Identification of a major Quantitative Trait Locus determining resistance to the organophosphate temephos in the dengue vector mosquito Aedes aegypti.

Organophosphate insecticides (OP) have extensively been used to control mosquitoes, such as the vector Aedes aegypti. Unfortunately, OP resistance has hampered control programs worldwide. We used Quantitative Trait Locus (QTL) mapping to evaluate temephos resistance in two F1 intercross populations derived from crosses between a resistant Ae. aegypti strain (RecR) and two susceptible strains (MoyoD and Red). A single major effect QTL was identified on chromosome 2 of both segregating populations, named rtt1 (resistance to temephos 1). Bioinformatics analyses identified a cluster of carboxylesterase genes (CCE) within the rtt1 interval. qRT-PCR demonstrated that different CCEs were up-regulated in F2 resistant individuals from both crosses. However, none exceeded the 2-fold expression. Primary mechanisms for temephos resistance may vary between Ae. aegypti populations, yet also appear to support previous findings suggesting that multiple linked esterase genes may contribute to temephos resistance in the RecR strain as well as other populations.

Circulation of DENV2 and DENV4 in Aedes aegypti (Diptera: Culicidae) mosquitoes from Praia, Santiago Island, Cabo Verde.

Arthropod-borne viruses, such as Dengue (DENV), Chikungunya (CHIKV), and Zika (ZIKV), pose a challenge to public health, due to their worldwide distribution and large-scale outbreaks. Dengue fever is currently one of the most important diseases and it is caused by four serotypes of DENV and is mainly transmitted by the mosquito Aedes aegypti. It is estimated that 50-100 million cases are reported every year worldwide. More recently, CHIKV and ZIKV, which are also transmitted by Ae. aegypti, have caused epidemics in countries in the Caribbean region, the Pacific region, and Americas. Cabo Verde faced its first dengue outbreak in 2009, with more than 21,000 reported cases and four registered deaths. The epidemic was caused by DENV-3 transmitted by Ae. aegypti mosquitoes. In addition, the country faced a Zika outbreak with more than 7,500 notified cases from October 2015 to May 2016. In the present study, we conducted a survey in mosquito samples to detect arboviruses circulating in the local vector population. Collections were performed from November 2014 to January 2015, in the City of Praia, the capital of Cabo Verde, using aspirators and BG-sentinel traps. Samples were examined by multiplex Reverse Transcription-polymerase chain reaction. A total of 161 Ae. aegypti adult females were analyzed (34 pools) and from these samples, eight pools were found positive for DENV-2 and DENV-4. Our results revealed a very high natural infection rate in the vector population and showed two different serotypes co-circulating in the island that differ from the one detected in the 2009 outbreak in Cabo Verde.

Mapping Aedes aegypti indoor resting behavior reveals a preference vulnerable to householder-led vector control.

Many mosquito vectors rest inside human habitations, a behavioral trait that is exploited for vector control by indoor residual spraying (IRS) of interior walls with insecticide. Although IRS and its refined version targeted IRS are very effective against Aedes aegypti, they are expensive and logistically challenging to deliver in densely populated urban areas where outbreaks of dengue and other arboviruses are the greatest challenge. In experiments in Recife, Brazil, we set out to quantify the indoor resting behavior of Ae. aegypti at a level beyond that previously reported. We found that significantly more Ae. aegypti males, unfed and fed females visited the base of walls (height 0-20 cm, corresponding to 12.3% of the total wall surface) more frequently than upper wall areas, with the difference more pronounced at higher temperatures. When the lowest 20 cm of the walls was treated with an appropriate insecticide and colored black, we recorded up to 85% cumulative mortality after 24-h exposure in the experimental room. The findings are significant because feasibly, householders could treat this small and accessible target zone manually, without the need for visits by costly IRS teams or equipment, reducing insecticide use and enabling communities to actively protect their own indoor environment.

The V410L knockdown resistance mutation occurs in island and continental populations of Aedes aegypti in West and Central Africa.

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.

An unsettling explanation for the failure of skatole-baited ovitraps to capture Culex mosquitoes.

Culex mosquitoes are primarily found in temperate and tropical regions worldwide where they play a crucial role as main vectors of filarial worms and arboviruses. In Recife, a northeast city in Brazil, high densities of Culex quinquefasciatus are often found in association with human populated areas. In marked contrast to another part of the city, field tests conducted in the neighborhood of Sítio dos Pintos showed that trapping of mosquitoes in skatole-baited ovitraps did not differ significantly from captures in control (water) traps. Thus, classical and molecular taxonomic approaches were used to analyze the Culex species circulating in Sítio dos Pintos. Results obtained from both approaches agreed on the cocirculation of Culex quinquefasciatus and Culex nigripalpus in three different areas of this neighborhood. What was initially considered as an unexpected failure of this lure turned out to be a more unsettling problem, that is, the first report in Recife of Culex nigripalpus, a vector of Venezuelan equine encephalitis virus and West Nile virus. Unplanned urbanization processes close to remnants of the Atlantic forest, such as observed in Sítio dos Pintos, may have contributed to the introduction of Cx. nigripalpus in urban areas.

Expression of defensin, cecropin, and transferrin in Aedes aegypti (Diptera: Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae), and the abnormal development of nematodes in the mosquito.

The temporal expression of defensin, cecropin and transferrin was assessed in Aedes aegypti naturally refractory to Wuchereria bancrofti upon infection with this worm, in parallel to analysis of filarial development in the insect. Compared to controls, transcription of defensin and cecropin was higher in infected mosquitoes as soon as 2h post infection and peaked before 48h. Transferrin transcription was higher in infected mosquitoes at 24h, and at 48h was almost leveled to controls. At 72h and 7 days post infection, levels of all transcripts in infected insects decreased gradually and were similar to controls in most cases. Worm development in A. aegypti was visually abnormal from the beginning of infection. Here, we report, for the first time, the up-regulation of endogenous immune molecules in A. aegypti infected with W. bancrofti and provide a description of the worm development inside the insect. The specificities of A. aegypti-W. bancrofti model compared to other mosquito-filaria systems are discussed.

Does Zika virus infection affect mosquito response to repellents?

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject's arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays.

Climate Change Influences on the Global Potential Distribution of the Mosquito Culex quinquefasciatus, Vector of West Nile Virus and Lymphatic Filariasis.

Rapid emergence of most vector-borne diseases (VBDs) may be associated with range expansion of vector populations. Culex quinquefasciatus Say 1823 is a potential vector of West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. We estimated the potential distribution of Cx. quinquefasciatus under both current and future climate conditions. The present potential distribution of Cx. quinquefasciatus showed high suitability across low-latitude parts of the world, reflecting the current distribution of the species. Suitable conditions were identified also in narrow zones of North Africa and Western Europe. Model transfers to future conditions showed a potential distribution similar to that under present-day conditions, although with higher suitability in southern Australia. Highest stability with changing climate was between 30°S and 30°N. The areas present high agreement among diverse climate models as regards distributional potential in the future, but differed in anticipating potential for distribution in North and Central Africa, southern Asia, central USA, and southeastern Europe. Highest disparity in model predictions across representative concentration pathways (RCPs) was in Saudi Arabia and Europe. The model predictions allow anticipation of changing distributional potential of the species in coming decades.

Exaggerated blood pressure response during exercise treadmill testing: functional and hemodynamic features, and risk factors.

The factors which contribute to an exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) are not wholly understood. The association between the insertion/deletion polymorphisms of the angiotensin-converting enzyme (ACE) and M235T of the angiotensinogen with EBPR during ETT still remains unstudied. To identify and compare the risk factors for hypertension between normotensive subjects with EBPR and those who exhibit a normal curve of blood pressure (BP) during ETT. In a series of EBPR cases from a historical cohort of normotensive individuals, a univariate analysis was performed to estimate the association of the studied factors with BP behavior during ETT. Additionally, logistic multivariate regression was conducted to analyze the joint effects of the variables. P-values above 0.05 were considered statistically significant. From a total of 10,027 analyzed examinations, only 219 met the criteria employed to define EBPR, which resulted in a prevalence of 12.6%. For the systolic component of the BP, hyperreactive subjects displayed a mean age and body mass index (BMI) significantly higher than the others (P=0.002 and <0.001, respectively). No association was observed between the polymorphisms cited above and EBPR. An analysis of the joint effect of variables has indicated that only age (P< 0.001) and BMI (P=0.001) were specifically associated with systolic BP during exercise. Age and BMI were the only factors that independently influenced EBPR during ETT.

Loss of genetic diversity in Culex quinquefasciatus targeted by a lymphatic filariasis vector control program in Recife, Brazil.

Recife is one of the largest cities in north-eastern Brazil and is endemic for lymphatic filariasis transmitted by Culex quinquefasciatus. Since 2003 a control program has targeted mosquito larvae by elimination of breeding sites and bimonthly application of Bacillus sphaericus. To assess the impact of this program on the local vector population we monitored the genetic diversity and differentiation of Cx. quinquefasciatus using microsatellites and a B. sphaericus-resistance associated mutation (cqm1(REC)) over a 3-year period. We detected a significant but gradual decline in allelic diversity, which, coupled with subtle temporal genetic structure, suggests a major impact of the control program on the vector population. Selection on cqm1(REC) does not appear to be involved with loss of neutral diversity from the population, with no temporal trend in resistant allele frequency and no correlation with microsatellite differentiation. The evidence for short-term genetic drift we detected suggests a low ratio of effective population size: census population size for Cx. quinquefasciatus, perhaps coupled with strong geographically-restricted population structure. Spatial definition of populations will be an important step for success of an expanded vector control program.

Comparative genomics of the anopheline glutathione S-transferase epsilon cluster.

Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated, gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (ψAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, ψAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera, though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site within GSTE5 may represent adaptations to new habitats.