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

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.Fiji (ImageJ)Funding provided by: National Institute of General Medical SciencesCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000057Award Number: R00GM135583Candidate gene selection Leveraging what is known about wMel's abilities to rescue essential maintenance and differentiation genes with empirical data on protein-protein and protein-mRNA interactions among these genes (esyN3 networks in fig S1A-D and Fig 8; references in table S1) and data on interactions between host genes and wMel titers 4, we identified the essential germline translational regulator meiotic P26 (mei-P26) as a potential target of of bacterial influence over germline stem cell (GSC) maintenance and differentiation pathways (fig S1A,B). 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. 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 two 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 Bloomington Drosophila Stock Center 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. 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. 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 two 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 ten 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., 1) have been characterized. 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 ~2.5 kb of flanking sequence. According to Page et al. 2000, these three 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 3002 fecundity crosses continuously between October 2020 and January 2022, in batches based on their eclosion date (raw data in table S2). 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 Table S2 and plotted in Fig. S3. Rescue 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 (1.2x 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 four 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 (three 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 3–7 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 zero or 3–7 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, one male and one 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 two days. After approximately 40 hours, we counted the number of hatched and unhatched eggs. The exact times and dates for all steps in all crosses were recorded (table S2) and plotted to show consistent results across the timeframe (fig S3). 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 3–7 day old female plots) and 10 or more eggs (for age vs. hatch rate plots). Egg lay and offspring production rates were calculated from raw counts, divided by the fraction of days spent laying (metadata in table S2). Crosses with zero laid eggs (and offspring) were not rejected, as the fertility mutants often laid zero to few eggs. Our cross conditions were highly consistent and run daily, lending confidence to these zero-lay/offspring samples (Figure S3). Drosophila fecundity vs 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 seven or eight vials of 1–10 females mated to a count-matched 1-10 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 10. Ovary fixation and immunocytochemistry Within a day or two or eclosion, flies were transferred to fresh food and aged 3–7 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 ~10 flies from each cross in 1xPBS 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 1x PBS), mixed with strong agitation, and rotated at room temperature for 20 min. Oocytes were then washed 5x in PBS-T (1% Triton X-100 in 1x PBS), and treated with RNAse A (10 mg/ml) overnight at room temperature. After washing six times in PBS-T, we blocked the oocytes in 1% bovine serum albumin in PBS-T for one hour 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 six times in PBS-T and incubated them in secondary antibodies diluted 1:500 in PBS-T overnight at 4C. On the final day, we washed the oocytes a final six times in PBS-T and incubated them over two nights in PI mounting media (20 μg/ml propidium iodide (PI, Invitrogen #P1304MP) in 70% glycerol and 1x 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) [11], anti-Vas 1:50 [12], anti-Orb 1:20 (4H8)13, anti-Bam 1:5 [14], and anti-Sxl 1:10 (M18) [15]. 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 63x 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 4x magnification and ovarioles and cysts were imaged at a 1.5x 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. Propidium iodide 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 16 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 17. 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-phospho-Histone H3 (pHH3) staining. The number of pHH3-positive cystocytes in region 1 was also quantified across germaria. Oocyte cyst tumor quantification: Using a 10x objective, we manually counted the number of nurse cells in each stage 6-10b cyst. Each count was repeated three 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 18 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