Complex effects of environment and Wolbachia infections on the life history of Drosophila melanogaster hosts

Abstract Wolbachia bacteria are common endosymbionts of many arthropods found in gonads and various somatic tissues. They manipulate host reproduction to enhance their transmission and confer complex effects on fitness‐related traits. Some of these effects can serve to increase the survival and transmission efficiency of Wolbachia in the host population. The Wolbachia–Drosophila melanogaster system represents a powerful model to study the evolutionary dynamics of host–microbe interactions and infections. Over the past decades, there has been a replacement of the ancestral wMelCS Wolbachia variant by the more recent wMel variant in worldwide D. melanogaster populations, but the reasons remain unknown. To investigate how environmental change and genetic variation of the symbiont affect host developmental and adult life‐history traits, we compared effects of both Wolbachia variants and uninfected controls in wild‐caught D. melanogaster strains at three developmental temperatures. While Wolbachia did not influence any developmental life‐history traits, we found that both lifespan and fecundity of host females were increased without apparent fitness trade‐offs. Interestingly, wMelCS‐infected flies were more fecund than uninfected and wMel‐infected flies. By contrast, males infected with wMel died sooner, indicating sex‐specific effects of infection that are specific to the Wolbachia variant. Our study uncovered complex temperature‐specific effects of Wolbachia infections, which suggests that symbiont–host interactions in nature are strongly dependent on the genotypes of both partners and the thermal environment.


| INTRODUC TI ON
The Rickettsia-like Alphaproteobacteria Wolbachia are endosymbionts of many arthropod taxa that have profound effects on reproduction and other life-history traits of the host (reviewed in Kaur et al., 2021;Landmann, 2019). They are transmitted vertically through the mother's egg to offspring and have evolved strategies that benefit females in the host population, thereby enhancing their own transmission (Werren et al., 2008). The microbe can manipulate the host reproductive system via different strategies like feminization (Rousset et al.,| 789 STRUNOV eT al. 1992), parthenogenesis (Huigens et al., 2000), male killing (Jiggins et al., 2000) or cytoplasmic incompatibility (CI; Caspari & Watson, 1959), which altogether can result in up to 100% infected individuals in some species (Landmann, 2019). Wolbachia symbionts are prevalent and persistent in nature (Bykov et al., 2019;Solignac et al., 1994;Verspoor & Haddrill, 2011) as well as in established long-term lab strains of the fruit fly Drosophila melanogaster (Clark et al., 2005).
Several studies on fruit flies documented potential fitness benefits of Wolbachia infections in the form of higher fecundity or increased longevity (Fry et al., 2004;Fry & Rand, 2002;Olsen et al., 2001), while other studies showed no or even negative fitness consequences of infections (Harcombe & Hoffmann, 2004). Moreover, Wolbachia infections can induce resistance against RNA viruses in D. melanogaster (Hedges et al., 2008;Teixeira et al., 2008) and other Drosophila hosts (Martinez et al., 2014). Together, the mutualistic effects of Wolbachia in D. melanogaster may explain its pervasiveness in natural fly populations (Teixeira et al., 2008). However, when considering RNA virus protection, it is important to account for differences between laboratory populations and flies collected in nature (Cogni et al., 2021;Shi et al., 2018).
There are two bacterial variants that are most commonly found in natural D. melanogaster populations, named wMel and wMelCS (Riegler et al., 2005), which diverged approximately 80 000 fly generations (Chrostek et al., 2013;Early & Clark, 2013;Richardson et al., 2012), that is, approximately 5.300 years ago (assuming 15 generations per year; Pool, 2015). Genetic analyses of long-term lab strains and recently collected samples indicate a worldwide replacement of the ancestral wMelCS by the more recent wMel variant within a few decades (Riegler et al., 2005). The reasons for this global turnover remain unknown. Since CI is generally weak in natural D. melanogaster populations (Hoffmann et al., 1994), differential fitness effects imposed by the two variants are the most plausible cause for this recent global turnover.
In D. melanogaster, fitness effects can differ with respect to Wolbachia variants (Chrostek & Teixeira, 2015). Fly strains infected with the wMelCS variant exhibit higher resistance against RNA viruses than strains infected with the closely related wMel variant (Chrostek et al., 2013). This phenotypic difference may be caused by an overall higher titre of wMelCS (Chrostek et al., 2013): elevated titres may impose fitness costs on infected flies that result in a shorter lifespan due to competition for cellular resources (Chrostek et al., 2013). In its most extreme form, the wMelPop variant, a labgenerated subvariant of wMelCS, induces the highest infection titre and significantly reduces lifespan at higher temperatures (Min & Benzer, 1997;Strunov et al., 2013).
Most of the aforementioned studies focused on intrinsic factors, such as different Wolbachia or host genotypes, which may influence the homoeostatic host-symbiont interactions. Much less is known about the influence of extrinsic environmental factors, such as temperature, nutrition or population density (Christensen et al., 2019;Hoffmann et al., 1998;Pontier & Schweisguth, 2015;Ponton et al., 2015;Reynolds et al., 2003). Recent studies indicate that individuals infected with wMelCS prefer cooler temperatures than uninfected individuals (Truitt et al., 2019;Arnold et al., 2019;but see Hague et al., 2020), suggesting that Wolbachia-infected hosts may avoid higher temperatures to alleviate fitness costs resulting from bacterial infections. A new study of natural D. melanogaster populations from Ukraine infected with wMel and wMelCS found that the effect of bacteria on fitness components and stress-related phenotypes is highly condition-dependent and influenced by the host genotype (Serga et al., 2021). However, most work focusing on phenotypic effects of Wolbachia infections used highly inbred, long-term Drosophila lab strains, which were often generated by de novo introgression via backcrossing (Chrostek et al., 2013;Teixeira et al., 2008) or transinfection with non-native Wolbachia variants (Martinez et al., 2014

| Crosses among isofemale fly strains from Portugal
Phenotypic variation among the two strains with wMel (RP1) and wMelCS (RP2) infections from Portugal may be a result of differences in Wolbachia infection types and/or their nuclear genetic background. In addition, high levels of inbreeding in the isofemale strains may result in inbreeding depression, which may negatively affect fitness. To homogenize the autosomal genetic background of RP1 and RP2, we took advantage of the strict maternal transmission of Wolbachia to obtain hybrid F1 offspring infected with either wMel (derived from the wMel female × wMelCS male cross) or wMelCS (from the wMelCS female × wMel male cross). We further eliminated Wolbachia infections in the natural strains by treating flies from a subset of each strain with antibiotic (0.1% Rifampicin) for three generations and subsequently restored their gut flora (GFR) by placing the flies into food vials with freshly deposited fly faeces from untreated males of the same strain for two generations. We then used these antibiotic-treated Wolbachia-free strains to set up F1 flies similar to the crosses above (wMel female GFR × wMelCS male GFR and wMelCS female GFR × wMel male GFR) to compare flies with homogeneous genetic background in the presence or absence of Wolbachia infections.
Importantly, the direction of the cross may additionally influence phenotypic effects independently of Wolbachia infections. For example, males have only one (hemizygous) copy of the X-chromosome, which they inherit from their mothers. Moreover, mitochondria are only transmitted maternally and thus differ in the F1 with respect to the direction of the cross. To account for this statistically, we decomposed the four possible Wolbachia infection outcomes from the crosses described above (wMel+, wMel−, wMelCS+, wMelCS−) into two main factors cross and infection in our statistical analyses. Cross is a factor with two levels (wMel and wMelCS) which describe the direction of the crosses among the two pure isofemale strains, that is, RP1 × RP2 (which we denote wMel) and RP2 × RP1 (which we denote wMelCS), irrespective of their infection status. This factor thus accounts for differences with respect to the direction of the cross among the two strains independently of the Wolbachia infection status, which may stem from maternally transmitted mtDNA or host sex-linked effects. Conversely, the factor infection is a fixed factor with two levels (+, −), which describes the presence or absence of Wolbachia infections irrespective of the Wolbachia type. Thus, only a significant interaction between cross and infection indicates different effects of the two Wolbachia variants on the investigated phenotype.

| Confirmation of Wolbachia infection with polymerase chain reaction (PCR)
To confirm the infection status of all experimental strains, we used PCR with Wolbachia-specific primers and conditions as described by Riegler et al. (2005). In brief, we extracted genomic DNA of five pooled flies using the Qiagen DNeasy kit (Qiagen, Hilden, Germany) and amplified a sequence of the wsp gene, a well-established maker for Wolbachia infections. Upon positive results for all infected strains for the wsp locus, we further amplified a variable tandem repeat region of the Wolbachia genome (VNTR-141) which is characterized by length polymorphisms that are diagnostic for different Wolbachia variants (Riegler et al., 2012). PCR amplification was set up in 10 µl reaction volumes with 0.3 μM primers in 1× reaction buffer (Promega 5x Green GoTaq), 2.5 mM MgCl 2 , 150 µM dNTPs and 0.025 U/µl DNA-Polymerase (Promega GoTaq). Following the protocol, we ran PCR reactions with the following conditions: 2 min at 94°C for initial denaturation followed by 30 cycles of 45 s at 94°C (denaturation), 45s at 67°C (annealing) and 30s at 72°C (elongation).
The run finished with a final extension at 72°C for 10 min. To quantify length polymorphisms, we visualized and separated PCR bands by gel electrophoresis using a 0.8% agarose gel (see supplementary data file for gel images).

| Development time
We measured egg to adult development time by counting eclosed F1 hybrids from all crosses described above (wMel × wMelCS, wMelCS × wMel, wMelCS GFR × wMel GFR, wMel GFR × wMelCS GFR). For each of the four crosses and each temperature regime (20°C, 24°C, 28°C), we allowed 15 individual females at an age of 4-7 days to lay eggs in individual vials (i.e. one female/vial) in one 24-h interval. These vials were then placed into incubators at the respective temperatures. To measure development time, each vial was checked for newly eclosed adults three times per day (8:00, 14:00, 20:00) for 1 week starting from the day the first flies eclosed. Flies were sexed and subsequently used for a longevity experiment (see below).

| Body size
Femur length is a reliable proxy for adult body size in Drosophila (Siomava et al., 2016), a fitness-related adult trait that is determined during larval development, which is positively correlated with female fecundity (e.g. see Flatt, 2020), To investigate if Wolbachia influences female body size, we dissected and mounted the left foreleg of the F1 females emerging from all crosses described above on glass slides with Euparal mounting medium (Roth), to be photographed at 40x magnification using a Leica DFC490 digital camera attached to a Leica MZ12 microscope to estimate femur length with ImageJ (http://imagej.nih.gov/ij/; v.1.53c) based on two landmarks (as described in Debat et al., 2011), in triplicate to minimize measurement error (see Supplementary data file). Raw images of female forelegs can be obtained from DataDryad (https://doi.org/10.5061/ dryad.sxksn 035v).

| Oogenesis and ovariole number
To obtain insight into the developmental mechanisms conferring fecundity differences between wMel and wMelCS variants, we compared the number of ovarioles in females at different temperatures.
We also counted the number of mature eggs in the ovarioles 24 and 48 h after eclosion. From each cross, we collected 20 freshly eclosed virgin females and paired each with two uninfected (RP3) males in incubators at 20, 24 and 28°C. To count ovarioles, we dissected female abdomens in phosphate-buffered saline (PBS) and stained ovaries for ca. 5 min in potassium dichromate solution. Ovarioles and mature eggs in each ovariole were counted by eye at 16-fold magnification under a Leica MZ12 microscope.

| Fecundity
To assess the effect of Wolbachia variant and infection type on female fecundity, we used F1 virgin females that were generated from the four crosses described above (wMel × wMelCS, wMelCS × wMel, wMelCS GFR × wMel GFR, wMel GFR × wMelCS GFR). We paired single F1 virgin females (within 24h post eclosion) with two naturally uninfected males of strain RP3 and propagated these trios at 20, 24 or 28°C. For each temperature, cross and infection status, we set up 30 replicate triads to investigate their fecundity for 10 consecutive days. Every day, all flies were transferred to a new vial, and their offspring was counted every second day as the number of eclosing adults. Males that died during this time were not replaced.

| Longevity
We assessed strain-and environment-specific lifespans for each Flies that drowned in the food media or escaped were not included in the analysis.
Figures were created with the ggplot2 R package (Wickham, 2016).
We analysed all outcome variables (listed above and below) as a fully crossed factorial design with three main fixed factors (temperature, cross, infection), including all possible higher order interactions, and replicate trial as random factor (only for the outcome variables development time, body size, ovariole number, mature eggs and longevity). Temperature was a (fixed) factor with three levels (20, 24, 28°C), cross a factor with two levels (wMel and wMelCS, see above for a more detailed description) and infection a factor with two levels denoting the presence (+) versus absence (−) of Wolbachia infection.
Additionally, for some outcome variables, sex was added as a fixed factor with two levels (male and female; for development time and longevity only), and age a fixed factor with two levels (24 and 48 h after adult eclosion) in case of ovariole number, and with five levels (1, 3, 5, 7, 9 days after eclosion) in case of fecundity (detailed below).
Body size at emergence was analysed with a regular general linear mixed model (LMM; including temperature, cross, infection) and normal error distribution and ovariole number with a generalized linear mixed model (GLMM; including temperature, cross, infection, age) with Poisson-distributed errors to account for the statistical properties of count data, using the R package lme4 (Bates et al., 2015). Development time and longevity (i.e. age at death) were analysed using proportional hazards (Cox regression) analysis with the R package coxme (Therneau, 2020;including temperature, cross, infection, sex). When scoring fecundity of females, we unfortunately not fully tracked the identity of individual females over time (female age when laying: 1, 3, 5, 7 and 9 days post eclosion). It was thus not possible to conduct the appropriate repeated-measures analysis with female ID as random factor. To avoid inflated type-II errors (i.e. falsely rejecting the null hypothesis) caused by the dependence of females across consecutive days, we did not enter female age as an additional factor in our analysis but analysed each day separately using general linear models (GLM; including temperature, cross, infection, age) with Poisson-distributed errors to account for the statistical properties of count data. Due to an excess of females that did not produce offspring on day 1 (75.5%), this first time point was excluded from further statistical analyses to avoid excessive zero inflation. To further account for the non-independence of the data across the four time points, we conservatively applied Bonferroni correction for multiple testing, that is, we only considered an effect significant if the p-value was smaller than the corrected significance threshold α' = 0.0125 (0.05/4). Similarly, for the number of mature eggs in ovarioles, we investigated the two time points of the assay (24 and 48 h posteclosion) separately. Since most females at 24 h had not developed mature eggs (only 18% = 52 females carried one or more mature eggs), we only describe this subset qualitatively. We statistically analysed only the data of 48-h-old females (when ca. 82% = 239 of all females carried at least one mature egg) with a GLMM (including temperature, cross, infection) and a negative binomial error structure as implemented in the R package glmmTMB (Brooks et al., 2017) to account for zero inflation in the data set.
For GLMMs and cox regression, we tested for significance with type-III analysis of deviance based on Wald χ 2 -tests as implemented in the R package car (Fox & Weisberg, 2019). For LMM and GLMs, we applied Satterthwaite's method to estimate degrees of freedom and tested for significance with Kenward-Roger F-tests using the car package (Fox & Weisberg, 2019). We performed pairwise comparisons among levels for significant response variables with more than two factor levels using Tukey's honest significant difference (HSD) post hoc tests as implemented in the R package emmeans (Lenth, 2021). All raw data, R scripts with complete models, the code for statistical analyses and the original output files are provided as a zipped Supplemental Data file.

| Development time
Neither Wolbachia infection nor direction of the cross influenced the development time of the host (Infection: χ 2 = 0.2, p = 0.63 and Cross: χ 2 = 0.28, p = 0.6; Table 1; Figure 1 and Figure S1). However, we found significant effects of temperature and sex on development time in all strains (Table 1). Females eclosed before males (Sex; χ 2 = 208.2, p < 2.2e-16) and developed faster at higher temperatures (Temp; χ 2 = 1650.9, p < 2.2e-16; Figure 1, Table 1). Accordingly, we found highly significant interactions between temperature and sex (χ 2 = 36.4, p = 1.3e-08). In addition, there was a significant interaction between sex and cross (χ 2 = 5.1, p = 0.024), which hints at sex-linked effects on development time determined by the direction of the cross. Lastly, there was a marginally significant higher order interaction between sex, temperature and infection (χ 2 = 6.2, p = 0.044), suggesting very subtle differential effects of Wolbachia infections at different temperature and the sexes.

| Ovariole number
We further tested if differences in larval development potentially affect female fecundity via the number of ovarioles. While temperature had a significantly positive effect on the total number of ovarioles (X 2 = 27.2, p = 1.3e-06, Table 3, Figure 3), we neither found an effect of Wolbachia infection (X 2 = 0.5, p = 0.474) nor differences with respect to direction of the crosses (X 2 = 0.9, p = 0.335) nor age of the female (X 2 = 2.8, p = 0.096).

| Female fecundity
We measured female fecundity as the number of eclosed adult offspring per female in 24-h intervals at different maintenance temperatures. Since Female IDs were not fully tracked during the experiment, we analysed each time point separately to avoid pseudoreplication, which may result in an inflated type-II error (i.e. false rejection of the null hypothesis). At all four time points (aged 3-9 days), temperature  Figure 4a). We further found significant interactions between infection and cross on day 3 (F 1/329 = 9.6, p = 0.002; Table 4) and day 9 (F 1/247 = 12, p = 0.001; Table 4). Particularly on day 3, wMelCS-infected females produced more offspring at 24 and 28°C than both uninfected females and females infected with wMel (Tukey HSD; p < 0.001 for all comparisons), indicating that Wolbachia infections have a positive effect on fecundity and that this effect differs for Wolbachia variants. Since such a significant interaction was not found at days 5 and 7, we speculate that wMelCS may stimulate an early onset of oogenesis. On day 9, in contrast, wMelCS+ fecundity did only differ from wMel+ and wMel− at 20°C (Tukey HSD; p < 0.001). However, wMelCSinfected flies were nevertheless more fecund than uninfected flies (wMelCS−) of the same crossing direction, irrespective of temperature (Tukey HSD; p ≤ 0.0001 for all comparisons). In addition, peak fecundity of wMelCS + females appears to be strongly temperaturedependent ( Figure 4a): while peak fecundity was not reached within 9 days at 20°C, it averaged 7 days at 24°C and 3 days at 28°C.
To test the hypothesis that wMelCS infections stimulate an early onset of oogenesis, we counted the number of mature eggs in ovaries of young females 24 h and 48 h after eclosion. Only 52 (18%) 24-h-old females had produced at least one mature egg. Due to excessive zero inflation, we did not statistically analyse this data subset. However, we noted that most of the females with mature eggs (33) were infected with wMelCS. For flies aged 48 h, we found a highly significant increase in number of mature eggs with temperature (χ 2 = 59.3, p < 1.3e-13; Table 5). In addition, we found significant interactions between temperature and infection (χ 2 = 12.3, p = 0.002; Table 5); cross and infection (χ 2 = 8.0, p = 0.005); and between temperature, cross and infection (χ 2 = 10.9, p = 0.004; onset of oogenesis in wMelCS-infected females begins earlier than in uninfected flies and flies infected with wMel, and that this effect is particularly pronounced at higher temperatures.

| Longevity
A well-described trade-off in life-history evolution is decreased longevity in case of higher early fecundity (cost of reproduction), which is commonly explained by allocation trade-offs due to limited resources (Flatt, 2011). We found highly significant effects of temperature and sex on longevity (temperature; χ 2 = 419.8, p < 2.2e-16; sex; χ 2 = 78.1, p < 2.2e-16; Table 6), but also significant twoway interactions between sex and infection (χ 2 = 35.8, p = 2.2e-09; Table 6) and temperature and infection (χ 2 = 46.4, p < 8.4e-11; Table 6). The significant interaction between sex and cross (χ 2 = 6.3, P 0.012; Table 6) indicates sex-linked effects with respect to the direction of the cross but independently of Wolbachia infections.

| DISCUSS ION
Systematic studies of D. melanogaster long-term lab strains and worldwide populations have uncovered a recent global turnover of two Wolbachia variants resulting in the replacement of wMelCS by wMel within half a century (Riegler et al., 2005).
However, it is still unknown which mechanisms underlie such rapid evolutionary change. This motivated us to disentangle the fitness consequences of these two Wolbachia variants on natural D. melanogaster populations while taking into account temperature, which is one of the most important environmental factors influencing the physiology and life history of all organisms (Angilletta et al., 2004;Thomas & Blanford, 2003). Temperature further affects the interaction dynamics between host and microbe (reviewed in Corbin et al., 2017). Moderate temperatures between 22 and 26°C are usually considered comfortable for both partners of the Wolbachia-Drosophila association (Gora et al., 2020;Hague et al., 2020;López-Madrigal & Duarte, 2019).
In general, high temperatures lead to depletion of bacteria from the host, while lower temperatures slow down the replication of the symbiont and alleviate potential fitness costs (Chrostek et al., 2021;Hague et al., 2020;Strunov et al., 2013), although there are exceptions (Min & Benzer, 1997;Mouton et al., 2005

| Developmental life-history traits are not influenced by Wolbachia infections
Consistent with earlier reports from many species, temperature had a major impact on development time and body size of D. melanogaster (see Flatt, 2020 for a comprehensive review). By contrast, there were no direct effects of Wolbachia infection nor variant, nor interactions with temperature on juvenile development time and resulting adult body size, which is also in line with a previous study (Harcombe & Hoffmann, 2004). It is known that environmental conditions and physiological interactions with an endosymbiont during development may directly affect the resulting adult phenotype (Grenier & Leulier, 2020). For instance, rearing D. simulans larvae infected with Wolbachia at high temperatures increased cytoplasmic incompatibility in males (Clancy & Hoffmann, 1998), which is beneficial for the spread of Wolbachia (Turelli & Hoffmann, 1995).
The absence of effects observed here in the juvenile life stages of

D. melanogaster hosts might therefore be the result of overall low
Wolbachia titre (infection) levels of juveniles (Stevanovic et al., 2015;Strunov et al., 2013). In line with this hypothesis, a previous study showed that wMel-infected larvae with low titre levels did not exhibit any resistance to the Drosophila C virus, (DCV; Stevanovic et al., 2015), possibly due to subthreshold physiological effects induced by the endosymbiont on the host. This interpretation is supported by a recent study of Chrostek et al. (2021), who found that infected individuals reared at 18°C did not exhibit enhanced resistance against DCV, contrary to those reared at 25°C. Low-rearing temperatures thus seem to suppress replication of Wolbachia during development (Hague et al., 2020), and might subsequently influence the life history of the adult in terms of fecundity and lifespan.
Alternatively, the absence of Wolbachia effects on developmental life-history traits might be explained by differential activity of the endosymbiont during this early period of the host life cycle. A comprehensive analysis of Wolbachia gene expression across the D.
melanogaster life cycle shows that the bacteria have significantly distinctive expression patterns in early larvae, late pupae and adults (Gutzwiller et al., 2015). A follow-up study that reanalysed previously published Wolbachia RNA-Seq transcriptomic data uncovered that wMel variant genes which affect ribosome biosynthesis and translation of the host are consistently upregulated during early life relative to adult stages (Chung et al., 2020). In this context however, and infection status on female fecundity (measured as number of adult offspring that emerged from eggs that were laid in 24-h intervals from single females) in four data sets collected at consecutive time points Drosophila species (Martinez et al., 2014(Martinez et al., , 2015, showing that high titre infections often result in negative fitness effects such as reduced fecundity, egg hatching rate or male fertility. By contrast, our study showed positive effects of wMelCS, and to a lesser extent also of wMel, on fecundity and longevity in D. melanogaster populations from Portugal. Thus, our findings are not consistent with previous data (Chrostek et al., 2013), which might be explained by differences among the fly strains investigated. Chrostek et al. (2013) and Martinez et al. (2014Martinez et al. ( , 2015 used long-term and highly inbred  (Charlesworth & Charlesworth, 1987

or instead represent artefacts from non-native infections with
Wolbachia variants that are not specific to their hosts.
The only negative effect associated with Wolbachia infections found in our study was observed at higher temperatures (24 and 28°C) in males infected with wMel, which died sooner than uninfected males. In line with our results, Fry and Rand (2002) similarly found that the influence of Wolbachia on survival differs for the sexes. Since Wolbachia is maternally transmitted to enhance its own transmission, it is advantageous for the endosymbiont to have a positive effect on the survival of infected females but not necessarily on that of males (Werren et al., 2008). Interestingly, the negative effect on longevity was not observed in wMelCS-infected males. The variant wMelCS is considered the ancestral infection type of D. melanogaster, which was more recently replaced by wMel globally (Early & Clark, 2013;Ilinsky, 2013;Richardson et al., 2012;Riegler et al., 2005 and decreased the programmed cell death in the germarium (Fast et al., 2011). However, in a more recent study, this finding could not be reproduced (Meany et al., 2019). Increased fecundity through GSCs manipulation by Wolbachia was reported in another insect, the hemipteran Laodelphax striatellus (Guo et al., 2020), suggests the existence of a common mechanism of endosymbiont interference promoting female host reproduction. Thus, it is possible that the wMelCS variant acts similarly in wild-caught D. melanogaster.
Enhanced reproduction is costly and might lead to a reduced lifespan of the female due to energetic trade-offs (Flatt, 2011). However, we did not observe any additional costs of wMelCS infection for females in our study, which might have remained undetected under laboratory conditions of sufficient food supply and controlled temperature, or may manifest in traits that were not investigated here.
In nature, the cost of producing a higher number of progeny might lead to otherwise reduced performance of wMelCS-infected flies, and eventually to replacement by a variant such as wMel with milder effects. Further studies investigating Wolbachia-borne trade-offs under natural conditions are necessary to test this hypothesis (e.g. see Utarini et al., 2021).  (Utarini et al., 2021).