Effect of Photoperiod On Permethrin Resistance In Aedes aegypti.

Living organisms have been exposed to light-dark cycles that allowed them to adapt to different ecological niches. Circadian cycles affect hormone release, metabolism, and response to xenobiotic compounds. Current studies have shown that insect susceptibility to toxic agents depends on circadian cycles, mainly because the biochemical processes involved in detoxification and responses to oxidative stress are modulated by this process. The goal of this study was to determine the effect of photoperiod on resistance to permethrin in Aedes aegypti . Collections of Ae. aegypti from 4 locations in Yucatan, southern Mexico, were subjected to 2 different photoperiod schemes: dark (0 h light:24 h dark) and natural photoperiod (12 h light:12 h dark). The comparison of both photoperiods was evaluated with respect to permethrin resistance using bottle bioassays and by monitoring the possible mechanism related such as enzymatic activity and by the frequency of 2 knockdown resistance mutations in the voltage-dependent sodium channel gene (V1016I and F1534C). The susceptible strain was used as a reference. The mosquitoes in dark photoperiod showed a reduction in resistance to the pyrethroid. The α-esterases and glutathione S-transferase enzymatic activities showed lower levels in the dark photoperiod, and the frequencies of V1016I knockdown resistance mutation showed significant difference between photoperiod schemes.

Susceptibility to chlorpyrifos in pyrethroid-resistant populations of Aedes aegypti (Diptera: Culicidae) from Mexico.

Resistance to the organophosphate insecticide chlorpyrifos was evaluated in females from six strains of Aedes aegypti (L.) that expressed high levels of cross-resistance to eight pyrethroid insecticides. Relative to LC50 and LC90 at 24 h of a susceptible New Orleans (NO) strain, three strains were highly resistant to chlorpyrifos (Coatzacoalcos, resistance ratio [RRLC90 = 11.97; Pozarica, RRLC90 = 12.98; and Cosoleacaque, RRLC50 = 13.94 and RRLC90 = 17.57), one strain was moderately resistant (Veracruz, RRLC90 = 5.92), and two strains were susceptible (Tantoyuca and Martinez de la Torre, RRLC50 and RRLC90 < 5) in bottle bioassays according to Centers for Disease Control and Prevention. Furthermore, high levels of alpha- or beta-esterase activity in the sample populations were correlated with resistance, suggesting that esterase activity may be a mechanism causing the development of organophosphate resistance in these populations. Overall, the populations in this study were less resistant to chlorpyrifos than to pyrethroids. Rotation of insecticides used in control activities is recommended to delay or minimize the occurrence of high levels of resistance to chlorpyrifos among local populations of Ae. aegypti. The diagnostic dose and diagnostic time for chlorpyrifos resistance monitoring was determined to be 85 microg per bottle and 30 min, respectively, using the susceptible NO strain.

Susceptibility to insecticides and resistance mechanisms in Aedes aegypti from the Colombian Caribbean Region.

We determined the susceptibility to insecticides and the biochemical and molecular mechanisms involved in resistance in nine populations of Aedes aegypti (L.) of the Colombian Caribbean region. Bioassays were performed on larvae for susceptibility to temephos and on adults to the insecticides malathion, fenitrothion, pirimiphos-methyl, permethrin, deltamethrin, λ-cyhalothrin and cyfluthrin. The resistance ratio (RR) for each insecticide in the populations was determined, using the susceptible Rockefeller strain as a susceptible control. Additionally, we evaluated the response of the populations to the diagnostic dose (DD) of the organochlorine pesticide DDT. The following biochemical mechanisms associated with resistance were studied: α-esterases, ß-esterases, mixed-function oxidases (MFO), glutathione s-transferases (GST) and insensitive acetylcholinesterase (iAChE) as well as the presence of kdr I1,016 mutation and its frequency. All populations studied showed susceptibility to the organophosphates evaluated (RR < 5-fold), except for the Puerto Colombia and Soledad populations which showed high resistance (RR 15-fold) and moderate resistance (RR 5-fold) to temephos, respectively, and Sincelejo (Sucre) with moderate resistance to pirimiphos-methyl (RR 5-fold). All populations evaluated with DD of DDT were found to be resistant with 2-28% of mortality. Variability was observed in the resistance to pyrethroids: permethrin (RR 1.2- to 30.8-fold), deltamethrin RR 0.9- to 37.8-fold), λ-cyalothrin (RR 3.4- to 83-fold) and cyfluthrin (RR 0.3- to 33.8-fold). Incipiently α-esterases and MFO levels were found in the Valledupar population; MFO showed the same profile in Cienaga and GST in the Sincelejo population, all other populations showed unaltered profiles of the enzymes evaluated. The kdr I1,016 mutation was found in all populations evaluated with variability in its allelic and genotypic frequencies.

Widespread Resistance to Temephos in Aedes aegypti (Diptera: Culicidae) from Mexico.

Organic synthetic insecticides continue to be part of the arsenal for combating vector-borne diseases in Mexico. Larvicides are a fundamental part of the process in programs for mosquito control, temephos being one of the most widely used in Mexico. In the present study, we analyzed the frequency of temephos resistance in twenty-three Aedes aegypti populations using the discriminating concentration (DC) of 0.012 mg/L. We also tested 5× DC (0.6 mg/L) and 10× DC (0.12 mg/L) of temephos. The resistance distribution to temephos was interpolated to unsampled sites using the inverse distance weighting (IDW) method. The populations of Ae. aegypti showed a high frequency of resistance (1× DC) with mortality rates below 93% in 22 of the 23 populations analyzed. Moderate resistance intensity (5× DC) was found in 78% of the populations, and high intensity (10× DC) in 30%. Predicted mortality was below 60% in the populations of the Pacific Coast, along the Gulf of Mexico, and in the state of Coahuila in Northeastern Mexico in relation to 1× DC; the Pacific Coast and Northeast patterns hold for 5× and 10× DC. The results suggest the need for rotation of the larvicide to effectively control the larval populations of the vector in the country.

Resistance to malathion and deltamethrin in Aedes aegypti (Diptera: Culicidae) from western Venezuela.

Resistance to the insecticides deltamethrin and malathion and the enzymes associated with metabolic resistance mechanisms were determined in four field populations of Aedes aegypti (L.) from western Venezuela during 2008 and 2010 using the bottle assay and the microplate biochemical techniques. For deltamethrin, mortality rates after 1 h exposure and after a 24-h recovery period were determined to calculate the 50% knock-downconcentration (KC50) and the lethal concentration (LC50), respectively. For malathion, mortality was recorded at 24 h to determine the LC50. For deltamethrin, resistance ratios of knock-down resistance and postrecovery were determined by calculating the RRKC50 and RRLC50, comparing the KC50 and LC50 values of the field populations and those of the susceptible New Orleans strain. Knock-down resistance to deltamethrin was moderate in the majority of the populations in 2008 (RRKC50 values were between 5- and 10-fold), and only one population showed high resistance in 2010 (RRKC50 > 10-fold). Moderate and high postrecovery resistance to deltamethrin was observed in the majority of the populations for 2008 and 2010, respectively. There was significantly increased expression of glutathione-S-tranferases and mixed-function oxidases. All populations showed low resistance to malathion in 2008 and 2010 with significantly higher levels of alpha-esterases for 2008 and 2010 and beta-esterases for 2008.

Genetic variation in Bactericera cockerelli (Hemiptera: Triozidae) from Mexico.

Bactericera cockerelli (Sulc) is a significant pest of several solanacious crops in Mexico and the United States since 1970. In 2001 significant outbreaks of outstanding importance were observed for the first time in areas where infestations of this insect were historically rare. Molecular studies revealed that this was because of the development of a new biotype of B. cockerelli that had become adapted to south-western United States, further demonstrating that this genetic differentiation was reflected in the survival, development cycle, fertility, and growth rate of both the native biotype as well as the one recently reported. To determine genetic variation in populations of B. cockerelli from Mexico, inter simple sequence repeat were used. Results showed that populations of B. cockerelli from central and northeastern Mexico (Guanajuato, Morelos, Estado de Mexico, and Nuevo Leon states) are genetically similar, meanwhile B. cockerelli from northwest, southwest, and southeast of the country (Sinaloa, Michoacan, and Oaxaca states) are genetically distinct from each other and from the rest of the populations included in the study.

Wide spread cross resistance to pyrethroids in Aedes aegypti (Diptera: Culicidae) from Veracruz state Mexico.

Seven F1 strains of Aedes aegypti (L.) were evaluated by bottle bioassay for resistance to the pyrethroids d-phenothrin, permethrin, deltamethrin, lambda-cyalothrin, bifenthrin, cypermethrin, alpha-cypermethrin, and z-cypermethrin. The New Orleans strain was used as a susceptible control. Mortality rates after a 1 h exposure and after a 24 h recovery period were determined. The resistance ratio between the 50% knockdown values (RR(KC50)) of the F1 and New Orleans strains indicated high levels of knockdown resistance. The RR(KC50) with alpha-cypermethrin varied from 10 to 100 among strains indicating high levels of knockdown resistance. Most of the strains had moderate resistance to d-phenothrin. Significant but much lower levels of resistance were detected for lambda-cyalothrin, permethrin, and cypermethrin. For zeta-cypermethrin and bifenthrin, only one strain exhibited resistance with RR(KC50) values of 10- and 21-fold, respectively. None of the strains showed RR(KC50) >10 with deltamethrin, and moderate resistance was seen in three strains, while the rest were susceptible. Mosquitoes from all strains exhibited some recovery from all pyrethroids except d-phenothrin. Regression analysis was used to analyze the relationship between RR(LC50) and RR(KC50). Both were highly correlated (R2 = 0.84-0.97) so that the slope could be used to determine how much additional pyrethroid was needed to ensure lethality. Slopes ranged from 0.875 for d-phenothrin (RR(LC50) approximately equal to RR(KC50)) to 8.67 for lambda-cyalothrin (-8.5-fold more insecticide needed to kill). Both RR(LC50) and RR(KC50) values were highly correlated for all pyrethroids except bifenthrin indicating strong cross-resistance. Bifenthrin appears to be an alternative pyrethroid without strong cross-resistance that could be used as an alternative to the current widespread use of permethrin in Mexico.

Evaluation of susceptibility to organophosphates in populations of Aedes aegypti in La Guajira, Colombia / Evaluación de la sensibilidad a organofosforados en poblaciones de Aedes aegypti (L.) (Diptera: Culicidae) del departamento de La Guajira, Colombia

Introduction. Dengue is a public health problem in La Guajira region. Control has focused on the vector using insecticides, including organophosphates. Objective. To evaluate the state of susceptibility to organophosphates insecticides in fifteen Aedes aegypti (L.) populations in La Guajira, Colombia. Materials and methods. We collected samples of third-instar larvae and adult mosquitoes of Ae. aegypti in the municipalities of Albania, Barrancas, Dibulla, Distracción, El Molino, Fonseca, Hatonuevo, La Jagua del Pilar, Maicao, Manaure, Riohacha, San Juan del Cesar, Uribia, Urumita, Villanueva. Bioassays for temefos, malathion, and pirimiphos-methyl were carried out following the methodology of the World Health Organization, and the bottle technique using the guidance of the Centers for Disease Control and Prevention. Susceptibility to temefos was determined through the resistance ratio between lethal concentration 50 and lethal concentration 95; for the compounds temefos, malathion and pirimiphos-methyl, susceptibility was calculated using diagnostic dose and diagnostic time in the populations evaluated. Rockefeller susceptible strain was used as a control. Results: All evaluated populations of Ae. aegypti from La Guajira were found to be susceptible to temefos (ratio resistance to CL50<5.0; ratio resistance to CL95<5.0; 98 - 100 % mortality); pirimiphosmethyl (99 - 100 % mortality), and malathion (100 % mortality). Conclusion. Based on the results, the use of temefos, malathion, and pirimiphosmethyl is feasible for the control of Ae. aegypti in the evaluated populations.

Introducción. El dengue es un problema de salud pública para el departamento de La Guajira. El control se ha enfocado en el vector con el uso de insecticidas, entre ellos los organofosforados. Objetivo. Evaluar el estado de la sensibilidad a insecticidas organofosforados de quince poblaciones de Aedes aegypti (L.) en el departamento de La Guajira, Colombia. Materiales y métodos. Se realizaron bioensayos para temefos, malatión y metil-pirimifos en larvas de tercer estadio y mosquitos adultos de Ae. aegypti en los municipios de Albania, Barrancas, Dibulla, Distracción, El Molino, Fonseca, Hatonuevo, La Jagua del Pilar, Maicao, Manaure, Riohacha, San Juan del Cesar, Uribia, Urumita y Villanueva, siguiendo la metodología de la Organización Mundial de la Salud (OMS) y la técnica de botellas usando la guía de los de los Centers for Disease Control and Prevention, respectivamente. Se determinó la sensibilidad por medio de la razón de resistencia a CL50 y CL95 (RRCL50, RRCL95) para temefos y a dosis y tiempo diagnóstico para temefos, malatión y metilpirimifos en las poblaciones de campo evaluadas, usando como control la cepa sensible Rockefeller. Resultados. Las 15 poblaciones del departamento de La Guajira son sensibles a: temefos (razón de la resistencia a RRCL50<5,0; relación de resistencia a CL95<5,0; 98 a 100 % de mortalidad); metil-pirimifos (99 a 100 % de mortalidad) y malatión (100 % de mortalidad). Conclusión. Con base en los resultados obtenidos, es factible el uso de temefos, malatión y metil-pirimifos para el control de Ae. aegypti en las poblaciones evaluadas.

Status of Acaricide Resistance and Detecting the Knockdown Resistance Mutation T2134A in the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae) from Northeastern Mexico.

Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Assessing the Impact of Insecticide Resistance on Vector Competence: A Review.

The primary strategy to avoid adverse impacts from insect-mediated pathogen transmission is the chemical control of vector populations through insecticides; its continued use has led to insecticide resistance and unknown consequences on vector competence. This review aims to systematically analyze and synthesize the research on the influence of insecticide resistance (IR) on vector competence (VC). Thirty studies met the inclusion criteria. Twenty studies, conducted either in laboratory or field settings, described the influence of phenotypic insecticide resistance and mechanisms on VC in vectors of human pathogens. Seven studies showed the effect of exposure to insecticides on VC in vectors of human pathogens. Three studies reported the influence of phenotypic resistance and mechanisms on VC in crop pests. The evidence shows that IR could enhance, impair, or have no direct effect on VC in either field or laboratory-designed studies. Similar positive and negative trends are found in pest vectors in crops and studies of insecticide exposure and VC. Even though there is evidence that exposure to insecticides and IR can enhance VC, thus increasing the risk of pathogen transmission, more investigations are needed to confirm the observed patterns and what implications these factors could have in vector control programs.

Susceptibility to Organophosphates in Culex quinquefasciatus from Colombia.

Susceptibility to organophosphates was evaluated in 2 populations of Culex quinquefasciatus from the department of Atlantico, Colombia. Bioassays for temephos, malathion, and pirimiphos-methyl were performed with 3rd-stage larvae and adult females of Cx. quinquefasciatus from the municipalities of Soledad and Puerto Colombia, following the methods of the World Health Organization and Centers for Disease Control and Prevention, respectively. The median lethal concentration (LC50) and 90% lethal concentration (LC90) resistance ratios (RRLC50 and RRLC90) were determined for each insecticide in the field populations evaluated, using the Cartagena strain as the susceptible control. Relative to LC50 and LC90 of the Cartagena strain, the population from Puerto Colombia was moderately resistant to temephos (RRLC50 5.7-fold) and malathion (RRLC50 8.6-fold, RRLC90 9-fold) and susceptible to pirimiphos-methyl (RRLC50 and RRLC90 < 5-fold). The population from Soledad was susceptible to temephos and pirimiphos-methyl (RRLC50 and RRLC90 < 5-fold) and showed moderate resistance to malathion (RRLC50 7.5-fold). It is important to emphasize that routine monitoring of insecticide resistance in Cx. quinquefasciatus helps us detect resistance early and improve the effectiveness of control strategies.

Spatial Distribution of Pyrethroid Resistance and kdr Mutations in Aedes aegypti from La Guajira, Colombia.

Dengue, chikungunya, and Zika are of great concern to the public health of Colombia. One of the main control strategies for these diseases is the application of insecticides directed at the Aedes aegypti vector. However, insecticide resistance has been increasingly recorded in the country, making control measures difficult. Here, we evaluated the resistance profiles for pyrethroids in populations of Ae. aegypti from La Guajira, Colombia. The frequency (diagnostic dose, DD) and intensity (2×, 5×, and 10× DD) of resistance to permethrin, deltamethrin, and lambda-cyhalothrin were determined in 15 populations of Ae. aegypti from La Guajira, Colombia, using the bottle bioassay. The kdr mutations V1016I, F1534C, and V410L, were identified, and their allele and genotype frequencies were calculated. Finally, the mortality values for the analyzed pyrethroids were interpolated following the IDW method for predicting pyrethroid resistance. The populations of Ae. aegypti showed a high frequency of resistance to permethrin with a low to moderate intensity, which was associated with the triple-resistant haplotype LL410/II1016/CC1534. They remain susceptible to deltamethrin and, in some populations, expressed the risk of developing resistance to lambda-cyhalothrin.

Fitness Cost of Sequential Selection with Deltamethrin in Aedes aegypti (Diptera: Culicidae).

In Mexico, Aedes aegypti (L.) is the primary dengue vector, chikungunya, and Zika viruses. The continued use of synthetic pyrethroids has led to the development of resistance in target populations, which has diminished the effectiveness of vector control programs. Resistance has been associated with disadvantages that affect the biological parameters of resistant mosquitoes compared to susceptible ones. In the present study, the disadvantages were evaluated by parameters related to survival and reproduction ('fitness cost') after selection with deltamethrin for five generations. The parameters analyzed were the length of the development cycle, sex ratio, survival, longevity, fecundity, egg viability, preoviposition, oviposition and postoviposition periods, and growth parameters. In the deltamethrin-selected strain, there was a decrease in the development cycle duration, the percentage of pupae, the oviposition period, and eggs viability. Although mean daily fecundity was not affected after the selection process, this, together with the decrease in the survival and fecundity levels by specific age, significantly affected the gross reproductive rate (GRR), net reproductive rate (Ro), and intrinsic growth rate (rm) of the group selected for five generations with deltamethrin compared to the group without selection. Identifying the 'cost' of resistance in biological fitness represents an advantage if it is desired to limit the spread of resistant populations since the fitness cost is the less likely that resistant individuals will spread in the population. This represents an important factor to consider in designing integrated vector management programs.

Update on the frequency of Ile1016 mutation in voltage-gated sodium channel gene of Aedes aegypti in Mexico.

We analyzed 790 Aedes aegypti from 14 localities of Mexico in 2009 to update information on the frequency of the Ile1016 allele in the voltage-gated sodium channel gene that confers resistance to pyrethroids and DDT. The Ile1016 mutation was present in all 17 collections, and was close to fixation in Acapulco (frequency = 0.97), Iguala (0.93), and San Nicolas (0.90). Genotypes at the 1016 locus were not in Hardy-Weinberg proportions in collections from Panuco, Veracruz, Cosoleacaque, Coatzacoalcos, Tantoyuca, and Monterrey due in every case to an excess of homozygotes. The high frequencies of this mutation in Ae. aegypti are probably due to selection pressure from pyrethroid insecticides, particularly permethrin, which has been used in mosquito control programs for >10 years in Mexico.

Insecticide-treated house screening protects against Zika-infected Aedes aegypti in Merida, Mexico.

BACKGROUND: The integration of house-screening and long-lasting insecticidal nets, known as insecticide-treated screening (ITS), can provide simple, safe, and low-tech Aedes aegypti control. Cluster randomised controlled trials in two endemic localities for Ae. aegypti of south Mexico, showed that ITS conferred both, immediate and sustained (~2 yr) impact on indoor-female Ae. aegypti infestations. Such encouraging results require further validation with studies quantifying more epidemiologically-related endpoints, including arbovirus infection in Ae. aegypti. We evaluated the efficacy of protecting houses with ITS on Ae. aegypti infestation and arbovirus infection during a Zika outbreak in Merida, Yucatan, Mexico. METHODOLOGY/PRINCIPAL FINDINGS: A two-arm cluster-randomised controlled trial evaluated the entomological efficacy of ITS compared to the absence of ITS (with both arms able to receive routine arbovirus vector control) in the neighbourhood Juan Pablo II of Merida. Cross-sectional entomological surveys quantified indoor adult mosquito infestation and arbovirus infection at baseline (pre-ITS installation) and throughout two post-intervention (PI) surveys spaced at 6-month intervals corresponding to dry/rainy seasons over one year (2016-2017). Household-surveys assessed the social reception of the intervention. Houses with ITS were 79-85% less infested with Aedes females than control houses up to one-year PI. A similar significant trend was observed for blood-fed Ae. aegypti females (76-82%). Houses with ITS had significantly less infected female Ae. aegypti than controls during the peak of the epidemic (OR = 0.15, 95%CI: 0.08-0.29), an effect that was significant up to a year PI (OR = 0.24, 0.15-0.39). Communities strongly accepted the intervention, due to its perceived mode of action, the prevalent risk for Aedes-borne diseases in the area, and the positive feedback from neighbours receiving ITS. CONCLUSIONS/SIGNIFICANCE: We show evidence of the protective efficacy of ITS against an arboviral disease of major relevance, and discuss the relevance of our findings for intervention adoption.

A novel amino acid substitution in the para-sodium channel gene in Rhipicephalus microplus (Acari: Ixodidae) associated with knockdown resistance.

Resistance acquired by the tick Rhipicephalus microplus (Canestrini) to different types of ixodicides in Mexico has had a negative impact on national and local livestock, mainly due to the transmission of diseases such as babesiosis and anaplasmosis, among others. The technique used for the diagnosis of resistance was that in the bioassays noted in the Norma Oficial Mexicana (NOM-006-ZOO-1994). The purpose of this investigation was the determination of resistance to pyrethroids through isoleucine-phenylalanine mutation in the gene KDR, in a population of ticks from Montemorelos, NL, Mexico. Preliminary bioassays demonstrated resistance to cypermethrin and deltamethrin (27.4%) and flumethrin (36.7-34.7%). To identify the mutation, DNA was extracted from 100 mg of larvae (pools), 10 pools were assessed by PCR, in which a pair of primers designed with the program Oligo 2.0 and Amplify 1.2 amplified a 136 bp fragment containing the mutation. The PCR product was subsequently sequenced to confirm the presence of the mutation. A strain susceptible to pyrethroid insecticides (Mora strain) was used as control, but it did not show the mutation. However, the mutation was detected in 4 out of 10 samples of the strain Montemorelos.

Susceptibility to Pyrethroids and the First Report of L1014F kdr Mutation in Culex quinquefasciatus (Diptera: Culicidae) in Colombia.

The use of insecticides for the control of Aedes aegypti (L.) (Diptera: Culicidae) in Colombia has indirectly influenced the susceptibility status of Culex quinquefasciatus Say populations. We evaluated pyrethroid susceptibility in two populations of Cx. quinquefasciatus in the Atlantico Department of Colombia and its possible resistance mechanism (kdr mutation). Bottle bioassays were performed for permethrin, deltamethrin, and λ-cyhalothrin in female mosquitoes of Cx. quinquefasciatus. The resistance ratios (RRs) for KC50 and LC50 for each insecticide in the field populations examined were determined, using the Cartagena strain as the susceptible control. The L1014F kdr mutation was identified in the para gene of the voltage-gated sodium channel (vgsc), along with its allelic and genotypic frequency. Low knockdown resistance (RRKC50) to deltamethrin was found in Puerto Colombia and Soledad populations as well as low resistance to λ-cyalothrin in this latter population. Moderate knockdown resistance to permethrin was found in both populations. At 24 h post-exposure on the other hand, there was low resistance (RRLC50) to permethrin in Puerto Colombia and moderate resistance in Soledad. Moderate resistance to deltamethrin was found in Puerto Colombia and low resistance in Soledad. Low resistance to λ-cyhalothrin was seen in Puerto Colombia and moderate resistance in Soledad. Variability was found in the susceptibility to the pyrethroids in the populations of Cx. quinquefasciatus evaluated, and the L1014F kdr mutation is reported for the first time as a possible pyrethroid resistance mechanism in this species in Colombia.

Impact of deltamethrin selection on kdr mutations and insecticide detoxifying enzymes in Aedes aegypti from Mexico.

BACKGROUND: Insecticide resistance is a serious problem for vector control programmes worldwide. Resistance is commonly attributed to mutations at the insecticide's target site or increased activity of detoxification enzymes. METHODS: We determined the knockdown concentration (KC50) and lethal concentration (LC50) of deltamethrin in six natural populations of adult Aedes aegypti from southeastern Mexico. These populations were then selected over five generations using the LC50 from the preceding generation that underwent selection, and the heritability of deltamethrin resistance was quantified. For each generation, we also determined the frequency of the kdr alleles L410, I1016 and C1534, and the levels of activity of three enzyme families (α- and ß-esterases, mixed-function oxidases and glutathione S-transferases (GST)) associated with insecticide detoxification. RESULTS: There was an increase in KC50 and LC50 values in the subsequent generations of selection with deltamethrin (FS5vs FS0). According to the resistance ratios (RRs), we detected increases in LC50 ranging from 1.5 to 5.6 times the values of the parental generation and in KC50 ranging from 1.3-3.8 times the values of the parental generation. Triple homozygous mutant individuals (tri-locus, LL/II/CC) were present in the parental generations and increased in frequency after selection. The frequency of L410 increased from 1.18-fold to 2.63-fold after selection with deltamethrin (FS5vs FS0) in the populations analyzed; for I1016 an increase between 1.19-fold to 2.79-fold was observed, and C1534 was fixed in all populations after deltamethrin selection. Enzymatic activity varied significantly over the generations of selection. However, only α- esterase activity remained elevated in multiple populations after five generations of deltamethrin selection. We observed an increase in the mean activity levels of GSTs in two of the six populations analyzed. CONCLUSIONS: The high levels of resistance and their association with high frequencies of kdr mutations (V410L, V1016I and F1534C) obtained through artificial selection, suggest an important role of these mutations in conferring resistance to deltamethrin. We highlight the need to implement strategies that involve the monitoring of kdr frequencies in insecticide resistance monitoring and management programmes.

Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1016I and F1534C in Aedes aegypti (L.).

BACKGROUND: Knockdown resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of non-synonymous mutations in the voltage-gated sodium channel (vgsc) gene, and these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1016I and F1534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. METHODS: A multiplex PCR was developed to detect the V410L, V1016I and F1534C mutations in Ae. aegypti. The validation of the technique was carried out by DNA sequencing using field populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. RESULTS: The standardized protocol for multiplex end-point PCR was highly effective in detecting 15 genotypes considering the three mutations V410L, V1106I and F1534C, in 12 field populations of Ae. aegypti from Mexico. A complete concordance with AS-PCR and DNA sequencing was found for the simultaneous detection of the three kdr mutations. CONCLUSIONS: Our diagnostic method is highly effective for the simultaneous detection of V410L, V1016I and F1534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.