Molecular diversity of genes related to biological rhythms (period and timeless) and insecticide resistance (Na V and ace-1) in Anopheles darlingi

BACKGROUND Malaria is a public health concern in the Amazonian Region, where Anopheles darlingi is the main vector of Plasmodium spp. Several studies hypothesised the existence of cryptic species in An. darlingi, considering variations in behaviour, morphological and genetic aspects. Determining their overall genetic background for vector competence, insecticide resistance, and other elements is essential to better guide strategies for malaria control. OBJECTIVES This study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation in An. darlingi populations from Amazonian localities in Brazil and Pacific Colombian region. METHODS We amplified, cloned and sequenced fragments of genes related to behaviour: timeless (tim) and period (per), and to insecticide resistance: voltage-gated sodium channel (Na V ) and acetylcholinesterase (ace-1) from 516 An. darlingi DNA samples from Manaus, Unini River, Jaú River and Porto Velho - Brazil, and Chocó - Colombia. We discriminated single nucleotide polymorphisms (SNPs), determined haplotypes and evaluate the phylogenetic relationship among the populations. FINDINGS The genes per, tim and ace-1 were more polymorphic than Na V . The classical kdr and ace-1 R mutations were not observed. Phylogenetic analyses suggested a significant differentiation between An. darlingi populations from Brazil and Colombia, except for the Na V gene. There was a geographic differentiation within Brazilian populations considering per and ace-1. CONCLUSIONS Our results add genetic data to the discussion about polymorphisms at population levels in An. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.

Liaisons dangereuses: cross-border gene flow and dispersal of insecticide resistance-associated genes in the mosquito Aedes aegypti from Brazil and French Guiana

BACKGROUND In recent years, South America has suffered the burden of continuous high impact outbreaks of dengue, chikungunya and Zika. Aedes aegypti is the main mosquito vector of these arboviruses and its control is the only solution to reduce transmission. OBJECTIVES In order to improve vector control it is essential to study mosquito population genetics in order to better estimate the population structures and the geneflow among them. METHODS We have analysed microsatellites and knockdown resistance (kdr) mutations from a trans-border region in Amazonia between the state of Amapá (Brazil) and French Guiana (overseas territory of France), to provide further knowledge on these issues. These two countries have followed distinct vector control policies since last century. For population genetic analyses we evaluated variability in 13 well-established microsatellites loci in Ae. aegypti from French Guiana (Saint Georges and Cayenne) and Brazil (Oiapoque and Macapá). The occurrence and frequency of kdr mutations in these same populations were accessed by TaqMan genotype assays for the sites 1016 (Val/Ile) and 1534 (Phe/Cys). FINDINGS We have detected high levels of gene flow between the closest cross-border samples of Saint-Georges and Oiapoque. These results suggest one common origin of re-colonisation for the populations of French Guiana and Oiapoque in Brazil, and a different source for Macapá, more similar to the other northern Brazilian populations. Genotyping of the kdr mutations revealed distinct patterns for Cayenne and Macapá associated with their different insecticide use history, and an admixture zone between these two patterns in Saint Georges and Oiapoque, in accordance with population genetic results. MAIN CONCLUSIONS The present study highlights the need for regional-local vector surveillance and transnational collaboration between neighboring countries to assess the impact of implemented vector control strategies, promote timely actions and develop preparedness plans.

Evaluation of insecticide resistance in Aedes aegypti populations connected by roads and rivers: the case of Tocantins state in Brazil

BACKGROUND The longstanding application of insecticides for vector control without periodic monitoring of the population response to these chemicals can directly drive the selection of resistant populations of vector mosquitoes. Tocantins is the newest state of the Brazilian Federation. Despite a historically high number of dengue cases, studies and monitoring data concerning insecticide resistance in the state are lacking. OBJECTIVES To verify the resistance status of Aedes aegypti from 10 populations distributed throughout the state connected by rivers and roads. METHODS Between 50 and 150 ovitraps were installed in house gardens within each municipality. Collection points were established based on the importance of the towns and on geographic aspects. Dose response bioassays were performed in accordance with World Health Organization guidelines. Molecular assays were conducted to detect kdr mutations, which are related to pyrethroid resistance. FINDINGS Of the 3,200 ovitrap paddles analysed, 25.8% contained eggs, with a total of 55,687 eggs collected. With the exception of Caseara, all evaluated populations were considered to be resistant to temephos. The data showed different levels of resistance to deltamethrin among the samples. Caseara and Guaraí showed the lowest RR95 values. On average, the NaVR1 kdr allele was most frequent (40.3%), followed by NaVS (38.1%), and NaVR2 (21.6%). Palmas, the capital of the state, had the highest frequency of kdr alleles (87.5%). MAIN CONCLUSIONS With the exception of Palmas, the towns with the highest indexes (ovitrap positivity, number and density of eggs), as well with high levels of resistance and kdr alleles were located along the BR-153 road, indicating that the flow of people and cargo can contribute to the dispersion of the vector and potentially resistance. This study contributes substantially to knowledge regarding the insecticide resistance profile of Tocantins mosquito populations; the data generated via the study could facilitate the judicious use of insecticides by vector control programs.

Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos

In Brazil, decades of dengue vector control using organophosphates and pyrethroids have led to dissemination of resistance. Although these insecticides have been employed for decades against Aedes aegypti in the country, knowledge of the impact of temephos resistance on vector viability is limited. We evaluated several fitness parameters in two Brazilian Ae. aegypti populations, both classified as deltamethrin resistant but with distinct resistant ratios (RR) for temephos. The insecticide-susceptible Rockefeller strain was used as an experimental control. The population presenting the higher temephos resistance level, Aparecida de Goiânia, state of Goiás (RR95 of 19.2), exhibited deficiency in the following four parameters: blood meal acceptance, amount of ingested blood, number of eggs and frequency of inseminated females. Mosquitoes from Boa Vista, state of Roraima, the population with lower temephos resistance level (RR95 of 7.4), presented impairment in only two parameters, blood meal acceptance and frequency of inseminated females. These results indicate that the overall fitness handicap was proportional to temephos resistance levels. However, it is unlikely that these disabilities can be attributed solely to temephos resistance, since both populations are also resistant to deltamethrin and harbour the kdr allele, which indicates resistance to pyrethroids. The effects of reduced fitness in resistant populations are discussed.

Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures

Despite its vector importance little attention is given to Aedes aegypti embryonic development. In this study, temperature influence on time course of Ae. aegypti larvae hatching and egg viability were evaluated. The dormancy state at the end of embryogenesis could be interrupted with a proper stimulus. Temperatures tested ranged between 12-36°C; the maximum temperature limit is 35°C and the minimum one is below 12°C. Egg viability between 16-31°C was above 80 percent. The definition of physiological embryonic parameters at this temperature range corroborates Ae. aegypti presence on tropical and subtropical world regions.

Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti

The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0 percent males) compared to the controls (50.2 percent males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2 percent of males and 13.8 percent of females were alive, in contrast with 94 percent survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4 percent and 48.4 percent of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30 percent less blood and laid 25 percent fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.