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. Region spp An aspects competence resistance region amplified (tim , (per) voltagegated voltage gated ace1 ace 1 (ace-1 51 Manaus Colombia SNPs, SNPs (SNPs) ace- observed gene ace1. 1. resistancerelated scenario (per (ace- 5 (SNPs (ace

BACKGROUND Aedes aegypti is the primary transmitter of several arbovirus with great impact in human health. Controlling vector mosquitoes is an essential and complex task. One promising control method is to use mosquitoes as a vehicle to disseminate tiny particles of juvenile-killing insecticides, such as pyriproxyfen (PPF), to breeding sites. OBJECTIVES We aimed to investigate the capacity of Ae. aegypti to disseminate two new formulations of PPF in two sites of Rio de Janeiro city for assessment of the efficacy of these products. METHODS Dissemination stations impregnated with powder and liquid new formulations of PPF were installed in two test sites. Ovitraps were used in the test sites and in a control site for monitoring the presence of Ae. aegypti throughout eggs collection. FINDINGS Entomological indices indicated that the new formulations of PPF were efficient in reducing eggs abundance. Liquid formulation performed better than powder formulation. Ready-to-use formulations of PPF can be quickly applied in the field and can be replaced after a few months. MAIN CONCLUSIONS New formulations of PPF associated with mosquito dissemination approach make a valuable vector control strategy, managing to cover places of difficult access for whatever reason. New formulations application requires less labour, being economically attractive.

Abstract In Brazil, malaria is an important public health problem first reported in 1560. Historically, fluctuations in malaria cases in Brazil are attributed to waves of economic development; construction of railroads, highways, and hydroelectric dams; and population displacement and land occupation policies. Vector control measures have been widely used with an important role in reducing malaria cases. In this review article, we reviewed the vector control measures established in the Brazilian territory and aspects associated with such measures for malaria. Although some vector control measures are routinely used in Brazil, many entomological and effectiveness information still need better evidence in endemic areas where Plasmodium vivax predominates. Herein, we outlined some of the needs and priorities for future research: a) update of the cartography of malaria vectors in Brazil, adding molecular techniques for the correct identification of species and complexes of species; b) evaluation of vector competence of anophelines in Brazil; c) strengthening of local entomology teams to perform vector control measures and interpret results; d) evaluation of vector control measures, especially use of insecticide-treated nets and long-lasting insecticidal nets, estimating their effectiveness, cost-benefit, and population acceptance; e) establishment of colonies of malaria vectors in Brazil, i.e., Anopheles darlingi, to understand parasite-vector interactions better; f) study of new vector control strategies with impacts on non-endophilic vectors; g) estimation of the impact of insecticide resistance in different geographical areas; and h) identification of the relative contribution of natural and artificial breeding sites in different epidemiological contexts for transmission.

BACKGROUND Aedes aegypti populations in Brazil have been subjected to insecticide selection pressures with variable levels and sources since 1967. Therefore, the Brazilian Ministry of Health (MoH) coordinated the activities of an Ae. aegypti insecticide resistance monitoring network (MoReNAa) from 1999 to 2012. OBJECTIVES The objective of this study was to consolidate all information available from between 1985 and 2017 regarding the resistance status and mechanisms of Brazilian Ae. aegypti populations against the main insecticide compounds used at the national level, including the larvicide temephos (an organophosphate) and the adulticide deltamethrin (a pyrethroid). METHODS Data were gathered from two sources: a bibliographic review of studies published from 1985 to 2017, and unpublished data produced by our team within the MoReNAa between 1998 and 2012. A total of 146 municipalities were included, many of which were evaluated several times, totalling 457 evaluations for temephos and 274 for deltamethrin. Insecticide resistance data from the five Brazilian regions were examined separately using annual records of both the MoH supply of insecticides to each state and the dengue incidence in each evaluated municipality. FINDINGS Ae. aegypti resistance to temephos and deltamethrin, the main larvicide and adulticide, respectively, employed against mosquitoes in Brazil for a long time, was found to be widespread in the country, although with some regional variations. Comparisons between metabolic and target-site resistance mechanisms showed that one or another of these was the main component of pesticide resistance in each studied population. MAIN CONCLUSIONS (i) A robust dataset on the assessments of the insecticide resistance of Brazilian Ae. aegypti populations performed since 1985 was made available through our study. (ii) Our findings call into question the efficacy of chemical control as the sole methodology of vector control. (iii) It is necessary to ensure that sustainable insecticide resistance monitoring is maintained as a key component of integrated vector management. (iv) Consideration of additional parameters, beyond the supply of insecticides distributed by the MoH or the diverse local dynamics of dengue incidence, is necessary to find consistent correlations with heterogeneous vector resistance profiles.

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.

ABSTRACT Culicidae colonization in laboratory is paramount to conduct studies aiming at a better understanding of mosquitoes' capacity to transmit pathogens that cause deadly diseases. Colonization requires female blood feeding, a necessary step for maturation of female's oocytes. Direct blood feeding on anesthetized mammals implies in a number of disadvantages when compared to artificial blood feeding. Consequently, laboratories worldwide have been trying to -feed female mosquitoes artificially in order to replace direct feeding. In this study, we compared the effects of direct blood feeding and artificial blood feeding on important life traits of three Culicidae species. Artificial feeding was performed using citrated or defibrinated sheep blood and citrated or defibrinated rabbit blood. Direct feeding was performed using anesthetized guinea pigs as the blood source and the experiment control. Results indicated that artificial feeding using sheep blood was not good enough to justify its use in the maintenance of laboratory colonies of Culicidae. However, artificial feeding using rabbit blood maintained a recovery rate always very close to the control, especially when blood was citrated. We concluded that artificial feeding using citrated rabbit blood can substitute direct feeding on mammals reducing the use of animals, eliminating the need to maintain a bioterium in the laboratory and reducing costs in scientific researches involving Culicidae vectors.

ABSTRACTINTRODUCTION:The mosquito Aedes aegypti has evolved resistance to pyrethroid insecticides. The present study evaluated Ae. aegypti from Goiânia for the resistant phenotype and for mutations associated with resistance.METHODS:Insecticide dose-response bioassays were conducted on mosquitoes descended from field-collected eggs, and polymerase chain reaction (PCR) was used to genotype 90 individuals at sites implicated in pyrethroid resistance.RESULTS:All mosquito populations displayed high levels of resistance to deltamethrin, as well as high frequencies of the 1016Ile kdr and 1534Cys kdrmutations.CONCLUSIONS:Aedes aegypti populations in the Goiânia area are highly resistant to deltamethrin, presumably due to high frequencies of kdr(knockdown-resistance) mutations.

Currently, sticky traps are regularly employed to assist in the surveillance of Aedes aegypti infestation. We tested two alternative procedures for specimen identification performed by local health agents: directly in the field, as recommended by certain manufacturers, or after transportation to the laboratory. A total of 384 sticky traps (MosquiTRAP) were monitored monthly during one year in four geographically representative Brazilian municipalities. When the same samples were inspected in the field and in the laboratory, large differences were noted in the total number of mosquitoes recorded and in the number of specimens identified as Ae. aegypti by both procedures. Although field identification has the potential to speed vector surveillance, these results point to uncertainties in the evaluated protocol.

Distribution, abundance, feeding behaviour, host preference, parity status and human-biting and infection rates are among the medical entomological parameters evaluated when determining the vector capacity of mosquito species. To evaluate these parameters, mosquitoes must be collected using an appropriate method. Malaria is primarily transmitted by anthropophilic and synanthropic anophelines. Thus, collection methods must result in the identification of the anthropophilic species and efficiently evaluate the parameters involved in malaria transmission dynamics. Consequently, human landing catches would be the most appropriate method if not for their inherent risk. The choice of alternative anopheline collection methods, such as traps, must consider their effectiveness in reproducing the efficiency of human attraction. Collection methods lure mosquitoes by using a mixture of olfactory, visual and thermal cues. Here, we reviewed, classified and compared the efficiency of anopheline collection methods, with an emphasis on Neotropical anthropophilic species, especially Anopheles darlingi, in distinct malaria epidemiological conditions in Brazil.

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides.

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% males) compared to the controls (50.2% males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2% of males and 13.8% of females were alive, in contrast with 94% survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4% and 48.4% of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30% less blood and laid 25% fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.

RESUMO Resistência do vetor de dengue, Aedes aegypti, a temephos estimulou sua substituição por Bacillus thuringiensis var. israelensis (Bti) desde 2001 no Brasil. A persistência de duas formulações de Bti empregadas naquele ano pelo Ministério da Saúde, Vectobac G e Aquabac G, foi testada em condições externas e de laboratório. Ambas formulações foram testadas a 0,2 g/10 litros de água, a mesma concentração recomendada para o controle do vetor no campo. Os testes foram realizados com larvas de Ae. aegypti de terceiro estádio (linhagem Rockefeller). No laboratório, Vectobac G e Aquabac G induziram pelo menos 95% de mortalidade até 101 e 45 dias depois do tratamento, respectivamente. Nos testes externos, recipientes de diferentes materiais foram tratados com cada formulação e colocados em local coberto. Larvas foram introduzidas a cada três a seis dias e a mortalidade foi observada após 24 e 48 horas. Na primeira série de ensaios (junho 2001) mortalidade de 70% ou mais foi alcançada por duas a cinco semanas em todos os recipientes. A exceção foi o recipiente de metal que oxidou, resultando em baixos níveis de mortalidade após sete dias. Na segunda série de ensaios (agosto 2001), 70% de mortalidade foi obtida por apenas uma a duas semanas para todos os recipientes e para ambas formulações.

Resistance of the dengue vector to temephos stimulated its substitution for Bacillus thuringiensis var. israelensis (Bti) since 2001 in Brazil. The persistence of the two Bti formulations employed at that time by the Health Ministry, Vectobac G and Aquabac G, was assayed under laboratory and outdoor conditions. Both formulations were tested at 0.2 g/10 liters of water, the same concentration applied in the field for vector control. The tests were done against Ae. aegypti third instar larvae (Rockefeller strain). In the laboratory, Vectobac G and Aquabac G caused at least 95% mortality until 101 and 45 days after treatment, respectively. In the outdoor assays, test containers of different materials were treated with either formulation and placed in a shaded area. Larvae were introduced each 3-6 days and mortality was recorded 24 and 48 hours later. In the first set of assays, performed in June 2001, mortality levels of 70% or more were attained for 2-5 weeks for both formulations in all containers. The exception was for the iron one that rusted, resulting in low mortality after seven days. In the second set of assays (August 2001), 70% mortality was attained for just 1-2 weeks for all the containers and both formulations.

A persistência de Bacillus thuringiensis var. israelensis (Vectobac WDG) e de Metoprene (Metoprag S-2G) contra larvas de terceiro estadio de Aedes aegypti (linhagem Rockefeller) foi avaliada em ensaios simulados de campo. Os testes foram realizados no Rio de Janeiro, em recipientes domésticos para estoque de água de plástico, ferro, cimento ou amianto, instalados em área sombreada. As formulações foram usadas nas concentrações de 0.2g / 100 l (Vectobac-WDG) e 1g / 100 l (Metoprag S-2G). Vectobac WDG foi submetido a dois testes, em março e abril/maio, 2002. Em março (temperaturas entre 21.5 e 39.3 ºC), 70-100% de mortalidade foi observada no sétimo dia, declinando posteriormente. Não houve diferença significativa entre os recipientes. Em abril / maio (18.6 a 34.8 ºC) a mortalidade foi superior a 70% até 30-36 dias em todos os casos, exceto no recipiente de ferro (40% de mortalidade no 12° dia). Metoprag S-2G, avaliado em abril / maio, 2002, induziu mortalidade acima de 70% durante 15 dias nos recipientes de plástico e de ferro e por apenas sete dias naquele de cimento. No recipiente de amianto, nunca se atingiu 70% de mortalidade. Estes resultados apontam para uma baixa persistência de ambas formulações nas condições climáticas do Rio de Janeiro.

Persistence of Bacillus thuringiensis var. israelensis (Vectobac WDG) and methoprene (Metoprag S-2G) was evaluated against Aedes aegypti late third instar larvae of the Rockefeller strain in a semi-field bioassay. Tests were performed in Rio de Janeiro, using containers made of plastic, iron, concrete and asbestos, placed in a shaded area. The formulations used were 0.2 g of Vectobac-WDG and 1g of Metoprag S-2G per 100 liters of water in house storage containers. Vectobac WDG was tested twice, in March and in April/May, 2002. In March (temperature ranging from 21.5 to 39.3 ºC), 70-100% mortality was observed by the 7th day and declined abruptly thereafter. No significant differences were observed among the container types. In April/May (18.6 to 34.8 ºC) mortality was higher than 70% to 30-36 days in all cases, except in the iron container (40% mortality on the 12th day). Metoprag S-2G was evaluated in April/May, 2002, and induced mortality higher than 70% up to 15 days in the plastic and iron containers and only seven days in the concrete container. In the asbestos container, maximal mortality was achieved on day one post-treatment (66%). Our results point to a low persistence of both formulations in the weather conditions of Rio de Janeiro.

For more than 30 years temephos, an organophosphate insecticide, has been the sole larvicide used in Brazil in the control of Aedes aegypti. Organophosphates were also used for adult control, being replaced by pyrethroids since l999. In this same year, the Brazilian Health Foundation started the coordination of the Ae. aegypti Insecticide Resistance Monitoring Program. In the context of this program, our group was responsible for the detection of temephos resistance in a total of 12 municipalities in the states of Rio de Janeiro (RJ), Alagoas (AL), and Sergipe (SE) during 2001. In each municipality, a pool of mosquitoes collected from different districts was used, with the exception of Rio de Janeiro city, where eight districts have been separately evaluated. Exposure of larvae to the diagnostic dose of temephos revealed resistance in all localities examined, with mortality levels ranging from 4% (Pilares district, Rio de Janeiro, RJ) to 61.9% (Campos dos Goytacazes, RJ). Quantification of mortality showed resistance ratios from 6.1 (Aracaju, SE) to 16.8 (São Gonçalo, RJ and Penha district, Rio de Janeiro, RJ). The national dengue control program is presently using these data to subside insecticide resistance management.