4G and S13 Table). The elevated relative Bam/pMad expression ratio in uninfected mei-P26[1] GSCs (p< = 2.5e-4 to 4.0e-4 Wilcoxon rank sum test, Fig 4H and 4I and S13 Table) may explain this departure from expectations. Although GSC Bam expression is lower than in wild-type, Bam expression is clearly dysregulated and up-regulated relative to pMad expression, as expected for germline cells that have lost their stem cell identity [57,58]. Normalizing against pMad expression also reinforces the finding that wMel elevates Bam expression even higher than wild-type levels in mei-P26[1] hypomorphs at the CB-to-16-cell stage (p< = 1.2e-6 to 2.0e-2 Wilcoxon rank sum test, Fig 4H and 4I and S13 Table).

wMel rescues normal germline cyst and oocyte development impaired by mei-P26 knockdown

Mei-P26-deficient flies over-proliferate nurse cells and partially differentiate GSCs to produce tumorous germline cysts ([38,39] and Fig 5A–5D versus 5E–5H, Fig 5I and S14 Table). In RNAi knockdown ovaries, the formation of tumorous germline cysts is significantly mitigated by wMel infection (35.3% versus 16.3% of cysts with more than 15 nurse cells, respectively; p = 4.5e-3 Fisher’s exact test, Fig 5E and 5F versus 5G and 5H and S14 Table). In contrast, germline cyst tumors found in mei-P26[1] allele germaria were not rescued by wMel infection, despite the bacterium’s ability to rescue the rate at which these eggs hatch into progeny (Fig 5I versus Fig 1A and 1C), but consistent with their dysregulation of Bam expression in germarium regions 2a and 2b (Fig 4G). The stronger nature of the mei-P26[1] allele relative to the nos-driven RNAi knockdown likely underlies the difference in the number of tumorous cysts. Lack of tumor rescue indicates that either tumorous cysts produce normal eggs at some rate or wMel infection rescues tumors later in oogenesis, perhaps through some somatic mechanism [59–62].

Overexpression of mei-P26 produces tumors in both uninfected and infected ovaries; however, infected ovaries exhibit significantly more tumors than uninfected ovaries (p = 1.6e-3 Fisher’s exact test, Fig 5I and S14 Table). Finding that an excess of mei-P26 recapitulates the tumorous phenotype induced by a lack of mei-P26 indicates that this gene exerts a concentration-dependent dominant negative effect on its own function. Given that infection with wMel makes this dominant negative phenotype more severe, we hypothesize that wMel may make a factor that acts to enhance Mei-P26 function, potentially through a direct interaction or redundant function.

Infection with wMel increases the rate of oocyte-specific Orb localization in mei-P26[1] germline cysts (Figs 5J–5R and S7G and S15 Table), suggesting that wMel rescues functional cyst formation. Oocyte specification in region 2a of the germarium (see diagram in Fig 1P) is inhibited by the loss of mei-P26 through the derepression of orb translation [38]. Mei-P26 binds to the orb 3′ UTR and prevents its translation. Following cyst patterning and development, one of the 16 germline-derived cells is designated as the oocyte through specific oo18 RNA-binding protein (Orb) expression [63]. In region 2a, the germline cyst cell fated to become the oocyte activates orb translation [38]. Loss of mei-P26 results in orb derepression and nonspecific expression [38]. Infection with wMel restores oocyte-specific Orb expression in mei-P26[1] cysts relative to uninfected cysts (p = 5.9e-9 Fisher’s exact test, Fig 5L–5N’ versus 5O–5Q’ and S15 Table). We observed oocyte-specific Orb staining 93% to 94% of the time in wild-type cysts, whereas mei-P26[1] reduced this frequency to 25%, and wMel infection recovered mei-P26[1] cysts to 61% (counts in S15 Table). Oocyte-specific expression of Orb in wMel-infected cysts is robust, even in those that are developmentally aberrant (e.g., the double oocyte in Fig 5M–5M’). These results suggest that wMel rescues oocyte-specific Orb translation enhancing or duplicating Mei-P26’s function in orb translational repression.

OreR wild-type host fertility is beneficially impacted by wMel infection

Consistent with wMel’s ability to rescue the partial to complete loss of essential germline maintenance genes encompassing a range of functions, we discovered that this intracellular symbiont can reinforce fertility in D. melanogaster stocks displaying full wild-type fertility (Figs 1C and S8A–S8C and S4–S6 Tables). We analyzed egg lay, egg hatch, and overall offspring production rates independently to detect wMel effects on multiple components of fertility. Overall offspring production per female per day was elevated in wMel-infected nos:Gal4/CyO females relative to uninfected females by 49% (27 (uninfected) versus 40 (infected) offspring/female/day, p< = 2.2e-3 Wilcoxon rank sum test, S8A Fig and S4 Table). Infection with wMel increased the number of eggs laid per female per day for the nos:Gal4/CyO balancer stock by 49% (42 versus 28 eggs/female/day, p< = 2.2e-3 Wilcoxon rank sum test, S8B Fig and S5 Table), but did not have any detectable effect on egg lay rates in other genotypes with wild-type fertility. Following embryogenesis, infection with wMel elevated the rate that OreR and nos:Gal4/CyO wild-type eggs hatch into L1 stage larvae by 5.2% and 2.5%, respectively (88% and 91% (infected) versus 83% and 89% (uninfected) of eggs hatched, p< = 1e-4 to 2.1e-3 Wilcoxon rank sum test, Figs 1C and S8C and S6 Table). These results suggest that beneficial impacts of wMel infection are evident, yet variable among D. melanogaster stocks with wild-type fertility.

We measured OreR fertility across its lifetime (approximately 50 days) and found that wMel may produce higher lifetime fecundity relative to uninfected flies (Fig 6A–6C and S7 Table). OreR females were aged as indicated along the x-axis of Fig 6A–6C and mated to 3 to 7 days old males of matching infection status. wMel-infection maintains elevated OreR egg hatch rates as flies age (p< = 7.1e-9 Kolmogorov–Smirnov test, Fig 6C and S7 Table), while maintaining similar levels of egg production as uninfected flies (Fig 6B and S7 Table). This culminates in age ranges in which infected flies may produce an excess of offspring relative to uninfected flies (see gap between the regression confidence intervals when flies were approximately 20 to 30 days old, Fig 6A). The efficiency gains imparted on hosts by infection-induced elevated hatch rates could potentially accumulate for higher lifetime fitnesses, as resources are more efficiently converted to offspring.

Fig 6 — (A–C) D. *melanogaster* OreR fecundity versus female age, fit with a local polynomial regression (dark gray bounds = 95% confidence intervals). Infection with wMel may elevate (A) offspring and (B) egg offspring production at time points during the host lifespan through continuously elevating (C) hatch rate. (D–F’) Confocal mean projections of D. *melanogaster* (D–D’) infected and (E–E’) uninfected germaria and (F–F’) an infected ovariole. Intracellular wMel FtsZ localizes to germline (Vas+, outlined in dashed yellow) and somatic (Vas-) cells at high titers in infected OreR wild-type flies, with low background in uninfected flies. Bacteria in the GSC (* near the Hts-bound spectrosome) are indicated with arrows. Fluorescence channels were sampled serially and overlaid as indicated on each set of images as follows: cyan = anti-Hts, yellow = anti-Vas, blue = anti-FtsZ, and red = PI. Scale bars F–G = 10 μm, H = 50 μm. The data underlying this figure can be found in S3 Table and on Dryad at doi.org/10.7291/D1DT2C. GSC, germline stem cell; OreR, Oregon R; PI, propidium iodide.

Immunostaining of anti-FtsZ confirms that wMel exhibits high titers in the OreR wild-type germarium and concentrates in the oocyte in early cysts (Fig 6D–6F’). The intracellular bacteria are continuously located in the germline, starting in the GSC and proceeding through the end of embryogenesis and fertilization. Thus, wMel are located in the right place at the right time to reinforce female host fertility through interactions with the GSC and oocyte cyst, as well as to correct for perturbations caused by male CI-related alterations [64].

The wMel strain of Wolbachia in its native D. melanogaster host exhibits weak CI that decreases with age (S9A–S9D Fig), suggesting low maintenance of CI mechanisms [8]. Mating wMel-infected OreR males to infected (a “rescue cross”) and uninfected OreR virgin females (a “CI cross”) revealed that wMel reduces uninfected egg hatch by 26% when males are 0 to 1 day old (p< = 4.6e-4 Wilcoxon rank sum test, S9A Fig and S6 Table), but this moderate effect is eliminated by the time males are 5 days old, on average (S9C Fig and S6 Table). Only young males significantly impact offspring production (p< = 1.7E-2 Wilcoxon rank sum test, S9B and S9D Fig and S4 Table). In contrast, the Wolbachia wRi strain that naturally infects Drosophila simulans (the Riv84 line) and induces strong CI [10,65], reduces uninfected egg hatch by 95% when males are 0 to 1 day old (p< = 5.6e-8 Wilcoxon rank sum test, S9E Fig and S6 Table) and only loses some of this efficacy by 5 days (75% hatch reduction, p< = 3.6e-4, Wilcoxon rank sum test; S9G Fig, S6 Table). This significantly impacts overall offspring production (p< = 6.5E-7 and 5.0E-2 Wilcoxon rank sum test, S9F and S9H Fig and S4 Table). Paternal grandmothers of CI offspring were 3 to 7 days old (i.e., relatively young), contributing to weak CI in the D. melanogaster crosses [17]. Overall, these results suggest that wMel’s beneficial reproductive manipulations may affect its fitness more than its negative reproductive manipulations in nature.

wMel-mediated mei-P26[1] rescue may be enabled through rescuing and perturbing host transcription

Exploring wMel’s effect on host gene expression in OreR and mei-P26[1] ovaries with dual-eukaryotic and bacterial RNA sequencing (dual-RNAseq) revealed that infection significantly alters host gene expression in a variety of ways (Fig 7A–7C and S16–S19 Tables), which indicate a few potential mechanisms for wMel-mediated fertility rescue. Of the 35,344 transcripts in the D. melanogaster genome, 10,720 were expressed at high enough levels in at least 5 of 6 samples of each genotype+infection group to be included in the analysis. Of the 1,286 genes in the wMel genome, 663 were expressed in D. melanogaster ovaries (Fig 7D and S20 Table).

Fig 7 — (A–D) Volcano plots of log2-fold change in gene expression versus log10-FDR adjusted p-value (padj) for the test performed in each panel. (E–K) Normalized transcript count plots for (E) the 3 top genotype-associated (~G) Wald Test hits by padj, and (F–K) D. *melanogaster* genes significantly associated (padj< = 1e-3) with infection (~I) or jointly, genotype and infection (*~G*I*), organized by the differential expression pattern. See S10–S15 Figs for other significant and insignificant count plots. In all barplots, dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected *mei-P26[1]*, light pink = uninfected *mei-P26[1]*. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602. OreR, Oregon R.

Both uninfected and wMel-infected hypomorphic mei-P26[1] ovaries exhibited a 5-fold depletion in mei-P26 expression relative to OreR ovaries (S2B and S2D Fig) and significant dysregulation of 1,044 other genes due to the mei-P26[1] allele after FDR correction (Wald test FDR-adjusted p-value (padj)< = 0.05, Figs 7A, 7E and S10A and S17 Table). The changes in gene expression due to the loss and dysfunction of mei-P26 far outnumber and outpace the changes in gene expression due to infection, in either D. melanogaster genotype, and highlight how important mei-P26 is as a global regulator of gene expression. This number is well in excess of the 366 genes thought to be involved in GSC self-renewal [66], underscoring how many processes mei-P26 is involved in, from germline differentiation [50] to somatic development [61]. Across biological process, cell component, and molecular function GO categories, loss of mei-P26 impacted processes involving chromatin, recombination, protein–protein interactions, and muscle cell differentiation (S10B–S10G Fig), supporting mei-P26’s known role in genetic repression, differentiation, and meiosis [38,40,50].

Infection with wMel significantly alters the expression of hundreds of D. melanogaster genes to either restore or compensate for mei-P26-regulated gene expression. We tested for genes significantly associated with infection state in DESeq2 under the full model: ~genotype + infection + infection*genotype, revealing 422 significantly differentially expressed genes (Wald test padj< = 0.05, Fig 7B and S18 Table). Testing for an effect of the interaction between infection and genotype yielded another 20 significantly differentially expressed genes (Wald test padj< = 0.05; Fig 7C and S19 Table). The most significantly differentially expressed genes are featured in Fig 7F–7K and other significant genes are plotted in Figs 8, S11 and S12. Binning gene expression patterns revealed that there are at least 6 main rescue gene expression phenotypes induced by wMel-infection in mutant ovaries: full rescue, overshoot, undershoot, wild-type effect, inverse, and novel (Figs 7F–7K, 8A, 8C, S11A, S11B, S12A and S12B). In “full rescue” phenotypes (Figs 7F and S11A), mei-P26[1] wMel gene expression is indistinguishable from OreR levels and significantly different from uninfected mei-P26[1] levels. “Overshoot” phenotypes (Fig 7G) exhibit expression that is significantly in the opposite direction of the uninfected mei-P26[1] knockdown effect relative to OreR expression. Oppositely, “undershoot” phenotypes (Fig 7H) are partial corrections towards OreR expression in mei-P26[1] wMel ovaries. The one “wild-type effect” gene, an ER and Golgi cytoskeletal membrane anchor protein, demonstrated differential expression only between wMel-infected and uninfected OreR ovaries (Fig 7I). Five genes exhibit “inverse” differential expression phenotypes, such as a sodium symporter (Fig 7J) and the transcription factor Chronophage (S12A Fig), in which mei-P26[1] and OreR ovaries show opposite changes in differential expression relative to the uninfected genotype. In total, only these 6 “wild-type effect” and “inverse” genes were differentially expressed due to infection in OreR ovaries. Lastly, “novel” genes (Fig 7K) are those that only exhibit differential expression in wMel-infected mei-P26[1] ovaries, suggesting a novel rescue or compensation pathway. For example, suppression of retinal degeneration disease 1 upon overexpression 2 (sordd2) ubiquitin ligase mRNA expression is up-regulated, without rescue, in both infected and uninfected mei-P26[1] ovaries (Fig 7E). One of the novel rescue candidates, SKP1-related C (skpC), is a down-regulated component of SCF ubiquitin ligase (Fig 7K), which might compensate for the up-regulation of sordd2.

Fig 8 — (A, B) Predicted *mei-P26* (A) protein and mRNA physical interactions and (B) genetic interactions. Symbols near gene names indicate significant DE interactions: * = significance by genotype; r = significance by infection, indicating a rescue phenotype; _ = significance by uncorrected p-values only. Red “X”s in A indicate genes that were not expressed in our dataset (CG6304 and mir-137). (C) Normalized transcript count plots for the genes designated with symbols in A and B. In all barplots, dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected *mei-P26[1]*, light pink = uninfected *mei-P26[1]*. (D) Cellular component GO terms for the 87 *(~G*I)* DE genes reveal an enrichment for early germline cytoskeletal structures and membrane proteins. Wald test association for listed padj and p-values are given in parentheses, as in Fig 7: genotype (~G), infection (~I), genotype*infection (*~G*I*). The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602. OreR, Oregon R.

GO terms associated with infection and the interaction between infection and genotype (Figs S11C–S11G, S12C–S12G and 8D) were consistent with wMel’s known ability to associate with host actin [67], microtubules, motor proteins [68–70], membrane-associated proteins [71], and chromatin [72]. Infection-associated genes were enriched in GO terms suggesting a cytoskeletal developmental function with chromatin interactions (S11C–S11G Fig). Across biological processes, cellular compartment, and molecular function categories, genes were enriched in terms suggesting interactions with actin, myosin, contractile fibers, and muscle cell differentiation. Chromatin interactions were indicated by associations with condensin complexes, centromeres, and mitotic chromosomes. Genotype-by-infection interaction-associated genes were also enriched in cytoskeletal and chromatin-interaction GO terms, in addition to plasma membrane/cell junction-associated terms such as cell junction and synapse structure maintenance, cell cortex, and lipid and calmodulin binding (S12C–S12G Fig).

Interestingly, 2 chorion proteins (Fig 7F), an ecdysteroid 22-kinase associated protein (S11A Fig), and an estrogen-related receptor (ERR) (S12A Fig), which are likely involved in chorion formation [73], were rescued by wMel. Considering that we have found mei-P26[1] embryos to have weaker corions then OreR wild-type embryos when treated with bleach, this finding suggests a novel chorion-associated function for mei-P26 and a novel rescue phenotype for wMel.

None of the 663 transcribed wMel genes were significantly differentially expressed between the OreR and mei-P26[1] host genotypes, after p-values were FDR-corrected (Figs 7D, S13 and S14 and S20 Table), suggesting host regulation by wMel’s constitutively expressed genes. On average, wMel transcriptomes were sequenced at 7.8 to 42.4× depth of coverage and D. melanogaster transcriptomes were sequenced at 25.9 to 35.9× (S16 Table). While these coverage ranges overlap, the 663 wMel genes detected only spanned −1.5-fold depletion to 1.3-fold up-regulation (S20 Table). In contrast, the 10,720 expressed D. melanogaster genes spanned an order of magnitude more differential expression, with −10.041-fold depletion to 9.866-fold up-regulation (S17–S19 Tables). Thus, wMel genes are weakly differentially transcribed compared to D. melanogaster genes, suggesting that wMel may alter host phenotypes through downstream impacts of constitutively expressed proteins. To explore the constitutive effects of wMel expression, we tested for GO pathway enrichment in the expressed transcriptome (S13 Fig). Pathways for DNA replication, protein expression, membrane transport, and heterotrophy (e.g., oxidative phosphorylation) were detected in abundance. While many of these genes are likely involved in normal bacterial cell maintenance and replication, a significant enrichment in pathways for oxidative phosphorylation, translation, transcription, and membrane functions (S13A Fig) suggest that some of these genes could be co-opted for host manipulation or processing host resources.

Among the 1,044 significantly differentially regulated D. melanogaster genes impacted by mei-P26 knockdown and infection were 6 genes (of 24) predicted in the literature (padj< = 0.05 Wald test and 9 of 24 by p-value< = 0.05; Figs 8 and S1 and S1 and S2 Tables). Loss of mei-P26 function caused dysregulation in oo18 RNA-binding protein (orb), gawky (gw), sex-lethal (sxl), benign gonial cell neoplasm (bgcn), tumorous testis (tut), and bantam (mir-ban) (padj< = 0.05 Wald test, Fig 8C and * in Fig 8A and 8B). Bam, Pumilio (pum), and Justice Seizure (jus) expression may be altered by mei-P26 knockdown, but additional samples are needed to resolve high infection variance (e.g., mir-ban) and low differential expression change (e.g., gw) (p-value< = 0.05 Wald test, Fig 8C and “*” in Fig 8A and 8B). Two of 6 genes exhibited evidence of wMel rescue with an “undershoot” rescue phenotype (“r” in Fig 8A and 8B): bgcn and tut by padjust (Fig 8C). If we consider genes with significant unadjusted p-values, gw may also exhibit evidence of “undershoot” rescue (Fig 8C, “r” in Fig 8A). Bantam or mir-ban expression suppression may be rescued as well, but high variance among wMel mei-P26[1] ovaries precludes DE rescue detection with this dataset (Fig 8C). In contrast, orb, sxl, bam, and nos transcript levels are not altered by mei-P26 knockdown or wMel infection, and therefore must be regulated posttranscriptionally. In total, these differential expression results reinforce and expand our understanding of how wMel rescues and reinforces host reproduction at transcriptional and posttranscriptional levels.

Discussion

The wMel strain of Wolbachia is a successful and widespread symbiont: naturally in D. melanogaster populations [19,20] and novelly through lab-generated trans-infections of wMel into non-native hosts used for biological control [3]. However, we know remarkably little about the processes that shape how symbiont and host coevolve and find mutually beneficial mechanisms for their reproduction. In this work, we confirm that wMel’s CI strength is typically weak in D. melanogaster, (Fig 9A–9D, [14–18]) and show that wMel confers benefits on host fertility through reinforcing host GSC maintenance and gamete differentiation (Figs 1–8).

Fig 9 — (A) In *mei-P26* mutants, wMel restores normal Bam, Sxl, and Orb expression through various mechanisms, most of which are unknown (??? = unknown wMel factors). Protein and mRNA expression rescue correlates with cytological rescue from GSC maintenance through germline cyst differentiation. (B) Sxl rescue in the GSC is mediated through TomO’s interaction with RNPs containing *nos* mRNA, up-regulating Nos translation. Later, in stage 8 of cyst oogenesis, TomO was found to bind *orb* mRNA, displacing the translational repressor Cup, and up-regulating Orb translation [34,35]. Dashed arrow in A indicates TomO’s predicted function in enabling Orb derepression in early cyst differentiation. The factor that restores *mei-P26* repression of Orb translation in *mei-P26* mutants has not been identified. GSC, germline stem cell; RNP, ribonucleoprotein; TomO, toxic manipulator of oogenesis.

Here, we demonstrate that wMel infection can reinforce host fertility in both OreR wild-type and mei-P26 mutants by correcting perturbed gene expression patterns, and we shed light on the long-standing mystery of how wMel beneficially impacts host reproduction. Previously, wMel was shown to rescue the partial loss of Sxl [31] and Bam [32], 2 essential genes for 2 very different stages of early oogenesis: GSC maintenance and CB differentiation and TA-mitosis, respectively. These findings left unresolved how wMel suppresses these mechanistically and temporally distinct cellular processes and suggested that mei-P26 was not involved [31]. We show with an array of alleles and developmental assays that infection with wMel rescues defects associated with mutant mei-P26, a gene required for both GSC maintenance and differentiation. Infection with wMel rescues all stages of oogenesis in mei-P26 RNAi knockdowns, as well as hypomorphic and null alleles (Figs 1–5, 7, 8, S4–S7 and S10–S12 and S4–S15 and S18 and S19 Tables). Mei-P26 is a TRIM-NHL protein that regulates gene expression via mRNA translational inhibition through the Nos mRNA-binding complex, interactions with the RNA-induced silencing complex (RISC), and protein ubiquitination [38,74]. Importantly, Mei-P26 interacts with Sxl in the GSC and CB and Bam in the CB and cystocytes [36,50]. These interactions suggest that the mechanism wMel employs to rescue mei-P26 function may also be responsible for rescuing aspects of Sxl-dependent GSC maintenance and CB differentiation and Bam-dependent cyst differentiation.

In this in-depth molecular and cellular analysis of wMel-mediated manipulation of GSC maintenance and differentiation, we discovered that wMel rescues mei-P26 germline defects through correcting perturbed pMad, Sxl, Bam, and Orb protein expression and bgcn and tut mRNA expression towards OreR wild-type levels (diagrammed in Fig 9A). Infection with wMel partially rescued GSC-specific pMad expression relative to wild-type flies (Fig 2A–2E), suggesting that the bacteria restored BMP signaling from the somatic GSC niche [38]. Finding that these cells are also able to divide (Fig 2F–2J), further supports their functionality and stemness. Given that mei-P26 is likely involved in Drosophila Myc (dMyc) regulation, and overexpression of dMyc induces competitive GSCs [75], wMel’s GSC rescue mechanism may involve dMyc up-regulation. Although, our transcriptomic data indicate that dMyc is not significantly repressed at the transcriptional level (S15 Fig). In the germline cells that left the niche, wMel recapitulated the properly timed changes in pMad, Sxl, and Bam expression that are normally influenced by mei-P26 and are required during cystoblast differentiation to form the 16-cell germline cyst [39,40,50,74] (Figs 3, 4, and S7E–S7F). While Bam down-regulation is not fully rescued by wMel (Fig 4), finding that tut mRNA expression is partially reduced in wMel-infected mei-P26[1] germaria (Fig 8) suggests that there may be an unreported regulatory interaction between Tut, Bam, and Mei-P26 in females (as in males [76]), which could rescue the overexpression of Bam in late germline cyst development.

This is the first time wMel has been found to rescue oocyte differentiation post-cyst formation, suggesting that wMel has pervasive impacts throughout oogenesis that may reinforce embryogenesis. We confirmed the downstream consequences of Bam expression rescue in wMel-infected mei-P26[1] ovaries by finding fewer germline cyst tumors in infected flies (Fig 5A–5I). These germline cysts appeared to be functional, based on restored oocyte-specific expression of the essential oocyte differentiation protein Orb in infected mei-P26[1] cysts, relative to uninfected cysts (Fig 5J–5R). Finding that wMel rescues Cp16 and Cp19 chorion protein, tut, bgcn, and other transcript expression in ovary tissues (Figs 7F, 8, S11 and S12) further supports the conclusion that wMel can rescue the downstream impacts of mei-P26 loss. Future work is needed to explore how the overshoot and novel DE categories mitigate the loss of mei-P26, which may occur through some alternate or compensatory pathway. In addition to the mechanism discussed previously for ubiquitin ligase rescue, the loss of Ionotropic receptor 76a (Ir76a) or nicotinic Acetylcholine Receptor α5 (nAChRα5) expression (S10A Fig) could be rescued by the novel up-regulation of the Wunen-2 lipid phosphate phosphatase as a receptor or membrane transporter in wMel-infected mei-P26[1] ovaries (S12B Fig). Functions like these could underlie wMel-induced developmental resilience in OreR embryogenesis, as demonstrated by elevated hatch rates in wMel-infected OreR flies (Figs 1C and 6C).

The wMel strain’s rescue of D. melanogaster mei-P26 is likely independent of the Wolbachia TomO protein. Prior work on TomO indicates that the bacterial protein’s primary mode of action is through destabilizing ribonucleoprotein (RNP) complexes [35] (diagrammed in Fig 9B). This is how TomO elevates Nos expression in the GSC [34] and how TomO inhibits the translational repressor Cup from repressing Orb translation in mid-oogenesis (stage 8) [35,77]. In contrast, Mei-P26 interacts with Ago1 RISC to inhibit Orb translation through binding the orb ′3-UTR miRNA-binding sites [38]. Although Mei-P26 and TomO produce opposite outcomes for Orb expression and TomO is not significantly up-regulated in mei-P26[1] ovaries (S14C Fig), TomO-orb binding may underlie wMel’s ability to rescue Orb repression, if wMel produces other factors that modulate the interaction. Alternatively, wMel may make another RNA-binding protein. Given that Sxl is also repressed through the sxl 3′-UTR by Bruno [78], such a protein could be involved in restoring Sxl regulation in mei-P26 mutants. Neither orb nor sxl expression is rescued at the mRNA level by wMel infection (Fig 8C), lending further support for rescue at the translational or protein interaction/modification level.

Additional bacterial factors besides TomO must be involved in reinforcing host fecundity in wild-type flies and fertility mutants. First, Mei-P26 encodes 3 functional domains that confer at least 3 different functional mechanisms for modulating gene expression (mRNA-binding, miRNA-mediated, and ubiquitination). In a bacterial genome, these domains/functions are likely to be encoded by more than 1 protein [79]. Second, TomO rescues Sxl’s GSC maintenance functions by binding nos mRNA, increasing levels of Nos translation, but it cannot fully rescue Sxl loss [34]. Immediately after the GSC divides to produce the CB, Sxl interacts with Bam, Mei-P26, Bgcn, and Wh to inhibit Nos translation [36,74]. Thus, a wMel factor that inhibits Nos translation, counteracting TomO’s function to destabilize Nos translational repressors through RNA binding [34,35], remains to be identified. Furthermore, Nos up-regulation cannot explain Bam rescue because Nos and Bam exhibit reciprocal expression patterns during TA mitosis [36]. Third, exacerbation of the tumor phenotype induced by nos:Gal4-driven Mei-P26 overexpression by wMel infection (Fig 5I) suggests that wMel synthesizes a protein that directly mimics Mei-P26’s functions in differentiation and can reach antimorphic levels. Misregulated expression of a mei-P26-like factor could explain why wMel-infected mei-P26 RNAi females produce more eggs and offspring than OreR wild-type flies (Fig 1A and 1B). While TomO may recapitulate some of Mei-P26’s functions, such as orb-binding, at least one other factor is needed to recapitulate proper Orb, Nos, and Bam regulation.

Understanding how Wolbachia interacts with host development at the molecular level is essential to deploying these bacteria in novel hosts and relying on vertical transmission to maintain them in host populations. CI has been a powerful tool for controlling host populations [80] and driving Wolbachia to high infection frequencies [3]. The continuous presence of bacterial reproductive manipulators in host GSCs opens the opportunity for compensatory developmental functions to evolve, similar to what we have shown for mei-P26. While it is not known if any of the naturally occurring mei-P26 alleles [32,81] confer loss of function that is remedied by the presence of Wolbachia, natural variation in developmental genes theoretically could have provided the selection pressure for wMel to evolve its beneficial reproductive functions. Given that selection will favor the loss of CI if conflicting beneficial impacts on host fertility are realized [8], it is imperative that we understand the mechanisms underlying beneficial reproductive manipulations such as fertility reinforcement.

Materials and methods

Russell and colleagues Wolbachia endosymbionts manipulate GSC self-renewal and differentiation to enhance host fertility

Candidate gene selection

Leveraging what is known about wMel’s abilities to rescue essential maintenance and differentiation genes [31,32] with empirical data on protein–protein and protein–mRNA interactions among these genes (esyN [82] networks in Figs S1A–S1D and 8; references in S1 Table) and data on interactions between host genes and wMel titers [37], we identified the essential germline translational regulator meiotic P26 (mei-P26) as a potential target of bacterial influence over GSC maintenance and differentiation pathways (S1A and S1B Fig). Loss of mei-P26 in uninfected flies produces mild to severe fertility defects through inhibiting GSC maintenance, meiosis, and the switch from germline cyst proliferation and differentiation [38–40].

Drosophila stocks and genetic crosses

Flies were maintained on white food prepared according to the Bloomington Drosophila Stock Center (BDSC) Cornmeal Food recipe (aka “white food,” see https://bdsc.indiana.edu/information/recipes/bloomfood.html). The wMel strain of Wolbachia was previously crossed into 2 D. melanogaster fly stocks, one carrying the markers and chromosomal balancers w[1]; Sp/Cyo; Sb/TM6B, Hu and the other carrying the germline double driver: P{GAL4-Nos.NGT}40; P{GAL4::VP16-Nos.UTR}MVD1. These infected double balanced and ovary driver stocks were used to cross wMel into the marked and balanced FM7cB;;;sv[spa-pol], null/hypomorphic mutants, and UAS RNAi TRiP lines to ensure that all wMel tested were of an identical genetic background. The D. melanogaster strains obtained from the BDSC at the University of Indiana were: Oregon-R-C (#5), w1118; P{UASp-mei-P26.N}2.1 (#25771), y[1] w[*] P{w[+mC] = lacW}mei-P26-1 mei-P26[1]/C(1)DX, y[1] f[1]/Dp(1;Y)y[+]; sv[spa-pol] (#25716), y[1] w[1] mei-P26[mfs1]; Dp(1;4)A17/sv[spa-pol] (#25919), y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8] = TRiP.GL01124}attP40 (#36855). We obtained the UASp-mRFP/CyO (1-7M) line from Manabu Ote at the Jikei University School of Medicine. Drosophila simulans w[–] stocks infected with the Riv84 strain of wRi and cured with tetracycline were sourced from Sullivan Lab stocks [83,84]. All fly stocks and crosses were maintained at room temperature or 25°C on white food (BDSC Cornmeal Food) because the sugar/protein composition of host food affects Wolbachia titer [84].

Wild-type fertility stocks: Paired wMel-infected (OreR_wMelDB) and uninfected Oregon R (OreR_uninf) stocks were made by crossing males of Oregon-R-C to virgin females from paired infected and uninfected balancer stocks of the genotype w[1];CyO/Sp;Hu/Sb. To ensure that no differences arose between these 2 D. melanogaster genotypes during the balancer cross or subsequently, we backcrossed males of the OreR_wMelDB stock to females of the OreR_uninf stock for 10 generations and repeated egg lay and hatch assays (F10_OreR_uninf). Paired infected and uninfected nosGal4/CyO and nosGal4/Sb flies were made by crossing to paired infected and uninfected balancer stocks, as described above.

The genomic and phenotypic consequences of the mei-P26 alleles studied here and previously (e.g., [31]) have been characterized [40]. The hypomorphic mei-P26[1] allele was created by the insertion of a P{lacW} transposon in the first intron of mei-P26, which codes for the RING domain. The mostly null mei-P26[fs1] allele tested by Starr and Cline in 2002 was generated by a second P{lacW} insertion in mei-P26[1]’s first insertion. The fully (male and female) null mei-P26[mfs1] allele arose by deleting the P{lacW} insertion from mei-P26[1], along with approximately 2.5 kb of flanking sequence. According to Page and colleagues (2000), these 3 alleles form a series, with increasing severity: mei-P26[1] < mei-P26[fs1] (and other fs alleles) < mei-P26[mfs1].

Fecundity crosses

Overview

SLR performed the 3,002 fecundity crosses continuously between October 2020 and January 2022, in batches based on their eclosion date (Table 1). Infected and uninfected OreR lines were maintained continuously to (1) control the age of grandmothers of CI males; (2) regularly have OreR virgins for mei-P26 and CI fecundity assays; (3) collect and age WT females for the aged fertility assay; and (4) obtain large sample sizes from mei-P26 fertility mutants. The full fecundity dataset is contained in S2 Table and plotted in S3 Fig.

Table 1. Fecundity cross genotypes, sexes, ages, and control crosses.

Fecundity cross category	Female age	Male age	Parallel control crosses
wMel-infected mei-P26 rescue	3–7 days old	3–7 days	Uninfected and wild-type
wMel-infected OreR wild-type	3–7 days old	3–7 days	Uninfected OreR wild-type and other “wild-type” fertility stocks (e.g., nos:Gal4/CyO)
Aged wMel-infected mei-P26 rescue	2–25 days old	3–7 days	Uninfected and wild-type
Aged wMel-infected OreR wild-type	2–45 days old	3–7 days	Uninfected OreR wild-type
CI in wMel-D. melanogaster OreR	3–7 days old	0 or 3–7 days (from 3- to 7-day-old mothers)	Rescue, reciprocal, and uninfected
CI in wRi-D. simulans w[–]	3–7 days old	0 or 3–7 days (from 3- to 7-day-old mothers)	Rescue

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CI, cytoplasmic incompatibility; OreR, Oregon R.

Cross conditions

Food (vials of Bloomington’s white food recipe), laying media (grape food), and incubation conditions were made in-house in large batches monthly. Grape spoons: Approximately 1.5 mL of grape agar media (1.2× Welch’s Grape Juice Concentrate with 3% w/v agar and 0.05% w/v tegosept/methylparaben, first dissolved at 5% w/v in ethanol) was dispensed into small spoons, allowed to harden, and stored at 4°C until use. Immediately prior to use in a cross, we added ground yeast to the surface of each spoon.

Preparation of flies for fecundity crosses

Paired wMel-infected and uninfected genetic crosses were performed in parallel for the 4 mei-P26 mutants (RNAi, [mfs1], [1], [mfs1/1]) to produce homozygous virgin females of both infection states for parallel fecundity crosses. Mutant genetic crosses were performed multiple (3 or more) times across the 14 months to produce homozygotes from many different parents and matings. CI crosses were performed with males from infected grandmothers that were 3 to 7 days old. Males were aged either 0 (collected that day) or 5 days, depending on the cross. We collected males from vials over multiple days, so the majority of males were not the first-emerged.

Virgin female flies collected for fecundity crosses were transferred to fresh food and aged an average of 5 days at room temperature (from 3 to 7), or longer for the aged OreR, mei-P26 RNAi, and mei-P26[1] fecundity crosses. Males and virgin female flies were stored separately, and males were aged 0 or 3 to 7 days. Long-term aged virgin females were kept as small groups in vials of fresh white food. Every few days, the vials were inspected for mold and flies were moved on to fresh food.

Fecundity cross protocol

In the afternoon of the first day of each cross, 1 male and 1 female fly were knocked out on a CO₂ pad, added to a vial containing a grape spoon, allowed to recover, and then transferred to a 25°C constant humidity incubator on a 12 light/dark cycle to allow for courting and mating. The following day, each spoon was replaced with a fresh spoon, and the vials were returned to 25°C for more mating. On the third day, we removed the flies from the vials, counted the number of eggs laid on the spoon’s grape media, and replaced the spoon in the vial at 25°C for 2 days. After approximately 40 h, we counted the number of hatched and unhatched eggs. The exact times and dates for all steps in all crosses were recorded (S2 Table) and plotted to show consistent results across the time frame (S3 Fig).

Fecundity cross analysis

Fecundity data were parsed using perl scripts and plotted in R. Individual crosses were treated as discrete samples. Hatch rates were calculated from samples that laid 20 or more eggs (for 3- to 7-day-old female plots) and 10 or more eggs (for age versus hatch rate plots). Egg lay and offspring production rates were calculated from raw counts, divided by the fraction of days spent laying (metadata in S2 Table). Crosses with 0 laid eggs (and offspring) were not rejected, as the fertility mutants often laid 0 to few eggs. Our cross conditions were highly consistent and run daily, lending confidence to these zero-lay/offspring samples (S3 Fig). Drosophila fecundity versus female age data were fit in R using the stat_smooth loess method, formula y~x, with 0.95 level confidence interval.

Non-disjunction assays

We placed females homozygous for the mei-P26[1] allele bearing the yellow mutation (y[1] w[*] mei-P26[1];;; sv[spa-pol]) in vials with males bearing a wild-type yellow allele fused to the Y chromosome (y[1] w[1] / Dp(1;Y)y+). We made 7 or 8 vials of 1 to 10 females mated to a count-matched 1 to 10 males for infected and uninfected mei-P26 hypomorphs, respectively. After eclosion, we screened for non-disjunction in the progeny by the presence of yellow males (XO) and normally colored females (XXY). Rates of non-disjunction (NDJ) were calculated to account for the inviable progeny (XXX and YO) with the equation NDJ rate = 2 *NDJ offspring/all offspring = (2XXY + 2X0)/(XX+XY+2XXY+2X0), as in [41].

Ovary fixation and immunocytochemistry

Within a day or 2 or eclosion, flies were transferred to fresh food and aged 3 to 7 days, or longer as indicated in the text. For long aging experiments, flies were transferred to fresh food every week and investigated for mold every few days. We dissected the ovaries from approximately 10 flies from each cross in 1× PBS and separated the ovarioles with pins. Ovaries were fixed in 600 μl heptane mixed with 200 μl devitellinizing solution (50% v/v paraformaldehyde and 0.5% v/v NP40 in 1× PBS), mixed with strong agitation, and rotated at room temperature for 20 min. Oocytes were then washed 5× in PBS-T (1% Triton X-100 in 1× PBS) and treated with RNAse A (10 mg/ml) overnight at room temperature.

After washing 6 times in PBS-T, we blocked the oocytes in 1% bovine serum albumin in PBS-T for 1 h at room temperature, and then incubated the oocytes in the primary antibodies diluted in PBS-T overnight at 4°C (antibodies and dilutions listed below). The following day, we washed the oocytes 6 times in PBS-T and incubated them in secondary antibodies diluted 1:500 in PBS-T overnight at 4°C. On the final day, we washed the oocytes a final 6 times in PBS-T and incubated them over 2 nights in PI mounting media (20 μg/ml PI (Invitrogen #P1304MP) in 70% glycerol and 1× PBS) at 4°C. Overlying PI medium was replaced with clear medium, and oocytes were mounted on glass slides. Slides were stored immediately at −20°C and imaged within a month. Infected and uninfected, as well as experimental and control samples were processed in parallel to minimize batch effects. Paired wMel-infected and uninfected OreR stocks were used as wild-type controls.

Primary monoclonal antibodies from the Developmental Studies Hybridoma Bank (University of Iowa) were used at the following dilutions in PBS-T: anti-Hts 1:20 (1B1) [45], anti-Vas 1:50 [44], anti-Orb 1:20 (4H8) [63], anti-Bam 1:5 [36], and anti-Sxl 1:10 (M18) [85]. Primary monoclonal antibodies from Cell Signaling Technology were used at the following dilutions: anti-Phospho-Smad1/5 (Ser463/465) (41D10) 1:300 (#9516S) and anti-Phospho-Histone H3 (Ser10) Antibody 1:200 (#9701S). Anti-Wolbachia FtsZ primary polyclonal antibodies were used at a 1:500 dilution (provided by Irene Newton). Secondary antibodies were obtained from Invitrogen: Alexa Fluor 405 Goat anti-Mouse (#A31553), Alexa Fluor 488 Goat anti-Rabbit (#A12379), and Alexa Fluor 647 Goat anti-Rat (#A21247).

Confocal imaging

Oocytes were imaged on a Leica SP5 confocal microscope with a 63× objective. Optical sections were taken at the Nyquist value for the objective, every 0.38 μm, at variable magnifications, depending on the sample. Most germaria were imaged at 4× magnification and ovarioles and cysts were imaged at a 1.5× magnification. Approximately 10 μm (27 slices) were sampled from each germarium and 25 μm (65 slices) were sampled from each cyst for presentation and analysis.

PI was excited with the 514 and 543 nm lasers, and emission from 550 to 680 nm was collected. Alexa 405 was imaged with the 405 nm laser, and emission from 415 to 450 nm was collected. Alexa 488 was imaged with the 488 laser, and emission from 500 to 526 nm was collected. Alexa 647 was imaged with the 633 laser, and emission from 675 to 750 nm was collected.

Image analysis

Germaria and ovarioles were 3D reconstructed from mean projections of approximately 10 μm-thick nyquist-sampled confocal images (0.38 μm apart) in Fiji/ImageJ. Germline cyst developmental staging followed the standard conventions established by Spradling [86] and the criteria described below. We also scored and processed the confocal z-stacks for analysis as follows in the sections below.

GSC quantification

GSCs were scored by the presence of pMad and an Hts-labeled spectrosome in cells with high cytoplasmic volumes adjacent to the somatic germ cell niche and terminal filament [56]. When antibody compatibility prevented both pMad and Hts staining (e.g., with anti-pHH3 staining), only one was used for GSC identification. The spectrosome was distinguished from the fusome by its position anterior of the putative GSC nucleus and the presence of a posterior fusome that continues into a posteriorly dividing cystoblast. We calculated the number of GSCs per germarium as the average number of pMad and Hts-spectrosome-expressing cells. Non-whole increments of GSCs indicate situations where a putative GSC expresses one attribute, but not the other.

Mitotic GSC quantification

Putative GSCs were identified as described above and scored for the presence of anti-pHH3 staining. The number of pHH3-positive cystocytes in region 1 was also quantified across germaria.

Oocyte cyst tumor quantification

Using a 10× objective, we manually counted the number of nurse cells in each stage 6 to 10b cyst. Each count was repeated 3 times consistently and the cyst tallied as having less than 15, 15, or more than 15 nurse cells per cyst.

Bam and Sxl expression measured by fluorescence

We summed 27-slice nyquist-sampled z-stacks of each germarium in Fiji/ImageJ. Fluorescence intensity was measured by setting ImageJ to measure: AREA, INTEGRATED DENSITY, and MEAN GRAY VALUE. Germarium regions were delimited as in [42] using Vas expression to indicate the germline. Three representative background selections were measured for subtraction. The corrected total cell fluorescence (CTCF) was calculated for each region of the oocyte as follows: CTCF = Integrated Density–Area of selected cell * Mean fluorescence of background readings. We controlled for staining intensity within a germarium and compared relative values among germaria.

Orb expression

Anti-Orb-stained oocyte cysts were manually scored for stage and Orb oocyte-staining in confocal z-stacks ImageJ.

Transcriptomics

We collected OreR and hypomorphic mei-P26[1] flies uninfected and infected with wMel and wMel-infected nos:Gal4>UAS:mei-P26 RNAi and nos:Gal4/CyO flies for RNAseq. Flies were collected after eclosion and moved to fresh food until they were 3 to 5 days old. Ovary dissections were performed in 1× PBS, in groups of 20 to 30 flies to obtain at least 10 mg of ovary tissue for each sample. After careful removal of all non-ovary somatic tissue, ovaries were promptly moved to RNAlater at room temperature, and then transferred to −80°C within 30 min for storage. Frozen tissue was shipped on dry ice to Genewiz Azenta Life Sciences for RNA extraction, cDNA synthesis, Illumina library preparation, and Illumina sequencing. The RNAi and control samples—both nos:Gal4>UAS:mei-P26 RNAi and nos:Gal4/CyO genotypes—were processed to cDNA with T-tailed primers, recovering only host transcripts. The mei-P26[1] and OreR samples were processed to cDNA using random hexamers and ribosomal sequences were depleted with sequential eukaryotic and bacterial rRNA depletion kits (Qiagen FastSelect). Illumina dual-indexed libraries were made from these cDNAs and sequenced as 2 × 150 bp reads.

We processed and analyzed RNAseq datasets for differential expression using standard computational approaches and custom perl parsing scripts. Briefly, following demultiplexing, we trimmed adapter fragments from the RNAseq reads with Trimmomatic [87]. To generate sitewise coverage data to examine read alignments directly, we used the STAR aligner [88] and samtools [89], and plotted read depths across samples in R. We quantified transcripts by pseudoalignment with Kallisto [90]. The choice of reference transcriptome was key to recovering the maximum number of alignments: we merged the NCBI RefSeq assemblies for the wMel reference genome CDSs and RNAs from genomic (accession GCF_000008025.1) and the D. melanogaster reference genome RNAs from genomic (accession GCF_000001215.4; Release_6_plus_ISO1_MT) to obtain alignments against the full, non-redundant host-symbiont transcriptome. Simultaneous mapping to both genomes was performed to avoid cross-species mismapping [91]. This reference transcriptome was indexed at a kmer length of 31 in Kallisto (version 0.45.1) [90] and reads were pseudoaligned against this reference with the “kallisto quant” command and default parameters. Host and symbionts have distinct transcriptome distributions [92], necessitating the separation of the 2 transcriptomes prior to transcript normalization and quantification in DESeq2 [93], which we performed with a custom perl script.

We imported the subset D. melanogaster and wMel Kallisto transcriptome quantifications separately into R with Tximport [94] for DESeq2 analysis [93]. Transcript-level abundances were mapped to gene IDs to estimate gene-level normalized counts. For each transcriptome, we filtered out low-count/coverage genes across samples by requiring at least 5 samples to have a read count of 10 or more. We modeled interactions among our experimental groups as a function of genotype, infection, and the interaction between genotype and infection (~genotype + infection + genotype*infection) and performed Wald Tests to detect differential expression. Briefly, the maximum likelihood gene model coefficient for each gene’s expression count was calculated and divided by its standard error to generate a z-statistic for each gene under the full and reduced models. These z-statistics were compared to the values obtained under standard normal distribution for p-value calculations. FDR/Benjamini–Hochberg corrections were performed on these p-values to reduce the number of false positives. Normalized counts for each gene were output with the plotCounts() function in DESeq2 for plotting in R.

We manually investigated the top hits for each test for evidence of germline expression in the Fly Cell Atlas project [95] through Flybase. GO categories for differentially expressed genes returned from each DESeq2 Wald Test and tested for significance against the reference transcriptome set of IDs with ShinyGO0.77 [96].

Transcriptomic data generated in this study are available through NCBI BioProject number PRJNA1007602.

Plotting and statistical analysis

Fecundity data were plotted, analyzed, and statistics were calculated in R. Differences in lay, hatch, and offspring production rates were evaluated with the nonparametric Wilcoxon rank sum test. When this was infeasible because non-zero samples were so few (e.g., null alleles), we compared the binomially distributed categorical groups of females who laid or did-not-lay eggs with the Fisher’s Exact test. All sample sizes, means, and p-values are presented in S2 Table. Fecundity was plotted across fly ages and fitted with local polynomial regression. Single age pots were made with base R and the beeswarm [97] package and fecundity-vs-time plots were made with the ggplot2 [98] package.

Confocal micrograph fluorescence intensities were analyzed and plotted, and statistics were calculated in R. Relative fluorescence intensities between oocyte genotypes were compared with the nonparametric Wilcoxon rank sum test. Counts of GSCs, mitotic GSCs, tumorous germline cysts, and orb-specific cysts were compared with Fisher Exact tests. Plots were made with the ggplot2 [98] package.

We analyzed and plotted the dual-RNAseq results from Kallisto and DESeq2 in R. Bar and scatter plots were made with the ggplot2 package [98] and volcano plots were made with the EnhancedVolcano package (release 3.17) [99].

Supporting information

S1 Fig. Key genetic regulators of germline stem cell (GSC) maintenance and differentiation in D. melanogaster oogenesis.

(A) Diagram of the GSC niche illustrating a subset of the genes that shift in expression in the cystoblast following GSC mitosis. (B) Model of the relative levels of protein expression during germline cyst development. (C, D) esyN interaction networks for (C) sxl and (D) bam gene products (supporting references in S1 Table).

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S2 Fig. Drosophila mei-P26 genetic resources and gene expression characterization.

(A) Genomic map and gene model for mei-P26 and the studied alleles. The insertion of a P{lacW} transposon in the first intron of mei-P26[1] impacts the RING domain. The mei-P26[mfs1] allele was generated by deletion of this insertion and 0.7–1.6 kb of DNA flanking each side of the insertion site. (B, C) mei-P26 transcript coverage and (D, E) Kallisto Kallisto normalized transcript counts for D. melanogaster mei-P26 transcripts from (B, D) mei-P26[1] and OreR wMel-infected vs. uninfected ovaries and (C, E) nos:Gal4>UAS:meiP26RNAi vs. OreR wMel-infected ovaries. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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S3 Fig. Fecundity data acquisition plots vs. time.

(A, B) Female mei-P26 RNAi, (C, D) Female mei-P26[1], and (E, F) CI assays. Both (A, C, E) egg lay rates and (B, D, F) hatch rates were consistent over time, across genetic crosses, and across fecundity crosses. A factor of 0.1 was added to the y-axis values as an offset to see zero-lay and zero-percent hatch data points.

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S4 Fig. Infection with wMel rescues mei-P26 function in females and males.

(A, B) Hypomorphic mei-P26[1] and (C, D) nos:Gal4>mei-P26RNAi D. melanogaster female fecundity vs. age, fit with a local polynomial regression (dark gray bounds = 95% confidence intervals). Infection with wMel elevates offspring production across the female lifespan through increasing the number of eggs laid and the proportion of those eggs that hatch. (E–G) Male mei-P26 rescue: wMel infection produced significantly higher rates of (D) overall offspring production, broken into (F) egg lay and (F) egg hatch, in RNAi, hypomorphic, and null mei-P26[1] knockdown male flies mated to wild-type females of the same age and infection status. Wilcoxon rank sum * = p < 0.05, ** = 0.01, **** = 1e-4. The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C. (H, I) Homozygous hypomorphic mei-P26[1] stocks (H) infected with wMel Wolbachia or (I) uninfected. Mold growth (green food vs. tan/brown food) is uninhibited in the uninfected stocks due to embryo and larval death, which both feeds and fails to stop mold. Infection enables stable robust stock persistence because larval production outruns mold growth. Both stocks were started at the same time (see 12/28 on the label). The wMel-infected stock never needed any adults added, whereas the uninfected stock produced too few offspring and had to be supplemented at every vial flip to keep the stock going artificially. We ended this after a few months and the uninfected stock fully died out.

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S5 Fig. Hypomorphic mei-P26[1] ovarioles and germaria exhibit a range of (A–I) wMel-infected and (J–R) uninfected phenotypes.

Red = PI DNA staining, yellow = anti-Vas staining, and cyan = anti-Hts staining. Scale bars A, D, G, H, I, J–L, N, O, Q, R = 50 μm; B, C, E, F, M, P = 25 μm.

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S6 Fig. Infection with wMel does not rescue mei-P26’s function in meiosis.

(A) Table containing X-chromosome nondisjunction experimental data. (B) Beeswarm boxplot of the rate of X-chromosome non-disjunction (NDJ) in each experiment. There was no significant difference between infected and uninfected mei-P26[1] females.

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S7 Fig. GSC maintenance and germline differentiation are rescued by wMel infection.

(A, B) Confocal mean projections of D. melanogaster germaria stained with antibodies against Hts and pMad. RNAi knockdown of mei-P26 does not affect GSC maintenance (Fig 2E). (C) Violin plots of the number of GSCs per germarium in 10- to 13-day-old females. As fully functional GSCs express pMad and have Hts-labeled spectrosomes, each was weighted by half and allows for partial scores. Wilcoxon rank sum * = p < 0.05, ** = 0.01. (D) F-K) Violin plots of the number of mitotic cystocytes per germarium detected by pH3 expression. (E, F) Bar-scatter plots of total (E) Sxl and (F) Bam fluorescence expression levels across the germarium, by region. (G) 1D barplots of oocyte-specific Orb staining among germline cysts, distributed across cyst developmental stages. The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C.

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S8 Fig. The wMel strain of Wolbachia is a beneficial manipulator of host reproduction.

(A–C) Beeswarm boxplots showing that wMel infection elevates wild-type D. melanogaster fertility relative to uninfected flies of the same genotype. (A) Overall offspring production, (B) egg lay, and (C) egg hatch were variably impacted in different “wild-type” genotypes. (D) D. melanogaster eggs laid per female per day plot against female age, fit with a local polynomial regression (dark gray bounds = 95% confidence intervals). The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C.

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S9 Fig. Cytoplasmic incompatibility (CI) differs in strength between Drosophila-Wolbachia associations and weakens with male age.

(A–D) Beeswarm box plots of (A, C) egg hatch rate and (B, D) offspring production of uninfected and wMel-infected D. melanogaster OreR females mated to (A, B) zero-day-old and (C, D) 5-day-old wMel-infected males. (E–H) Beeswarm box plots of (E, G) egg hatch rate and (F, G) offspring production in uninfected and wRi-infected D. simulans females mated to (A, B) zero-day-old and (C, D) 5-day-old wRi-infected males. Wilcoxon rank sum * = p < 0.05, ** = 0.01, *** = 0.001, **** = 1e-4. The data underlying this figure can be found on Dryad at doi.org/10.7291/D1DT2C.

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S10 Fig. D. melanogaster genes significantly differentially expressed due to genotype (Wald Test ~G vs. ~G+I+G*I) reveal that mei-P26 is required for the regulation of many genes.

(A) Kallisto normalized transcript counts for D. melanogaster genes (top 15 hits (including Fig 7E) padj< = 2.0E-30; see Figs 7 and S2 for other plots). Barplots are colored by group: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected mei-P26[1], light pink = uninfected mei-P26[1]. (B–G) GO analysis for mei-P26-associated DE genes reveal an enrichment for processes involving chromatin, recombination, protein–protein interactions, and muscle cell differentiation. GO enrichment (B–D) category plots and (E–G) term interaction networks for the categories of (B, E) biological process, (C, F) cellular component, and (D, G) molecular function. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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S11 Fig. D. melanogaster genes significantly differentially expressed due to infection state (Wald Test ~I vs. ~G+I+G*I) reveal an enrichment of rescue events, genes for which wMel-infected mei-P26[1] ovaries exhibit OreR expression levels.

(A, B) Kallisto normalized transcript counts for D. melanogaster genes exhibiting (A) rescue and (B) overshoot of OreR expression levels (padj< = 0.002; see Fig 7 for other plots). Barplots are colored by group: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected mei-P26[1], light pink = uninfected mei-P26[1]. (C–G) GO analysis for infection DE genes reveal an abundance of cytoskeletal and chromatin components. GO enrichment (C–E) category plots and (F, G) term interaction networks for the categories of (C, F) biological process, (D, G) cellular component, and (E) molecular function (no network for a single term). The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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S12 Fig. D. melanogaster genes significantly differentially expressed due to the joint infection-by-genotype state (Wald Test ~G*I vs. ~G+I+G*I) reveal an enrichment of reversal DEG, genes for which wMel-infected ovaries exhibit inverse DE patterns for mei-P26[1] and OreR ovaries.

(A–C) Kallisto normalized transcript counts for D. melanogaster genes exhibiting (A) inverse, (B) undershoot, and (C) novel regulation of OreR expression levels (padj< = 0.02; see Fig 7 for other plots). Barplots are colored by group: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected mei-P26[1], light pink = uninfected mei-P26[1]. (D–G) GO analysis for infection DE genes reveal cytoskeletal and membrane factors. GO enrichment (C–E) category plots and (F, G) term interaction networks for the categories of (C, G) biological process, (D) cellular component (see Fig 8D for network), and (E, F) molecular function. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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S13 Fig. wMel Wolbachia transcriptome differential expression GO categories of all expressed genes reveal cell maintenance, central metabolic, and membrane-associated processes.

GO enrichment (A) category plot, (B) hierarchical clustering tree, and (C) term interaction network the biological process category. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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S14 Fig. Kallisto normalized transcript counts for wMel Wolbachia (A, B) differential expression candidate genes (pending deeper sampling) and (C) genes of interest from the literature.

Barplots are colored by group: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected mei-P26[1], light pink = uninfected mei-P26[1]. Wald test genotype-association p-values and adjusted p-values (padj). The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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Click here for additional data file.^{(1MB, tif)}

S15 Fig. Nonsignificant Kallisto normalized transcript counts for a subset of essential D. melanogaster oogenesis genes from the literature selected based upon their known interactions with mei-P26, sxl, bam, or germ plasm formation (a mid-stage 9 of oogenesis).

Barplots are colored by group: dark gray = wMel-infected OreR, light gray = uninfected OreR, dark pink = wMel-infected mei-P26[1], light pink = uninfected mei-P26[1]. The data underlying this figure can be found at NCBI, under BioProject number PRJNA1007602.

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Click here for additional data file.^{(2.2MB, tif)}

S1 Table. esyN references for sxl and bam interactions in S1C Fig and mei-P26 interactions in Fig 9.

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Click here for additional data file.^{(100KB, pdf)}

S2 Table. Annotated references used to make S1A and S1B Fig and the table in Fig 9C.

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Click here for additional data file.^{(94.2KB, pdf)}

S3 Table. Full fecundity dataset (n = 3,002).

See S3 Table.

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Click here for additional data file.^{(253.8KB, tsv)}

S4 Table. Fecundity statistics: offspring produced per female per day in single female-by-single male crosses.

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Click here for additional data file.^{(78.3KB, pdf)}

S5 Table. Fecundity statistics: eggs produced per female per day in single female-by-single male crosses.

(PDF)

Click here for additional data file.^{(76.7KB, pdf)}

S6 Table. Fecundity statistics: percentage of eggs that hatched from single female-by-single male crosses that laid > = 20 eggs.

Experimental genotypes, infection statuses, and sexes are listed. The mate for each cross was OreR, of the same infection status, and of the opposite sex as the experimental fly. Males were aged 3–6 days, except for the young male CI crosses, which were aged zero days (distinguished with “-0d” and “-5d” labels). Sample counts (n1, n2) in parentheses are for Fisher exact tests (samples with hatched eggs vs. no hatched eggs, opposed to % hatch for samples with 20 or more eggs laid). P-values <0.01 are in light green and <0.05 are in dark green for clarity.

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Click here for additional data file.^{(79.1KB, pdf)}

S7 Table. Fecundity versus age statistics.

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Click here for additional data file.^{(71.9KB, pdf)}

S8 Table. Germline stem cell (GSC) counts per germarium.

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Click here for additional data file.^{(71.2KB, pdf)}

S9 Table. Number of GSCs in mitosis (anti-pHH3-positive staining), per germarium.

(PDF)

Click here for additional data file.^{(68.4KB, pdf)}

S10 Table. Number of cystocytes (CC) in mitosis (anti-pHH3-positive staining), per germarium.

(PDF)

Click here for additional data file.^{(68KB, pdf)}

S11 Table. Sxl expression by germarium region, measured by fluorescence intensity.

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Click here for additional data file.^{(69.4KB, pdf)}

S12 Table. Bam expression by germarium region, measured by fluorescence intensity.

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Click here for additional data file.^{(70.3KB, pdf)}

S13 Table. Relative Bam vs. pMad expression, measured by fluorescence, in GSCs.

(PDF)

Click here for additional data file.^{(68.6KB, pdf)}

S14 Table. Tumorous germline cyst counts.

Normal cysts contain 16 germline-derived cells, 15 nurse cells and 1 oocyte. Cysts containing greater or less than 15 nurse cells were scored as tumorous or abnormal, respectively.

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Click here for additional data file.^{(73.6KB, pdf)}

S15 Table. Counts of germline cysts exhibiting oocyte-specific Orb expression (Y), unclear staining (M), or no specific expression, indicating developmentally abnormal cysts lacking specified oocytes.

(PDF)

Click here for additional data file.^{(68.3KB, pdf)}

S16 Table. Transcriptomic dataset generated to test the impacts of mei-P26 knockdown and wMel infection.

Data deposited under NCBI BioProject number PRJNA1007602.

(PDF)

Click here for additional data file.^{(92.2KB, pdf)}

S17 Table. D. melanogaster genes Wald Test significant results for ~Genotype vs. ~Genotype+Infection+Genotype*Infection.

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Click here for additional data file.^{(267.5KB, pdf)}

S18 Table. D. melanogaster genes Wald Test significant results for ~Infection vs. ~Genotype+Infection+Genotype*Infection.

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Click here for additional data file.^{(154.2KB, pdf)}

S19 Table. D. melanogaster genes with Wald Test significant results for ~Genotype*Infection vs. ~Genotype+Infection+Genotype*Infection.

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Click here for additional data file.^{(94KB, pdf)}

S20 Table. wMel Wolbachia genes Wald Test significant results for ~Genotype vs. ~1.

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Click here for additional data file.^{(80.6KB, pdf)}

Acknowledgments

We thank the UCSC Life Sciences Microscopy Center (RRID:SCR_021135) and Ben Abrams for training and the use of their microscopes. We thank Irene Newton for aliquots of anti-Wolbachia FtsZ antibodies and Manabu Ote for a stock of UASp-mRFP/CyO (1-7M) flies. We thank Russ Corbett-Detig for his comments and feedback throughout this project.

Abbreviations

BDSC: Bloomington Drosophila Stock Center
BMP: bone morphogenic protein
CB: cystoblast
CC: cystocyte
CI: cytoplasmic incompatibility
CTCF: corrected total cell fluorescence
ERR: estrogen-related receptor
GSC: germline stem cell
NDJ: non-disjunction
OreR: Oregon R
pHH3: phospho-Histone H3
PI: propidium iodide
RISC: RNA-induced silencing complex
RNP: ribonucleoprotein
TA: transit-amplifying
TomO: toxic manipulator of oogenesis
UTR: untranslated region

Data Availability

All relevant data are contained within the paper, Supporting Information files, NCBI BioProject number PRJNA1007602 or on Dryad: https://doi.org/10.7291/D1DT2C.

Funding Statement

This work was supported by the UC Santa Cruz Chancellor’s Postdoctoral Fellowship and the NIH (R00GM135583 to SLR; R35GM139595 to WTS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Biol. doi: 10.1371/journal.pbio.3002335.r001

Decision Letter 0

Lucas Smith

26 Jan 2023

Dear Dr Russell,

Thank you for submitting your manuscript entitled "Wolbachia endosymbionts manipulate GSC self-renewal and differentiation to enhance host fertility" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review. Although we find the phenomenon reported here interesting, I should note that we have yet to make a firm call about whether the study provides a sufficient advance for PLOS Biology, as there is some question about the extent that this represents the basis for emerging mutualism and as the mechanism of wMel's effects is not fully elucidated. We will be looking for enthusiasm from the reviewers regarding the fit of your study for PLOS Biology.

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Kind regards,

Lucas

Lucas Smith, Ph.D.

Associate Editor

PLOS Biology

lsmith@plos.org

PLoS Biol. doi: 10.1371/journal.pbio.3002335.r002

Decision Letter 1

Lucas Smith

27 Mar 2023

Dear Dr Russell,

Thank you for your patience while your manuscript "Wolbachia endosymbionts manipulate GSC self-renewal and differentiation to enhance host fertility" was peer-reviewed at PLOS Biology. I apologize for the protracted review process for your study - in this case, for some reason, we had a bit of trouble lining up 3 reviewers in the initial stage and then I was at a conference all last week which delayed things further. Your manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by several independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports.

As you will see below, the reviewers find the work of potential interest and the analyses to be fairly comprehensive. However they have raised a number of important and overlapping concerns that would need to be thoroughly addressed before we can consider your manuscript for publication. The reviewers have highlighted that the writing and data presentation is a bit challenging to follow, noting, for example that the discussion and connection of the discussion to figures is loose, and that some statements do not seem to be aligned with relevant figures or statistics. Additionally, the authors raise concerns about the appropriateness of the control genotypes used in several experiments and the reviewers have noted that comparisons of infected and uninfected wt flies are needed as a contrast to what happens in the mutants. After discussion with the Academic Editor we think these trials should be done in parallel, which might require a repeat of the whole experiment on fertility effects.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Lucas

Lucas Smith, Ph.D.

Associate Editor

PLOS Biology

lsmith@plos.org

------------------------------------

REVIEWS:

Reviewer #1:

Since the early discovery that Wolbachia could rescue the sterility of some mutant alleles of the Sxl gene, additional studies have established Wolbachia as an important regulator of GSC maintenance and proliferation in the female germline. In this work, Russell et al. have now discovered that wMel is capable of rescuing several aspects of the complex oogenesis phenotype associated with mei-P26 knock-down or mutants in D. melanogaster. They performed a detailed analysis of these defects affecting GSC maintenance, germ cell proliferation and differentiation, as well as female fecundity and fertility. They also show that wMel is beneficial to the fertility of wild-type flies. One main interest of this work is to reveal the complexity of this critical host-symbiont interaction, with Wolbachia likely controlling several aspects of oogenesis through the expression of multiple proteins.

- Having confirmed that CI is very weak in D. mel, the authors speculate on the possibility that wMel is losing its ability to induce CI and instead develops a mutualistic interaction with its host based on increased female fertility. However, the authors should explain how they integrate in their model the fact that wMel is fully capable of inducing strong CI in D. simulans or Aedes mosquitoes.

- I found quite surprising that experiments in Figure 1A-C do not include a wild-type control. The authors refers to "wild-type" (l. 127) but it is actually missing in the Figure.

- The text alludes on several occasions on the "nature" of the mei-P26[1] allele (eg l.220, l.352). However, the mei-P26 alleles used in this work are not described. mei-P26[1] actually contains a transposon insertion in the first intron of the gene, so the actual impact of this mutation on the production of mei-P26 product is unclear. The authors could measure the amount of transcripts produced by this allele and the RNAi, compared to a control.

- It was previously reported in Starr & Cline Nature 2002 that Wolbachia is incapable of rescuing the sterility of homozygous mei-P26[fs1] females. The fact that a complete null of mei-P26 is not rescuable should be clearly mentioned in the text and discussed. Similarly, l. 120, "flies lacking Mei-P26" is likely an overstatement.

Additional comments:

- I am not sure if male-killing should be considered as a reproductive parasitism (l. 63).

- l. 70 "pipiensis"

- l.143-144: I had hard time to understand this Table E until I read l. 163

- Fig 1J shows a perfect ovariole, yet fecundity is severely affected. A more global view of the ovary with several ovarioles could perhaps better reflect the reality.

- L. 201: the Wolbachia titers in somatic vs germ cells cannot really be appreciated in this image.

- L. 202: (Fig 1L-L')

- L. 210: A description of GSC and other germ cells could help.

- L. 216: one extra "uninfected" word in the sentence.

- L. 230: I guess "PHH3" refers to phosphorylation of H3 serine 10.

- L. 242: Not sure to understand what the authors mean by "to recover some of their GSCs" or l. 245 "increased their GSCs abundance".

- L. 288: This diagram is beautiful but unfortunately wrong. It is known since King (1970) that the oocyte invariably forms from one of the two cells with four ring canals.

- L. 375: "this dominant-negative …". I don't see any obvious dominant-negative effect here. This should be clarified.

- L.404-405: the use of the term "wild-type" is confusing in this sentence.

- L. 416: a color code is missing for Fig. 6B

- L. 557: This genotype is wrong.

Reviewer #2: In this manuscript, Russell et al. demonstrate that Wolbachia infection in Drosophila melanogaster is capable of rescuing defects in germline stem cell self-renewal and differentiation. Wolbachia bacteria of arthropods are some of the world's most successful endosymbionts in terms of spread to new hosts. Although the host reproductive manipulations of Wolbachia are infamously tied to their global spread, there is doubt that these manipulations alone could fully account for their success and many have speculated or investigated other contributions of Wolbachia to host fitness. The authors show that compensation for host reproductive defects may be one such mechanism, which is an important finding for basic and applied Wolbachia research. In particular, the authors identify mei-P26 as a specific host factor where a loss of function causes various developmental defects in oocytes that are rescued by Wolbachia infection. The study is comprehensive in its analysis and presents ample evidence of an interaction between the symbiont and mei-P26. The results are important for answering key questions in the field. There are some comments and suggestions below:

Comments:

* It would help to explain more in the main text how and why mei-P26 was chosen as a candidate. There is a section in the methods on it, but it would help at the beginning of the results or somewhere in the introduction to go into more detail on this. Since the paper focuses so much on it, it would help the reader to walk through the logic of how this candidate was identified out of all other possibilities.

* How common are mei-P26 or similar GSC deficiencies in the wild? Would Wolbachia rescue of these defects be expected regularly, and would this ability commonly increase Wolbachia's fitness?

* Would you expect to see mei-P26 mutants maintained in the wild with Wolbachia infection?

* Some of the main comparisons of fertility across some fly genotypes are not quite appropriate. For example, the percentages highlighted in L124-130 compare the fertility of mei-P26 lines to wild-type lines. If the question being asked is whether Wolbachia rescues a mei-P26 deficit, then the main comparison should be between a mei-P26 deficient background either with or without the symbiont. It is ok to compare to the wild-type in other cases (for example, where the question being asked is how similar the Wolbachia rescue of defects is to wild-type). However, in the experiments with fertility, it would be most appropriate to focus primarily on within-genotype comparisons.

* Related to this, was there a control for the RNAi knockdown line that was used throughout the paper? I did not see one in the supplemental tables. For example, were RNAi knockdown results of mei-P26 ever compared to knockdown of a control gene like GFP? This is an important control to determine if there is an effect of the RNAi expression system on the phenotype. The inclusion of other mei-P26 deficient fly lines mitigates the concerns of the lack of this control somewhat, however, it may be beneficial (though perhaps not strictly necessary) to conduct one of the key assays with an RNAi control if there wasn't one already.

* It is somewhat difficult to follow the supplemental tables without spending significant time looking at them. This is primarily because there are many features that are not explained in the captions. Though many can be inferred by spending some time studying the tables closely, it would help readers to include more detail in the captions to avoid having to do this. For example, what do the colors mean? Why were the n values so different across comparison groups? What are the unfilled boxes? What are all of the shorthand notations for genotypes? Etc. It would help the readability of the tables to include more details on the meaning of each component.

* It would help to include a few more details on experimental design for the fertility crosses. Since these crosses are all being compared to each other, were they all conducted at the same time using similar controls and conditions? How many separate times were the experiments conducted/repeated? This is particularly important for the CI/rescue crosses, where they need to be performed on the same day for accurate comparison and other details are important to know: were the older and younger males all the first-emerged flies from their vials, did they all have similar-aged grandmothers, etc. It would help to have more of these details in the methods.

* There are some small details that would be good to include in figure captions that are missing. For example, what does each dot represent in the beeswarm boxplots? Are those hatch rates of individual females? Is this the combined data of multiple replicates?

* For the fertility assays as well, a couple of minor points are unclear. It would help to explain the term "allelic strengths" (L123) the first time it is brought up, as it is unclear until later in the paragraph.

* It may also help at the beginning of the results section to simply introduce the basic experimental setup, including which fly genotypes are being used and why, before going into the results. As is, the reader goes straight into the conclusion based on the results without being introduced to the assay that was performed.

* It is a little unclear what is meant by offspring production in Figure 1/S2/S5. Are the offspring produced not the same as the number of eggs laid or hatched?

* L155-156: It may help to add that FtsZ is a Wolbachia marker

* L179-181: It's stated here that wMel also rescues fertility impacts of mei-P26 in males, but the figure only shows a couple of significant differences, and in different genetic backgrounds across S2D-F, so it looks like the phenotype may not be as robust in males.

* FigS2G,H: It would help to have a few details on these vials, maybe in the methods or in the caption. Were these vials set up on similar days with similar numbers/ages of flies? It may help to label exactly what the most important differences are between the vials, particularly for people without fly experience. Is it just the color? If so, it may help to point out the color difference in the caption so readers know what to compare between the images.

* L272/Figure 3: It doesn't look like region 2a is significant between the two mei-P26 groups

* Figure 6: It would help to add details on components of the figure like color and shape meanings.

Minor comments:

* There are some typos and small formatting errors in the document. For example, species names are not always italicized and the w in wMel should be italicized as well

* "pipientis" is misspelled in the introduction (L70)

* Some of the references to figures or tables are incorrect. These should be doublechecked throughout the document, for example:

o L137: should say Fig S2E-G, not S3E-G

o L141: is S3 correct here?

o L179: missing table reference?

o L181: should be Table S3 not S4

* Table S1/S2 titles: should "females" be plural?

* The sentences in L168-171 seem to potentially be duplicates

Reviewer #3: Russell et al. have investigated how Wolbachia bacteria affect Drosophila fertility. They make a major discovery in finding that Wolbachia infection substantially rescues infertility caused by the mei-P26 mutation, and provide insight relating this to previous interactions observed with other germline genes. The data largely support the authors' conclusions (excepting point 4 below) but there are substantial issues with data presentation and organization that made this manuscript challenging to evaluate.

1) Results are often not connected to the relevant figure/table, or cite an incorrect table/figure. A wide range of figures is often cited at beginning of paragraphs with specific results that follow being not cited, leaving the reader to wade through multiple figures and tables to find the data. For examples, lines 124-130 appear to match to Fig 1E, but this is not stated. Lines 140-141 appear to match to Fig 1D but is cited as S3. Line 160 'differentiation defects' appears to refer to Fig 1H-K but is not cited. Data for the results in the paragraph starting on L160 are difficult to find. L244-246 - where is data?

2) Data presentation is confusing. Line 160/161 is redundant with L120/121. L161-164 is redundant with L124-125. L168-171 says the same thing twice.

There is confusion in some places whether the result being discussed is of mutant relative to wild type, or mutant with and without Wolbachia infection. For example, L166-167 appears to be making a statement about RNAi knockdown phenotype of mei-P26. But following sentence on Line 168-169 instead provides data on RNAi with and without infection.

The authors sometime start a paragraph stating a result, then give background/motivation on why doing the experiment, and then give more results. Which is confusing. Such as paragraphs on p. 15.

What's the difference between Fig 1G and S2A?

L378. What are the 'differences among these tumor phenotypes'?

L178 - "across the fly lifespan". Does not seem to match Fig S2A-C, which shows effects reversing around day 30.

L214-214. Unclear what 'young' means here.

L434. 'differences … are insignificant'. In Table S4, some results are significant and some are not.

LL547. FS2 should be FS1?

Fig 1. Should emphasize that all data is from days 2-3 only (I think).

Fig6A - what is parent age?

Fig 6B,C - presumably is also OR-R?

3) Comparisons of mutants vs wild type.

At many places mutant genotypes are compared for fertility phenotypes to wild type. One potential issue is experimental design. Presumably with and without Wolbachia comparisons within a genotype were done in parallel (at same time). Is that also true for different genotypes (e.g. OR-R and mei-P26 mutants)? If not, then that weakens the comparative power among genotypes. A second issue is the different genetic backgrounds. Different wild type backgrounds can have wide variation in fertility, and that will also occur here since mutants and wild type are different backgrounds. It therefore is unclear what the biological meaning is that some mutant conditions have higher fertility than wild-type (eg. L163-164). For these reasons, I would suggest de-emphasizing comparisons of fertility between mutants and wild-type, and focusing on paired comparisons of fertility phenotypes with and without Wolbachia within individual genotypes.

4) Wolbachia effects in wild type.

I wouldn't call Gal4-expressing lines "wild type". At some places the authors seem to agree (lines 403-405), but at other places including in the Highlights and Abstract, they conflate the genotypes. This is important to clear up because a major claim of the paper is that Wolbachia increase fitness even in wild type individuals. If I understand all the data on this point, in true wild type (OR-R), only egg hatch rate is significantly increased by Wolbachia infection (S5C). But egg lay is somewhat reduced (S5B). Thus, total offspring is not different (S5A), which is the phenotype that ultimately matters for fitness. So the general conclusion that fitness is increased by infection in wild type lines does not seem to be established.

Minor points.

L395. Not sure 'recapitulates' is correct word here.

L462-3. Better to say 'rescue the PARTIAL loss'. The description of Sxl and Bam with the 'respectively' is confusing.

L585-590. How were long-term experiments with collections over 60 days done?

PLoS Biol. 2023 Oct 24;21(10):e3002335. doi: 10.1371/journal.pbio.3002335.r003

Author response to Decision Letter 1

10 Jul 2023

Attachment

Submitted filename: R2R_meiP26rescue_PLoSBio.pdf

Click here for additional data file.^{(248.9KB, pdf)}

PLoS Biol. doi: 10.1371/journal.pbio.3002335.r004

Decision Letter 2

Lucas Smith

16 Aug 2023

Dear Dr Russell,

Thank you for your patience while we considered your revised manuscript "Wolbachia endosymbionts manipulate GSC self-renewal and differentiation to reinforce host fertility" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

The reviewers are largely satisfied by the revision and have all suggested that we accept your study. However I note they raise a number of minor suggestions that we think should be addressed before publication. Additionally, before we can accept your study, we need you to address a number of editorial requests detailed below. We therefore would like to invite one last revision that we do not think will take very long.

**EDITORIAL REQUESTS:

1) TITLE: We suggest that the title be edited to spell out GSC and specify the host, as this will make the study more broadly accessible. If you agree, we suggest you change the title to something like:

"Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host"

2) ABSTRACT: Similarly, GSC should be defined in the abstract

3) FINANCIAL DISCLOSURES: Please update the financial disclosures statement, in our editorial manager system, to describe the role of any sponsors or funders in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. If the funders had no role in any of the above, include this sentence at the end of your statement: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

4) DATA: Thank you for providing data on NCBI and Dryad related to your manuscript. For some reason, I could not access these datasets to verify that they meet our requirements. Can you provide me with a reviewer code? (sorry if I missed this somewhere)

>>Please do take a look at our Data Policy, which requires that all data be made available without restriction, and make sure that your data provided on these repositories meets our requirements. Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available either as a deposition on a repository or as a supplementary excel file.

>>Please also ensure that figure legends in your manuscript include information on where the underlying data can be found. For example, to each figure legend you can add the sentence "the data underlying this figure can be found at ____"

As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.

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Please do not hesitate to contact me should you have any questions.

Sincerely,

Luke

Lucas Smith, Ph.D.

Senior Editor,

lsmith@plos.org,

PLOS Biology

------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: My main requests have been addressed. As for my minor comment on "other germ cells", I meant nurse cells and the oocyte.

Reviewer #2: The authors have thoroughly addressed comments by the reviewers. In particular, they have made many clarifying statements regarding the methods, addressed concerns with figure/table citations in the text, and addressed concerns with the fertility assays. The new details on the methods for the fertility assays are helpful. The authors have also added comparisons of fertility within genotype which, paired with the comparisons of fertility to controls (and their justification for this is good), establish their points well. This addresses the questions raised by reviewers. I have only a couple of very minor suggestions remaining:

I suggest a few very minor clarifying details on the caption of Figure S4H,I (the stock images): 1) I would label the dates that each individual vial received adults (presumably older on the left to newer on the right of each image?). This would help readers know which vials should be compared to which other vials between images. 2) I would also suggest pointing out the mold growth is visible by different food colors/consistencies between comparably-aged vials in H and I. 3) I would clarify that the sentence starting with "Infection enables stable robust stock persistence..." is referring to the Wolbachia infection as opposed to the mold.

Also, very minor: if the authors have agreed to go with the convention of italicizing the w in wMel, then this should be also applied to supplemental documents (supplemental figure captions etc.)

Reviewer #3: The authors have clarified many of the concerns and queries about the fertility assays. They have also added extensive discussion of new RNA-seq experiments. What does "dual-RNAseq" mean - examining both Dmel and wMel genes, or combining Or-R and mei-P26 in a single analysis?

I didn't see the following in Fig 1, referring to the time frame of the eggs/offspring being analyzed.

Fig 1. Should emphasize that all data is from days 2-3 only (I think).

Thank you for pointing this out. We added this info to the figure legend to make sure it is clear.

PLoS Biol. 2023 Oct 24;21(10):e3002335. doi: 10.1371/journal.pbio.3002335.r005

Author response to Decision Letter 2

1 Sep 2023

Attachment

Submitted filename: R2R2_meiP26rescue_PLoSBio.pdf

Click here for additional data file.^{(96.8KB, pdf)}

PLoS Biol. doi: 10.1371/journal.pbio.3002335.r006

Decision Letter 3

Lucas Smith

14 Sep 2023

Dear Dr Russell,

Thank you for the submission of your revised Research Article "Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host" for publication in PLOS Biology, and thank you for addressing the last reviewer and editorial requests in this revision. On behalf of my colleagues and the Academic Editor, Nancy A. Moran, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

**IMPORTANT: As you address any formatting and reporting requests, to come, please also address the following editorial points which we discussed over email:

1) Please update the data availability statement in our editorial manager system, and the relevant sections of the manuscript, to reference the correct BioProject number PRJNA1007602

2) Please update the supplemental figure legends with a short sentence pointing readers to the relevant underlying datasets.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study.

Sincerely,

Luke

Lucas Smith, Ph.D.,

Senior Editor

PLOS Biology

lsmith@plos.org

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Key genetic regulators of germline stem cell (GSC) maintenance and differentiation in D. melanogaster oogenesis.

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S2 Fig. Drosophila mei-P26 genetic resources and gene expression characterization.

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S3 Fig. Fecundity data acquisition plots vs. time.

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S4 Fig. Infection with wMel rescues mei-P26 function in females and males.

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S5 Fig. Hypomorphic mei-P26[1] ovarioles and germaria exhibit a range of (A–I) wMel-infected and (J–R) uninfected phenotypes.

Red = PI DNA staining, yellow = anti-Vas staining, and cyan = anti-Hts staining. Scale bars A, D, G, H, I, J–L, N, O, Q, R = 50 μm; B, C, E, F, M, P = 25 μm.

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S6 Fig. Infection with wMel does not rescue mei-P26’s function in meiosis.

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S7 Fig. GSC maintenance and germline differentiation are rescued by wMel infection.

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S8 Fig. The wMel strain of Wolbachia is a beneficial manipulator of host reproduction.

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S9 Fig. Cytoplasmic incompatibility (CI) differs in strength between Drosophila-Wolbachia associations and weakens with male age.

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S10 Fig. D. melanogaster genes significantly differentially expressed due to genotype (Wald Test ~G vs. ~G+I+G*I) reveal that mei-P26 is required for the regulation of many genes.

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S13 Fig. wMel Wolbachia transcriptome differential expression GO categories of all expressed genes reveal cell maintenance, central metabolic, and membrane-associated processes.

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S14 Fig. Kallisto normalized transcript counts for wMel Wolbachia (A, B) differential expression candidate genes (pending deeper sampling) and (C) genes of interest from the literature.

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S1 Table. esyN references for sxl and bam interactions in S1C Fig and mei-P26 interactions in Fig 9.

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S2 Table. Annotated references used to make S1A and S1B Fig and the table in Fig 9C.

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S3 Table. Full fecundity dataset (n = 3,002).

See S3 Table.

(TSV)

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S4 Table. Fecundity statistics: offspring produced per female per day in single female-by-single male crosses.

(PDF)

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S5 Table. Fecundity statistics: eggs produced per female per day in single female-by-single male crosses.

(PDF)

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S6 Table. Fecundity statistics: percentage of eggs that hatched from single female-by-single male crosses that laid > = 20 eggs.

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S7 Table. Fecundity versus age statistics.

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S8 Table. Germline stem cell (GSC) counts per germarium.

(PDF)

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S9 Table. Number of GSCs in mitosis (anti-pHH3-positive staining), per germarium.

(PDF)

Click here for additional data file.^{(68.4KB, pdf)}

S10 Table. Number of cystocytes (CC) in mitosis (anti-pHH3-positive staining), per germarium.

(PDF)

Click here for additional data file.^{(68KB, pdf)}

S11 Table. Sxl expression by germarium region, measured by fluorescence intensity.

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Click here for additional data file.^{(69.4KB, pdf)}

S12 Table. Bam expression by germarium region, measured by fluorescence intensity.

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S13 Table. Relative Bam vs. pMad expression, measured by fluorescence, in GSCs.

(PDF)

Click here for additional data file.^{(68.6KB, pdf)}

S14 Table. Tumorous germline cyst counts.

Normal cysts contain 16 germline-derived cells, 15 nurse cells and 1 oocyte. Cysts containing greater or less than 15 nurse cells were scored as tumorous or abnormal, respectively.

(PDF)

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S16 Table. Transcriptomic dataset generated to test the impacts of mei-P26 knockdown and wMel infection.

Data deposited under NCBI BioProject number PRJNA1007602.

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Click here for additional data file.^{(92.2KB, pdf)}

S17 Table. D. melanogaster genes Wald Test significant results for ~Genotype vs. ~Genotype+Infection+Genotype*Infection.

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S18 Table. D. melanogaster genes Wald Test significant results for ~Infection vs. ~Genotype+Infection+Genotype*Infection.

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S19 Table. D. melanogaster genes with Wald Test significant results for ~Genotype*Infection vs. ~Genotype+Infection+Genotype*Infection.

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S20 Table. wMel Wolbachia genes Wald Test significant results for ~Genotype vs. ~1.

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Click here for additional data file.^{(80.6KB, pdf)}

Attachment

Submitted filename: R2R_meiP26rescue_PLoSBio.pdf

Click here for additional data file.^{(248.9KB, pdf)}

Attachment

Submitted filename: R2R2_meiP26rescue_PLoSBio.pdf

Click here for additional data file.^{(96.8KB, pdf)}

Data Availability Statement

All relevant data are contained within the paper, Supporting Information files, NCBI BioProject number PRJNA1007602 or on Dryad: https://doi.org/10.7291/D1DT2C.

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PERMALINK

Wolbachia endosymbionts manipulate the self-renewal and differentiation of germline stem cells to reinforce fertility of their fruit fly host

Shelbi L Russell

Jennie Ruelas Castillo

William T Sullivan

Roles

Abstract

Introduction

Results

Infection with wMel rescues female D. melanogaster fertility in mei-P26 deficient flies

Fig 1. wMel infection rescues the loss of essential host fertility gene mei-P26 in females (and males, see S4E–S4G Fig).

Female germline morphology is rescued by wMel infection in mei-P26-deficient flies

Host GSCs are maintained at higher rates in wMel-infected than uninfected mei-P26-deficient flies

Fig 2. wMel infection rescues mei-P26-induced defects in female GSC maintenance.

Regulation of host Sxl expression is rescued in wMel-infected germaria

Fig 3. Infection with wMel mitigates the consequences of dysfunctional mei-P26 on Sxl expression in the germarium.

Bam expression is partially restored in wMel-infected mei-P26 mutant germaria

Fig 4. Infection with wMel mitigates the consequences of dysfunctional mei-P26 on Bam expression in the germarium.

wMel rescues normal germline cyst and oocyte development impaired by mei-P26 knockdown

Fig 5. wMel infection rescues cyst tumors and restores oocyte specification in mei-P26 mutants.

OreR wild-type host fertility is beneficially impacted by wMel infection

Fig 6. The wMel strain of Wolbachia reinforces OreR wild-type fertility.

wMel-mediated mei-P26[1] rescue may be enabled through rescuing and perturbing host transcription

Fig 7. Differential expression analysis of wMel and D. melanogaster genes from host ovaries reveal that mei-P26 knockdown and infection influence global expression patterns.

Fig 8. Mei-P26 interactions predicted from the literature are supported by our ovary dual-RNAseq results.

Discussion

Fig 9. Model of wMel’s interactions with essential host genes in early GSC maintenance and germline cyst formation.

Materials and methods

Candidate gene selection

Drosophila stocks and genetic crosses

Fecundity crosses

Overview

Table 1. Fecundity cross genotypes, sexes, ages, and control crosses.

Cross conditions

Preparation of flies for fecundity crosses

Fecundity cross protocol

Fecundity cross analysis

Non-disjunction assays

Ovary fixation and immunocytochemistry

Confocal imaging

Image analysis

GSC quantification

Mitotic GSC quantification

Oocyte cyst tumor quantification

Bam and Sxl expression measured by fluorescence

Orb expression

Transcriptomics

Plotting and statistical analysis

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Lucas Smith

Roles

Decision Letter 1

Lucas Smith

Roles

Author response to Decision Letter 1

Decision Letter 2

Lucas Smith

Roles

Author response to Decision Letter 2

Decision Letter 3

Lucas Smith

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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