Highlights d The wAlbB strain of Wolbachia bacteria was established in Aedes aegypti populations d Wolbachia frequencies remained high and stable at some sites d Dengue incidence decreased at release sites

Aedes aegypti mosquitoes infected with Wolbachia bacteria are currently being released for arbovirus suppression around the world. Their potential to invade populations and persist will depend on interactions with environmental conditions, particularly as larvae are often exposed to fluctuating and extreme temperatures in the field. We reared Ae. aegypti larvae infected with different types of Wolbachia (wMel, wAlbB and wMelPop-CLA) under diurnal cyclical temperatures. Rearing wMel and wMelPop-CLA-infected larvae at 26–37°C reduced the expression of cytoplasmic incompatibility, a reproductive manipulation induced by Wolbachia. We also observed a sharp reduction in the density of Wolbachia in adults. Furthermore, the wMel and wMelPop-CLA infections were not transmitted to the next generation when mosquitoes were exposed to 26–37°C across all life stages. In contrast, the wAlbB infection was maintained at a high density, exhibited complete cytoplasmic incompatibility, and was transmitted from mother to offspring with a high fidelity under this temperature cycle. These findings have implications for the potential success of Wolbachia interventions across different environments and highlight the importance of temperature control in rearing.

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

ABSTRACTWe have developed and validated two new fluorescence-based PCR assays to detect theWolbachia wMel strain inAedes aegyptiand thewRi andwAu strains inDrosophila simulans. The new assays are accurate, informative, and cost-efficient for large-scaleWolbachiascreening.

Summary1. Field release of endosymbiotic Wolbachia bacteria into wild Aedes aegypti mosquito populations is a promising strategy for biocontrol of dengue. This strategy requires successful Wolbachia invasion through the mosquito vector population. Natural variation in mosquito fitness due to density-dependent competition for limited food resources may influence Wolbachia invasion. We know little about these effects, largely because our understanding of density-dependent dynamics in mosquito populations is limited. 2. We developed an empirical model of A. aegypti-Wolbachia dynamics where food resources available to the developing larvae are limited. We assessed the extent of density-dependent regulation in our A. aegypti population using a Bayesian statistical model that estimates the temporal variation in mosquito fitness components. We monitored the spread of Wolbachia and assessed the effect of the bacteria on larval fitness components. 3. We demonstrate that mosquito population growth is regulated by strong larval densitydependent variation in mosquito fitness components. Wolbachia spread was slowed by this heterogeneity in mosquito fitness, which reduces the capacity of the bacteria to invade. However, we found no evidence that Wolbachia affects larval fitness components. 4. Synthesis and applications. We demonstrate that the extent and form of density-dependent dynamics in the host population can have a major influence on Wolbachia invasion. These findings help explain slow Wolbachia invasion rates and indicate that the success of field release strategies for dengue control can depend on attaining high Wolbachia frequencies in the mosquito population.

BackgroundArbovirus transmission by the mosquito Aedes aegypti can be reduced by the introduction and establishment of the endosymbiotic bacteria Wolbachia in wild populations of the vector. Wolbachia spreads by increasing the fitness of its hosts relative to uninfected mosquitoes. However, mosquito fitness is also strongly affected by population size through density-dependent competition for limited food resources. We do not understand how this natural variation in fitness affects symbiont spread, which limits our ability to design successful control strategies.ResultsWe develop a mathematical model to predict A. aegypti–Wolbachia dynamics that incorporates larval density-dependent variation in important fitness components of infected and uninfected mosquitoes. Our model explains detailed features of the mosquito–Wolbachia dynamics observed in two independent experimental A. aegypti populations, allowing the combined effects on dynamics of multiple density-dependent fitness components to be characterized. We apply our model to investigate Wolbachia field release dynamics, and show how invasion outcomes can depend strongly on the severity of density-dependent competition at the release site. Specifically, the ratio of released relative to wild mosquitoes required to attain a target infection frequency (at the end of a release program) can vary by nearly an order of magnitude. The time taken for Wolbachia to become established following releases can differ by over 2 years. These effects depend on the relative fitness of field and insectary-reared mosquitoes.ConclusionsModels of Wolbachia invasion incorporating density-dependent demographic variation in the host population explain observed dynamics in experimental A. aegypti populations. These models predict strong effects of density-dependence on Wolbachia dynamics in field populations, and can assist in the effective use of Wolbachia to control the transmission of arboviruses such as dengue, chikungunya and zika.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0319-5) contains supplementary material, which is available to authorized users.

Aedes aegypti mosquitoes infected with Wolbachia bacteria are currently being released for arbovirus suppression around the world. Their potential to invade populations and persist will depend on interactions with environmental conditions, particularly as larvae are often exposed to fluctuating and extreme temperatures in the field. We reared Ae. aegypti larvae infected with different types of Wolbachia (wMel, wAlbB and wMelPop-CLA) under diurnal cyclical temperatures. Rearing wMel and wMelPop-CLA-infected larvae at 26-37˚C reduced the expression of cytoplasmic incompatibility, a reproductive manipulation induced by Wolbachia. We also observed a sharp reduction in the density of Wolbachia in adults. Furthermore, the wMel and wMelPop-CLA infections were not transmitted to the next generation when mosquitoes were exposed to 26-37˚C across all life stages. In contrast, the wAlbB infection was maintained at a high density, exhibited complete cytoplasmic incompatibility, and was transmitted from mother to offspring with a high fidelity under this temperature cycle. These findings have implications for the potential success of Wolbachia interventions across different environments and highlight the importance of temperature control in rearing. Author SummaryThere is currently great interest in using the bacterium Wolbachia to reduce the burden of dengue and Zika; viruses which infect millions of people globally each year. Aedes aegypti mosquitoes with Wolbachia infections can invade natural populations and interfere with the transmission of these viruses. However, we find that the wMel strain of Wolbachia which is currently being used for dengue and Zika control in several countries may have reduced effectiveness at invading populations when mosquitoes experience heat stress. Since mosquito larvae experience extreme temperatures in their natural habitat, our results have implications for current and future releases of Wolbachia-infected mosquitoes and highlight the need for further investigation into alternative strains of Wolbachia.

The distribution of Aedes aegypti (L.) in Australia is currently restricted to northern Queensland, but it has been more extensive in the past. In this study, we evaluate the genetic structure of Ae. aegypti populations in Australia and Vietnam and consider genetic differentiation between mosquitoes from these areas and those from a population in Thailand. Six microsatellites and two exon primed intron crossing markers were used to assess isolation by distance across all populations and also within the Australian sample. Investigations of founder effects, amount of molecular variation between and within regions and comparison of FST values among Australian and Vietnamese populations were made to assess the scale of movement of Ae. aegypti. Genetic control methods are under development for mosquito vector populations including the dengue vector Ae. aegypti. The success of these control methods will depend on the population structure of the target species including population size and rates of movement among populations. Releases of modified mosquitoes could target local populations that show a high degree of isolation from surrounding populations, potentially allowing new variants to become established in one region with eventual dispersal to other regions.