Evaluation of a polymerase chain reaction assay for detection of pyrethroid insecticide resistance in the malaria vector species of the Anopheles gambiae complex

A published polymerase chain reaction assay proved reliable for detecting nerve-insensitivity (kdr-type) resistant and susceptible alleles to the pyrethroid insecticide permethrin in the malaria vectors Anopheles gambiae s.s. and An. arabiensis and the nonvector species An. quadriannulatus. The assay detects pyrethroid susceptible and resistance alleles mediated by a mutation in region II of the para-type sodium channel gene, showing that resistance is conferred by pyrethroid target site insensitivity. Analysis of results of the assay suggests that more than 1 mechanism conferring pyrethroid resistance is operating in an An. gambiae s.s. strain from C?te d'Ivoire. The value of the assay as a tool for monitoring this mode of insecticide resistance in wild populations is discussed.     

A new tactic for Triatoma infestans control: fabrics impregnated with beta-cypermethrin.

Triatoma infestans is the most important vector of Chagas' disease in Argentina. Experiences from Argentine national campaigns have shown indoor spraying to control triatomine bugs is both expensive and difficult. An alternative control method may be the use of pyrethroid-impregnated fabrics (PIFs), which could be both a practical and cheap complement to conventional control measures. In this study, a formulation of emulsifiable concentrate of beta-cypermethrin [alpha-cyano-3-phenoxybenzyl-3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropanecarboxilate] pyrethroid was used to impregnate different fabrics. The fabrics were then tested for their efficacy and persistence in killing Triatoma infestans. Insects attempting to penetrate or grasp the fabric in search of blood meals were poisoned by the exposure to the absorbed pyrethroid. Laboratory bioassays showed that the insecticidal effect of the PIFs with a dosage of 0.2 g/m2 lasted longer on wool than on cotton or rayon. Eight months after impregnation, a residual efficacy of 100% was found for wool, 80% for rayon-cotton fabrics, and 50% for rayon-polyester fabrics. In addition, beta-cypermethrin-impregnated fabrics showed a better repellency effect than did fabrics impregnated with either deltamethrin or cypermethrin. For field trials, fabrics were impregnated either in the laboratory or "in situ" at a dosage of 1 g beta-cypermethrin/m2 and then dried. The PIFs were placed inside homes, either under the roof or under the bed mattress. The field trials showed the PIF approach to be very effective in keeping dwellings free of triatomine bugs for at least one year and found a high degree of acceptability among the houses' residents.     

Evidence of congenital transmission of Trypanosoma cruzi in a vector-free area of Bolivia

A seroprevalence survey of Trypanosoma cruzi was carried out in two areas of South Bolivia. Triatoma infestans, the main vector of Tryp. cruzi, was abundant in the first area, but absent in the second one. Titration of Tryp. cruzi antibodies was carried out in children aged 6-24 months and their mothers. The seroprevalence of Chagas' disease was significantly higher in the area with the vector, but also high in the second area. Chagas' infection in children under 2 years old could be linked to congenital transmission of parasites during pregnancy and/or delivery, despite active vector control in both areas.     

Partial characterization of the voltage-gated sodium channel gene and molecular detection of permethrin resistance in Rhipicephalus annulatus (Say, 1821)

The cattle tick, Rhipicephalus annulatus (Say) is a vector of bovine babesiosis and responsible for direct and indirect losses to cattle producing areas located in temperate and subtropical dry regions. Resistance against pyrethroids has been reported for this species in Asia and Africa, but never before in North America. An outbreak strain, Rio Lado, collected close to the border between Mexico and the United States, in Maverick County, Texas, showed low level of resistance to permethrin, a pyrethroid pesticide. We used genomic material from different strains of cattle ticks collected within the Permanent Quarantine Zone (Rio Lado, Vega and Klein Grass strains) to partially characterize the coding gene of the voltage-gated sodium channel (Na-channel), target-site of pyrethroid pesticides, and search for putative mutations associated with resistance using quantitative PCR high resolution melt (HRM) analysis. The two amplified fragments, corresponding to domains II and III of the Na-channel, were 100 % identical to its ortholog in Rhipicephalus microplus (Canestrini). No nucleotide polymorphisms in the Na-channel gene were observed in the pyrethroid-resistant Rio Lado strain, when compared to the susceptible strains Klein Grass and Vega. This study reports the first case of pyrethroid resistance in R. annulatus collected in the United States. Also, we provide new genomic data for this species of tick that allows for the development of a new method to screen for mutations associated with pyrethroid resistance.     

The elimination of Chagas' disease from Brazil

On 9 June 2006 the Pan American Health Organization (PAHO) presented the Minister of Health of Brazil with the International Elimination of Transmission of Chagas' Disease Certificate. This act was the culmination of an intensive process that began in 1991 with the Southern Cone Initiative, a joint agreement between the governments of Argentina, Bolivia, Brazil, Chile, Paraguay, Uruguay and Peru, to control Chagas' disease by the elimination of the main vector, Triatoma infestans. This initiative has been highly successful and the prevalence area of the vector diminished rapidly in the last years. As a consequence, the current seroprevalence in children aged between 0 and 5 years is of the order of 10(-5), a clear indication that transmission, if it is occurring, is only accidental. In this review I calculate the basic reproduction number, R0, for Chagas' disease and demonstrate that its relatively low value (1.25) explains why vectorial transmission was interrupted relatively easily. In addition, I used a mathematical model to forecast how long the remaining cases of the disease, as well as the additional vertically transmitted cases will last.     

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.     

Characterisation of DDT, pyrethroid and carbamate resistance in Anopheles funestus from Obuasi, Ghana

Indoor-resting anopheline mosquitoes were collected from Obuasi, Ghana, and were identified morphologically and by PCR as Anopheles funestus Giles. Wild-caught females were induced to lay eggs. Samples of F1 progeny from each family were divided into cohorts and were either exposed to DDT and permethrin or were stored for biochemical analysis. Bioassay data by family show evidence of DDT and pyrethroid resistance in the parent A. funestus population. The sodium channel gene of DDT survivors and DDT-susceptible individuals was PCR amplified and sequenced to determine whether any kdr-type mutations were present. Molecular analysis of the IIS5-IIS6 segment of the sodium channel gene gave no indication of any kdr-type mutations associated with resistance phenotypes. Biochemical analysis suggests that DDT and pyrethroid resistance may be metabolically mediated, although there were no clear correlations between enzyme levels/activities and insecticide resistance across families. Furthermore, an altered acetylcholinesterase conferring carbamate resistance was evident. These results can be used to plan an effective malaria control strategy in the Obuasi region.     

Insights into the evolution and dispersion of pyrethroid resistance among sylvatic Andean Triatoma infestans from Bolivia

Sylvatic populations of Triatoma infestans represent a challenge to Chagas disease control as they are not targeted by vector control activities and may play a key role in post-spraying house re-infestation. Understanding sylvatic foci distribution and gene flow between sylvatic and domestic populations is crucial to optimize vector control interventions and elucidate the development and spread of insecticide resistance. Herein, the genetic profiles of five Andean T. infestans populations from Bolivia with distinct insecticide susceptibility profiles were compared. Multilocus genotypes based on eight microsatellites and the DNA sequence of a fragment of the cytochrome B (cytB) gene were obtained for 92 individuals. CytB haplotypes were analyzed with previously reported Bolivian T. infestans haplotypes to evaluate putative historical gene flow among populations. Each specimen was also screened for two nucleotide mutations in the sodium channel gene (kdr), related to pyrethroid resistance (L1014 and L9251). Significant genetic differentiation was observed among all populations, although individuals of admixed origin were detected in four of them. Notably, the genetic profiles of adjacent domestic and sylvatic populations of Mataral, characterized by higher levels of insecticide resistance, support their common ancestry. Only one sylvatic individual from Mataral carried the kdr mutation L1014, suggesting that this mechanism is unlikely to cause the altered insecticide susceptibility observed in these populations. However, as the resistance mutation is present in the area, it has the potential to be selected under insecticidal pressure. Genetic comparisons of these populations suggest that insecticide resistance is likely conferred by ancient trait(s) in T. infestans sylvatic populations, which are capable of invading domiciles. These results emphasize the need for stronger entomological surveillance in the region, including early detection of house invasion, particularly post-spraying, monitoring for resistance to pyrethroids and the design of integrative control actions that consider sylvatic foci around domestic settings and their dispersion dynamics.     

Risk of transmission of Trypanosoma cruzi by wild Triatoma infestans (Hemiptera: Reduviidae) in Bolivia supported by the detection of human blood meals

We analyzed the food sources of Bolivian wild Triatoma infestans (the main vector of Chagas disease in this country), to assess the role of these populations in the epidemiological context of Chagas disease. Ninety-eight blood meals were identified by heteroduplex assay and sequencing. Most of them were from wild mammals but surprisingly 27 were from humans. This brings to light the occurrence of human–vector contacts at risk of Trypanosoma cruzi transmission in the wild environment by highly infected insects.     

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma infestans (Heteroptera: Reduviidae).

Triatoma infestans, the main vector of Chagas disease in the southern cone countries, is the principal target of a regional elimination program. A better understanding of its dispersal, sources of reinfestation, and insecticide resistance is key to an effective control program. To address such problems, we identified and characterized 13 microsatellite loci of T. infestans. For each locus, primer sequences and PCR conditions are presented. Allele variability and frequency were analyzed in 59 T. infestans specimens from different rural communities in northwestern Argentina; nine loci were considered suitable for population genetic studies. Departure from Hardy-Weinberg equilibrium was detected in 10/13 loci with F(IS) values ranging from 0.04 to 0.91, indicating heterozygote deficit and a possible grade of sub-structure in the sample analyzed. Presence of null alleles in some loci cannot be discarded. The present work provides a promising tool to develop a population genetic study of natural populations of T. infestans in tandem with field studies and analyses of bug dispersal and the reinfestation process.     

Genomic changes of Chagas disease vector, South America

We analyzed the main karyologic changes that have occurred during the dispersion of Triatoma infestans, the main vector of Chagas disease. We identified two allopatric groups, named Andean and non-Andean. The Andean specimens present C-heterochromatic blocks in most of their 22 chromosomes, whereas non-Andean specimens have only 4-7 autosomes with C-banding. These heterochromatin differences are the likely cause of a striking DNA content variation (approximately 30%) between Andean and non-Andean insects. Our study, together with previous historical and genetic data, suggests that T. infestans was originally a sylvatic species, with large quantities of DNA and heterochromatin, inhabiting the Andean region of Bolivia. However, the spread of domestic T. infestans throughout the non-Andean regions only involved insects with an important reduction of heterochromatin and DNA amounts. We propose that heterochromatin and DNA variation mainly reflected adaptive genomic changes that contribute to the ability of T. infestans to survive, reproduce, and disperse in different environments.     

A Preliminary Toxicology Study on Eco-friendly Control Target of Spodoptera frugiperda

Pyrethroid and indoxacarb are commonly used pesticides to control the fall armyworm (Spodoptera frugiperda) in the crops. There are a series of consequences caused by the use of pyrethroid and indoxacarb pesticides under emergency control, such as pest resistance development, contamination of soil, water, and farm products. This study analyzed the structure and physiological function of the sodium channel in S. frugiperda, which is an important step to elaborate the resistance mechanism of S. frugiperda to indoxacarb and pyrethroid pesticides. According to genetic analysis, the cloned cDNA sequences of sodium channel in S. frugiperda (SfNa_v) showed the shortest genetic distance with that of the sodium channel in Helicoverpa armigera. Under the induction of three pesticides, the expression of SfNa_v decreased in the first 12 h and then increased after 24 h. It was concluded that SfNa_v had a typical structure of the sodium channel of insects and its down-regulated expression can decrease the combination of S. frugiperda with pyrethroid and indoxacarb pesticides. The up-regulated expression of SfNa_v was conducive to the enhancement of the pesticide resistance.

     

Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria

Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major concern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa and are currently being investigated in West Africa. Here we report the co-occurrence of both resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s. from the same geographical area revealed >50% of the West African kdr mutation in the resistant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using pyperonyl butoxide before permethrin exposure showed a significant increase in mortality compared with the non-synergized. Biochemical assays showed an increased level of monooxygenase but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-expressed genes in the resistant strain when compared with the susceptible one. Two of these, CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae s.s, and could constitute a novel set of candidate genes that warrant further investigation.     

Fluctuating asymmetry and exposure to pyrethroid insecticides in Triatoma infestans populations in northeastern Argentina

Fluctuating asymmetry (FA), a phenotypic marker used as indicator of developmental stress or instability, is sometimes associated with insecticide application and resistance. Here we investigated the occurrence and amount of wing size and wing shape FA in Triatoma infestans females and males collected before and 4 months after a community-wide pyrethroid spraying campaign in a well-defined rural area of Pampa del Indio, Argentina. Moderate levels of pyrethroid resistance were previously confirmed for this area, and postspraying house infestation was mainly attributed to this condition. In the absence of insecticide-based selective pressures over the previous 12 years, we hypothesized that 1- if postspraying triatomines were mostly survivors to insecticide spraying (pyrethroid resistant), they would have higher levels of FA than prespraying triatomines. 2- if postspraying triatomines have a selective advantage, they would have lower FA levels than their prespraying counterparts, whereas if postspraying infestation was positively associated with immigrants not exposed to the insecticide, prespraying and postspraying triatomines would display similar FA levels. For 243 adult T. infestans collected at identified sites before insecticide spraying and 112 collected 4 months postspraying, wing size and wing shape asymmetry was estimated from landmark configurations of left and right sides of each individual. At population level, wing size and shape FA significantly decreased in both females and males after spraying. Males displayed greater wing size and shape FA than females. However, at a single peridomestic site that was persistently infested after spraying, FA declined similarly in females whereas the reverse pattern occurred in males. Our results suggest differential survival of adults with more symmetric wings. This pattern may be related to a selective advantage of survivors to insecticide spraying, which may be mediated or not by their pyrethroid-resistant status or to lower triatomine densities after insecticide spraying and the concomitant increase in feeding success.     

The relationship between Triatoma infestans, poultry, and humans in a rural settlement in Santiago del Estero, Argentina

An association was determined between Triatona infestans, poultry, and humans in Trinidad, in the Province of Santiago del Estero, Argentina. To collect triatomines, four samples were taken at the area immediately surrounding six houses by the one hour/man capture method (December 1991 - October 1992). Peridomiciliary ecotopes were classified as arthropic (where humans carry out daily activities) or non-anthropic. Tratomine feeding habits were also determined. Of 134 biotopes, 21% had T. infestans, 22% had poultry, and 54% were anthropic. Some 25% of the latter harboured both poultry and T. infestans. Poultry were the only domestic animals associated with T. infestans, a finding that was exclusive to anthropic ecotopes. The proportion of feedings on individual fowl (61/146) was highly significant. Chicken coops are not used in Trinidad, and poultry brood in anthropic structures. Due to the materials used for making their nests and their repeated use, a periodic bug flow can be established from the intra- to the peridomiciliary area and vice versa, through passive transport. The close relationship among T, infestans, poultry, and humans in anthropic biotopes where other T. cruzi reservoirs such as dogs are also present contributes to the maintenance of domestic triatomine colonies and transmission of Chagas' disease to humans.     

Chagas' disease epidemiology in the province of General Paz, Argentina

OBJECTIVE: The aim of this study was to evaluate triatomine colonization in domestic and peridomestic ecotopes and to assess the seroprevalence of Chagas' disease in rural areas. METHODS: The study was carried out in the province of General Paz, Corrientes, Argentina. Human dwellings and peridomestic ecotopes were randomly selected. Triatomines were collected using the man/hour/capture method. The insects were taxonomically determined and classified by age and sex. Trypanosoma cruzi infection was detected through direct microscopy of feces and polymerase chain reaction (PCR). Diagnosis of Chagas infection in volunteer residents was performed using indirect hemagglutination (IHA) and immunoenzimatic assay (ELISA). Infestation, density, colonization, natural infection and dispersion indexes were recorded. RESULTS; A total of 42 households and 50 peridomestic areas were examined. Triatoma infestans domestic infestation and infection indexes were 23.8 and 19.4; respectively. Density, colonization and dispersion indexes were 2.1; 47.0 and 50.0; respectively. Of all, 5.9% of peridomiciles were infested by T. infestans and 11.8% by Triatoma sordida. Trypanosoma cruzi infection was observed in 2.0% of T. sordida. The seroprevalence rate obtained from 85 human sera analyzed was 22.3%. CONCLUSIONS: The results suggest a halt in T. cruzi transmission in the study area. Nevertheless the presence of seroreactive individuals and infected T. infestans vectors might facilitate the resurgence of the transmission cycle. Control actions with the use of insecticides might be considered in order to eliminate vector populations.     

Global trends in the use of insecticides to control vector-borne diseases

Background: Data on insecticide use for vector control are essential for guiding pesticide management systems on judicious and appropriate use, resistance management, and reduction of risks to human health and the environment.Objective: We studied the global use and trends of insecticide use for control of vector-borne diseases for the period 2000 through 2009.Methods: A survey was distributed to countries with vector control programs to request national data on vector control insecticide use, excluding the use of long-lasting insecticidal nets (LNs). Data were received from 125 countries, representing 97% of the human populations of 143 targeted countries.Results: The main disease targeted with insecticides was malaria, followed by dengue, leishmaniasis, and Chagas disease. The use of vector control insecticides was dominated by organochlorines [i.e., DDT (dichlorodiphenyltrichloroethane)] in terms of quantity applied (71% of total) and by pyrethroids in terms of the surface or area covered (81% of total). Global use of DDT for vector control, most of which was in India alone, was fairly constant during 2000 through 2009. In Africa, pyrethroid use increased in countries that also achieved high coverage for LNs, and DDT increased sharply until 2008 but dropped in 2009.Conclusions: The global use of DDT has not changed substantially since the Stockholm Convention went into effect. The dominance of pyrethroid use has major implications because of the spread of insecticide resistance with the potential to reduce the efficacy of LNs. Managing insecticide resistance should be coordinated between disease-specific programs and sectors of public health and agriculture within the context of an integrated vector management approach.     

Voltage-gated sodium channel gene mutations and pyrethroid resistance in Rhipicephalus microplus

Pyrethroid pesticides are extensively used to manage animal and human disease vectors including the southern cattle tick Rhipicephalus microplus (Canestrini). The indiscriminate and incorrect use of pyrethroids has led to the almost ubiquitous development of resistance to this pesticide class for this tick species. Voltage-gated sodium channels (Na-channels) are the primary target-site of pyrethroids and several studies on the involvement of mutations in the coding gene among pyrethroid-resistant R. microplus populations from different parts of the world have shown that there are various single nucleotide polymorphisms (SNPs) that are associated with resistance to pyrethroids. Identification of the exact location of the mutations in the protein coding regions of the targeted gene facilitates the design of various molecular tools for genotyping the resistant populations and thus promotes the rapid detection of resistance. This review aims to provide an update on the identification of pyrethroid resistance-associated Na-channel mutations from R. microplus.The database of diverse mutations from different regions of the world helped us to develop the molecular markers for resistance monitoring in a rapid and efficient manner. Their role and the development of different forms of molecular tools for genotyping ticks for mutations in the Na-channel gene are also discussed. In this review, the word mutation is used interchangeably with SNP.