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.

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.

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.

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.

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.

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.

Distribution and Frequency of the kdr Mutation V410L in Natural Populations of Aedes aegypti (L.) (Diptera: Culicidae) From Eastern and Southern Mexico.

Aedes aegypti (L.) is the primary vector of the viruses that cause dengue, Zika, and chikungunya, for which effective vaccines and drugs are still lacking. Current strategies for suppressing arbovirus outbreaks based on insecticide use pose a challenge because of the rapid increase in resistance. The widespread and excessive use of pyrethroid-based insecticides has created a large selection pressure for a kdr-type resistance, caused by mutations in the para gene of the voltage-gated sodium channel (vgsc). Our objective was to evaluate the allelic frequency of natural populations of Ae. aegypti of Mexico at codon 410 of the para gene. Twenty-six Ae. aegypti populations from east and southern Mexico were genotyped for the codon 410 using allele-specific PCR. The frequencies of the L410 allele in Ae. aegypti ranged from 0.10 to 0.99; however, most of the frequencies were in the range of 0.36 to 0.64. The highest frequencies were found in three populations from the state of Veracruz, namely, Minatitlan with 0.99, Poza Rica with 0.82, and Jose Cardel with 0.97, along with populations from Cancun in Quintana Roo with 0.93, Frontera in Tabasco with 0.91, and Ciudad del Carmen in Campeche with 0.86. The frequency of the L410 allele was high in all populations of Ae. aegypti with higher values in populations of the southeast of the country. The knowledge of specific substitutions in vgsc and their interaction to confer resistance is essential to predict and develop future strategies for resistance management in Ae. aegypti in Mexico.

Efficacy of novel indoor residual spraying methods targeting pyrethroid-resistant Aedes aegypti within experimental houses.

Challenges in maintaining high effectiveness of classic vector control in urban areas has renewed the interest in indoor residual spraying (IRS) as a promising approach for Aedes-borne disease prevention. While IRS has many benefits, application time and intrusive indoor applications make its scalability in urban areas difficult. Modifying IRS to account for Ae. aegypti resting behavior, named targeted IRS (TIRS, spraying walls below 1.5 m and under furniture) can reduce application time; however, an untested assumption is that modifications to IRS will not negatively impact entomological efficacy. We conducted a comparative experimental study evaluating the residual efficacy of classically-applied IRS (as developed for malaria control) compared to two TIRS application methods using a carbamate insecticide against a pyrethroid-resistant, field-derived Ae. aegypti strain. We performed our study within a novel experimental house setting (n = 9 houses) located in Merida (Mexico), with similar layouts and standardized contents. Classic IRS application (insecticide applied to full walls and under furniture) was compared to: a) TIRS: insecticide applied to walls below 1.5 m and under furniture, and b) Resting Site TIRS (RS-TIRS): insecticide applied only under furniture. Mosquito mortality was measured eight times post-application (out to six months post-application) by releasing 100 Ae. aegypti females /house and collecting live and dead individuals after 24 hrs exposure. Compared to Classic IRS, TIRS and RS-TIRS took less time to apply (31% and 82% reduction, respectively) and used less insecticide (38% and 85% reduction, respectively). Mortality of pyrethroid-resistant Ae. aegypti did not significantly differ among the three IRS application methods up to two months post application, and did not significantly differ between Classic IRS and TIRS up to four months post application. These data illustrate that optimizing IRS to more efficiently target Ae. aegypti can both reduce application time and insecticide volume with no apparent reduction in entomological efficacy.

Novel Kdr mutations (K964R and A943V) in pyrethroid-resistant populations of Triatoma mazzottii and Triatoma longipennis from Mexico and detoxifying enzymes.

Although having five different ways of transmission the vector-borne is the principal way of transmission of Chagas disease, which involves insects of the subfamily Triatominae (Hemiptera: Reduviidae). Nineteen of the 31 species that occur in Mexico are associated with humans, and all are capable of transmitting the disease. Pyrethroids are the insecticides recommended for the control of these vectors in Mexico. We determined the susceptibility to the pyrethroids deltamethrin and permethrin of peridomestic populations of Triatoma mazzottii Usinger and two populations of Triatoma longipennis Usinger in comparison with a reference strain for each species. Bioassays were performed for the determination of the LD50 for both field populations and reference strains. A maximum of 27 fold resistance to deltamethrin was observed in T. mazzottii, meanwhile, for permethrin, T. longipennis from Jalisco show the highest value of 3.19 fold. There was significantly increased activity of esterases in field populations in comparison with their corresponding reference strain. The results of the search of kdr mutations related to the resistance to deltamethrin and permethrin in the evaluated species show the presence of mutations in the field populations, as is the case with individuals of T. mazzottii, for which the mutation was found A943V, and for the two populations of T. longipennis included in this study, we report the presence of the kdr mutation K964R. Evaluation of the various mechanisms involved in resistance to pyrethroids in triatomines from Mexico could guide us to the real justification for insecticide resistance monitoring.

Bioefficacy of Two Nonpyrethroid Insecticides for Targeted Indoor Residual Spraying Against Pyrethroid-Resistant Aedes aegypti.

We evaluated the efficacy of bendiocarb (Ficam W® 80%) and pirimiphos-methyl (Actellic 300CS® 28.16%), applied to different surfaces potentially sprayable within houses during the application of a targeted indoor residual spraying (TIRS) against a field pyrethroid-resistant strain of Aedes aegypti. Bioassays with cones were performed on cement (walls), wood (doors), and textile (cloth) surfaces within typical houses in the Mexican city of Merida (n = 10). Optimal residual efficacy (>80% of mean mortality) of bendiocarb ranged from 3 months (cement) to 2 months (wood and textiles). Residual efficacy of pirimiphos-methyl ranged from 5 months (cement) to 2 months (wood and textiles). Both insecticides proved to be effective as adulticides against field Ae. aegypti and may be useful in mosquito control programs implementing TIRS with pyrethroid-resistant populations.

Frequency and intensity of pyrethroid resistance through the CDC bottle bioassay and their association with the frequency of kdr mutations in Aedes aegypti (Diptera: Culicidae) from Mexico.

BACKGROUND: The control of Aedes aegypti (L.), the main urban vector that causes arboviral diseases such as dengue, Chikungunya and Zika, has proved to be a challenge because of a rapid increase in insecticide resistance. Therefore, adequate monitoring of insecticide resistance is an essential element in the control of Ae. aegypti and the diseases it transmits. We estimated the frequency and intensity (Resistance Frequency Rapid Diagnostic Test [F-RDT] and Resistance Intensity Rapid Diagnostic Test [I-RDT]) of pyrethroid resistance in populations of Ae. aegypti from Mexico using the bottle bioassay and results were related to the frequencies of knockdown resistance (kdr) mutations V1016I and F1534C. RESULTS: All populations under study were resistant to the pyrethroids: bifenthrin (99%), d-(cis-trans)-phenothrin (6.3% cis, 91.7% trans) and permethrin (99.5%) according to F-RDT, and showed moderate to high-intensity resistance at 10× the diagnostic dose (DD) in I-RDT. Frequencies of the kdr mutation V1016I in Ae. aegypti populations were correlated with moderate permethrin resistance at 10× DD, whereas F1534C mutation frequencies were correlated with high bifenthrin resistance at 5× DD. Both I1016 and C1535 were highly correlated with high-intensity phenothrin resistance at 1× to 10× DD. CONCLUSIONS: This study showed that high frequencies of kdr mutations V1016I and F1534C are reflected in the results of F-RDT and I-RDT tests. Bioassays in conjunction with the characterization of genetic resistance mechanisms are indispensable in the strategic and rational management of resistance in mosquitoes. © 2018 Society of Chemical Industry.

Report of Chikungunya Virus in Wild Populations of Aedes aegypti in Guerrero State, Mexico.

We detected vertical transmission of chikungunya virus (CHIKV) in wild populations of Aedes aegypti from San Marcos, Guerrero, Mexico, with real-time reverse transcriptase-polymerase chain reaction. A total of 20 pools (1-11 specimens/pool) of larvae, male, and female mosquitoes were tested. We report the detection of CHIKV in 2 of 11 larval pools, 4 of 5 male pools, and 1 of 4 female pools, from field-collected mosquitoes.

Assessing the effect of selection with deltamethrin on biological parameters and detoxifying enzymes in Aedes aegypti (L.).

BACKGROUND: Resistance to insecticides through one or several mechanisms has a cost for an insect in various parameters of its biological cycle. The present study evaluated the effect of deltamethrin on detoxifying enzymes and biological parameters in a population of Aedes aegypti selected for 15 generations. The enzyme activities of alpha- and beta-esterases, mixed-function oxidases and glutathione-S-transferases were determined during selection, along with biological parameters. RESULTS: Overexpression of mixed-function oxidases as a mechanism of metabolic resistance to deltamethrin was found. There were decreases in percentages of eggs hatching, pupation and age-specific survival and in total survival at the end of the selection (F16 ). Although age-specific fecundity was not affected by selection with deltamethrin, total fertility, together with lower survival, significantly affected gross reproduction rate, gradually decreasing due to deltamethrin selection. Similarly, net reproductive rate and intrinsic growth rate were affected by selection. CONCLUSION: Alterations in life parameters could be due to the accumulation of noxious effects or deleterious genes related to detoxifying enzymes, specifically those coding for mixed-function oxidases, along with the presence of recessive alleles of the V1016I and F1534C mutations, associating deltamethrin resistance with fitness cost in Ae. aegypti. © 2017 Society of Chemical Industry.

First Detection of the Kdr Mutation T929I in Head Lice (Phthiraptera: Pediculidae) in Schoolchildren of the Metropolitan Area of Nuevo Leon and Yucatan, Mexico.

The head louse Pediculus humanus capitis (De Geer) is a hematophagous ectoparasite that inhabits the human scalp. Infestations by this insect are commonly known as pediculosis, which is more common in younger groups. These infestations are asymptomatic; however, skin irritation from scratching occasionally may cause secondary bacterial infections. In recent years, the prevalence of pediculosis has increased in children; this increase has been attributed to louse resistance to the insecticides used as a control measure for infestation. The aim of the present study was to determine the presence and frequency of the knockdown resistance mutation (kdr) T929I in 468 head lice collected from 32 elementary schools in the metropolitan area of Nuevo Leon (24) and Yucatan (8), Mexico. This is the first report of a knockdown resistance (kdr) mechanism in head lice from Mexico. The T929I mutation was present in all of the sampled schools, with variability observed in its allelic and genotypic frequencies.

Profiles of Amino Acids and Acylcarnitines Related with Insecticide Exposure in Culex quinquefasciatus (Say).

Culex quinquefasciatus Say is a vector of many pathogens of humans, and both domestic and wild animals. Personal protection, reduction of larval habitats, and chemical control are the best ways to reduce mosquito bites and, therefore, the transmission of mosquito-borne pathogens. Currently, to reduce the risk of transmission, the pyrethroids, and other insecticide groups have been extensively used to control both larvae and adult mosquitoes. In this context, amino acids and acylcarnitines have never been associated with insecticide exposure and or insecticide resistance. It has been suggested that changes in acylcarnitines and amino acids profiles could be a powerful diagnostic tool for metabolic alterations. Monitoring these changes could help to better understand the mechanisms involved in insecticide resistance, complementing the strategies for managing this phenomenon in the integrated resistance management. The purpose of the study was to determine the amino acids and acylcarnitines profiles in larvae of Cx. quinquefasciatus after the exposure to different insecticides. Bioassays were performed on Cx. quinquefasciatus larvae exposed to the diagnostic doses (DD) of the insecticides chlorpyrifos (0.001 µg/mL), temephos (0.002 µg/mL) and permethrin (0.01 µg/mL). In each sample, we analyzed the profile of 12 amino acids and 31 acylcarnitines by LC-MS/MS. A t-test was used to determine statistically significant differences between groups and corrections of q-values. Results indicates three changes, the amino acids arginine (ARG), free carnitine (C0) and acetyl-carnitine (C2) that could be involved in energy production and insecticide detoxification. We confirmed that concentrations of amino acids and acylcarnitines in Cx. quinquefasciatus vary with respect to different insecticides. The information generated contributes to understand the possible mechanisms and metabolic changes occurring during insecticide exposure.

Susceptibility to Temephos and Spinosad in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

We examined the susceptibility to temephos and spinosad (Natular EC) of eight Aedes aegypti (L.) populations from Puerto Rico, following WHO method (WHO 2005). Enzyme activity was measured for alpha- and beta-esterases, multiple function oxidases, glutathione-s-transferases, and insensitive acetylcholinesterase and was tested for correlation with the susceptibility level. The results showed that larval populations from Puerto Rico obtained during 2014 were found to be susceptible to both larvicides, with low (resistance factor) RRLC50 values (<5 fold) and altered and incipiently altered enzyme expression for all populations, except the insensitive acetylcholinesterase enzyme, where only the population of Ponce showed overexpression (53.3%) above the threshold established with the New Orleans susceptible strain. We recommend the use of both larvicides for mosquito control in the study area and encourage further susceptibility monitoring.

First report of L1014F kdr mutation in Culex quinquefasciatus in Mexico.

The L1014F mutation in the voltage-sodium channel gene has been associated with resistance to DDT and pyrethroids in various arthropod species including mosquitoes. We determined the frequency of the L1014F kdr mutation in 16 field populations of Culex quinquefasciatus from Northeastern Mexico collected between 2008 and 2013. The L1014F was present in all populations analyzed with the lowest frequency (3.33%) corresponding to the population from Monclova collected in 2012, and the highest frequency (63.63%) from the Monterrey population collected in 2012. The presence of a kdr mutation in populations of Cx. quinquefasciatus from northeastern Mexico provides evidence of pyrethroid resistance. This requires a special attention, considering that pyrethroid-based insecticides are commonly used in vector-control campaigns, especially against Aedes aegypti (L.).