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. 2022 Oct 18;24(12):5749–5759. doi: 10.1111/1462-2920.16235

Fitness costs of Wolbachia shift in locally‐adapted Aedes aegypti mosquitoes

Perran A Ross 1,✉, Ary A Hoffmann 1
PMCID: PMC10947380  PMID: 36200325

Abstract

Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavourable conditions. A. aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We found that the cost of wMel infection differed consistently between mosquitoes collected from different locations and became weaker across laboratory generations, suggesting environment‐specific adaptation of mosquitoes to the wMel infection. Reciprocal crossing experiments show that differences in the cost of wMel to quiescent egg viability were mainly due to mosquito genetic background and not Wolbachia origin. wMel‐infected mosquitoes hatching from long‐term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long‐term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and mosquito genetic backgrounds. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection.

INTRODUCTION

Aedes aegypti is well adapted to anthropogenic environments across the world, that include not only the ability to tolerate pesticides in these environments (Moyes et al., 2017) but also life history strategies that allow it to breed under variable conditions (Clements, 1992). One adaptation to variable conditions is the ability for its eggs to enter a quiescent stage, where they remain viable for periods without water before hatching following rainfall or human‐associated activities that increase water availability (Holmes & Benoit, 2019). This phenotype is likely to be particularly important in climates with an extended dry season where populations may otherwise not persist (Rašić et al., 2014). A. aegypti show local adaptation to quiescence (Faull & Williams, 2015), where populations from locations that experience longer dry seasons or infrequent water availability have longer viability as quiescence eggs.

While egg quiescence is expected to contribute to fitness under many natural conditions, it will be less important in the laboratory, at least when mosquitoes are maintained under conditions that lead to a constant turnover of generations that maximize population productivity. This raises the issue of whether mosquito populations under constant turnover might adapt to laboratory conditions in terms of quiescence as well as other traits like mating behaviour and stress tolerance (Hoffmann & Ross, 2018). While there will often be a lack of selection to maintain long‐term quiescent egg viability, effects of laboratory adaptation on this phenotype have rarely been tested (Faull & Williams, 2015).

Wolbachia endosymbionts are being released in various locations around the world and having substantial localized impacts on dengue transmission (Nazni et al., 2019; Ryan et al., 2019; Utarini et al., 2021). One challenge is that Wolbachia can have a substantial impact on quiescent egg viability; this was first recognized for the wMelPop Wolbachia infection (McMeniman & O'Neill, 2010; Yeap et al., 2011) but also applies to wAlbB (Axford et al., 2016; Lau et al., 2021a). These costs may have contributed to unsuccessful establishment of both wMelPop and wAlbB in some locations (Nazni et al., 2019; Nguyen et al., 2015). In fact, quiescent egg viability costs have been proposed as a tool for seasonal population suppression (Rašić et al., 2014; Ritchie et al., 2015). The widely released wMel strain also reduces egg viability (Farnesi et al., 2019; Fraser et al., 2017) but the effects are usually weaker compared to other strains, and this has not prevented its establishment in several locations, even in places with long dry seasons such as Cairns, Australia (Hoffmann et al., 2011).

Releasing a Wolbachia strain that induces fitness costs is expected to cause evolutionary changes in either the mosquito or Wolbachia to attenuate these effects (Bull & Turelli, 2013; Correa & Ballard, 2016). While the wMel genome has remained unchanged (Dainty et al., 2021; Huang et al., 2020; Ross et al., 2022) there is currently a limited understanding of mosquito adaptation to Wolbachia infections. The A. aegypti genome shows minor changes across the decade in which Wolbachia have been released in North Queensland, Australia and some of these changes might relate to fitness effects (Lau et al., 2021b). Selection experiments show that the quiescent egg viability costs of wMelPop can be ameliorated through selection due to changes in the nuclear genome (Ritchie et al., 2015) and costs to some traits also appear to have shifted over time though long‐term lab rearing (Ross et al., 2020a).

Currently, there is no evidence that wMel fitness costs have shifted much under field conditions (Hoffmann et al., 2014; Ross et al., 2022). There is also no evidence for differences in fitness costs of wMel between mosquito genetic backgrounds that have had long‐term coadaptation versus naïve backgrounds (Ross et al., 2022). However, phenotypic comparisons have covered only a limited number of traits, most of which are only minimally influenced by wMel infection, and quiescent egg viability costs have not been compared between backgrounds. In field populations, we might expect to see Wolbachia costs associated with quiescence become weaker over time. This could have impacts on population dynamics; wMel is expected to reduce the A. aegypti population size relative to competing container species due to its fitness costs (Ross, 2021), thus indirectly reducing virus transmission, but evolution could mitigate this benefit. Data from long‐term post‐release monitoring of wMel in Yogyakarta, Indonesia suggest minor or absent fitness costs of wMel infection under these conditions, as the ratio of A. aegypti to competing Aedes albopictus decreased only marginally after Wolbachia establishment (Tantowijoyo et al., 2022).

In this study, we measured the effects of wMel infection on A. aegypti quiescent egg viability in mosquito populations originating from different locations. We then performed reciprocal backcrossing to identify the cause of differences in fitness costs. Costs were driven strongly by mosquito genetic background, with surprisingly higher costs in field‐collected mosquitoes and consistent differences between populations from different locations. In the absence of Wolbachia, there was no evidence for evolution of quiescent egg viability in long‐term laboratory populations despite the lack of selection to maintain this trait. The differential fitness costs of wMel in the backgrounds observed here provide some of the first direct evidence of genetic differences in mosquito populations affecting Wolbachia‐related phenotypes. We also identify additional cross‐generational costs of wMel in quiescent eggs. Our results have implications for the spread of Wolbachia in different local environments, and the use of laboratory studies to test fitness costs of Wolbachia.

EXPERIMENTAL PROCEDURES

Ethics statement

Blood feeding of female mosquitoes on human volunteers for this research was approved by the University of Melbourne Human Ethics Committee (approval 0723847). All adult subjects provided informed written consent (no children were involved).

Field collections and colony maintenance

We established A. aegypti populations from eggs collected from ovitraps placed in suburban Cairns in 2016, 2018, 2019 and 2020 as described previously (Ross et al., 2020b). Collections focused on two suburbs: Yorkeys Knob (YK) and Gordonvale (GV), where wMel‐infected A. aegypti were first released in 2011 (Hoffmann et al., 2011), as well as suburbs in central Cairns, where staggered releases took place from 2014 (Ryan et al., 2019). Each A. aegypti population was established from a pool of 15–50 ovitraps and at least 200 founding individuals. Laboratory populations were maintained at a census size of ~450 individuals each generation at 26°C and a 12:12 light: dark cycle as described previously (Ross et al., 2017a). Female mosquitoes were fed on the forearm of a single human volunteer for egg production. Repeated collections from the field enabled us to establish wMel‐infected populations from the same origin but with different numbers of generations of laboratory rearing, with approximately 12 generations per year. We also performed experiments on the same populations at different times allowing us to track changes over time, though experiments were not directly comparable due to potential differences in egg storage conditions. In all experiments, we included a long‐term laboratory population (wMel Lab), which was collected from Cairns in 2014 and had spent at least 60 generations in the lab at the time of the first experiment.

Antibiotic curing

To generate wMel‐infected and uninfected mosquitoes with matching genetic backgrounds, populations were split in two, with one population treated with tetracycline and the other left untreated. Treated populations were provided with 10% sucrose containing 2 mg/ml of tetracycline hydrochloride for two consecutive generations, followed by two generations of recovery (with no tetracycline) before experiments. Populations collected from the field began treatment within the first 1–2 generations in the laboratory to minimize potential effects of laboratory adaptation. In the generation following treatment, 30 individuals from each treated population and untreated population were screened for the presence of wMel according to previously described methods (Lee et al., 2012). Populations were only used in experiments if wMel was absent from all individuals in treated populations and present in all individuals in untreated populations. wMel‐infected populations were cured each time they were used in experiments; wMel‐infected and uninfected counterparts were not maintained as separate populations for more than five generations to minimize potential effects of genetic drift (Ross et al., 2019a).

Quiescent egg viability

We measured the quiescent egg viability of A. aegypti in several experiments using a standardized approach. Females in colony cages (aged 5–7 days, starved of sugar for 1 day) were blood fed and six plastic cups (250 ml) filled with larval rearing water and lined with sandpaper strips (Norton Master Painters P80; Saint‐Gobain Abrasives Pty. Ltd., Thomastown, Victoria, Australia) were placed in the cages. Sandpaper strips were removed 4 days after blood feeding, wrapped in paper towel and placed in zip‐lock bags. The next day, sandpaper strips were labelled and placed in a single sealed chamber with a saturated solution of potassium chloride to maintain the relative humidity ~80%. The chamber was placed in a controlled temperature room at 26°C with a 12:12 light: dark cycle. To measure quiescent egg viability, batches of >40 eggs were removed from the chamber and submerged in 500 ml rectangular plastic trays filled with 300 ml of water. Each tray was provided with a small amount of fish food (TetraMin tropical fish food tablets, Tetra, Melle, Germany) and a few grains of yeast to stimulate hatching and provide food for larvae. Egg hatch proportions were scored at least 3 days after hatching by dividing the number of hatched eggs (with a clearly detached cap) by the total number of eggs in each batch. Trays that were not scored for egg hatch after 3 days were stored at 4°C to prevent further hatching and larval development. For all experiments, batches of eggs were hatched on Weeks 1 and 2, then every 2 weeks until Week 24. The number of replicate batches of eggs per population and timepoint varied between experiments (see below).

Variation in quiescent egg viability across laboratory A. aegypti populations

We performed two experiments to assess variation in quiescent egg viability in wMel and uninfected laboratory and near‐field populations. The first experiment was performed in November 2018 with Lab (~F60) and YK (F11) populations, both wMel‐infected and uninfected. Each population (wMel Lab, uninfected Lab, wMel YK F11 and uninfected YK F11) was maintained as two separate replicate lines. We scored egg viability for six replicate batches of eggs per time point, per replicate line, for a total of 12 replicate batches per population at each time point. In August 2019, we performed a second experiment with a single replicate line of each population from the first experiment (wMel Lab, uninfected Lab, wMel YK F18 and uninfected YK F18). We also included additional populations (both wMel‐infected and uninfected) collected from YK and GV in April 2019 which were at F3 at the time of the experiments, and a population from central Cairns at F11. In this experiment, eight replicate batches of eggs were tested per population, per time point.

Effects of laboratory adaptation on quiescent egg viability

To further investigate the effects of laboratory rearing and wMel infection on quiescent egg viability, we performed a third experiment to compare populations collected at two time points from two locations. wMel‐infected populations were collected from YK and GV in April 2019 and March 2020 and uninfected populations were generated according to the procedure above (see ‘antibiotic curing’). The experiment was performed in August 2020, with the wMel and uninfected YK and GV populations at F4 and F15 in the laboratory at the time of experiments. In this experiment, eight replicate batches of eggs were tested per population, per time point. Data for Weeks 10 and 20 were discarded due to fungal growth preventing accurate scoring of egg hatch proportions following cold storage.

wMel origin and genetic background contributions to quiescent egg viability

To estimate the contribution of wMel origin and genetic background to quiescent egg viability, we performed reciprocal backcrossing between wMel Lab and wMel‐infected populations from YK and GV collected in March 2020. Total 100 females from each population (wMel YK, wMel GV and wMel Lab) were crossed to 100 males from each population (wMel YK, wMel GV and wMel Lab). Female progeny were crossed to non‐backcrossed males from the same population for two additional generations. This resulted in the partial introgression of wMel from each origin (Lab, YK and GV) to the target background (Lab, YK and GV), with an estimated 87.5% similarity to the target background. Backcrosses were performed after the first generation of laboratory rearing so that populations were at F4 at the time of experiments. This set of comparisons was performed as part of Experiment 3, therefore data for wMel Lab, wMel YK F4 and wMel GV F4 (the non‐backcrossed populations) were included in two sets of analyses.

Cytoplasmic incompatibility and female infertility

We tested cytoplasmic incompatibility and female infertility in wMel‐infected individuals hatching from long‐term quiescence. Larvae hatching from wMel Lab, wMel YK F4 and wMel GV F4 eggs stored for 1, 20 or 24 weeks were reared to the pupal stage and separated by sex. We then performed the following crosses: (1) stored wMel‐infected females were crossed to non‐stored uninfected males, (2) non‐stored uninfected females were crossed to stored wMel‐infected males and (3) stored wMel‐infected females were crossed to non‐stored wMel‐infected males. This design allowed us to test for (1) effects of storage on female fertility, (2) effects on storage on the strength of cytoplasmic incompatibility induced by wMel‐infected males and (3) effects of storage on the compatibility of wMel‐infected females with wMel‐infected males. Uninfected females stored for 1 or 20 weeks were also crossed to non‐stored uninfected males to test for effects of egg storage on female fertility in the absence of wMel. Females in each cross were blood fed and isolated for oviposition 5 days after crosses were established. Four days after blood feeding, eggs were collected on sandpaper strips from individual females, partially dried, then hatched 3 days after collection. We scored fecundity by counting the total number of eggs laid per female and scored egg hatch proportions according to the procedure above for quiescent egg viability. Females that did not lay eggs were scored as infertile. Up to 20 individuals from each wMel‐infected population were measured per cross, but due to low egg hatch following long‐term storage we were left with fewer than 20 individuals per cross for some wMel‐infected populations.

Statistical analysis

Most changes in egg hatch proportions were evident from a visual evaluation of the populations across the egg storage period and we used changes across time in comparing across populations. However we were also interested in comparing the size of different effects on hatch rates (including laboratory rearing, wMel infection and genetic background) and for this purpose we focused on two period of storage between 1 and 8 weeks when there was a relatively consistent decline in viability, and between 12 and 18 weeks when population effects were strong based on preliminary experimental data. We also had relatively complete data sets for these periods. We computed logits for all the independent egg hatch proportion measures (Warton & Hui, 2011) and then ran general linear models comparing populations across these periods including weeks in storage as a covariate to reflect the continued decrease in hatch rates. We included interaction terms with weeks in storage in the models testing for population effects. General linear models were analysed with IBM SPSS Statistics 26. For the population term, we computed the effect size (partial Eta squared) to allow comparisons between periods and treatments.

For the assays on cytoplasmic incompatibility, the data was usually highly skewed towards near‐complete hatching or no hatching. We therefore undertook pairwise comparisons between relevant treatments with non‐parametric Mann–Whitney U tests. Egg hatch proportions did not differ between different wMel‐infected populations within the same cross according to Mann–Whitney U tests, so these were pooled for analysis. For the fertility comparisons we used Fisher's exact tests using RStudio version 2022.02.2 and the rcompanion package (Mangiafico, 2015) to compare the number of infertile and fertile females across the populations for each time point tested.

RESULTS

Variable effects of wMel infection on quiescent egg viability in A. aegypti laboratory populations

We tested the quiescent egg viability of A. aegypti populations that were either wMel‐infected or uninfected (cured) and had been reared in the laboratory for different numbers of generations. In a pilot experiment (Figure S1), we found that wMel had a weaker cost to quiescent egg viability in a long‐term laboratory population (Lab), compared to a population collected from the field more recently (YK F11). To further investigate this variability, we collected fresh populations from two locations, GV and YK, which represent areas where wMel was first released in Australia (Hoffmann et al., 2011). wMel infection reduced quiescent egg viability in all populations (Figure 1), but there was a high level of variation in the wMel populations compared (general linear model [GLM], Weeks 1–8: F 4175 = 30.407, p < 0.001, partial Eta squared = 0.410; Weeks 12–18: F 4140 = 175.707, p < 0.001, partial Eta squared = 0.834). The main effect of week was also significant in both periods (p < 0.001) and there was a week by population interaction for Weeks 12–18 (p < 0.001) but not for Weeks 1–8 (p = 0.379). The wMel Lab population had more than twice the egg viability of wMel GV F3 at most time points and the other populations fell between these extremes (Figure 1(A)). In contrast, the five matched uninfected populations showed similar patterns of quiescent egg viability (Figure 1(B)), with no significant effect of population in Weeks 1–8 (F 4173 = 0.722, p = 0.578, partial Eta squared = 0.016) but a significant week effect (p < 0.001) but no interaction between week and population (p = 0.975). In Weeks 12–18, there was a population effect (F 4140 = 7.485, p < 0.001) but this was much smaller (partial Eta squared = 0.176) than for the earlier period and other effects were not significant (p > 0.078). The lack of consistent differences between uninfected populations from the near‐field and laboratory counterparts indicate that laboratory adaptation has only a minor effect on this trait. This suggests that the differences between the wMel‐infected populations are due to Wolbachia infection or its interaction with the genetic background of those populations.

FIGURE 1.

FIGURE 1

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Quiescent egg viability of (A) wMel‐infected and (B) uninfected Aedes Aegypti across different laboratory generations. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage

Laboratory adaptation reduces the cost of wMel infection to quiescent egg viability

To further investigate the effect of laboratory rearing, we collected wMel‐infected mosquitoes from both GV and Yorkey's Knob at two time points (1 year apart) and generated uninfected counterparts. Consistent with the first experiment (Figure 1) and the pilot experiment (Figure S1), the uninfected populations had similar quiescent egg viability (Figure 2), though significant population effects were detected across all uninfected populations (GLM, Weeks 1–8: F 4162 = 8.607, p < 0.001, partial Eta squared = 0.175; Weeks 12–18: F 4140 = 8.840, p < 0.001, partial Eta squared = 0.202, Figure S2). wMel‐infected populations showed larger differences than uninfected populations (Weeks 1–8: F 4174 = 66.124, p < 0.001, partial Eta squared = 0.603; Weeks 12–18: F 4139 = 72.650, p < 0.001, partial Eta squared = 0.676). There were no significant interactions between week and population in these comparisons (all p > 0.154). In both the YK (Figure 2(A)) and GV (Figure 2(B)) populations, quiescent eggs from the F15 populations had higher viability than those from the F4 populations collected more recently from the field. The effects of Wolbachia infection were stronger in F4 populations (Weeks 1–8: F 1138 = 345.048, p < 0.001, partial Eta squared = 0.714; Weeks 12–18: F 1111 = 773.583, p < 0.001, partial Eta squared = 0.875) compared to F15 populations (Weeks 1–8: F 1135 = 108.100, p < 0.001, partial Eta squared = 0.445; Weeks 12–18: F 1112 = 345.214, p < 0.001, partial Eta squared = 0.755). This suggests that adaptation to laboratory conditions reduces the cost of the wMel infection to quiescent egg viability.

FIGURE 2.

FIGURE 2

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Effects of laboratory rearing on quiescent egg viability in Aedes Aegypti populations from (A) Yorkeys Knob and (B) Gordonvale. Populations were either wMel‐infected (solid lines) or uninfected (dashed lines). Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage

Costs of wMel infection to quiescent egg viability depend on genetic background and not Wolbachia origin

We performed reciprocal backcrosses between the wMel YK, wMel GV and wMel Lab populations to introduce wMel from different origins to different genetic backgrounds and assess the contributions of Wolbachia origin and background to quiescent egg viability. When Wolbachia from different origins was introduced to the same background, we found minor effects of wMel origin on quiescent egg viability (Figure 3(A–C)). In contrast, wMel had consistent differences in costs when introduced to different backgrounds (Figure 3(D–F)). Regardless of the origin of the wMel infection, wMel‐infected populations with a lab background performed better than the YK background, which performed better than GV. Overall, the effects of background (Weeks 1–8: F 2311 = 157.361, p < 0.001, partial Eta squared = 0.503; Weeks 12–18: F 2249 = 238.517, p < 0.001, partial Eta squared = 0.657) were much larger than those of wMel origin (Weeks 1–8: F 2311 = 19.234, p < 0.001, partial Eta squared = 0.110; Weeks 12–18: F 2249 = 27.036, p < 0.001, partial Eta squared = 0.178). Differences between YK and GV may indicate adaptation to local conditions that differ between these locales, and a pattern that is consistent with that of the earlier experiments (Figures S1 and 2) showing that costs are weaker in lab backgrounds compared to those of recently established populations. Note that differences between the backgrounds were more substantial for the experiments undertaken with Wolbachia from the field populations, however it should be noted that replacement of the genetic background was incomplete (estimated at 87.5%) so the wMel origin of the different backgrounds also included a residual nuclear component.

FIGURE 3.

FIGURE 3

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Effects of Wolbachia origin (A–C) and genetic background (D–F) on quiescent egg viability in reciprocally‐backcrossed wMel‐infected Aedes aegypti. Panels (A–C) show comparisons between populations with wMel from Lab (black), Yorkeys Knob (YK) (yellow) or Gordonvale (GV) (blue) origins when introduced into the same genetic background: Lab (A), YK (B) or GV (C). Panels (D–F) show comparisons between populations with wMel from a single origin: Lab (D), Yorkey's Knob (E) or GV (F) that has been introduced to the genetic backgrounds from the Lab (black), Yorkey's Knob (yellow) and GV (blue) populations. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage

Long‐term egg storage weakens cytoplasmic incompatibility by wMel‐infected males and rescue by wMel‐infected females

We measured the ability of wMel‐infected mosquitoes to induce cytoplasmic incompatibility following long‐term egg storage. In non‐stored eggs, wMel induced complete cytoplasmic incompatibility (no eggs hatching) when compared to controls that were all compatible, mostly to a high level (Figure 4(A)). When eggs were stored for 20 (Figure 4(B)) or 24 (Figure 4(C)) weeks, the cytoplasmic incompatibility induced by wMel‐infected males was incomplete, with uninfected females producing some viable progeny across both time points. Egg hatch proportions in the incompatible cross were significantly higher when males were stored for 20 (Mann–Whitney U: Z = 2.750, p = 0.006) but not 24 (Z = 1.047, p = 0.294) weeks compared to 1 week. wMel‐infected females also showed a reduced ability to restore compatibility with wMel‐infected males that had not been stored, with reduced hatch proportions when wMel stored females were crossed to wMel males compared to uninfected males (Mann–Whitney U: 20 weeks: Z = 2.750, p = 0.006, 24 weeks: Z = 4.307, p < 0.001).

FIGURE 4.

FIGURE 4

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Cytoplasmic incompatibility and compatibility restoration in wMel‐infected Aedes aegypti hatching from 1 (A), 20 (B) and 24 (C) weeks of egg quiescence. Coloured text indicates sexes that were stored as eggs for 20 (red) or 24 (purple) weeks. Dots show egg hatch proportions for individual females while vertical lines and error bars show medians and 95% confidence intervals

wMel‐infected females hatching from long‐term stored eggs become infertile

We showed previously that the wAlbB Wolbachia strain causes female mosquitoes to become infertile if they hatch from stored eggs (Lau et al., 2021a, 2022). We scored the proportion of females used in crosses in Figure 4 that laid eggs as a measure of infertility, although we note that the storage period to induce this effect is much longer than described previously for wAlbB (Lau et al., 2021a).

The data show a dramatic effect of egg storage on fertility, much stronger than the effects on incompatibility described in the previous section. About half the wMel females stored for 20 or 24 weeks did not lay eggs (Table 1). Across all wMel‐infected populations, the proportion of infertile females was 0.54 (binomial confidence interval: 0.44–0.65, n = 94), while for 24 weeks it was 0.51 (0.40–0.63, n = 76). In contrast, no infertile females were observed for any uninfected population or for any wMel population stored for 1 week (upper binomial confidence interval: 0.17).

TABLE 1.

Proportion of females from wMel‐infected and uninfected Aedes aegypti populations hatching from stored eggs that did not lay eggs (i.e. were infertile)

Egg storage duration Population Proportion infertile (n tested)* Fisher's exact test p value*
1 week Uninfected Lab 0 (97) a –
wMel Lab 0 (20) a 1.000
wMel YK F4 0 (20) a 1.000
wMel GV F4 0 (20) a 1.000
20 weeks Uninfected Lab 0 (20) a –
wMel Lab 0.58 (38) bc <0.001
wMel YK F4 0.39 (36) b 0.002
wMel GV F4 0.75 (20) c <0.001
24 weeks Uninfected Lab 0 (20) a –
wMel Lab 0.71 (35) b <0.001
wMel YK F4 0.30 (33) c 0.013
wMel GV F4 0.50 (8) bc 0.007
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*

For each egg storage duration, populations with the same letter are not significantly different by Fisher's exact test, with p values adjusted by the false discovery rate (FDR) method for multiple comparisons (Benjamini–Hochberg false discovery rate). p Values are shown for pairwise comparisons with the Uninfected Lab population.

DISCUSSION

It is well known that Wolbachia infections reduce the viability of quiescent A. aegypti eggs, including the wMel strain (Allman et al., 2020). Here we show that this cost has shifted in mosquito populations that are adapted to different environments, with consistent differences between the two field populations and shifts with laboratory adaptation. Differences in egg viability were not due to genetic background or Wolbachia infection alone; uninfected populations showed similar patterns of egg viability and Wolbachia infections from different origins had similar effects when introduced to the same background. However, we found interactions between genetic background and Wolbachia infection which may reflect mosquito adaptation to Wolbachia infection. Our study provides some of the first direct evidence for phenotypic changes associated with Wolbachia in mosquitoes adapted to different environments.

Post‐release monitoring of mosquito populations is an important part of any Wolbachia release programme (Ritchie et al., 2018). Previous studies have monitored the phenotypic effects of Wolbachia in field‐collected populations and found them to be largely consistent with pre‐release laboratory populations. Gesto et al. (2021) and Ahmad et al. (2021) show an increase in Wolbachia density and sustained virus blocking after Wolbachia releases in Brazil (wMel) and Malaysia (wAlbB), respectively. In Cairns, Australia, post‐release monitoring found persistent costs of the wMel infection to female fertility (Hoffmann et al., 2014) and sustained virus blocking (Frentiu et al., 2014) after 1 year. Phenotypic effects have largely persisted in the longer term, with one exception being development time where costs were only apparent in a laboratory background (Ross et al., 2022). However, no studies to date have identified the basis of population differences. Here we used a combination of repeated field collections, antibiotic curing and reciprocal backcrossing to show that fitness costs depend on genetic background and shift with laboratory adaptation. This finding is consistent with previous studies demonstrating the potential for host genetic changes to mediate fitness costs (Ford et al., 2019; Ritchie et al., 2015) and the lack of changes in the wMel genome itself (Dainty et al., 2021; Huang et al., 2020; Ross et al., 2022). We have previously found that the genomes of A. aegypti have not changed much across the 10 years period since releases of wMel started, although some outliers were detected that may reflect adaptive responses (Lau et al., 2021b). This study is the first case where shifts in Wolbachia‐affected host traits have been linked to host genetic differences under field conditions.

The patterns of quiescent egg viability costs we observed were puzzling, given that we found higher costs of wMel in field‐collected populations which experience long dry seasons (www.bom.gov.au) and where selection for increased quiescent egg viability is expected and the costs of wMel should attenuate. Costs were unexpectedly weaker in laboratory‐adapted populations where eggs were usually stored for less than 2 weeks, therefore with little selection to maintain long‐term quiescent egg viability. It is possible that these patterns are due to trade‐offs, where wMel provides a benefit under field conditions at the cost of reducing quiescent egg viability. In natural Wolbachia‐insect associations, Wolbachia infections often provide context‐dependent fitness costs and benefits (Correa & Ballard, 2016). We also found consistent differences in wMel effects between the YK and GV populations. While container surveys have not been performed in these suburbs, there are clear differences in their built environment, with GV having a higher proportion of older houses on stilts and YK having newer houses on flat ground, which may influence the types of larval habitats available.

Wolbachia infections typically induce greater fitness costs in novel mosquito hosts than in natural hosts, but these costs are expected to attenuate over time (Ross et al., 2019b). In a previous study, we found that the costs of wMel to quiescent egg viability were consistently low when wMel was introduced to a naïve laboratory genetic background (Ross et al., 2022). While the experiments are not directly comparable, together they suggest that costs may depend more on local mosquito adaptation than the novelty of the Wolbachia‐mosquito association. It is therefore possible that our findings reflect innate differences in mosquito populations rather than adaptation to Wolbachia infection, but confirming this would require Wolbachia‐free field populations which are no longer present in our study region due to widespread wMel coverage (O'Neill et al., 2018; Ryan et al., 2019). We also lack data on the costs of wMel on quiescent egg viability before its release into natural populations. Future work investigating mosquito host genome responses to Wolbachia infection should consider effects in both naïve and ‘Wolbachia‐adapted’ backgrounds and in both field‐ and laboratory‐adapted populations across time.

Our study identified novel and substantial fitness costs of wMel infection following long‐term storage of eggs. When eggs from wMel‐infected populations were stored for extended periods (20 or 24 weeks), males partially lost their ability to induce cytoplasmic incompatibility and around half of all females became infertile, with fertile females partially losing their compatibility with Wolbachia infected males. This effect was Wolbachia‐specific because uninfected females stored for the same amount of time never became infertile. The effects of egg storage on infertility are similar to those described previously for the wAlbB infection (Lau et al., 2021a, 2022) but substantially weaker, with ~50% infertility observed after only 9 weeks for wAlbB. The weakening of cytoplasmic incompatibility is likely due to partial or complete loss of Wolbachia during egg quiescence as demonstrated previously for wMel (Lau et al., 2021a). While the additional costs of wMel infection were only apparent after long‐term storage, interactions with other environmental factors such as high temperatures, which also reduce cytoplasmic incompatibility by wMel (Ross et al., 2017b), could exacerbate these costs. These cumulative effects on fitness may influence Wolbachia spread and persistence in locations with long dry seasons where these costs will be most apparent.

Host genomic variation in response to novel Wolbachia infection is poorly understood and our study provides a foundation for future investigations. It is worth exploring host responses to Wolbachia strains with more substantial effects on fitness, for instance wAlbB which is established in natural populations in Malaysia (Nazni et al., 2019). The consistent differences in wMel effects between Yorkey's Knob and GV populations also highlight the potential for local adaptation to influence fitness costs; a factor which is rarely considered (Carvalho et al., 2020). Host genotype effects on fitness costs have important implications for Wolbachia releases given that the strength of fitness costs drives Wolbachia establishment potential (Nguyen et al., 2015). Our work also prompts further investigation into the extent to which adaptation to artificial rearing conditions masks the fitness costs of Wolbachia, particularly under stressful conditions where Wolbachia infections have clear costs and where selective pressures differ greatly between the laboratory and the field. Our results here suggest that fitness comparisons between mosquitoes with laboratory genetic backgrounds may not always predict Wolbachia fitness costs in wild mosquito populations. When planning a local Wolbachia release program it is therefore important to use locally‐sourced mosquitoes that have spent minimal time in the laboratory for phenotypic assessments.

CONFLICT OF INTEREST

The authors declare that no competing interests exist.

Supporting information

Figure S1. Quiescent egg viability of wMel‐infected and uninfected Aedes aegypti from laboratory (Lab) and Yorkeys Knob (YK F11) populations. Data are pooled from two replicate populations. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage.

EMI-24-5749-s002.pdf (57.4KB, pdf)

Figure S2. Quiescent egg viability of uninfected Aedes aegypti populations included in experiments to test effects of laboratory rearing and genetic background. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage.

EMI-24-5749-s001.pdf (83.2KB, pdf)

ACKNOWLEDGEMENTS

We thank Marianne Coquilleau for assistance with experiments and Ashley Callahan and Qiong Yang for routine screening of Wolbachia‐infected mosquito populations. We also thank Kyran Staunton and Scott Ritchie from James Cook University, Cairns for providing field‐collected Aedes aegypti. Finally, we thank an anonymous reviewer for their comments which improved the interpretation of our results. Open access publishing facilitated by The University of Melbourne, as part of the Wiley ‐ The University of Melbourne agreement via the Council of Australian University Librarians.

Ross, P.A. & Hoffmann, A.A. (2022) Fitness costs of Wolbachia shift in locally‐adapted Aedes aegypti mosquitoes. Environmental Microbiology, 24(12), 5749–5759. Available from: 10.1111/1462-2920.16235

Funding information National Health and Medical Research Council, Grant/Award Numbers: 1118640, 1132412

DATA AVAILABILITY STATEMENT

All data are contained within the manuscript and its supporting information files.

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Associated Data

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

Supplementary Materials

Figure S1. Quiescent egg viability of wMel‐infected and uninfected Aedes aegypti from laboratory (Lab) and Yorkeys Knob (YK F11) populations. Data are pooled from two replicate populations. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage.

EMI-24-5749-s002.pdf (57.4KB, pdf)

Figure S2. Quiescent egg viability of uninfected Aedes aegypti populations included in experiments to test effects of laboratory rearing and genetic background. Symbols and error bars represent median egg hatch proportions and 95% confidence intervals at each duration of egg storage.

EMI-24-5749-s001.pdf (83.2KB, pdf)

Data Availability Statement

All data are contained within the manuscript and its supporting information files.

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