How does competition among wild type mosquitoes influence the performance of Aedes aegypti and dissemination of Wolbachia pipientis?

Background Wolbachia has been deployed in several countries to reduce transmission of dengue, Zika and chikungunya viruses. During releases, Wolbachia-infected females are likely to lay their eggs in local available breeding sites, which might already be colonized by local Aedes sp. mosquitoes. Therefore, there is an urgent need to estimate the deleterious effects of intra and interspecific larval competition on mosquito life history traits, especially on the duration of larval development time, larval mortality and adult size. Methodology/principal findings Three different mosquito populations were used: Ae. aegypti infected with Wolbachia (wMelBr strain), wild Ae. aegypti and wild Ae. albopictus. A total of 21 treatments explored intra and interspecific larval competition with varying larval densities, species proportions and food levels. Each treatment had eight replicates with two distinct food levels: 0.25 or 0.50 g of Chitosan and fallen avocado leaves. Overall, overcrowding reduced fitness correlates of the three populations. Ae. albopictus larvae presented lower larval mortality, shorter development time to adult and smaller wing sizes than Ae. aegypti. The presence of Wolbachia had a slight positive effect on larval biology, since infected individuals had higher survivorship than uninfected Ae. aegypti larvae. Conclusions/significance In all treatments, Ae. albopictus outperformed both wild Ae. aegypti and the Wolbachia-infected group in larval competition, irrespective of larval density and the amount of food resources. The major force that can slow down Wolbachia invasion is the population density of wild mosquitoes. Given that Ae. aegypti currently dominates in Rio, in comparison with Ae. albopictus frequency, additional attention must be given to the population density of Ae. aegypti during releases to increase the likelihood of Wolbachia invasion.


Methodology/principal findings
Three different mosquito populations were used: Ae. aegypti infected with Wolbachia (wMelBr strain), wild Ae. aegypti and wild Ae. albopictus. A total of 21 treatments explored intra and interspecific larval competition with varying larval densities, species proportions and food levels. Each treatment had eight replicates with two distinct food levels: 0.25 or 0.50 g of Chitosan and fallen avocado leaves. Overall, overcrowding reduced fitness correlates of the three populations. Ae. albopictus larvae presented lower larval mortality, shorter development time to adult and smaller wing sizes than Ae. aegypti. The presence of Wolbachia had a slight positive effect on larval biology, since infected individuals had higher survivorship than uninfected Ae. aegypti larvae.

Conclusions/significance
In all treatments, Ae. albopictus outperformed both wild Ae. aegypti and the Wolbachiainfected group in larval competition, irrespective of larval density and the amount of food resources. The major force that can slow down Wolbachia invasion is the population density of wild mosquitoes. Given

Introduction
Infectious diseases caused by arboviruses are a growing global health concern. Among the disease vectors, mosquitoes from the genus Aedes and mostly important Ae. aegypti (Linnaeus, 1762) and Ae. albopictus (Skuse, 1894) have a prominent role in transmitting several arboviruses to humans. In the last 50 years, dengue virus (DENV) has shown a 30-fold increase in global incidence, with around 400 million new infections yearly [1][2][3]. In the last decade, chikungunya became pandemic after spreading from limited regions of Africa and Asia and arriving into the Americas. Two CHIKV genotypes were detected in Brazil: The Asian genotype has probably invaded the country through the Caribbean and the East-Central-South African (ECSA) genotype was first detected in the Bahia State [4,5]. Most recently, Zika virus (ZIKV) emerged in the Pacific and later in the Americas, causing a public health emergency due to its association with microcephaly in newborns [6][7][8].
The Ae. aegypti mosquito is more frequently observed in highly urbanized areas. It is extremely well adapted to live in close association with human dwellings. Females blood feed preferentially on humans and lay eggs on man-made containers often located on the surroundings of residences [9][10][11][12]. Conversely, Ae. albopictus is more frequently collected in wooded areas next to humans and tends to bite on a variety of vertebrates outdoors [13]. Both species overlap their spatial distribution in suburban areas, especially in those districts with high vegetation coverage [11,[14][15][16]. Thus, eventually, Ae. aegypti and Ae. albopictus share the use of the same breeding sites, which triggers a series of ecological interactions due to the limited resources available.
Many studies have investigated the negative outcomes of competing environment on adult life history traits. Inadequate nutrition during the larval stage of mosquitoes can be associated with reduced wing size, shorter longevity and flight performance, higher susceptibility to arboviral infections and replication under laboratory conditions [17][18][19][20][21]. Density-dependent competition in larval stages causes mortality and thus reduced recruitment to the adult stage, showing that Ae. aegypti vectorial capacity is strongly dependent on the larval habitat quality.
Effective vector control activities are used as the primary approach to mitigate arbovirus transmission, especially in the absence of vaccines. Ae. aegypti control still relies massively on source reduction and on using chemicals such as insecticides. However, maintaining infestation level below a theoretical threshold to avoid outbreaks requires a constant and somehow utopic military discipline of field health agents over time [22]. Moreover, the overuse of insecticides favors the dissemination of alleles that confer resistance among wild Ae. aegypti populations, jeopardizing insecticide efficiency as tools for vector control [23,24]. Due to the low capacity of traditional control measures to reduce mosquito populations, new approaches to mitigate transmission must be tested.
One of the innovative approaches currently being tested is the deployment of the maternally inherited endosymbiont Wolbachia pipientis into wild mosquito populations [25]. The use of Wolbachia as a natural control agent is supported by findings showing that Ae. aegypti females infected with the wMel strain are able to block DENV, CHIKV and ZIKV [26-28]. Accordingly, Wolbachia releases may be used to mitigate arbovirus transmission by two different strategies: suppression of mosquito population by massive male-releases or the substitution of a highly susceptible population by one that blocks arbovirus transmission [29,30].
Wolbachia deployments are taking place in five countries, including Brazil (www. eliminatedengue.com). When Wolbachia-infected mosquitoes are released, females tend to behave as their wild counterparts, i.e., will blood feed on local householders and lay eggs in the available breeding sites. Considering that in Rio de Janeiro city there is a strong co-occurrence of Ae. aegypti and Ae. albopictus [11,16], females carrying Wolbachia will eventually lay eggs in containers already colonized by wild Aedes sp. mosquitoes. Therefore, our main objectives were to estimate the deleterious effects of larval competition on mosquito life history traits, but also to determine to what extent larval competition of wMel-Ae. aegypti mosquitoes with wild Ae. aegypti and Ae. albopictus may jeopardize Wolbachia invasion.

Mosquitoes
We used three different mosquito populations: Ae. aegypti infected with Wolbachia (wMelBr strain), wild Ae. aegypti and wild Ae. albopictus.

Ae. aegypti infected with Wolbachia strain wMelBr
The lineage of Ae. aegypti with wMel was imported from Australia to Brazil (IBAMA license 11BR005873/DF). Briefly, a backcrossing with 250 virgin females (wMel) and 200 wild males was conducted for nine consecutive generations, producing wMelBr [31]. After that period, the wMelBr colony was outcrossed every five generations with 10% wild males from a pool of four districts (Jurujuba, Tubiacanga, Urca and Vila Valqueire) with high nuclear genome homogeneity across Rio to refresh the genetic pool [32]. We used the F19 of wMelBr generation.
Wooden paddles were brought to Fiocruz, eggs were hatched and larvae were classified using taxonomic keys [33]. Larvae of each species were pooled and reared in dechlorinated water and fed with TetraMin (fish food), maintained in a climate controlled insectary, at 26 ± 1˚C and 70 ± 10% relative humidity. Adult females were kept under a 12:12 hour light:dark cycle, ad libitum access to sugar solution (10%) and blood fed twice a week using anesthetized mice (CEUA L-0007/09). Eggs were stored under insectary conditions until the experiment. We used the F1 of wild Ae. aegypti and F2 of Ae. albopictus.

Semi-field conditions
The larval competition experiments were performed in a semi-field setting, an open building located at the Army Institute of Biology in Rio de Janeiro, Brazil (22˚53'34"S, 43˚14'33"W), but with limited control access to unauthorized personnel. The experiment was subject to the influences of climate variation, such as humidity and air temperature, as well as rainfall. These conditions were continuously recorded by means of a weather station (Instrutemp, ITWH model 1080) installed on site.

Experimental design
The intraspecific larval competition of Ae. aegypti (with and without wMelBr) and interspecific competition with Ae. albopictus were investigated by monitoring the development of larvae at different densities, species proportions, and food levels in containers. Twenty-one treatments were set and used different proportions of wild Ae. aegypti: wMelBr-Ae. aegypti: Ae. albopictus  [19] and represent larval crowding in nature [12,34]. Larvae were placed as L1 in 400 ml of tap water into black plastic containers (9.5 cm in height, 8.5 cm base diameter). Each treatment had eight containers with two distinct food levels. The food consisted of 0.25 or 0.50 g of Chitosan (an analogue of insect chitin used to simulate the remains of arthropods), and fallen avocado leaves (extra source of natural nutrition commonly used in Aedes competition assays), in the same proportion, that were collected, washed, dried, broken into small pieces and weighed. Therefore, our experimental design consisted of 200 plastic containers (12 cm in diameter x 15 cm in height).
Each container was identified and received the appropriate quantity of Chitosan and leaf litter, with 400 ml of tap water, three days before the addition of larvae. Containers were covered with black tulle to prevent oviposition by wild mosquitoes. One hour after eggs were hatched, larvae were counted with the help of a stereo microscope and then added to their appropriate containers. Each container was monitored daily for the presence of pupae, which were collected and placed in small covered vials (6.5 cm height x 2.5 cm diameter) and kept until adult emergence. On the day of emergence, adults were killed with acetyl acetate and, after being sexed, one wing was removed. Wing length was defined as the distance from the axillary incision to the apical margin excluding the fringe [35]. The experiment ended when the last pupa became adult.

Detection of wMelBr through molecular assays
In treatments with the presence of wMelBr and wild Ae. aegypti simultaneously, all adults and dead pupae were screened for Wolbachia. Screening was performed using the Taqma n multiplex Real Time-Polymerase Chain Reaction. Adult mosquitoes and dead pupae were individually screened on ViiA7 Real Time PCR machine (Life Technologies). The genomic DNA was extracted using a squash buffer (0.1 M NaCl; 10 mM Tris Base; 1 mM EDTA; pH 8.2) supplemented with 9 μg of Proteinase K per mosquito (Qiagen). After macerating the mosquitoes with a 2mm glass-bead on a Mini-beadbeater (Biospec Products), samples were placed on a thermocycler following the thermal cycle: 56˚C for 5 minutes and 98˚C for 15 minutes. Genomic DNA was diluted 1:10 in ultra-pure water and then used as the template for Wolbachia screening. We used the WD0513 gene that amplifies a fragment of 110 bp with the following primers: TM513-Forw: CAA ATT GCT CTT GTC CTG TGG and TM513-Rev: GGG TGT TAA GCA GAG TTA CGG and TM513-probe 5'-/FAM Cy5/ TGA AAT GGA AAA ATT GGC GAG GTG TAG G --BHQ-1/-3'. In the same reaction, a ribosomal gene from Ae. aegypti that amplifies a fragment of 68 bp was analyzed with the primers: RPS17-Forw: RPS17-Forw: 5'-TCC GTG GTA TCT CCA TCA AGC T-3' and RPS17-Rev: 5'-CAC TTC CGG CAC GTA GTT GTC-3', and RPS17-probe: 5'-/FAM/CAG GAG GAG GAA CGT GAG CGC AG/ 3BHQ_1/-3'. Negative and positive controls of Ae. aegypti (with and without wMelBr) and Ae. albopictus were used in all reactions.
Reagents used in the qPCR were: 5 μL of TaqMan Universal PCR Master Mix (Thermo Fisher), 0.5mM of RPS17 primers, 0.6mM of TM513 primers, 0.1mM of RPS17 probe, 0.25mM of TM513 probe and 1μL of diluted DNA. Water was added to complete a final volume of 10 μL.

Data analyses
Three biological aspects were observed throughout the experiment: larval survivorship, developmental time and wing length. Survivorship was calculated, for each container, by the frequency of larvae that reached the adult stage. Developmental time per container was calculated as the average number of days from hatching until the emergence of the adult was observed in the plastic vial.
An important parameter in population ecology is the performance index λ', related to the growth rate r' by λ' = exp(r´). We calculate λ' using values of observed biological aspects, such as survival of immature, development time and adult size of cohorts of mosquitoes, for each replicate. An estimate of the performance index has been adapted by [36] from the equation established by [37] using r' as a measurement of population growth. According to this index, the condition λ 0 > 1.0 represents an increase in the population, whereas condition λ 0 < 1.0 points to a population decrease. The λ' index was calculated for each replicate as follows: where N 0 is the initial number of females in a cohort, which we assumed to be 50% of the added larvae, since the sex ratio of the species studied here is generally 1:1 [38,39]; A x is the number of adult females on day x; w x is the average size of the female wing on day x; fecundity of females is modeled by a function ƒ (w x ) of the wing size, as proposed for Ae. aegypti [40] and Ae. albopictus [41]. No significant differences in fecundity have been found due to Wolbachia infection [31], thus we assumed the same relationship between mosquito size and fecundity for infected and uninfected mosquitoes. D is the time required (in days) for a newly hatched female to mate, blood feed and lay eggs. In our experiments, D is typically equal to the number of days that a female takes to reach the adult stage plus four days, the length of the first gonotrophic cycle [42].
The effects of competition conditions on the performance of Ae. aegypti infected with wMelBr were analyzed using a Generalized Linear Model (GLM). Development time, wing length, survival proportion and the performance index were analyzed each separately as outcomes using as explanatory variables: species, nutrients, competing numbers of wild Ae. aegypti, Ae. albopictus, and Ae. aegypti with wMelBr. For development time, performance index, and wing length we used a normal distribution and logarithmic link function. For survivorship we used logistic regression models with a binomial family/logit link function. For each of the outcomes we selected the model with lowest Akaike Information Criterion (AIC). P-values lower than 0.05 were considered significant. We used R 3.0.1 software for these analyzes.
The index values of λ' were used to make a model to simulate the impact of different levels of infestation of Ae. aegypti wild type and Ae. albopictus in the performance of Ae. aegypti with wMelBr in larval competition. Three nonlinear regressions were applied to each of the indices λ' for the three populations: wild Ae. aegypti, Ae. albopictus and Ae. aegypti with wMelBr, with the number (n x ) of individuals in each cohort x (aeg for wild Ae. aegypti, albo for Ae. albopictus, wmel for Ae. aegypti with wMelBr) in competition, according to the following model: log (λ')~n aeg + n albo + n wmel . These analyses allowed us to evaluate the effect on the performance index when increasing both interspecific and intraspecific competition. The values obtained in the regressions were used to simulate the interspecific competition among the three populations.
Once coefficients for interspecific competition were obtained we evaluated the intensity of interspecific competition that makes the growth rate negative, i.e., r´= log (λ') < 0. For instance, if population of Ae. aegypti with wMelBr suppresses the wild Ae. aegypti population, this permitted us to evaluate the frequency of Ae. albopictus that causes a severe interspecific competition that might compromise sustained growth of Ae. aegypti with wMel. In this case, we find value for r´w Mel = log (λ' wMel ) = β wmel + α wmel n albo + γ wmel n wmel < 0, where β, α wmel , and γ wmel are coefficients obtained in the regression analysis.

Ethical issues
The use of anesthetized mice to blood feed mosquitoes was authorized by Fiocruz Ethical Committee for Animal Use (CEUA L-0007/09), which follows the National guidelines for the scientific use of animals disposed on the Law 11.794/2008.

Survivorship
Under intraspecific competition, survival was inversely proportional to larval density in the three tested populations, as expected (Ae. albopictus: t = -27.2, P<0.05, Ae. aegypti: t = -28.3, P<0.05, Ae. aegypti with wMelBr: t = -26.3, P<0.05). Ae. albopictus presented higher tolerance for increasing competition than wild Ae. aegypti and Ae. aegypti with wMelBr. On the other hand, Ae. aegypti presented a significant decrease in survivorship when larval density per container doubled. This pattern was observed independently of Wolbachia presence (Fig 1,  Table 1).
Under interspecific competition, the survival of Ae. albopictus and Ae. aegypti with wMelBr larvae was significantly higher than survival of wild Ae. aegypti, irrespective of the amount of food resources (Table 1). Nonetheless, competitive advantage of Ae. albopictus over wild Ae. aegypti seemed slightly more evident in the most stressful and competitive treatments. Ae. aegypti with wMelBr also survived less than Ae. albopictus, although its survival is marginally higher than that observed for wild Ae. aegypti. In some particular treatments, Ae. aegypti larvae infected with wMelBr presented better survival than wild Ae. aegypti.

Developmental time
Under intraspecific competition, overcrowding was directly related to the increase in developmental time (DT) (P<0.05). Wild Ae. aegypti and those infected with wMelBr have a longer DT starting at 40 larvae per container, while Ae. albopictus DT was notably affected only at a higher density, i.e., 60 larvae per container (Fig 2, Table 2). Wild Ae. aegypti presented the longest DT at high densities with an average duration of 57.17 and 47.99 days in low and high food resources, respectively. Under interspecific competition, Ae. aegypti with wMelBr had a similar DT to Ae. albopictus at high food resources, but was outcompeted when food resources were scarce. Interestingly, Ae. albopictus presented a shorter DT than wild Ae. aegypti (t = 4.33, P<0.05), but the presence of Wolbachia did not alter the DT in Ae. aegypti.

Female wing size
The three populations had a significant decrease in wing size due to overcrowding (Table 3, Fig 3). Ae. albopictus had a sharper decrease than wild Ae. aegypti and Ae. aegypti with wMelBr. The presence of Wolbachia did not seem to influence mosquito wing size. The amount of nutrients had a positive effect for wild Ae. aegypti and a negative effect for Ae. albopictus.

Performance index (λ')
Overall, overcrowding had a significant effect on the performance of the three populations (Fig 4, Table 4). The value of λ' for wild Ae. aegypti and Ae. aegypti with wMelBr suffered a reduction from 1.2 to 1.0 when the larval density was doubled (treatment 2). On the other hand, the λ' for Ae. albopictus was only reduced to 1.0 when the larval density was tripled (treatment 3). Remarkably, all populations tested were able to maintain λ' above 1 under low densities, meaning that they could be successfully sustained in the wild. Under these experimental settings, Ae. albopictus showed superior performance to wild Ae. aegypti and the presence of Wolbachia did not seem to affect Ae. aegypti performance (Fig 4, Table 4).

Effects of competition intensity on the performance index (λ') of competing species
We simulated interspecific competition among the three different populations applying the results from the nonlinear regression analyzes for each of the Ae. aegypti, Ae. albopictus, Ae. aegypti with wMel performance indices. As expected, increasing either intraspecific or interspecific competition makes the performance index smaller for the three populations. In Fig 5, when performance indices reach values below lines for λ = 1, the interspecific competition does not allow a positive growth rate. We generally observe that the values under which Ae. albopictus can sustain a positive growth rate are larger than values for both Ae. aegypti and Ae. aegypti with wMel populations. We also studied the frequency of Ae. albopictus that could make interspecific competition intense enough to make Ae. aegypti/wMel performance index λ wmel <1, i.e. impacting severely on the sustained growth assuming only larval competition (Fig 6). As the number of Ae. aegypti/wMel larvae increases (intraspecific competition), the frequency of Ae. albopictus that causes the performance index to reach an unsustainable level λ wmel <1 decreases.

Discussion
The deployment of Wolbachia to reduce dengue transmission is currently being undertaken in several regions of the world. During releases, Ae. aegypti mosquitoes infected with Wolbachia will lay eggs in breeding sites in which wild mosquitoes previously colonized, i.e., intra-specific competition with local Ae. aegypti and other native mosquitoes such as Ae. albopictus might be an important issue to determine the pace of Wolbachia invasion. Due to the co-occurrence of Ae. aegypti and Ae. albopictus in several countries of Southeast Asia and Latin America, we investigated how the intra-and interspecific competition with Ae. albopictus might undermine wMel invasion.
We explored three critical aspects of mosquito biology under different competition scenarios: larval survivorship, developmental time and wing length. Using these estimates, we calculated a performance index that is related to growth rate for wild Ae. aegypti, Ae. albopictus, and Ae. aegypti with wMelBr.
Overcrowding significantly reduced larval survivorship in the three populations tested, as expected [18,19,[43][44][45]. Our data show that under intraspecific competition settings, larval survivorship decreased more intensely for Ae. aegypti than for Ae. albopictus. Also, the presence of Wolbachia did not affect this pattern (Fig 1). The effects of Ae. albopictus density on Ae. aegypti mortality and vice-versa has been evaluated elsewhere [19,34,46,47]. In summary, one of these populations gets more severely affected when the density of the other increases, and this increase in mortality might be seen in the larval or adult stage [48,49]. Ae. albopictus is frequently pointed as a better competitor than Ae. aegypti [19,34,36,46,50,51] as well as other species such as Ae. japonicus [52] and Culex pipiens [53]. Larvae survival under interspecific competition conditions may vary due to the difference in efficacy with which larvae exploit food resources [54]. Ae. albopictus showed a larval survivorship higher than that observed for  55], but interestingly, a superior survivorship of infected larvae was reported in a competitive environment when compared with uninfected larvae [39]. On the other hand, Wolbachia infection reduced the tolerance of Ae. aegypti larvae to starvation, probably due to an increasing rate of depletion of accumulated energy reserves [45]. Our data support an overall beneficial impact of Wolbachia infection on Ae. aegypti larval survivorship, since infected larvae present a superior survival rate than their uninfected counterparts (the exception being observed in starvation scenarios, in which Wolbachia reduced larval survivorship).
The development time from egg hatch to adult is a critical fitness aspect of mosquito biology under field conditions. Delayed larvae are more subject to external factors such as predation [56], water evaporation and breeding site treatment or removal. Our results show that nutrient levels caused longer development time for all three populations when food resources were scarce [19,48,57]. Ae. albopictus had a rapid development time when compared with Ae. aegypti [11] even in the more competitive treatments. The presence of Wolbachia did not accelerate Ae. aegypti developmental time. Differences in larvae development time were highest in treatments with interspecific competition with 0.25 g of litter. The exception was the Ae. albopictus/Ae. aegypti with wMel, in which larvae of both populations have distinct development time differences only at 0.50 g litter, with the former developing faster than the latter.
Overall, results regarding the influence of Wolbachia on larval development time are conflicting. The experimental design and settings established in our competitive assays were unable to detect any changes on Ae. aegypti development time due to Wolbachia presence. At intermediate (50 larvae/tray) and high densities (250 larvae/tray), wMel infection led to more rapid larval development for both males and females, with no effect under a less crowded and more stressful condition [28]. Opposing results to our data, a slight delay was observed in wMel infected larvae related to their uninfected counterparts [39]. Despite these findings in disagreement, few strains of Wolbachia are known to modify adult feeding behavior, and might interfere with larval foraging capability as well [26,56,58,59]. Potential explanations for the effects of Wolbachia on mosquito larval development time involve immune up-regulation or increased metabolism observed in the adults, which may also influence larval development rate [26,60]. Other aspects still need an evaluation to better understand the effect of Wolbachia on immature development time, such as the effects of the population genetic background, Wolbachia strain and experimental design.
Mosquito body size is ultimately a manifestation of larval habitat quality and can produce significant effects on an insect's fitness and then alter mosquito vectorial capacity [12,61,62]. Physiological stress in juvenile stages produces negative effects that may pass into adulthood [63]. For instance, highly competitive environments produce mosquitoes with a small wing length, which are less likely to promote Wolbachia invasion since they should blood feed more often, possess shorter longevity and lower flight performance than bigger mosquitoes [18][19][20][21]. Hence, Ae. aegypti vectorial capacity is strongly dependent on the larval habitat quality [11,[17][18][19][20][21]. Previous reports have shown an inversely proportional correlation between wing size and larval density in Ae. aegypti, as we observed [28,45]. Our results indicate reduction in mosquito size due to overcrowding in all three populations, which is highly expected [19,49]. Ae. albopictus wing size was consistently smaller than Ae. aegypti in almost all treatments, with visible differences when competition was intra or interspecific. The interaction between nutrients and population produced unexpected results. Ae. albopictus body size decreased at 0.5 g litter when compared with the 0.25 g treatment. Interestingly, body size of Ae. aegypti with wMel was not affected by availability of food resources. Hence, from the perspective of Wolbachia deployment, the infection with wMel strain does not pose a significant disadvantage during competition against wild mosquitoes [39].
We used three population growth correlates, i.e. larval survivorship, time to adulthood and adult wing size to estimate a composite index of mosquito performance (λ') for each container [36,37]. Overall, larval density negatively affected the performance index λ' of the three populations, but remarkably only Ae. albopictus population growth was positive in all treatments. In fact, population growth of Ae. albopictus was significantly superior from the observed for wild Ae. aegypti, while the presence of Wolbachia provided no advantage to infected Ae. aegypti. Interspecific assays using Ae. aegypti and Ae. albopictus at different densities have shown a superior competitive ability of the the latter [19,34,36]. Despite being frequently described as a superior larval competitor to Ae. aegypti, these two species coexist in much of Brazil and in southeast US and Southeast Asia [16,64]. Part of the explanation for coexistence may rely on life-history trade-offs and abiotic factors [40, [65][66][67] Coexistence between Ae. aegypti and Ae. albopictus may be possible due to dry and warm climates that would favor the former and mitigate effects of larval competition via differential mortality of Ae. albopictus [67]. This hypothesis was reinforced by Camara et al. (2016) [34], observing that intensity of competition at the larval stage may vary seasonally, with harsh effects on development time during warmer Summer. Abiotic factors may also contribute to habitat segregation since urbanized areas tend to be warmer than arborized surrounding areas [68]. Additionally, one force that can impact Ae. aegypti/wMelBr invasion is the asymmetric reproductive interference among mosquitoes, in which male Ae. albopictus can inseminate and thus sterilize Ae. aegypti females. The act of reducing the reproductive success of a different species by mating a female of an incompatible species is called satyrization [69][70][71]. Evidence of satyrization of Ae. aegypti females seems to be more likely than on Ae. albopictus females, although still low (less than 5%), biasing the asymmetric nature of cross matings in favor of the latter [72][73][74]. Therefore, although still not observed in Brazilian sites where Wolbachia deployment is ongoing, additional concern would be required if invasion is lagging. The major force that can affect Wolbachia invasion is the population density of wild mosquitoes [75,76]. This concern is even more important if we consider that mosquitoes from other species can lay eggs in the same breeding sites of Ae. aegypti. Therefore, during Wolbachia deployment, infected mosquitoes will lay their eggs in breeding sites already colonized by local mosquitoes, such as uninfected Ae. aegypti and Ae. albopictus. Assuming Ae. albopictus is a better competitor and the presence of Wolbachia does not increase mosquito performance at the larval stage, the natural density of Ae. albopictus may become an additional obstacle to slow invasion. However, we observed a negative growth rate of Ae. aegypti/wMelBr only when Ae. albopictus frequency was high. In Rio de Janeiro, we selected four neighborhoods with different landscapes and performed adult mosquito collections with BG-Sentinel Traps installed at the peridomestic area of local householders on a weekly basis for 104 consecutive weeks [31]. We observed that the frequency of Ae. albopictus was lower than 5% in the four sites during the 104 weeks of trapping. In fact, during approximately four consecutive months, no Ae. albopictus mosquitoes were collected in any trap from any field site (Eliminate Dengue Program). Therefore, Ae. albopictus is more likely to slow down Wolbachia invasion, rather than to stop it. Density-dependent traits can promote strong effects on Wolbachia dynamics in Ae. aegypti field populations [77]. Therefore, an estimation of the population sizes of Ae. aegypti and other mosquito populations that can occasionally lay eggs in the same breeding sites, such as Ae. albopictus, Culex quinquefasciatus and Limmatus durhami, might provide important information on the Wolbachia invasion pattern in highly infested field sites [31, [76][77][78][79][80].