{'fraction_non_alphanumeric': 0.03324808184143223, 'fraction_numerical': 0.023310193642674462, 'mean_word_length': 5.056870989156893, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics

The endosymbiotic bacterium Wolbachia shows viral blockage in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11-19°C and 22-30°C. Eggs stored at 11-19°C had higher hatch proportions than those stored at 22-30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. wAlbB-infected males continued to show complete CI. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22-30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected Ae. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.


Introduction
Aedes aegypti is a major vector of dengue, Zika, chikungunya and yellow fever viruses and is distributed mainly in tropical and subtropical areas [1]. Aedes aegypti abundance is determined by seasonal fluctuations in temperature, humidity and precipitation [2]; temperature influences mortality and development rates while rainfall affects the availability of larval habitats and egg hatching success. Long term survival of eggs is the main factor influencing population persistence especially in areas where there is a cold, dry season [3,4]. Under ideal laboratory conditions, Ae. aegypti eggs can be stored for ten weeks under 27C with more than 80% of eggs hatching when submerged [5]. In nature when eggs are exposed to harsh environments as well as predators and parasites [6], egg survival can still be around 50% after overwintering [7,8], but high temperature and low humidity conditions increase egg mortality [9].
Wolbachia, a common endosymbiont in arthropods, has antiviral effects on its host and was artificially introduced into Ae. aegypti to help suppress arbovirus transmission [10][11][12]. Wolbachia-infected populations have been established in the field including the wMel [13][14][15] and wAlbB strains [16]. This approach relies on the ability of Wolbachia infections to invade through cytoplasmic incompatibility (CI), when infected males cause the death of offspring when mated with uninfected females [17]. However, the Wolbachia infection can also induce fitness costs on its host to reduce the host population [18], while environmental conditions can influence infection dynamics in populations by affecting Wolbachia density, leading to other impacts like CI leakage [19] and maternal transmission failure [20]. Both these effects can increase the uninfected proportion of mosquitoes in a population across generations. A reduction in Wolbachia frequency in a population will reduce efficiency whereby Wolbachia invades a population [21][22][23] with consequences for successful disease control.
Given that Wolbachia can play an important role in arboviral disease control [13,16], it is important to determine environmental conditions when invasion dynamics are more likely to favor Wolbachia to persist at a high level. In areas where Ae. aegypti occurs, factors such as temperature and rainfall variability will impact population abundance, through influencing egg quiescence [15,24,25]. Egg quiescence can be affected by high temperatures and Wolbachia infection status under laboratory conditions. The effect of wMelPop on Ae. aegypti eggs is particularly strong [26,27], whereas wMel-and wAlbB-infected eggs can survive much longer [28,29], although fitness costs under some conditions are apparent. For example, wMel-infected eggs are more sensitive than uninfected eggs to high temperatures, with lower hatch proportions when eggs are stored at 26-36 C [19]. The impacts of rainfall variability, which is an important factor in the seasonal outbreak of mosquitoes [30], has received less attention. Moreover, environmental and Wolbachia effects on egg quiescence have so far been characterized within the egg life stage, whereas Wolbachia invasion depends on effects on host phenotypes across life stages.
In this study, we tested the viability of wMel-infected, wAlbB-infected and uninfected eggs after storage under cold (11-19°C) and warm (22-30°C) cycling temperatures for different durations. We then tested Wolbachia infection density and fitness of the adults emerging from these eggs as well as on maternal transmission and CI. These experiments led to the surprising finding that many wAlbB-infected females showed sterility when emerging from warm-stored eggs, and females with either infection showed a reduction in fecundity. Such effects could influence the local population dynamics of Wolbachia, and the seasonal size of outbreaks of Ae. aegypti populations in areas once Wolbachia has established, with secondary effects on disease transmission. Our results also provide guidance on efficient accumulation of Wolbachia-infected eggs prior to release which can help release programs [16,31] and they point to novel options for suppressing mosquito populations by Wolbachia strains in some contexts.

Mosquito strains and maintenance
Uninfected, wMel-infected and wAlbB-infected Ae. aegypti were used in this work. The wMel-infected and uninfected populations were derived from eggs collected from Cairns, Queensland, Australia [29] while the wAlbB-infected population was generated by Xi et al [32] and then crossed to an Australian background [29]. Mosquitoes were maintained at 26  1 °C in a controlled temperature room [33] until experiments commenced. Wolbachia-infected populations were backcrossed to an uninfected population at least three times a year to maintain a similar genetic background in all the stocks [26]. Another three generations of backcrossing were undertaken directly before this experiment. Female mosquitoes were blood fed by a volunteer as described by the University of Melbourne Human Ethics Committee (approval 0723847). After a week, egg batches were collected on sandpaper strips, rolled up in paper towels and sealed in a plastic bag for three days before the viability of eggs was tested.

Egg viability across storage time
We cut egg batches into small 1 cm  3 cm pieces. Each piece contained 50 to 150 eggs which were hatched to calculate the initial hatch rate (week 0) or stored egg hatch rate under cycling temperatures of 11-19  1 °C or 22-30  1 °C in two incubators with a 12:12 h photoperiod. The eggs were labelled and sealed in a plastic box with a cup of saturated sodium chloride solution to balance the humidity at approximately 75% [34]. Temperature and humidity in the box were checked by data loggers (Thermochron; 1-Wire, iButton.com, Dallas Semiconductors, Sunnyvale, CA, USA) (Supplementary Figure 1). After storage for 0, 1, 4, 8, 11, 14 or 16 weeks, four to six batches of eggs per population and temperature cycle were submerged in reverse osmosis (RO) water with TetraMin® fish food tablets (Tetra, Melle, Germany) ad libitum and yeast (to stimulate the hatching) and a second immersion was carried out with a one-day drying interval to hatch any viable eggs that had failed to hatch in the first immersion [35]. Intact and hatched eggs (with a detached egg cap) were counted to indicate dead or surviving larvae. After hatching, larvae were reared until the adult stage for further testing.

Wolbachia density across storage time
Wolbachia-infected eggs before (0 weeks) and after cycling temperature treatments (8, 11, 14, or 16 weeks) were hatched. Two-day-old larvae were density controlled to a maximum of 30 larvae in trays of 300 mL RO water in each treatment and reared to adulthood. Eleven 2-day-old females per treatment were stored in 100 % ethanol for Wolbachia screening. Individual DNA was extracted in 200 µL 5% Chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA) [36], and diluted ten times for quantitative PCR analyses. Mosquito-specific (mRpS6) primers, Ae. aegypti-specific (aRpS6) primers and Wolbachia-specific primers (w1 primers for wMel-infected or wAlbB primers for wAlbB-infected) were used in the same run for each sample [29,37,38]. Three technical replicates were completed for each sample with two requirements: first, the difference of the crossing point (∆Cp) between mRpS6 and aRpS6 primers, which was used to indicate the quality of each run, had to be less than 1; and second, the variance of ∆Cp between the three runs was expected to be less than 0.1. Relative density was calculated by power 2 transformation of ∆Cp differences between aRpS6 and Wolbachia primers and technical replicates were averaged before testing treatment differences (storage time, Wolbachia infection type, temperature).

Cytoplasmic incompatibility
wMel-and wAlbB-infected eggs that not been stored (preserved under constant 26 C for less than three weeks, regarded as untreated) or had been stored (treated) for 14 weeks at 11-19 C and 10 (wAlbB) or 11 (wMel) weeks at 22-30 C were hatched and larvae were reared to adulthood for crossing experiments. Wing lengths were measured for seven to ten randomly selected females from each treatment as a measure of mosquito size. Eight replicate cages, each consisting of five uninfected untreated females crossed with five Wolbachia-infected treated males, were set up to test cytoplasmic incompatibility (CI), while four replicates involving crosses with Wolbachia-infected untreated males were set up as controls. Eight replicates, each with five Wolbachia-infected treated females crossed with five Wolbachia-infected untreated males, were set up to test for CI rescue, while four replicates of Wolbachia-infected untreated females crossed with untreated males were set up as controls. After adults emerged, they were left with a 10% sucrose cup and water cup for four or five days to ensure that females had matured and mated. After blood feeding, females were allowed to oviposit on Norton®Master Painters P80 sandpaper (3.8 × 18 cm; Saint-Gobain Abrasives Pty. Ltd., Thomastown, Victoria, Australia) which was half submerged into water which had been used to rear larvae. These egg strips were collected at the fifth and seventh day and hatched on the ninth day after blood feeding.

Maternal transmission
To test for maternal transmission, 20 Wolbachia-infected female mosquitoes that had been stored for 14 weeks at 11-19 C and 10 (wAlbB) or 11 (wMel) weeks at 22-30 C were crossed to uninfected males that had not been stored before blood feeding. Females were individually isolated in a 70 mL cup after blood feeding and allowed to lay eggs for one week. After laying eggs, mothers were stored in ethanol for Wolbachia screening. Mothers who died before being stored were excluded from the experiment. We selected six to seven mothers per treatment which each had more than 30 offspring alive, and we screened the mother and eight to ten offspring for Wolbachia infection. wAlbB-infected females from the 22-30 C treatment that failed to lay any eggs were dissected after being stored in ethanol to look for the presence of mature eggs. Since no wAlbB-infected females stored for 10 weeks at 22-30 C laid eggs in this experiment, we instead tested maternal transmission from wAlbB-infected females stored for 11 weeks at 22-30 C in a second experiment (see below).

Female fertility
We further investigated the loss of fertility in Wolbachia-infected females derived from warm (22-30 C) stored eggs by hatching batches of wAlbB-infected (stored for 0, 6, 9 or 11 weeks), wMel-infected (0, 9 and 11 weeks) and uninfected eggs (0, 6, 9 and 11 weeks). To understand the role males played, females hatched at each time point were (1) crossed to males from the same treatment, (2) crossed to males with the same Wolbachia infection type that were not stored, and (3) crossed to uninfected untreated males. Between 30-42 females from each cross were isolated in a 70 mL cup and provided with a strip of sandpaper to lay eggs. Eggs were collected once at the sixth day and hatched at the ninth day after blood feeding to determine fecundity and egg hatch proportions. Females that died before egg collection were excluded from analyses.
We determined the prevalence of female infertility by scoring the proportion of females in each cross that did not lay eggs on the collection day. We dissected female mosquitoes that failed to produce eggs from each cross at 9 weeks, and at 11 weeks we randomly selected and dissected up to ten female mosquitoes from each cross. We identified female insemination status by looking at their spermathecae under a compound light microscope (Motic B1 series, Australian Instrument Services Pty. Ltd., Australia). Each female has three spermathecae, one larger one and two smaller ones. The spermathecae containing sperm were distinguished from those that did not based on their texture and transparency, with spermathecae containing sperm having a dark and solid appearance with visible sperm swirling around the perimeter (Supplementary Figure 2, Supplementary video 1).

Statistical analysis
All data analysis and visualization were undertaken with R v.3.6.0. We performed Kaplan-Meier survival tests and log rank tests [39] to compare treatment effects on changes in egg viability across time, and used the Benjamini p-value adjustment method for pairwise comparisons. We used ANOVAs to compare treatment effects for normally distributed data including fecundity. We log transformed Wolbachia density and used logistic regression to analyze treatment and storage time effects on proportional data such as egg hatch and female infertility. We ran Kruskal-Wallis rank tests to analyze treatment effects of hatch proportions in the maternal transmission experiment. Hatch proportions were arcsine square root transformed before computing 95% confidence intervals but these were then back transformed before being presented (Supplementary Table 3).

Cytoplasmic incompatibility
The wAlbB-infected males caused complete CI with uninfected females (no eggs hatched) regardless of egg storage duration. For wMel-infected Ae. aegypti, incomplete CI occurred when males were reared from eggs stored at either 11-19 C or 22-30 C. No eggs hatched from crosses where wMel-infected males were hatched from unstored eggs. However, hatch proportions in CI crosses were 0.35% and 0.26% when wMel-infected fathers were stored at 11-19 C and 22-30 C respectively (Supplementary Table 1), suggesting a minor loss of CI.
We performed crosses between Wolbachia-infected treated females (hatched from eggs stored at 11-19 C or 22-30 C) and Wolbachia-infected untreated males to test for CI rescue. The number of eggs laid by females was impacted by their egg storage treatment (ANOVA, F2,34 = 39.593, p < 0.001), but not by their Wolbachia infection type (F1,34 = 1.423, p = 0.241). Wolbachia-infected females derived from eggs stored under 22-30 C for 10/11 weeks laid approximately 70% and 45% fewer eggs for wAlbB-infected and wMel-infected respectively than when stored under 11-19 C (Figure 3). Egg hatch proportions from these crosses were unaffected by the egg storage treatment of the female parents ( Figure 3B  We measured the wing length of female mosquitoes to estimate their body size. No significant differences were found for wMel or wAlbB between the two temperature treatments and untreated samples (ANOVA, wMel: F2,23 = 0.578, p = 0.569; wAlbB: F2,23 = 3.153, p = 0.062). There were weakly significant differences in wing length between untreated wMel-infected, wAlbB-infected and uninfected females (F2,19 = 4.62, p = 0.023), where wAlbB-infected females were somewhat larger than females from the other two populations (Supplementary Table 2).

Maternal transmission
Wolbachia-infected females stored at 22-30 C or 11-19 C transmitted the infection to the next generation at a frequency of 100% regardless of Wolbachia infection type and egg storage temperature. In our first test of wAlbB-infected females stored at 22-30 C, all twelve isolated females did not lay eggs, and no mature eggs were observed in their body upon dissection. We then counted the number of eggs laid by wAlbB-infected females stored at 11-19 C and wMel-infected females from both treatments and their hatch proportions (Supplementary Table 3

Female fertility
The results in the previous experiments suggest fertility effects from storage in that Wolbachia-infected females produced low egg numbers (or did not lay eggs at all) when stored as eggs under 22-30 C for a period. To further investigate the apparent loss of female fertility under egg storage, we determined the proportion of Wolbachia-infected and uninfected females that did not lay eggs when female parents were derived from eggs stored under 22-30 C. When Wolbachia infection type was considered separately, there were differences for the wAlbB-infected strain for both egg storage duration (F3,6 = 185.138, p < 0.001) and cross (F2,6 = 8.985, p = 0.016), whereas wMel-infected and uninfected strains showed no significant effects of these factors (all p > 0.05). The proportion of wAlbB-infected females not laying eggs increased dramatically with egg storage duration, with over 75% of wAlbBinfected females not producing eggs by the final assessment time (Figure 4). All dissected females were inseminated (Supplementary Figure 2) but lacked visible eggs in their ovaries, indicating that infertility was not due to a lack of mating.
For the fertility of females that laid eggs, there were effects of Wolbachia infection type (ANOVA, F2.537 = 18.361, p < 0.001) and egg storage duration (F1.537 = 90.650, p < 0.001) on the number of eggs per female laid but no effect of cross (F2.537 = 0.038, p = 0.962). Both wMel-and wAlbB-infected females laid fewer eggs with increasing egg storage duration in all crosses ( Figure 5A). In contrast, uninfected female fecundity did not decline. For the hatch proportion of these eggs, effects were induced by Wolbachia infection type ( Figure    derived from eggs stored under a cycling temperature of 22-30C for 0, 6, 9 or 11 weeks. Females hatched at each time point were crossed to males from the same treatment (Control), crossed to males with the same Wolbachia infection type that were not stored (CI rescue) and crossed to uninfected untreated males (Maternal). The data exclude females that did not lay eggs (see Figure 4). Error bars represent means ± standard errors.

Discussion
Aedes aegypti mosquitoes carrying the wMel or wAlbB strains of Wolbachia have the potential to reduce dengue transmission through decreased mosquito vector competence [16,40], and there is already good evidence that both strains are having such impacts in Wolbachia-invaded release areas [13,16]. These releases can involve adults or eggs. Releases involving eggs, where containers of eggs are hatched directly in the field, are desirable for Wolbachia release programs because they do not require local mass-rearing facilities [13,16,41] and allow eggs to be accumulated across several weeks before being transported for release. Although most releases to date have involved the wMel strain, wAlbB was used for releases in Malaysia [16] based on its desirable characteristic of being stable in its density and phenotypic effects under high temperatures [42].
In this study, we investigated the effects of long-term storage on Ae. aegypti eggs infected with Wolbachia strains wMel or wAlbB. Eggs survived longer under cooler environments; more than 50% of eggs from all strains hatched after storage for 14 weeks at 11-19C while hatch proportions were below 50% when stored at 22-30C for the same amount of time. Although the Wolbachia density of both wMel and wAlbB-infected adults decreased with storage time, density was not affected by storage temperature, suggesting that density did not contribute to the decrease in egg viability under warmer temperatures. Nevertheless, we suspect that the decreased density contributed to incomplete CI for wMel-infected males stored under both temperature treatments given the strong relationship between CI and Wolbachia density previously reported for wMel [19]. Despite decreased density, the maternal transmission of Wolbachia remained stable after eggs had passed through a quiescent phase, regardless of storage temperature.
However, Wolbachia infections induced substantial costs to fertility when adults were reared from eggs stored under 22-30°C for 10 or 11 weeks. Both wMel and wAlbB-infected females that were stored as eggs at 22-30°C produced fewer eggs. In a second experiment where we tested individual females, a high proportion of wAlbB-infected females suffered infertility when stored as eggs at 22-30°C, regardless of the male used in crosses. In contrast, wMel-infected and uninfected females retained high rates of fertility throughout the experiment. Dissection of wAlbB-infected females that failed to lay eggs showed that all females were inseminated but lacked mature eggs in their abdomens despite being blood fed. Previous studies suggest that wAlbB has only a minor negative impact on egg viability during the first two months of storage [43]. However, these costs may be underestimated because a high proportion of females hatching from stored eggs are infertile. Furthermore, both wAlbB and wMel-infected females laid fewer eggs when hatching from stored eggs though the hatch rate remain high. These effects appear to be unrelated to larval development, since body sizes were similar across Wolbachia infection types and egg storage treatments (Supplementary Table 2). Wolbachia infections compete with their hosts for key nutrients required for egg production [44] which may be exacerbated when female parents are stored as eggs. Egg provisioning or hormone modulation may also be impacted by Wolbachia under stress [43,45], but this requires further investigation.
The survival and fertility effects generated in the quiescent Ae. aegypti eggs seem likely to impact the spread and maintenance of Wolbachia in populations in certain situations. Mosquitoes with wAlbB may not invade readily if releases happen in areas where larval habitats used by Ae. aegypti are intermittent, requiring long periods of egg quiescence. In such environments, the threshold frequency of wAlbB in a population that needs to be exceeded to get invasion may increase above the currently estimated point (for wMel) of 20-30% [31]. Releases based on wAlbB eggs rather than adults (such as carried out at one site in Malaysia [16]) could also be much less effective than expected if they use eggs that have been stored too long, unless these have been stored under cool conditions. Aedes aegypti is an opportunistic breeder, taking advantage of locally suitable conditions which are expected to vary spatially and temporally depending on rubbish accumulation, defective building spaces and defective water and sewerage tanks [46][47][48]. Because of these differences, the contribution of mosquitoes from quiescent eggs to the adult population is expected to vary locally. Releases across different locations show that rates of Wolbachia invasion can vary for both wAlbB [16] and wMel [46]. While various factors may be responsible for site-to-site variation in invasion rates and success including density dependent processes [21], fitness costs associated with quiescence are also likely to be important, particularly for wAlbB.
Once Wolbachia invasions have been completed, there is the potential for Wolbachia infections to decrease the overall size of mosquito populations due to fitness costs [27,49]. Our data suggest that this is particularly likely in locations with strong seasonality, where quiescent eggs persisting during the dry season are more likely to contribute to Ae. aegypti population continuity. A strategy for crashing Ae. aegypti populations during the dry season based on the reduced hatch rate of infected quiescent eggs was originally proposed several years ago, based around the wMelPop infection which induces a particularly large fitness costs in quiescent eggs [26,50]. However, this strain cannot easily invade mosquito populations [10,18] whereas the wAlbB strain does establish successfully [16] and may carry fitness costs appropriate for such an intervention.
Given that we only tested infertility effects in females stored for up to 11 weeks under warm environment, it is unclear whether this effect will occur under cooler environments or occur in wMel as well when stored for a longer period. The original host of wAlbB, Aedes albopictus, has higher cold tolerance than Ae. aegypti and prefers cooler temperatures [51][52][53]. The wAlbB infection may provide fitness benefits to Ae. aegypti under cool environments, which points to the wAlbB-infected Ae. aegypti being relatively better adapted to cool environments than uninfected mosquitoes [54]. Under hot environments, wAlbB is relatively more stable than wMel in terms of density and its ability to induce cytoplasmic incompatibility [42]. The infertility effect we describe here was detected after only six weeks of egg storage under a milder temperature range than the hot temperatures tested previously. Nevertheless, the ability of wAlbB to maintain a relatively high density in quiescing eggs may result in the accumulation of damage in such eggs which leads to fitness effects at a later life stage, producing female infertility and a reduction in fecundity.
In summary, we have tested the performance of wMel, wAlbB-infected and uninfected Ae. aegypti after eggs were stored under cycling temperatures of 11-19°C and 22-30°C. We found that, compared to warm environments that are normally used to culture mosquitoes [33,55], cooler environments may be better for egg storage prior to Wolbachia releases. Long-term storage under warm environment greatly reduces the fertility of hatched females that carry wAlbB, with consequences for Wolbachia invasion success. Our study helps to guide conditions for appropriate storage of Wolbachia-infected eggs, inform release timing to coincide with periods where quiescent eggs contribute little to population dynamics, and help to explain Wolbachia invasion success or failure in different environments [16].