Stable phenotypic effects of an unstable deleterious Wolbachia infection

Wolbachia are being used to reduce dengue transmission by Aedes aegypti mosquitoes around the world. To date releases have mostly involved Wolbachia strains with limited fitness effects but strains with larger fitness costs could be used to suppress mosquito populations. However, such infections are expected to evolve towards decreased deleterious effects. Here we investigate potential evolutionary changes in the wMelPop infection transferred from Drosophila melanogaster to Aedes aegypti more than ten years (∼120 generations) ago. We show that most deleterious effects of this infection are stable despite strong selection to ameliorate them. The wMelPop infection is difficult to maintain in laboratory colonies, likely due to the persistent deleterious effects coupled with occasional maternal transmission leakage. Furthermore, female mosquitoes can be scored incorrectly as infected due to transmission of Wolbachia through mating. Infection loss in colonies was not associated with evolutionary changes in the nuclear background. These findings suggest that transinfections with deleterious effects may have stable phenotypes which could ensure their long-term effectiveness if released in natural populations to reduce population size.


Introduction
There is increasing interest in using Wolbachia bacterial infections for suppressing dengue transmission by mosquitoes, with field releases aimed at both replacing existing natural mosquito populations with those infected by Wolbachia O'Neill et al. 2018) and suppressing these populations through sterility induced by Wolbachia-infected males (Zheng et al. 2019). Replacement releases can be effective because the presence of Wolbachia in mosquitoes reduces transmission of arboviruses (Moreira et al. 2009a;Walker et al. 2011;Ant et al. 2018). In addition, Wolbachia decreases the fitness of its mosquito hosts (Ross et al. 2019c). While this might have a suppressive effect on dengue transmission, for instance, by shortening mosquito lifespan (Cook et al. 2008), it can make the infections more difficult to introduce into populations because the initial Wolbachia frequency has to be higher for the population to be invaded by Wolbachia (Hoffmann and Turelli 1997).
The wMelPop infection, which originated from a laboratory strain of Drosophila melanogaster, was one of the first Wolbachia strains successfully introduced into Aedes aegypti (McMeniman et al. 2009) where it is very effective at blocking transmission of dengue and other arboviruses (Moreira et al. 2009a). This strain reduces longevity in D. melanogaster (Min and Benzer 1997) and also in mosquitoes where it has additional deleterious effects, including reduced viability of eggs maintained in a quiescent state (McMeniman and O'Neill 2010;Yeap et al. 2011). The wMelPop infection was released in field trials in Vietnam and Australia but failed to establish (Nguyen et al. 2015), although it successfully invaded semifield cages . Because of these deleterious effects, wMelPop may represent an effective tool to reduce or even eliminate mosquito populations , particularly in isolated populations experiencing seasonal rainfall (Rašić et al. 2014).
One of the challenges in using wMelPop is that the strain can be difficult to maintain under laboratory conditions, with the infection occasionally being lost from colonies. For instance, on one occasion we found that 95.5% (43/45) of our colony was infected based on RT-PCR screening but this declined to 6.7% (2/30) four months later. Although the infection causes strong cytoplasmic incompatibility and shows near-complete maternal transmission, which allow Wolbachia infections to invade populations once an unstable equilibrium frequency dictated by deleterious fitness effects is exceeded , the infection may still be lost for unknown reasons even when it is detected at a high frequency with molecular assays. Environmental effects might reduce infection frequencies since high temperatures and low levels of antibiotics can clear Wolbachia infections (Ross et al. 2017b;Endersby-Harshman et al. 2019). However, there is normally careful control of temperature and antibiotics in laboratory cultures. Other factors that may contribute to infection loss are inappropriate storage of eggs coupled with sporadic incomplete maternal transmission.
While Wolbachia infections like wMelPop and wAu (Ant et al. 2018) reduce host fitness, their effects are expected to attenuate over time because any Wolbachia or host alleles that decrease deleterious fitness effects should be favoured by selection (Turelli 1994;Hoffmann and Turelli 1997). Evidence for such a process has been obtained for the wRi infection of Drosophila simulans where an initially deleterious effect on offspring production has attenuated to the extent that wRi infected D. simulans now have a higher production rate than uninfected females (Weeks et al. 2007). This could undermine any strategy that relies on maintaining deleterious fitness effects after Wolbachia are established in novel hosts, a process that has been documented for wMelPop after transfer to D. simulans (Carrington et al. 2009;Carrington et al. 2010). Evolutionary changes in the nuclear background may also suppress the phenotypic effects of Wolbachia, as demonstrated by the evolution of male-killing suppression in butterflies (Hornett et al. 2006). However, it is unknown if the deleterious effects of wMelPop on Ae.
aegypti and its ability to cause cytoplasmic incompatibility are stable.
To investigate these issues, we here consider whether there have been evolutionary changes in wMelPop or its Ae. aegypti host in the 10-year period since the infection was established by comparing recent and past data on phenotypic effects of the infection. We also investigate factors that may confound monitoring of wMelPop and contribute to instability of the infection in laboratory cultures.

Mosquito strains and colony maintenance
We performed experiments with our laboratory populations of wMelPop-infected (McMeniman et al. 2009), wMel-infected , wAlbB-infected (Xi et al. 2005) and uninfected Ae. aegypti mosquitoes. The wMelPop transinfection in Ae. aegypti was derived from D. melanogaster (Min and Benzer 1997) and was passaged in a mosquito cell line before being introduced into Ae. aegypti through embryonic microinjection (McMeniman et al. 2009). wMelPop-infected mosquitoes were collected from Babinda, Queensland, Australia in 2012, three months after releases commenced (Nguyen et al. 2015) and maintained in the laboratory since collection. All Wolbachia-infected populations were backcrossed to a common Australian nuclear background for at least five generations to ensure that backgrounds were >98% similar (Yeap et al. 2011). Stock populations were maintained through continued backcrossing to North Queensland material every six generations. Mosquitoes were reared in a temperature controlled laboratory environment at 26 °C ± 1 °C with a 12 hr photoperiod according to methods described previously (Axford et al. 2016;Ross et al. 2017a). An uninfected population (denoted wMelPop negative) was derived from wMelPop females that had lost their Wolbachia infection in June 2019. The wMelPop negative population was used in phenotypic stability experiments and to test for nuclear background evolution.

Wolbachia screening
Aedes aegypti females were tested for the presence of Wolbachia DNA using methods previously described with modifications (Lee et al. 2012;Axford et al. 2016). DNA extraction methods varied between experiments due to our research spanning several years. Mosquito DNA was extracted using 100-250 µL of 5% Chelex® solution (Bio-Rad Laboratories, Gladesville, NSW, Australia) and 2.5-5 µL of Proteinase K (20 mg/mL, Bioline Australia Pty Ltd, Alexandria, NSW, Australia) in either 96-well plates or 1.5 mL tubes. Polymerase chain reactions were carried out with a Roche LightCycler 480 system (384well format, Roche Applied Science, Indianapolis, IN, USA) using a RT/HRM (real-time PCR/highresolution melt) assay as described previously (Lee et al. 2012;Axford et al. 2016).
We used mosquito-specific (mRpS6), Aedes aegypti-specific (aRpS6) and Wolbachia-specific primers (w1 primers for the wMelPop and wMel infections and wAlbB primers for the wAlbB infection) to diagnose

Phenotypic stability of deleterious effects
The wMelPop infection induces a range of deleterious effects, including life shortening, reduced fertility, impaired blood feeding success and reduced quiescent egg viability as outlined below. To evaluate the stability of these deleterious effects, we performed experiments with the wMelPop infection over 10 years after its introduction to Ae. aegypti. Before experiments commenced, the wMelPop-infected colony was purified by pooling the offspring of isolated females that were strongly positive for Wolbachia (see Infection recovery). Female offspring were crossed to uninfected males, and the progeny were used in the following experiments. We compared fitness relative to two uninfected populations; a natively uninfected laboratory population (uninfected) and a population derived from uninfected individuals from the wMelPop colony that had lost their infection (wMelPop negative). Due to logistical constraints, the fertility experiment included the wMelPop and wMelPop negative populations only.

Longevity
Previous studies reported that wMelPop shortens adult lifespan by approximately 50% (McMeniman et al. 2009;Yeap et al. 2011). We performed longevity assays by establishing 8 replicate 3 L cages with 50 adults (25 males and 25 females) for each population. Cages were provided with 10% sucrose and water cups which were replaced weekly. Females were provided with blood meals for 10 minutes once per week and given constant access to an oviposition substrate. Mortality was scored three times per week by removing and counting dead adults from each cage until all adults had died. One replicate of wMelPop was discarded due to a sugar spill early in the experiment which caused high mortality. We used log-rank tests to compare adult longevity between populations. To evaluate Wolbachia density and infection frequencies with adult age, 16 females from separate cages that were 0, 7, 14, 21, 28 and 35 d old were screened for Wolbachia. We used a linear regression to test whether (log) Wolbachia density was affected by adult age. All data were analyzed using SPSS statistics version 24.0 for Windows (SPSS Inc, Chicago, IL).

Fertility
The wMelPop infection substantially reduces fertility as females age (McMeniman and O'Neill 2010); we therefore tested the fertility of wMelPop and wMelPop negative populations over successive gonotrophic cycles. We established two cages of approximately 500 individuals (equal sex ratio) for each population. Five-day old females (starved for 1 d) were blood fed and 35 engorged females were selected randomly from each population and isolated in 70 mL cups with sandpaper strips and larval rearing water to encourage oviposition. Eggs were collected 4 days after blood feeding, partially dried and hatched three days after collection. Fecundity and egg hatch proportions were determined by counting the number of unhatched and hatched eggs (hatched eggs having a clearly detached cap).
Following egg collection, females were returned to their respective cages for blood feeding. Successive gonotrophic cycles were initiated every 4-5 days with females selected randomly from cages. Cages were provided with oviposition substrates, however no sugar was provided to isolated females or the population cage during the experiment because sugar feeding influences fecundity (Naksathit and Scott 1998). We tested fertility for a total of 9 gonotrophic cycles. Females from the wMelPop population that were still alive after 9 gonotrophic cycles were tested with qPCR to confirm Wolbachia infection. Effects of gonotrophic cycle on egg hatch proportions were compared for the wMelPop and wMelPop negative populations. Egg hatch proportions were not normally distributed and were therefore analysed with Kruskal-Wallis tests.

Quiescent egg viability
The Larvae that had not completely eclosed and died in the egg were scored as unhatched. This experiment continued until eggs were 31 d old. To test for the potential loss of wMelPop infection with egg storage, we reared larvae hatching from 3, 13, 20, 24, 27 and 31 d old egg to adulthood and scored 16 females (< 24 hr old) for Wolbachia infection and density from each group. We used a linear regression to test whether (log) Wolbachia density was affected by egg storage duration.

Blood feeding success
The wMelPop infection reduces female blood feeding success and affects probing behaviour, particularly in older females (Moreira et al. 2009b;Turley et al. 2009). We evaluated blood feeding traits in 5 and 35 d old females according to methods described previously (Ross et al. 2019b). We recorded pre-probing duration (time from landing to insertion of the proboscis), feeding duration, blood meal weight and proportion feeding. Females that did not feed within 10 minutes were scored as not feeding. The proportion of females exhibiting a bendy or shaky proboscis phenotype (Moreira et al. 2009b;Turley et al. 2009) was also recorded. Feeding trials were performed on individual females by three experimenters. At least 32 individuals per population and age group were tested across the three experimenters. To confirm the infection status of wMelPop females, we screened all 35 d old females for Wolbachia infection. Pre-probing duration, feeding duration and blood meal weight data were analysed with general linear models. Pre-probing and feeding durations were log transformed for normality before analysis. Comparisons of proportional data (proportion feeding and the presence of a bendy or shaky proboscis) with previous studies were performed with two proportions Z-tests.

Loss of Wolbachia during colony maintenance
We carried out a series of experiments and monitoring exercises to understand the loss of the wMelPop infection in colonies during routine maintenance.

Infection recovery
In May 2019 we observed an apparent loss of wMelPop infection from our laboratory colony despite a high level of infection in previous generations. To return the population to a 100% infection frequency, one hundred blood-fed females were isolated for oviposition, screened for Wolbachia, then placed into categories of strongly positive, weakly positive or negative (see Wolbachia screening). We then pooled the offspring of females from each category and screened 30 offspring (15 males and 15 females) for Wolbachia per category. Female offspring from the strongly positive population were crossed to uninfected males before commencing the maternal transmission, nuclear background evolution and phenotypic stability experiments.

Maternal transmission
We estimated maternal transmission fidelity by crossing wMelPop-infected females to uninfected males, then screening ten offspring (4 th instar larvae) from the first gonotrophic cycle of ten females that had been separated individually for oviposition. Maternal transmission fidelity was expressed as the proportion of infected offspring produced by infected mothers, for which 95% binomial confidence intervals were calculated.

Nuclear background evolution
Loss of wMelPop infection in laboratory colonies may be explained by the evolution of resistance to Wolbachia infection by uninfected mosquitoes. We performed crossing experiments to test whether the wMelPop infection was maintained across generations when wMelPop-infected females were crossed to natively uninfected males or uninfected males that had lost their Wolbachia infection (wMelPop negative). We established two replicate populations for each cross with 200 adults of each sex. Crosses were performed for four consecutive generations. Thirty individuals from each replicate population per generation were then screened for Wolbachia infection. A wMelPop-infected colony (wMelPop-infected males crossed with wMelPop-infected females) was also monitored across the same time period.
To test for resistance to cytoplasmic incompatibility, we tested the ability of wMelPop-infected males to induce cytoplasmic incompatibility with uninfected and wMelPop negative females. For each cross, 30 males and 30 females were aspirated into a single 3 L cage. When adults were 5 d old, females were blood fed. Twenty females from each cross were isolated for oviposition and egg hatch proportions were determined according to the fertility experiment (see above).

Wolbachia mating transmission
Although Wolbachia in mosquitoes are maternally transmitted, it is possible that Wolbachia might also be transferred through seminal fluid, leading to the detection of Wolbachia in uninfected females that mate with infected males. To test for Wolbachia transmission through mating, we performed crosses between Wolbachia-infected males and uninfected females. Experiments were performed with the wMelPop, wMel and wAlbB strains. Control crosses were also performed, where both sexes were either infected (positive controls) or uninfected (negative control). Crosses were established with 160 virgin adults of each sex (4 -7 d old) in a single cage and left for two days to mate, after which males were removed. Females were blood-fed one week after crosses were established and provided with an oviposition substrate. Thirty females (whole adults) were stored 2, 9, 16 and 23 d after crosses were established and screened for Wolbachia. Females from the positive and negative controls were tested 2 and 23 d after crosses were established. Due to apparent differences in mating transfer between Wolbachia strains, this experiment was repeated with the wAlbB infection, but females were stored 5 d after crosses were established.
We conducted an additional cross between uninfected females and wMelPop-infected males to see if the detection of Wolbachia following transmission through mating was tissue-specific. Females and males were left to mate for five days, after which females were stored in ethanol. Heads and abdomens from 20 uninfected females were dissected and extracted separately for Wolbachia screening.

Relative fitness during laboratory maintenance
We compiled data on egg hatch proportions during our routine maintenance of wMelPop, wMel, wAlbB and uninfected colonies from July 2012 to April 2018. Egg hatch proportions were determined by hatching a subset of eggs collected from each colony during maintenance (>200 eggs per subset), then dividing the number of larvae counted by the number of eggs tested. We then divided the egg hatch proportions of Wolbachia-infected colonies by the egg hatch proportion of the uninfected colony to obtain relative egg hatch proportions. When multiple Wolbachia-infected colonies were maintained simultaneously, we included these as separate estimates. We used sign tests to compare relative hatch proportions of Wolbachia-infected and uninfected colony eggs. We used a general linear model to test for long-term changes in the relative egg hatch proportion of wMelPop-infected colonies.

Phenotypic stability of deleterious effects
We re-evaluated the deleterious fitness effects induced by wMelPop to test for attenuation. In previous experiments conducted more than 10 years ago, wMelPop infection shortened adult lifespan by ~50%  remained stable for over 10 years after transinfection. Loss of female fertility with age was due to declining fecundity rather than egg hatch, which was stable across gonotrophic cycles for both wMelPop (Kruskal-Wallis: χ 2 = 4.654, df = 7, P = 0.702) and wMelPop negative (χ 2 = 7.580, df = 8, P = 0.476) females ( Figure 2B).
As adult age increased, we observed an increasing proportion of wMelPop females that had a high fecundity but had zero eggs hatching ( Figure 2C). We excluded these individuals from the results since they may represent uninfected mosquitoes that mated with wMelPop infected males. Uninfected individuals may result from incomplete maternal transmission and become increasingly represented throughout the experiment due to having a longer lifespan ( Figure 1A  The wMelPop infection reduces female blood feeding success and affects probing behaviour, particularly in older females (Moreira et al. 2009b;Turley et al. 2009). Here we found no effect of population on preprobing and feeding duration or blood meal weight in 5 d old females (GLM: all P > 0.05, Figure 3).
Effects of wMelPop infection on blood feeding traits were weaker in comparison to previous studies with similar methods. For instance, Turley et al. (2009) observed a 50.3% (95% confidence interval: 37.5-63.1%) reduction in blood meal weight in 35 d old females due to wMelPop infection, while we observed a 29.5% (95% confidence interval: 12.1-46.7%) reduction relative to the two uninfected populations.
Aged wMelPop females had reduced feeding success (65% feeding compared to 91% for uninfected populations) and also displayed a bendy/shaky proboscis phenotype as characterized previously (Moreira et al. 2009b;Turley et al. 2009). However, these phenotypes occurred at a significantly lower frequency than previously reported (Moreira et al. 2009b

Infection recovery
Due to an apparent loss of Wolbachia from our wMelPop colony, we isolated females to restore the wMelPop infection in the population. Of the females that produced viable offspring, 20 were negative, 17 were strongly positive (median Cp 16.3, range 3.33) and 41 were weakly positive (median Cp 31.38, range 8.37). These results point to a polymorphic colony despite the colony having been scored as 100% infected prior to this time (all Cp values ≤ 23). All offspring tested from strongly positive females were strongly positive (females: median Cp 19.19, range 0.61), males: median Cp 19.12, range 5.83). No offspring from the weakly positive or negative females were infected (n = 30 each), thus females scored as weakly positive were unable to transmit wMelPop to the next generation.

Maternal transmission
We tested ten offspring from ten wMelPop-infected females and found that a single female produced two uninfected offspring, with an overall maternal transmission fidelity of 98% (binomial confidence interval: 92.96-99.76%). These results are consistent with previous studies that indicate a low level of maternal transmission failure (Yeap et al. 2011;Ross et al. 2017b).

Nuclear background evolution
We  wMelPop-infected females were crossed to wMelPop negative (gray), uninfected (gray) or wMelPop (pink) males each generation for four generations. Infection frequencies were determined for 30 individuals per population, per generation.
wMelPop-infected males induced complete cytoplasmic incompatibility with uninfected females (no eggs hatching, Table 1), suggesting that this phenotype has remained stable since transinfection over 10 years ago (McMeniman et al. 2009). Compatible crosses exhibited high hatch proportions, showing that the wMelPop infection is self-compatible. wMelPop-infected males also induced complete cytoplasmic incompatibility with wMelPop negative females, indicating that this population has not evolved resistance to cytoplasmic incompatibility.

Wolbachia mating transmission
We crossed Wolbachia-infected males with uninfected females to test the potential for Wolbachia to be transferred through mating. In control crosses, Wolbachia-infected females had a 100% infection frequency and high densities ( Figure 5), while Wolbachia were not detected when uninfected females were crossed to uninfected males. We detected Wolbachia in uninfected females that were crossed to wMelPop-( Figure 5A) and wMel-infected ( Figure 5B) males for up to 23 d post-mating, with the proportion scored as positive decreasing with time after mating. Wolbachia densities in uninfected females were distinctly lower than in females with a maternally-inherited Wolbachia infection. In an additional cross, we specifically tested for transfer of seminal fluid by crossing uninfected females to wMelPop-infected males and testing the heads and abdomens of females separately. All heads were negative for Wolbachia, while 19/20 abdomens were positive with a median Cp of 28.78 (range 4.44).
Uninfected females can therefore be incorrectly scored as infected if they have mated with a wMelPop or wMel-infected male.
In contrast to the other two infections, we did not detect Wolbachia in any uninfected females that were crossed to wAlbB-infected males ( Figure 4C). We detected no Wolbachia in a second independent experiment, indicating that this Wolbachia strain is not transferred through mating.

Relative fitness during laboratory maintenance
We monitored egg hatch proportions of our Wolbachia-infected laboratory colonies across multiple generations to assess variance in fitness costs. wMelPop-infected (Sign test: Z = 6.197, P < 0.001) and wMel-infected (Z = 3.900, P < 0.001) colonies tended to have lower egg hatch proportions relative to uninfected colonies ( Figure 6). wAlbB-infected colonies had similar hatch proportions to uninfected colonies overall (Z = 1.000, P = 0.317), though the sample size for this infection was much lower.

Discussion
Here we show that there has been little evolutionary attenuation of deleterious effects in wMelPop, either through changes in the host nuclear genome or the Wolbachia genome. This is despite an elapsed period of more than ten years or ~120 generations of rearing in the laboratory (and with an additional short period in the field). This contrasts sharply with the attenuation of wMelPop seen in D. simulans following its transfer from D. melanogaster. As in its native host, wMelPop reduced longevity when transferred to D. simulans (McGraw et al. 2001), Ae. aegypti (McMeniman et al. 2009) and Aedes albopictus (Suh et al. 2009). Other deleterious effects in D. simulans were also detected; however, many of these attenuated after around 20 generations, including effects on egg hatch (McGraw et al. 2002).
Moreover, after around 200 generations, wMelPop-infected D. simulans lines no longer showed a decrease in longevity in some genetic backgrounds (Carrington et al. 2010).
It is unclear why most deleterious effects in Ae. aegypti have not attenuated. It may be due to the nature of the mutation causing the high virulence; a duplication of a gene region has been identified in Drosophila (Chrostek and Teixeira 2015) which may be difficult to reverse. Instead, attenuation may require evolutionary changes in the host nuclear background. Selection experiments for increased quiescent egg viability in wMelPop-infected Ae. aegypti found evidence for attenuation due to nuclear background evolution but not Wolbachia evolution . Compared to studies performed over ten years ago, some deleterious effects of wMelPop appear weaker, particularly blood feeding traits (Moreira et al. 2009b;Turley et al. 2009) and male longevity (McMeniman et al. 2009;Yeap et al. 2011). Although this may indicate nuclear background evolution, direct comparisons with previous studies are difficult due to methodological differences and potential confounding effects of inbreeding, drift and laboratory adaptation that can occur during colony maintenance (Ross et al. 2019a).
Because Wolbachia are maternally inherited, selection acts to increase maternal transmission fidelity and not the ability of males to induce cytoplasmic incompatibility (Turelli 1994). Novel Wolbachia infections tend to induce much stronger cytoplasmic incompatibility than natural infections, suggesting that these effects can attenuate . Furthermore, theory predicts that resistance to cytoplasmic incompatibility may evolve if maternal transmission is incomplete (Bull and Turelli 2013).
Although hosts may evolve resistance to the effects of Wolbachia on reproduction, such as male killing in Hypolimnas bolina (Hornett et al. 2006) and cytoplasmic incompatibility in D. melanogaster (Hoffmann and Turelli 1997), effects can also remain stable despite intense selection pressure (Jaenike and Dyer 2008;Carrington et al. 2011). Over ten years after wMelPop was introduced to Ae. aegypti, the infection still induces complete cytoplasmic incompatibility. We therefore find no evidence to suggest that cytoplasmic incompatibility has attenuated or that Ae. aegypti has evolved to suppress cytoplasmic incompatibility. In crossing experiments, the wMelPop infection was lost from colonies regardless of whether infected females were crossed to uninfected males or males that had lost the wMelPop infection, suggesting that loss of wMelPop was not due to paternal factors that affect Wolbachia maternal transmission.
The lack of attenuation of deleterious fitness effects may contribute to the occasional loss of the wMelPop infection from Ae. aegypti laboratory populations. Following Hoffmann et al. (1990) the change in frequency of the infection (p f ) in a population is given by where u is the fraction of uninfected progeny produced by infected females, s f is the fecundity deficit (representing a combination of the number of eggs laid and that hatch) and s h is the incompatibility between infected and uninfected strains. In the presence of strong maternal transmission (u=0) the unstable point for invasion versus loss of the infection is given by the ratio of s f /s h (Hoffmann and Turelli 1997 wMelPop-infected mosquitoes compared to uninfected mosquitoes may fall to as low as 28% during routine maintenance, or even lower if adults are aged or eggs are stored before hatching. This will produce a situation where p (t+1) is less than p, and the infection will continue to drop out unless relative fitness is increased.
Our detection of Wolbachia at low densities in uninfected females that had mated with Wolbachiainfected males was unexpected, given that Wolbachia are absent from mature sperm in other insects (Yen and Barr 1971;Binnington and Hoffmann 1989;Clark et al. 2008). However, a recent report in

2019), determining infection status based on a threshold
Wolbachia density may be unreliable under field conditions. We therefore advise that during laboratory maintenance and field monitoring, infection frequencies are determined by screening immature stages or unmated adults. This issue appears to be specific to certain Wolbachia strains given that we found no evidence for the transmission through mating of wAlbB.
In conclusion, our findings have implications for the long-term effectiveness of Wolbachia releases and for the maintenance of wMelPop stocks in the laboratory. The stability of the deleterious effects detected here suggests that wMelPop can suppress populations for a long time once established.
However, field trials with this infection suggest that long-term persistence in natural populations is unlikely (Nguyen et al. 2015). wMelPop is difficult to maintain even under benign laboratory conditions due to a combination of incomplete maternal transmission, deleterious effects and monitoring issues, but a strict rearing schedule and regular Wolbachia screening will help to ensure its persistence in a colony. Due to its fitness costs, wMelPop may be suitable for temporary suppression or elimination of populations rather than population replacement which is now taking place in field populations with the wMel and wAlbB strains Nazni et al. 2019). Suppression through the release of wMelPop was proposed as a way of tackling mosquito incursions in isolated areas without the use of pesticides (Rašić et al. 2014). Establishing wMelPop in large semi-field cages and then imposing a dry period that required the persistence of quiescent eggs led to population elimination .
This approach to suppression does not require sex separation unlike strategies that rely on cytoplasmic incompatibility (Mains et al. 2019) and could be effective even if the infection does not persist in the long-term. Although research has shifted away from this deleterious Wolbachia infection, wMelPop may still prove to be useful when seasonal population suppression is desirable. Figure S1. Relative Wolbachia density of wMelPop-infected females with increasing (A) adult age or (B) egg storage duration. Each dot represents the Wolbachia density of a single female, while solid lines join the median densities for each time point.