insecticideresistancein mosquitoes . InMartinique island, temephos (AbateH) was used for decades for larval control (abandoned in 2009 to respect the recent European biocide legislation; European Commission Environment Biocidal Products, 1998) and now Bacillus thuringiensis var israelensis (Bti, Vectobac H) is the only insecticide used for such application. Space spraying treatments with vehicle-mounted or portable thermal fogger (aerial or inside application, respectively) are implemented during inter-epidemic periods (i.e. when high entomological indices are reported) and during outbreaks to rapidly kill infected adult mosquitoes . DDT and several OPs (e.g., malathion, fenitrothion) were used since the 1950s but there was a switch to PYRs inthe early 1990s  because of their rapid knockdown action and low mammalian toxicity (WHO, 2006b). Currently, deltamethrin (K-Othrine 15/5H) and to a lesser extend synergized natural pyrethrins (AquaPyH) are used for the control of adult mosquitoes. In addition, Martinique is an island with intensive agriculture practice (mainly sugar cane and bananas) where huge amount of pesticides have been applied for crop protection. Pesticides used in agriculture include organochlorines (OCs), OPs and carbamates. Recently, Bocquene´ and Franco  reported the widespread contamination of rivers and soils inMartiniquewith pesticides and particularly with high levels of the OCs chlordecone and lindane. The constant exposure of the mosquito populations to these pesticides associated withthe increasing urbanization may have led to the selection of particular detoxification genes and to an increased tolerance to pesticides [13,14,15,16].
Madeira is a famous touristic destination, mostly for Europeans, with daily flights from/to several European countries and regular stops of cruise ships 12,14 . This scenario increases the risk of exportation of both Ae. aegyptiand viremic individuals to Europe 12 . In fact, thedengue epidemic in Madeira was responsible for 78 imported cases that were notified in 13 different countries, the majority corresponding to tourists that had travelled to the island during the outbreak 15 . To mitigate the risk of importation/exportation of virus and vectors, local health authorities perform vector control activities at the International Airport and at the Funchal harbour, and coordinate an island-wide integrated Madeira Dengue Surveillance System (MDSS), responsible for the detection of imported cases. Despite its insular condition, Madeira has a considerable risk of importing exotic vectors and pathogens from tropical regions. This is mainly due to the strong socio-economic relations that the island maintains with South American countries, such as Brazil and Venezuela 14 . Coincidently, phylogenetic analysis and an importation index based on the air-travel interconnectivity withdengue-endemic countries revealed Venezuela as the most likely country of origin for the circulating DENV-1 inthe island 16 . In addition, previous studies showed that Ae. aegyptifrom Madeira is able to transmit dengue, chikungunya and Zika viruses 17,18 , pinpointing the potential risk of local arbovirus transmission.
In addition to detoxification gene families, eight transcripts encoding putative cuticular proteins were up-regulated possibly indicating alteration of the cuticle as a mechanism of resis- tance. Resistance related to the cuticle by lowering the amount or rate of insecticide penetrat- ing into the body has been reported in insects such as Helicoverpa armigera , Drosophila melanogaster , the Trypanosoma cruzi vector Triatoma infestans , andthe Plasmodium vector Anopheles funestus . Thickening of the whole cuticle, as well as the epicuticle layer, due to an increased number of cuticular hydrocarbons, has also been recently described in a multi-resistant strain of the malaria vector Anopheles gambiae . Furthermore, there were also three transcripts (one marginally non-significant) encoding hexamerins among the most highly overexpressed genes. The link of hexamerins to insecticideresistance is poorly under- stood, but previous studies suggest a role for these storage proteins in cuticle formation [45,46]. It is also plausible that composition and thickening of the cuticle might reflect adaptive responses to environmental challenges and/or seasonality, rather than insecticideresistance. Seasonal cuticular variations, mostly associated with adaptation to aridity (desiccation toler- ance), have been previously observed in other insect species, including scorpions , crickets  and more recently inthe malaria vector A. coluzzii . There is evidence that Ae. aegypti of Madeira derives from a tropical south American source population , thus, adaptation of this mosquito to the more temperate climate of Madeira might have involved changes in cuti- cle composition and thickening.
ber 2010. This situation reflects a worldwide problem of increasing insecticideresistance, while the number of chemicals available for public health use is continually reduced. Environmentaland toxic issues, difficulties in developing new, efficient compounds and registration constraints are the main causes of the reduced avail- ability of these chemicals. Therefore, it is fundamental to improve the management of resistancein particular by monitoring its level on a regular basis, identifying resistancemechanismsand developing targeting strate- gies. Multiple insecticideresistancein Ae. aegypti is not an isolated phenomenon; it has already been reported in latin America (Rodríguez et al. 2002, 2007) andthe French West Indies (Marcombe et al. 2009a). Studies from these regions demonstrated that multiple insecti- cide resistance may be due to the combination the over- expression of genes - such as mixed-function oxidases - involved in metabolic resistance (Flores et al. 2006, Strode et al. 2008, Marcombe et al. 2009b) and target site mutations on the sodium channel gene. Such mutations are extensively described in insects and are known to confer resistance to pyrethroids and DDT called knock- down resistance (kdr). Our results are congruent with these observations. Indeed, the implication in deltam- ethrin resistance of mixed-function oxidases, and to a lesser extent carboxylesterases, cannot fully explain the level of resistance we observed during the tube and field tests. In 2003, Brengues et al. (2003) found mutations inthe Domain II segment 6 of the sodium channel gene in a population from CAY, but no further studies have been conducted. Further investigation of both target site mutations and metabolic resistance of adult organisms must be implemented to better understand the resist- ance mechanisms involved and their implications for the choice of insecticides inthe future.
The archipelago of Cabo Verde is located on the west coast of Africa, and is composed of 10 islands clustered in two groups: the Barlavento group (comprising the islands of Santo Antão, São Vicente, Santa Luzia, São Nicolau, Sal, and Boavista) andthe Sotavento group (comprising the islands of Maio, Santiago, Fogo, and Brava). However, each island has specific topography and displays differences in microclimate and vegetation. Historically, the topography and geographical location of Cabo Verde has promoted and allowed for the active movement of population and goods . This increases the risk of pathogen circulation through infected travelers, which can cause the emergence or re-emergence of arboviral diseases if competent mosquito vectors are present andvector capacity is high . In 2015, Cabo Verde had a passenger volume of more than 7000 travelers from Zika-affected countries, including direct flights from Brazil . The modification of the environment by anthropic actions, disordered urban planning, population growth, and emergent factors related to the globalization process and climate change affect the bionomics of mosquito vectors, increasing their vectorial capacity [16–18].
The fight against theAedesaegypti mosquito vector is the only link that is susceptible to intervention for dengue control, since there is still no vaccine or specific treatment for the disease. Due to socio-environmental conditions andvector control inefficiency in many Brazilian cities, the endemic/epidemic process has been modulated by the exhaustion of susceptible individuals inthe population at risk, rather than by vector control itself.
Aedesaegypti is considered the main vector of arboviruses affecting humans. Nowadays, the only feasible measure to eradicate thedengue fever depends exclusively of vector control. The deep knowledge about the structure and dynamics of Ae. aegypti population in distinct environments is critical, since genetically different populations may present differences related to vector competence and capacity. Brazil has regions with different climatic and geographic characteristics, therefore, detailed knowledge about mosquito population that colonizes different habitats is extremely important. The present study evaluated the population structure of Ae. aegypti mosquitoes in five different Brazilian cities (i.e., Belo Horizonte, Botucatu, Campo Grande, Maringá and Rondonópolis) using a microsatellite markers. Those markers were also used to evaluate the oviposition dynamic andthe consequences of Ae. aegypti mosquitoes dispersion, of the city of Botucatu. Population macro-analysis, using DAPC evidenced, genetic clusters among individuals of the same locality, and population structure in mosquitoes of Belo Horizonte. Population microstructure analysis identified six sub-populations of Ae. aegypti mosquitoes among six sub-regions of Belo Horizonte. In addition, the microstructure analysis suggested intermediate population structure inthe sub-regions of Campo Grande, and lack of genetic structure among mosquitoes fromthe sub-regions of Botucatu. Therefore, these results indicate that genetic organization occurs in correlation with city size – where large towns seem to provide genetic structure to the populations of Ae. aegypti. PCA analysis of ovitraps obtained in Botucatu indicated population organization of Ae. aegypti mosquitoes in families. Pedigree analysis and inbreeding coefficient indicated that only for out of 30 mosquitoes analyzed inthe same ovitrap, keep restricted familial relationships. These results suggest that Ae. aegypti females mates with mosquitoes geographically close, and scatter their eggs in different breeding sites, supporting the maintenance and dissemination of mosquitoes and, consequently the etiologic agents conveyed by them. The results of the present study assist inthe understanding of Ae. aegipty mosquitoes dispersion. Also our study may facilitate the development of strategies for mosquito incidence reduction anddengue transmission reduction, helping solve the disease outbreaks that affects Brazil.
Após a realização dos trabalhos de controle visando à interrupção da transmissão do vírus do dengue, iniciou-se um trabalho de monitorização de Aedesaegypti e Aedes albopictus com dois métodos de vigilância entomológica: Índice de Breteau (IB) e ovitrampas. Pretendeu-se avaliar o tempo necessário para que as espécies envolvidas fossem novamente detectadas na área urbana do município de Catanduva, SP. As ovitrampas apresentaram positividade para Aedesaegypti dois meses após os trabalhos de controle, enquanto o Índice de Breteau veio a positivar-se somente no quarto mês após o término dos referidos trabalhos.
capture experiment in a highly endemic area of denguewith female mosquitoes with amputated proboscis to avoid ethical issues and they also eliminated or removed most small containers fromthe study area. These in- terventions may have infl uenced the dispersal pattern of Ae. aegypti females, which displaced at least 800 m from their release point. Maciel-de-Freitas et al 13
Chemical control of A. aegypti is a challenging issue anddengue outbreaks have repeatedly occurred in Brazil over the last 10 years since this vector species is resistant to insecticides such as organochlorines, organophosphates and onset of resistance to pyrethroids (Tauil, 2006). Besides toxicity, resistance reported in several areas under continuous application of insecticides is also an important issue and alternatives to conventional chemical control based on natural products from plants, including essential oils, stands as a potential source for new molecules for mosquito control. The genus Piper stands out as a source of active principles derived from plants has been described as antimicrobial and insecticidal (Bergo, et al. 2005).
Inhibition of DENV might be caused by the activation of certain host defense responses as a result of Wolbachia infection. Virus-inhibitory effects have been observed in human infected with a close relative of Wolbachia, Orientia tsutsugamushi; these effects appear to be caused by binding to the virus of antibodies against bacteria . In arthropods, innate immunity plays an important role in limiting pathogen infection. Such immune responses are largely regulated by two main pathways, the Toll and Imd pathways [17,18]. In Drosophila, the Toll pathway is mainly involved in defense against fungi and Gram-positive bacteria, while the Imd pathway affects resistance to Gram-negative bacteria. In response to either the Drosophila X virus (a member of the Birnaviridae) or E. coli infection, D. melanogaster induces the same antimicrobial peptide genes. This commonality suggests that these two diverse classes of pathogen can activate the same immune response pathway inthe insect host . More importantly, we have recently found that activation of the mosquito Toll pathway can suppress DENV infection . A recent genome-wide analysis of the Wolbachia-host interaction has revealed that Wolbachia infection has an impact on a broad range of physiological systems inthe host, including innate immunity .Inthe present study, we also observed an elevated mosquito immune response inthe WB1 strain. Thus, the observed inhibition
All told, we observed higher rates of gene flow for populations of the Brazilian Amazon when compared to the two previous studies undertaken in Brazil. Ayres et al. (2003) analyzed the Ae. aegypti populations of five Brazil- ian states with RAPD markers, and found high levels of ge- netic differentiation and reduced gene flow at both the macro-geographic (Nm = 0.54) and micro-geographic (Nm = 0.69) levels. Paduan et al. (2006) also used RAPD mark- ers in populations from six other Brazilian states and ob- served reduced gene flow among all of these (Nm = 0.65), even though fromthe same state (Nm = 0.83). Compared to the present study, these results suggest that levels of genetic variability and differentiation among Brazilian Ae. aegypti populations are relatively high. Nevertheless, most of the populations of the Brazilian Amazon analyzed here mani- fested higher gene flow, probably due to human migration and river or land trading movement, thereby favoring the dispersal of this vectorinthe region. Alternatively, this could indicate the recent expansion of Ae. aegypti through- out this area. The absence of isolation by distance, when the samples from Pacaraima and Rio Branco were removed fromthe analyses, supports these hypotheses. Identical re- sults were found in samples from Mexico (Gorrochotegui- Escalante et al., 2002), Venezuela (Herrera et al., 2006) and Brazil (Scarpassa et al., 2008). Furthermore, the active dis- persal patterns (flight) of Ae. aegypti have been estimated as being between 100 and 800 m (Ordonez-Gonzalez et al., 2001; Honorio et al., 2003), this dispersal mechanism obvi- ously not being a possible explanation for our results.
Lastly, in nearly all cases, Wolbachia densities declined during pathogen infection. This may be the direct result of innate immune effectors elicited by the pathogens in addition to those elicited by Wolbachia. Fold reductions in Wolbachia numbers are in keeping with those of the intracellular pathogens that would be exposed to the same aspects of the immune response. A related study inthe mosquito A. albopictus naturally infected with Wolbachia also reported reductions in symbiont density after co-infection withthe vectored virus Chikungunya . Alternatively, Wolbachia reductions may spring from indirect effects of innate immune priming. Mounting an immune response withthe production of AMPs and prophenoloxidases is costly . Infection by intracellular pathogens also carries with it the added cost of direct competition for resources within cells. Wolbachia is highly dependent on its host for nutrition and replication  and as such co-infection with pathogens may cause Wolbachia replication to slow due to resource limitation. Because change in Wolbachia numbers was measured over short time periods (8–26 hrs) and because estimates of Wolbachia’s dividing time are long (,14 hours) , however, our data are more likely to provide support for control of densities by the direct effect of the immune response on Wolbachia.
A striking finding that needs to be discussed here in is the reason why the DENV3 was the single serotype detected in our samples. We suppose that it is possible that DENV1 and DENV2 serotypes, that were responsible for the previous dengue epidemic episodes in Manaus, were displaced by DENV3, the last serotype introduced in Manaus. It is likely that part of the population had already been infected by the serotypes DENV1 and DENV2. However, the introduction of DENV3, found the whole population to be susceptible to it, which perhaps contributed to dispersion, thus becoming easier to be detected and isolated. In fact, earlier experiments showed that when a new dengue serotype is introduced into a community, occurs a natural decrease of circulation of other previously existing dengue serotypes (WHO 1997, Rigau-Pérez et al. 2002, Urdaneta et al. 2005). Moreover, the official data of the Ministry of Health, confirmed these findings, since only DENV3 isolations were officially re- corded in 2003 (MS 2003).
infested by thevector take the timely measures to overcome any administrative difficulties that may be hampering the progress of their campaigns, and that they give the highest priority to the provision of the funds, personnel, and supplies needed to complete those campaigns as soon as possible.
Roraima is the northernmost state of Brazil, bordering both Venezuela and Guyana. Appropriate climate andvector conditions for dengue transmission together with its proximity to countries where all four dengue serotypes circulate make this state, particularly the capital Boa Vista, strategically important for dengue surveillance in Brazil. None- theless, few studies have addressed the population dynamics of Aedesaegyptiin Boa Vista. In this study, we report temporal and spatial variations in Ae. aegypti population density using ovitraps in two highly populated neighbour- hoods; Centro and Tancredo Neves. In three out of six surveys, Ae. aegypti was present in more than 80% of the sites visited. High presence levels of this mosquito suggest ubiquitous human exposure to thevector, at least during part of the year. The highest infestation rates occurred during the peak of the rainy seasons, but a large presence was also observed during the early dry season (although with more variation among years). Spatial distribution of positive houses changed from a sparse and local pattern to a very dense pattern during the dry-wet season transi- tion. These results suggest that the risk of dengue transmission andthe potential for the new serotype invasions are high for most of the year.
between the two at the stage of the larva in order to direct eradication efforts. This work describes software developed to this end. It classifies a digitized image of the larva of Aedes mosquitoes into aegypti or albopictus species using a deterministic rule based on morphological characteristics of the larvae 2 .
Habitat characteristics - Ae. aegypti habitat suitability was assessed at different spatial scales; i.e. within a city, among microenvironments andin regard to types of con- tainer used as breeding sites. Ovitrap surveys conducted throughout Buenos Aires city suggested that downtown neighborhoods close to the river have lower levels of infestation than the rest of the city (Schweigmann et al. 2002, Carbajo et al. 2004). A posterior study involving spatial analysis found strong evidence that in Buenos Aires city mosquito proliferation is highest in medium urbanization levels and minimum in densely built areas (Carbajo et al. 2006). In this study, the variables positive- ly related to oviposition activity were house density and proximity to industries, flat density was negatively as- sociated, and vegetation cover was between the variables not significantly associated. At a more detailed scale, among cemeteries within the same city, vegetation cover was strongly correlated with Ae. aegypti infestation (Vez- zani et al. 2001) and at a microhabitat scale the presence of immatures was enhanced in sites less exposed to sun- light, with taller and closer vegetation, andwith shaded and vegetated surroundings (Vezzani et al. 2005). On the contrary, Almirón et al. (1999) found no difference inthe number of eggs collected in ovitraps located under dif- ferent conditions of light exposition in one premise of Córdoba city. Field experiments suggested that oviposi- tion response depends stronger on container color than on water surface area, andthe response to color would depend on the microenvironment lighting condition (Ba- dano & Regidor 2002). Several field studies identified Ae. aegypti habitat types (Schweigmann et al. 1997, Borda et al. 1999, Stein et al. 2002, Stein & Oria 2002, Zapata et al. 2002, Almirón & Asis 2003) but in general gave no information about availability of each container type or productivity, hampering key containers identification (i.e., those of higher productivity). Key containers identi- fied for Ae. aegypti control were tires and wide mouth containers in CH (Stein et al. 2002, Stein & Oria 2002). Studies restricted to cemeteries recorded mosquito imma- tures in flower vases made of plastic, metal, ceramic and glass (García et al. 2002) andthe use of each type of con- tainer varied according to sun exposure; in shaded areas it was proportional to the availability, andin sun exposed areas plastic and metal containers were the most and less frequent habitats, respectively (Vezzani & Schweigmann 2002). Also in cemeteries, Vezzani et al. (2004b) reported that Ae. aegypti productivity was higher in containers of 1-5 l than in those up to 1 l.