Matching the genetics of released and local Aedes aegypti populations is critical to assure Wolbachia invasion.

BACKGROUND: Traditional vector control approaches such as source reduction and insecticide spraying have limited effect on reducing Aedes aegypti population. The endosymbiont Wolbachia is pointed as a promising tool to mitigate arbovirus transmission and has been deployed worldwide. Models predict a rapid increase on the frequency of Wolbachia-positive Ae. aegypti mosquitoes in local settings, supported by cytoplasmic incompatibility (CI) and high maternal transmission rate associated with the wMelBr strain. METHODOLOGY/PRINCIPLE FINDINGS: Wolbachia wMelBr strain was released for 20 consecutive weeks after receiving >87% approval of householders of the isolated community of Tubiacanga, Rio de Janeiro. wMelBr frequency plateued~40% during weeks 7-19, peaked 65% but dropped as releases stopped. A high (97.56%) maternal transmission was observed. Doubling releases and deploying mosquitoes with large wing length and low laboratory mortality produced no detectable effects on invasion trend. By investigating the lab colony maintenance procedures backwardly, pyrethroid resistant genotypes in wMelBr decreased from 68% to 3.5% after 17 generations. Therefore, we initially released susceptible mosquitoes in a local population highly resistant to pyrethroids which, associated with the over use of insecticides by householders, ended jeopardizing Wolbachia invasion. A new strain (wMelRio) was produced after backcrossing wMelBr females with males from field to introduce mostly pyrethroid resistance alleles. The new strain increased mosquito survival but produced relevant negative effects on Ae. aegypti fecundity traits, reducing egg clutche size and egg hatch. Despite the cost on fitness, wMelRio successful established where wMelBr failed, revealing that matching the local population genetics, especially insecticide resistance background, is critical to achieve invasion. CONCLUSIONS/SIGNIFICANCE: Local householders support was constantly high, reaching 90% backing on the second release (wMelRio strain). Notwithstanding the drought summer, the harsh temperature recorded (daily average above 30°C) did not seem to affect the expression of maternal transmission of wMel on a Brazilian background. Wolbachia deployment should match the insecticide resistance profile of the wild population to achieve invasion. Considering pyrethroid-resistance is a widely distributed phenotype in natural Ae. aegypti populations, future Wolbachia deployments must pay special attention in maintaining insecticide resistance in lab colonies for releases.

The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions.

BACKGROUND: In the tropics, the utilization of insecticides is still an important strategy for controlling Aedes aegypti, the principle vector of dengue, chikungunya and Zika viruses. However, increasing insecticide resistance in Ae. aegypti populations might hinder insecticide efficacy on a long-term basis. It will be important to understand the dynamics and evolution of insecticide resistance by assessing its frequency and the mechanisms by which it occurs. METHODOLOGY/PRINCIPAL FINDINGS: The insecticide resistance status of four Brazilian Ae. aegypti populations was monitored. Quantitative bioassays with the major insecticides employed in the country was performed: the adulticide deltamethrin (a pyrethroid-PY) and the larvicides, temephos (an organophosphate) and diflubenzuron (a chitin synthesis inhibitor). Temephos resistance was detected in all populations although exhibiting a slight decrease over time probably due to the interruption of field use. All vector populations were susceptible to diflubenzuron, recently introduced in the country to control Ae. aegypti. Resistance against deltamethrin was extremely high in three populations. Molecular assays investigated substitutions in the voltage gated sodium channel (NaV), the PY target site, at positions 1011, 1016 and 1534. Elevated frequencies of substitutions Val1016Ile and Phe1534Cys related to high PY resistance levels were identified. Biochemical assays detected alterations in the activities of two detoxifying enzyme classes related to metabolic resistance, glutathion-S-transferases and esterases. The results obtained were evaluated in the context of both recent insecticide use and the records of dengue incidence in each locality. CONCLUSIONS/SIGNIFICANCE: The four Ae. aegypti populations evaluated were resistant to the neurotoxic insecticides, temephos and deltamethrin. However, they were still susceptible to diflubenzuron. A probable correlation between adult insect resistance to PY and the domestic application of insecticides is discussed, pointing to the need for awareness measures regarding the correct utilization by citizens. This work aims to contribute to the efficient and rational management of Ae. aegypti control of both larvae and adults.

Novel inference models for estimation of abundance, survivorship and recruitment in mosquito populations using mark-release-recapture data.

BACKGROUND: Experiments involving mosquito mark-release-recapture (MRR) design are helpful to determine abundance, survival and even recruitment of mosquito populations in the field. Obstacles in mosquito MRR protocols include marking limitations due to small individual size, short lifespan, low efficiency in capturing devices such as traps, and individual removal upon capture. These limitations usually make MRR analysis restricted to only abundance estimation or a combination of abundance and survivorship, and often generate a great degree of uncertainty about the estimations. METHODOLOGY/PRINCIPAL FINDINGS: We present a set of Bayesian biodemographic models designed to fit data from most common mosquito recapture experiments. Using both field data and simulations, we consider model features such as capture efficiency, survival rates, removal of individuals due to capturing, and collection of pupae. These models permit estimation of abundance, survivorship of both marked and unmarked mosquitoes, if different, and recruitment rate. We analyze the accuracy of estimates by varying the number of released individuals, abundance, survivorship, and capture efficiency in multiple simulations. These methods can stand capture efficiencies as low as usually reported but their accuracy depends on the number of released mosquitoes, abundance and survivorship. We also show that gathering pupal counts allows estimating differences in survivorship between released mosquitoes and the unmarked population. CONCLUSION/SIGNIFICANCE: These models are important both to reduce uncertainty in evaluating MMR experiments and also to help planning future MRR studies.

O papel da resistência a inseticidas e da densidade de Aedes aegypti na disseminação da Wolbachia em populações nativas do Rio de Janeiro, Brasil / The role of insecticide resistance and Aedes aegypti density in the spread of Wolbachia in native populations of Rio de Janeiro, Brazil

No Brasil encontra-se em andamento liberações de Aedes aegypti infectados com Wolbachia, uma bactéria que reduz, experimentalmente, a transmissão de arbovírus como os vírus dengue, chikungunya e Zika. Testes em áreas da Austrália, Indonésia e Vietnã confirmaram uma invasão rápida e estável de Ae. aegypti contendo a cepa wMel sobre a população selvagem. No Rio de Janeiro, liberações semanais de Ae. aegypti com Wolbachia (linhagem wMelBr) foram iniciadas em setembro de 2014. Entretanto, diferentemente do observado naqueles países, a primeira liberação de Ae. aegypti infectados com Wolbachia na América Latina não logrou êxito. Durante 20 semanas consecutivas, a linhagem wMelBr foi liberada no Rio, mas um platô inesperado (devido à introdução semanal de milhares de mosquitos e ao efeito da incompatibilidade citoplasmática) foi observado nas semanas 7 a 19. Primeiramente, realizamos experimentos de marcação-soltura-recaptura durante as liberações para verificar o número de mosquitos liberados e a densidade populacional dos mosquitos selvagens. Após esses resultados, dobramos o número de indivíduos liberados. Comparamos também a aptidão física (fitness) dos mosquitos wMelBr com a dos nativos. Os mosquitos wMelBr apresentaram tamanho de asas maior, menor mortalidade e uma razão sexual de 1,2: 1 para as fêmeas e, a análise de mtDNA, não mostrou falha na transmissão materna da bactéria. Todas estas características favorecem a invasão da wMelBr. Entretanto, apesar da frequência de wMelBr atingir 65% em campo na semana 20, ela caiu dramaticamente nas quatro semanas seguintes ao fim das liberações. Animado pelo aumento de venda de inseticidas spray, o dono da mercearia local manifestou desejo de que as liberações fossem incessantes, chamando-nos atenção para o possível status da resistência a inseticidas na linhagem liberada

A wMelBr permanecera em endocruzamento por 17 gerações em laboratório e os genótipos de resistência a piretroides (PI) diminuíram de 68% para 3,5%. Ou seja, havíamos liberado mosquitos altamente suscetíveis a PI em uma área cuja população selvagem que era altamente resistente. Após isso, uma segunda linhagem (wMelRio), com alelos de resistência a PI, foi gerada em laboratório e liberada simultaneamente em dois locais no Rio de Janeiro. Os alelos de resistência foram mantidos em wMelRio, em laboratório, por introdução de 50% de machos selvagens a cada duas gerações. Após 18 meses sem liberações adicionais, a frequência da wMelRio em campo era > 90%, em ambos os locais, evidenciando o sucesso em campo. A linhagem wMelRio é resistente a PI e ao larvicida temephos, e suscetível ao malathion e ao diflubenzuron, os compostos químicos atualmente utilizados no controle vetorial. Modelos matemáticos foram aplicados para testar como diferentes estratégias de liberação (variações na suscetibilidade a inseticidas das linhagens, custo de fitness da resistência a inseticidas) aumentam a chance de invasão da Wolbachia. Nossos resultados indicam uma invasão bem sucedida em duas situações: 1) liberando-se mosquitos suscetíveis em um ambiente sem uso de inseticidas (pode causar uma reversão nos níveis de resistência aos inseticidas); e 2) liberando-se mosquitos resistentes (wMelRio) em uma população resistente (como no Rio de Janeiro), mesmo com um elevado uso de inseticidas. Portanto, a implementação da tecnologia de liberação de Ae. aegypti infectado com Wolbachia como potencial estratégia para redução da transmissão de arbovírus deve considerar o perfil de resistência aos inseticidas da população selvagem, e da linhagem liberada, para alcançar uma invasão bem sucedida em campo. (AU)

Using Wolbachia Releases to Estimate Aedes aegypti (Diptera: Culicidae) Population Size and Survival.

Mosquitoes carrying the endosymbiont bacterium Wolbachia have been deployed in field trials as a biological control intervention due to Wolbachia effects on reducing transmission of arboviruses. We performed mark, release and recapture (MRR) experiments using Wolbachia as an internal marker with daily collections with BG-Traps during the first two weeks of releases in Rio de Janeiro, Brazil. The MRR design allowed us to investigate two critical parameters that determine whether Wolbachia would successful invade a field population: the probability of daily survival (PDS) of Wolbachia-infected Aedes aegypti females, and the wild population density during releases. Released females had a PDS of 0.82 and 0.89 in the first and second weeks, respectively, immediately after releases, which is well within the range of previous estimates of survivorship of wild mosquitoes in Rio de Janeiro. Abundance estimation of wild population varied up to 10-fold higher depending on the estimation method used (634-3565 females on the average-difference model to 6365-16188 females according to Lincoln-Petersen). Wolbachia-released mosquitoes were lower than the density estimation of their wild counterparts, irrespectively of the model used. Individually screening mosquitoes for the presence of Wolbachia reduced uncertainty on abundance estimations due to fluctuation in capturing per week. A successful invasion into local population requires Ae. aegypti fitness is unaffected by Wolbachia presence, but also reliable estimates on the population size of wild mosquitoes.