Permethrin resistance in Aedes aegypti: Genomic variants that confer knockdown resistance, recovery, and death.

Pyrethroids are one of the few classes of insecticides available to control Aedes aegypti, the major vector of dengue, chikungunya, and Zika viruses. Unfortunately, evolving mechanisms of pyrethroid resistance in mosquito populations threaten our ability to control disease outbreaks. Two common pyrethroid resistance mechanisms occur in Ae. aegypti: 1) knockdown resistance, which involves amino acid substitutions at the pyrethroid target site-the voltage-gated sodium channel (VGSC)-and 2) enhanced metabolism by detoxification enzymes. When a heterogeneous population of mosquitoes is exposed to pyrethroids, different responses occur. During exposure, a proportion of mosquitoes exhibit immediate knockdown, whereas others are not knocked-down and are designated knockdown resistant (kdr). When these individuals are removed from the source of insecticide, the knocked-down mosquitoes can either remain in this status and lead to dead or recover within a few hours. The proportion of these phenotypic responses is dependent on the pyrethroid concentration and the genetic background of the population tested. In this study, we sequenced and performed pairwise genome comparisons between kdr, recovered, and dead phenotypes in a pyrethroid-resistant colony from Tapachula, Mexico. We identified single-nucleotide polymorphisms (SNPs) associated with each phenotype and identified genes that are likely associated with the mechanisms of pyrethroid resistance, including detoxification, the cuticle, and insecticide target sites. We identified high association between kdr and mutations at VGSC and moderate association with additional insecticide target site, detoxification, and cuticle protein coding genes. Recovery was associated with cuticle proteins, the voltage-dependent calcium channel, and a different group of detoxification genes. We provide a list of detoxification genes under directional selection in this field-resistant population. Their functional roles in pyrethroid metabolism and their potential uses as genomic markers of resistance require validation.

Current enzyme-mediated insecticide resistance status of Aedes aegypti populations from a dengue-endemic city in southern Mexico.

OBJECTIVE: To identify the enzyme-mediated insecticide resistance in Aedes aegypti in Tapachula, Mexico. MATERIALS AND METHODS: Biochemical assays were undertaken to determine the enzyme levels in mosquitoes from 22 sites collected in 2018 and 2020 in Tapachula. Results of 2018 were correlated with the resistance to insecticides pub-lished. RESULTS: Mosquitoes had higher levels than those of the susceptible strain in 2018 and 2020 respectively of α-esterases in 15 and 12 sites; ß-esterases in 7 and 6 sites; glutathione-S-transferases in 11 and 19 sites; ρNPA-esterases in 21 and 17 sites; and cytochromes P450 in 20 and 22 sites. In mosquitoes of 2018, there was a moderate correlation between previously documented Malathion resistance ratios and the insensitive acetylcholinesterase (r=0.459, p= 0.03). CONCLUSIONS: The elevated enzyme levels found indicate its contribution to the resistance to pyrethroids and organo-phosphates already published in mosquitoes from Tapachula. Bioassays using enzyme inhibitors resulted in greater mor-tality, confirming that metabolism contributes to resistance.

Evaluation of commercial aerosol insecticides for control of Aedes aegypti susceptible or resistant to pyrethroids.

OBJECTIVE: To evaluate indoor use of commercial aerosols for dengue vector mosquito control, and estimate the number of treatable houses per can. MATERIALS AND METHODS: Four aerosol products containing combinations of pyrethroids (two containing propoxur and one containing synergists too), were evaluated with mosquitoes in a room of a Tapachulastyle house. Eight cages containing 20 insecticide susceptible or resistant females were hung from tripods, another set was placed in sheltered areas of the room. From the entrance of the room, one of 4-9 concentrations was sprayed for each aerosol, leaving the mosquitoes for 30 min after sprayed. Mortality was recorded after 24 h and lethal concentrations were calculated. RESULTS: Aerosol A had the highest LC50, with 0.308 g for mosquitoes hanging from tripods and 0.453 g for sheltered mosquitoes; followed by aerosols C, D and B, with statistical differences between types of exposure. CONCLUSIONS: Aerosols B-D could spray 20-25 3-room houses (56 m3-room), killing all resistant mosquitoes. Aerosols may become a good tool for indoor mosquito control, if the optimal concentration and correct spray method are used.

Entomopathogenic fungi for the control of larvae and adults of Aedes aegypti (Diptera: Culicidae) vector of dengue, chikungunya and Zika viruses in Mexico.

OBJECTIVE: To assess larvicide and adulticide activity of different native strains of fungi on Aedes aegypti. MATERIALS AND METHODS: Third instar larvae were exposed for 72 h at a concentration of 1x108 conidia/ml of 15 fungi; only fungi that significantly affected the larvae were evaluated against the adult phase at a concentration of 2x1010 conidia/ml. Mortality readings were performed at 24, 48, and 72 h for larvae, and every day to 30 days for adults. RESULTS: Trichoderma longibrachiatum, Aspergillus aculeatus, and Metarhizium anisopliae had the best larvicidal activity at 24 h of exposure (p<0.05), causing mortalities of 100, 72, and 62%, respectively. Adult mosquitoes were more affected by Gliocladium virens (45% mortality), M. anisopliae (30% mortality), and T. longibrachiatum (23.33% mortality). CONCLUSION: The larval stage of Ae. aegypti was more susceptible than the adult phase to the pathogenic action of native fungi, with T. longibrachiatum being with the highest virulence.

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata.

OBJECTIVE: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. MATERIALS AND METHODS: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. RESULTS: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and ß-eterase levels. CONCLUSIONS: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

OBJETIVO: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. MATERIAL Y MÉTODOS: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. RESULTADOS: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y ß-eterasas. CONCLUSIONES: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Composition and abundance of anopheline species according to habitat diversity in Mexico.

OBJECTIVE: To determine the abundance and geographic distribution of the main malaria vectors, which are influenced by habitat characteristics and ecological factors that directly impact adult density and the dynamics of malaria transmission in Mexico. MATERIALS AND METHODS: Samples of larvae were collected from 19 states in Mexico. Each larval habitat was characterized in situ determining the following parameters: water depth, turbidity, percentage of vegetation cover, amount of detritus, presence of algae, light intensity, type of vegetation, amount of predators, habitat stability, altitude, and hydrologic type. RESULTS: A total of 21 687 larvae corresponding to 13 anopheline species were obtained from 149 aquatic habitats. The most abundant species were Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) and An. franciscanus (5.29%). The multiple logistic regression analysis showed a negative association between An. pseudopunctipennis and water turbidity (ß=-1.342; Wald=6.122; p=0.013) and the amount of detritus (ß=-2.206; Wald=3.642; p=0.050). While in An. albimanus, there was a significant positive association with water turbidity (ß=1.344; Wald=4.256; p=0.039), a negative correlation was found with the altitude (ß=-3.445; Wald=5.407; p =0.020). The highest mosquito species diversity index was found in Chiapas (Fisher's α=1.20) and the lowest diversity in Chihuahua (Fisher's α=0.26). The greatest richness was found in streams (n=11). CONCLUSIONS: The two most abundant species were: An. albimanus and An. pseudopunctipennis. Detailed knowledge of the distribution and characteristics of their larval habitats will be useful for the effective implementation of control strategies in Mexico.

OBJETIVO: Determinar la abundancia y la distribución geográfica de los principales vectores de la malaria, las cuales están influenciadas por las características del hábitat y los factores ecológicos que afectan directamente la densidad de los adultos y la dinámica de la transmisión de la malaria en México. MATERIAL Y MÉTODOS: Se obtuvieron muestras de larvas de 19 estados de México. Cada hábitat larvario se caracterizó in situ determinando los siguientes parámetros: profundidad del agua, turbidez, porcentaje de cobertura vegetal, cantidad de detritus, presencia de algas, intensidad de luz, tipo de vegetación, cantidad de depredadores, estabilidad del hábitat, altitud y tipo hidrológico. RESULTADOS: Se identificaron un total de 21 687 larvas pertenecientes a 13 especies de anofelinos, de 149 hábitats acuáticos. Las tres especies más abundantes fueron Anopheles pseudopunctipennis (52.91%), An. albimanus (39.14%) y An. franciscanus (5.29%). El análisis de regresión logística múltiple mostró una asociación negativa para An. pseudopunctipennis y la turbidez del agua (ß=-1.342; Wald= 6.122; p=0.013) y la cantidad de detritus (ß=-2.206; Wald= 3.642; p=0.050). Para An. albimanus se encontró una asociación positiva significativa con la turbidez del agua (ß=1.344; Wald= 4.256; p=0.039) y una correlación negativa con la altitud (ß=-3.445; Wald=5.407; p=0.020). El índice de diversidad más alto se encontró en Chiapas (α de Fisher=1.20) y la diversidad más baja en Chihuahua (α de Fisher=0.26). La mayor riqueza se encontró en los arroyos (n=11). CONCLUSIONES: Las dos especies más abundantes fueron An. albimanus y An. pseudopunctipennis. El conocimiento detallado de la distribución y características de sus hábitats larvales será útil para la implementación efectiva de las estrategias de control en México.

[Insecticide resistance in Aedes aegypti and Ae. albopictus (Diptera: Culicidae) populations from Tapachula, Chiapas, Mexico]. / Resistencia a insecticidas en Aedes aegypti y Aedes albopictus (Diptera: Culicidae) de Tapachula, Chiapas, México.

OBJECTIVE: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México. MATERIALS AND METHODS: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively. RESULTS: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species. CONCLUSIONS: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur.

OBJETIVO: Determinar la resistencia a insecticidas en Ae. aegypti y Ae. albopictus de Tapachula, Chiapas, México. MATERIAL Y MÉTODOS: Se utilizaron ovitrampas para obtener huevos de mosquitos Aedes y se realizaron pruebas de susceptibilidad (CDC) y ensayos enzimáticos con la primera generación. RESULTADOS: Aedes aegypti mostró resistencia a deltametrina, permetrina, malatión, clorpirifos, temefos y a bendiocarb (CARB), mientras que Aedes albopictus a malatión y en menor grado a cloripirifos, temefos, permetrina y deltametrina. Ambas especies mostraron altos niveles de enzimas como citocomo P450 y glutatión S-tranferasa, mientras que los niveles de esterasas variaron por especie y sitio muestreado. Se detectó acetilcolinesterasa insensible a insecticidas en ambas especies. CONCLUSIONES: En un hábitat urbano de Tapachula, Chiapas, México donde se aplica control con insecticidas Ae. aegypti y Ae. albopictus sólo son susceptibles al propoxur.

[Indoor thermal fogging of the mixture of flupyradifurone and transflutrin on Aedes aegypti mosquitoes susceptible and resistant to pyrethroids in southern Mexico]. / Nebulización térmica intradomiciliar de la mezcla de flupyradifurona y transflutrina en mosquitos Aedes aegypti susceptibles y resistentes a piretroides en el Sur de México.

OBJECTIVE: To evaluate the efficacy of thermal fogging of a mixture of flupyrafirudone (26.3 g/L) and transfluthrin (52.5 g/L) against dengue, Zika y chikungunya Aedes mosquito vectors. MATERIALS AND METHODS: Groups of 15 caged Ae. aegypti (susceptible and pyrethroid resistant) mosquitoes were placed in living room, kitchen and bedroom inside houses, after which a dose of 2 and 4 mg/m3 of flupyradifurone and transfluthrine, respectively, was applied as thermal fog. After one hour of exposure mosquitoes were transferred to the laboratory and mortality was recorded after 24 h. RESULTS: The mixture killed 97 to 100% of mosquitoes from the strains and the efficacy was similar independently of their place within the premises. CONCLUSIONS: The mixture of flupyrafirudone and transfluthrin applied as thermal fog is a promising tool to control Aedes mosquito populations independently of the pyrethroid-insecticide resistance status.

OBJETIVO: Evaluar la efectividad de la mezcla de flupyradifurona 26.3 g/L y transflutrina 52.5 g/L aplicada como niebla térmica a mosquitos Aedes vectores de virus dengue, Zika y chikungunya. MATERIAL Y MÉTODOS: Se colocaron grupos de 15 mosquitos de Ae. aegypti (susceptibles y resistentes a piretroides) dentro de jaulas, en sala, recámara y cocina. Posteriormente, se aplicó la mezcla de flupyradifurona y transflutrina dentro de las viviendas a una dosis de 2 y 4 mg/m3, respectivamente. RESULTADOS: La mezcla de flupyradifurona y transflutrina causó mortalidades de 97 a 100% sobre las cepas de mosquitos Aedes y su efectividad fue la misma en los diferentes compartimentos de las viviendas. CONCLUSIONES: La mezcla de flupyradifurona y transflutrina, aplicada en niebla térmica, es una herramienta prometedora para el control de poblaciones de mosquitos Aedes independientemente de su estado de resistencia a insecticidas.

Larval habitat characterization of Anopheles darlingi from its northernmost geographical distribution in Chiapas, Mexico.

BACKGROUND: Anopheles darlingi is considered the most efficient malaria vector in the Neotropical region. In Mexico, its role as an incriminated vector of Plasmodium has not been confirmed in the Lacandon forest. Similarly, knowledge about bionomic and larval ecology is scarce. The study aim was to identify and describe the larval habitats of An. darlingi in Chiapas, México. METHODS: Standard larval collections were performed in the Lacandon forest region and in the Soconusco region of southern Chiapas from January 2010 to April 2014, including dry and rainy seasons. Mean larval density of An. darlingi was estimated according to hydrological types, and associations between the presence of An. darlingi and environmental factors including ecological parameters and geographic positions were statistically analysed. RESULTS: One hundred and twelve aquatic habitats were analysed, 80 from the Lacandon forest region and 32 from the Soconusco region; 94.64% of these sites presented anopheline larvae. In total, 10,977 larvae belonging to 11 Anopheles species were collected. The 19 (out of 112) larval habitats positive to An. darlingi were: rain puddles (26.32%), ground pools (21.05%), ponds (15.79%), ditches (15.79%), river margins (10.53%) and streams (10.53%). Overall, the average (±SD) larval density was 6.60 ± 2.41 larvae per dip. Multiple logistic regression analysis showed that temporary habitats, green algae presence and stagnant water were associated with An. darlingi larval presence. The positive habitats were found in the Lacandon forest region during the rainy season (May-September). No specimens were found in the Soconusco region of the coastal plain of Chiapas. CONCLUSION: The mosquito An. darlingi larval habitats were found in different hydrological types. The habitat stability, presence of algae and water current were the main factors for An. darlingi larval occurrence. The information on the characteristics of the larval habitats of An. darlingi will be useful in sustainable programmes for malaria control in the Lacandon forest region, Chiapas.

Socioeconomic factors, attitudes and practices associated with malaria prevention in the coastal plain of Chiapas, Mexico.

BACKGROUND: Mexico is in the malaria pre-elimination phase; therefore, continuous assessment and understanding of the social and behavioural risk factors related to exposure to malaria are necessary to achieve the overall goal. The aim of this research was to investigate socio-economic backgrounds, attitudes and practices related with malaria in rural locations from the coastal plain of Chiapas. METHODS: In January 2012, 542 interviews were conducted to householders from 20 villages across the coastal plain of Chiapas. Questions were about housing conditions, protection from mosquito bites and general information of householders. Chi2 analyses were performed to see whether there was a dependence of those reported having malaria with their house conditions and their malaria preventive practices. Results were discussed and also compared statistically against those obtained 17 years ago from the same area. RESULTS: Most households had 2-5 people (73.6%), 91.6% of houses had 1-3 bedrooms. The physical structure of the houses consisted of walls mainly made of block or brick 72.3%, the floor made of cement 90.0%, while the roof made of zinc sheet 43.9%, and straw or palm 42.2%. A 23.1% of the interviewed completed elementary school and 16.6% was illiterate. A 9.9% of the residents reported at least one family member having had malaria. A 98.1% of families used some method to prevent mosquito bites; those using bed nets were 94.3%. Almost 72% of families bought products for mosquito protection. A total of 537 out of 542 families agreed with the indoor residual spraying (IRS) of insecticide and a frequency of application as often as every two months was preferred. CONCLUSION: Housing conditions and malaria preventive practices have improved in these rural areas in 17 years, which could be in favor of malaria elimination in this area. Information generated by this study could help in the decision making about whether to use insecticide as indoor residual spraying or to implement massive distribution of long-lasting impregnated bed nets, considering the number of bedrooms and the structure of houses in the region, which may lead to a more efficient vector control for the coastal plain of Chiapas.

Pyrethroid susceptibility reversal in Aedes aegypti: A longitudinal study in Tapachula, Mexico.

Pyrethroid resistance in Aedes aegypti has become widespread after almost two decades of frequent applications to reduce the transmission of mosquito-borne diseases. Because few insecticide classes are available for public health use, insecticide resistance management (IRM) is proposed as a strategy to retain their use. A key hypothesis of IRM assumes that negative fitness is associated with resistance, and when insecticides are removed from use, susceptibility is restored. In Tapachula, Mexico, pyrethroids (PYRs) were used exclusively by dengue control programs for 15 years, thereby contributing to selection for high PYR resistance in mosquitoes and failure in dengue control. In 2013, PYRs were replaced by organophosphates-insecticides from a class with a different mode of action. To test the hypothesis that PYR resistance is reversed in the absence of PYRs, we monitored Ae. aegypti's PYR resistance from 2016 to 2021 in Tapachula. We observed significant declining rates in the lethal concentration 50 (LC50), for permethrin and deltamethrin. For each month following the discontinuation of PYR use by vector control programs, we observed increases in the odds of mosquitoes dying by 1.5% and 8.4% for permethrin and deltamethrin, respectively. Also, knockdown-resistance mutations (kdr) in the voltage-gated sodium channel explained the variation in the permethrin LC50s, whereas variation in the deltamethrin LC50s was only explained by time. This trend was rapidly offset by application of a mixture of neonicotinoid and PYRs by vector control programs. Our results suggest that IRM strategies can be used to reverse PYR resistance in Ae. aegypti; however, long-term commitment by operational and community programs will be required for success.

Aedes aegypti, Ae. albopictus and Culex quinquefasciatus Adults Found Coexisting in Urban and Semiurban Dwellings of Southern Chiapas, Mexico.

Tapachula, Mexico, a tropical city, is an endemic area for dengue, in addition to several outbreaks in the last decade with chikungunya and zika. As part of the migratory corridor from Central to North America and the risks of scattered infectious diseases that this implies, the identification and distribution of potential disease vectors in and around residential areas are essential in terms of entomological surveillance for the prevention of disease outbreaks. The identification of mosquito species of medical importance coexisting in houses and cemeteries in Tapachula and two semiurban sites in southern Chiapas was investigated. Adult mosquitoes were collected from May to December 2018, resting inside and outside houses and in the tombstones and fallen tree leaves in cemeteries. A total of 10,883 mosquitoes belonging to three vector species were collected across 20 sites; 6738 were from neighborhood houses, of which 55.4% were Culex quinquefasciatus, 41.6% Aedes aegypti, and 2.9% Ae. albopictus. Aedes aegypti was the most common mosquito resting inside houses (56.7%), while Ae. albopictus and Cx. quinquefasciatus were mostly found resting outside houses (75.7%). In the cemeteries, Cx. quinquefasciatus (60.8%) and Ae. albopictus (37.3%) were the most abundant, while Ae. aegypti (1.9%) was the least abundant. This is the first report to identify adults of three major disease vector species coexisting in the domestic environment of urban and semiurban sites and Ae. albopictus adult resting inside of urban houses in Mexico. It would be opportune to consider comprehensive strategies that can be applied in this region to control the three species at the same time and avoid outbreaks of the diseases they transmit.

Effectiveness of indoor ultra-low volume application of Aqua Reslin® Super during an emergency.

Indoor ultra-low volume (ULV) applications of Aqua Reslin Super (Permethrin + s-bioallethrin) were carried out using portable Solo Port 423 sprayers in an urban area of Reforma, Chiapas, Mexico. Sprayers were calibrated to discharge a flow rate of 110 ml/min with a dose range of approximately 0.55 to 0.792 mg Al/m3 space. Entomological evaluation, based on 3 cages per house, each containing 15 sugar-fed, 2-4-day-old Aedes aegypti females and placed in hidden locations in 4 randomly selected houses for 15 min, showed 98.8% to 100% mortality. After the spraying, ovitrap data showed no mosquito adults present 4 days after the applications, and only 1 ovitrap out of 60 positive 8 days after the intervention. This evaluation suggests that indoor ULV application can be useful during emergencies after disasters, during dengue outbreaks, or to prevent mosquito population outbreaks before rainy seasons and, therefore, the onset of dengue transmission in Mexican dengue transmission risk areas.

Insecticide resistance in Aedes aegypti from Tapachula, Mexico: Spatial variation and response to historical insecticide use.

BACKGROUND: Insecticide use continues as the main strategy to control Aedes aegypti, the vector of dengue, Zika, chikungunya, and yellow fever. In the city of Tapachula, Mexico, mosquito control programs switched from pyrethroids to organophosphates for outdoor spatial spraying in 2013. Additionally, the spraying scheme switched from total coverage to focused control, prioritizing areas with higher entomological-virological risk. Five years after this strategy had been implemented, we evaluated the status and variability of insecticide resistance among Ae. aegypti collected at 26 sites in Tapachula. METHODOLOGY/PRINCIPAL FINDINGS: We determined the lethal concentrations at 50% of the tested populations (LC50) using a bottle bioassay, and then, we calculated the resistance ratio (RR) relative to the susceptible New Orleans strain. Permethrin and deltamethrin (pyrethroids), chlorpyrifos and malathion (organophosphates), and bendiocarb (carbamate) were tested. The frequencies of the substitutions V1016I and F1534C, which are in the voltage-gated sodium channel and confer knockdown-resistance (kdr) to pyrethroid insecticides, were calculated. Despite 5 years having passed since the removal of pyrethroids from the control programs, Ae. aegypti remained highly resistant to permethrin and deltamethrin (RR > 10-fold). In addition, following 5 years of chlorpyrifos use, mosquitoes at 15 of 26 sites showed moderate resistance to chlorpyrifos (5- to 10-fold), and the mosquitoes from one site were highly resistant. All sites had low resistance to malathion (< 5-fold). Resistance to bendiocarb was low at 19 sites, moderate at five, and high at two. Frequencies of the V1016I ranged from 0.16-0.71, while C1534 approached fixation at 23 sites (0.8-1). Resistance profiles and kdr allele frequencies varied across Tapachula. The variability was not associated with a spatial pattern at the scale of the sampling. CONCLUSION/SIGNIFICANCE: Mosquito populations respond to selection pressure at a focal scale in the field. Spatial variation across sites highlights the importance of testing multiple sites within geographical regions.

Permethrin Resistance Status and Associated Mechanisms in Aedes albopictus (Diptera: Culicidae) From Chiapas, Mexico.

There are major public health concerns regarding the spread of mosquito-borne diseases such as dengue, Zika, and chikungunya, which are mainly controlled by using insecticides against the vectors, Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse). Pyrethroids are the primary class of insecticides used for vector control, due to their rapid knockdown effect and low toxicity to vertebrates. Unfortunately, continued use of pyrethroids has led to widespread insecticide resistance in Ae. aegypti; however, we lack information for Ae. albopictus-a sympatric species in Chiapas since 2002. In this study, we evaluated the permethrin resistance status of Ae. albopictus collected from Mexico and Texas. We also selected for permethrin resistance in the laboratory and investigated the potential mechanisms conferring resistance in this species. Knockdown resistance mutations, specifically F1534C, in the voltage-gated sodium channel gene, and increased activity of detoxifying enzymes were evaluated. Low levels of permethrin resistance (<2.4-fold) were observed in our field populations of Ae. albopictus and the F1534C mutation was not detected in any of the sites. Low levels of resistance were also observed in the artificially selected strain. There was significantly higher cytochrome P450 activity in our permethrin-selected and nonselected strains from Mexico compared to the control strain. Our results suggest the Ae. albopictus sampled from 2016 are mostly susceptible to pyrethroids. These results contrast with the high levels of permethrin resistance (>58-fold) found in Ae. aegypti from the same sites in Mexico. This research indicates the importance of continued monitoring of Ae. albopictus populations to prevent resistance from developing in the future.

Mechanisms of pyrethroid resistance in Aedes (Stegomyia) aegypti from Colombia.

In Colombia Aedes (Stegomyia) aegypti is the main vector of urban arboviruses such as dengue, chikungunya and Zika. This urban mosquito has a well-established capacity to develop insecticide resistance to different types of insecticides (pyrethroids, organochlorides, organophosphates), using multiple resistance mechanisms. An understanding of ongoing resistance mechanisms is critical to determining the activities of vector control programs. In order to identify the biochemical and molecular mechanisms associated with pyrethroid resistance in Colombia, three laboratory-selected strains resistant to DDT, Propoxur and lambdacyhalothrin, and 7 field-collected strains were evaluated. CDC bioassays were performed to measure the susceptibility status to pyrethroid type I (permethrin) and II (deltamethrin and lambdacyhalothrin), and potential cross-resistance to different types of insecticides; organochlorine (DDT), carbamates (propoxur) and organophosphates (malathion). The enzymatic activity of esterases, glutathione S-transferases (GST) and P450 monooxygenases were biochemically determined. Frequencies of kdr mutations Val1016Ile and Phe1534cys were determined through real-time PCR. The Rockefeller strain of Aedes (Stegomyia) aegypti was used as the susceptible control. The laboratory-selected strains "propoxur" and "lambdacyhalothrin" and one field population (Medellín (BF) F2 were resistant to all evaluated pyrethroids. Six of the seven field populations as well as the laboratory- selected "DDT" strain were resistant to permethrin. All the evaluated strains were resistant to DDT. Cross-resistance between lambdacyhalothrin and propoxur was observed in the laboratory-selected strains; however, all field-collected strains were susceptible to propoxur and no evidence of malathion resistance was found. The main biochemical mechanism for resistance observed in the field-collected strains was related to the enzyme GST. Further, the frequencies of kdr mutations alleles associated with insecticide resistance were high and ranged from 0.02 to 0.72 for Ile1016 and from 0.44 to 0.99 for Cys1534. Strains with high frequencies of both kdr mutations were resistant to both type I and II pyrethroids. These results suggest that Ae. aegypti from Colombia have developed multiple resistance mechanisms associated with pyrethroid resistance; therefore a resistance management strategy against these field populations of Ae. Aegypti, incorporating these findings is strongly recommended.

A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery.

Identifying novel viruses or assessing viral variation by NGS requires high sequencing coverage. More than 90% of total RNA is ribosomal (rRNA), making variant calling, virus discovery or transcriptomic profiling difficult. Current methods to increase informative reads suffer from drawbacks, either they cannot be used for some viruses, are optimized for a single species, or introduce bias. We describe a two-part approach combining reverse-transcription to create RNA/DNA hybrids which are then degraded with RNaseH/DNase sequentially that works for three medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate depletion of rRNA from different samples, including whole mosquitoes and midgut contents from FTA cards. We describe novel insect-specific virus genomes from field collected mosquitoes. The protocol requires only common laboratory reagents and small oligonucleotides specific to rRNA. This approach can be adapted for other organisms, aiding virus diversity analyses, virus discovery and transcriptomics in both laboratory and field samples.

Comparison of two DNA extraction methods from larvae, pupae, and adults of Aedes aegypti.

Mosquitoes are the most important arthropods from the point of view of public health, due to the fact that they can transmit a large number of pathogens which can cause diseases to humans and animals. Aedes aegypti (L.) is one of the most important vector species in the world, since it can transmit numerous pathogens such as dengue, Zika, and chikungunya. Therefore, studies involving the molecular aspects of this and other mosquitoes species are currently increasing. In this report, we describe the comparison between two DNA extraction techniques, Chelex and cetyltrimethylammonium bromide (CTAB), for carrying out DNA extraction in larvae, pupae and adult female of Ae. aegypti. The Chelex technique was superior in the amount and purity of DNA as compared to the CTAB technique in the three life stages we tested.

Estimating the effects of variation in viremia on mosquito susceptibility, infectiousness, and R0 of Zika in Aedes aegypti.

Zika virus (ZIKV) is an arbovirus primarily transmitted by Aedes mosquitoes. Like most viral infections, ZIKV viremia varies over several orders of magnitude, with unknown consequences for transmission. To determine the effect of viral concentration on ZIKV transmission risk, we exposed field-derived Ae. aegypti mosquitoes to four doses (10(3), 10(4), 10(5), 10(6) PFU/mL) representative of potential variation in the field. We demonstrate that increasing ZIKV dose in the blood-meal significantly increases the probability of mosquitoes becoming infected, and consequently disseminating virus and becoming infectious. Additionally, we observed significant interactions between dose and days post-infection on dissemination and overall transmission efficiency, suggesting that variation in ZIKV dose affects the rates of midgut escape and salivary gland invasion. We did not find significant effects of dose on mosquito mortality. We also demonstrate that detecting virus using RT-qPCR approaches rather than plaque assays potentially over-estimates key transmission parameters, including the time at which mosquitoes become infectious and viral burden. Finally, using these data to parameterize an R0 model, we showed that increasing viremia from 10(4) to 10(6) PFU/mL increased relative R0 3.8-fold, demonstrating that variation in viremia substantially affects transmission risk.

Variation in competence for ZIKV transmission by Aedes aegypti and Aedes albopictus in Mexico.

BACKGROUND: ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59-Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. CONCLUSIONS/SIGNIFICANCE: Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8-51% in Ae. aegypti and from 2-26% in Ae. albopictus.