Spatial and temporal country-wide survey of temephos resistance in Brazilian populations of Aedes aegypti

The organophosphate temephos has been the main insecticide used against larvae of the dengue and yellow fever mosquito (Aedes aegypti) in Brazil since the mid-1980s. Reports of resistance date back to 1995; however, no systematic reports of widespread temephos resistance have occurred to date. As resistance investigation is paramount for strategic decision-making by health officials, our objective here was to investigate the spatial and temporal spread of temephos resistance in Ae. aegypti in Brazil for the last 12 years using discriminating temephos concentrations and the bioassay protocols of the World Health Organization. The mortality results obtained were subjected to spatial analysis for distance interpolation using semi-variance models to generate maps that depict the spread of temephos resistance in Brazil since 1999. The problem has been expanding. Since 2002-2003, approximately half the country has exhibited mosquito populations resistant to temephos. The frequency of temephos resistance and, likely, control failures, which start when the insecticide mortality level drops below 80%, has increased even further since 2004. Few parts of Brazil are able to achieve the target 80% efficacy threshold by 2010/2011, resulting in a significant risk of control failure by temephos in most of the country. The widespread resistance to temephos in Brazilian Ae. aegypti populations greatly compromise effective mosquito control efforts using this insecticide and indicates the urgent need to identify alternative insecticides aided by the preventive elimination of potential mosquito breeding sites.

Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

Fitness evaluation of two Brazilian Aedes aegypti field populations with distinct levels of resistance to the organophosphate temephos

In Brazil, decades of dengue vector control using organophosphates and pyrethroids have led to dissemination of resistance. Although these insecticides have been employed for decades against Aedes aegypti in the country, knowledge of the impact of temephos resistance on vector viability is limited. We evaluated several fitness parameters in two Brazilian Ae. aegypti populations, both classified as deltamethrin resistant but with distinct resistant ratios (RR) for temephos. The insecticide-susceptible Rockefeller strain was used as an experimental control. The population presenting the higher temephos resistance level, Aparecida de Goiânia, state of Goiás (RR95 of 19.2), exhibited deficiency in the following four parameters: blood meal acceptance, amount of ingested blood, number of eggs and frequency of inseminated females. Mosquitoes from Boa Vista, state of Roraima, the population with lower temephos resistance level (RR95 of 7.4), presented impairment in only two parameters, blood meal acceptance and frequency of inseminated females. These results indicate that the overall fitness handicap was proportional to temephos resistance levels. However, it is unlikely that these disabilities can be attributed solely to temephos resistance, since both populations are also resistant to deltamethrin and harbour the kdr allele, which indicates resistance to pyrethroids. The effects of reduced fitness in resistant populations are discussed.

Impact of environmental temperatures on resistance to organophosphate insecticides in Aedes aegypti from Trinidad / Repercusión de las temperaturas ambientales sobre la resistencia de Aedes aegypti a los insecticidas organofosforados en Trinidad

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and β-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.

OBJETIVO: Examinar los efectos del aumento de las temperaturas de desarrollo larvario sobre el estado de resistencia a los insecticidas organofosforados de las poblaciones de Aedes aegypti en Trinidad. MÉTODOS: En 2007 y 2008 se llevaron a cabo ensayos biológicos y bioquímicos en larvas de A. aegypti recogidas en el 2006 de ocho áreas geográficamente separadas en Trinidad (Trinidad y Tabago). Las poblaciones larvarias se desarrollaron en cuatro temperaturas (28 ± 2 ºC, 32 ºC, 34 ºC y 36 ºC) antes de los ensayos biológicos con insecticidas organofosforados (fentión, malatión y temefós) y los análisis bioquímicos para las enzimas de esterasa. RESULTADOS: La mayoría de las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC fueron susceptibles al fentión (mortalidad > 98%) pero resistentes al malatión y al temefós (mortalidad < 80%). Se encontró una asociación positiva entre la resistencia a los insecticidas organofosforados y la mayor actividad de αy β-esterasas en las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC. Aunque las poblaciones larvarias que se desarrollaron a temperaturas mayores mostraron variaciones en la resistencia a los organofosforados, hubo un aumento general de la sensibilidad. Sin embargo, los aumentos o las disminuciones en los niveles de actividad de las enzimas no siempre se correspondieron con un aumento o disminución en la proporción de individuos resistentes desarrollados a las temperaturas más altas. CONCLUSIONES: Aunque el recalentamiento del planeta puede causar un aumento de la transmisión del dengue, según los resultados de este estudio el uso de insecticidas para la prevención y el control del dengue todavía puede ser eficaz si las temperaturas aumentan según lo proyectado.

Bottle and biochemical assays on temephos resistance in Aedes aegypti in Thailand.

The bottle bioassay measuring the time-mortality rate is a simplified procedure for detecting insecticide resistance. It can be used with a biochemical microplate assay to identify the mechanism involved. This integrated approach was used to detect temephos resistance in Aedes aegypti from Nonthaburi (lowest use) and Roi Et (highest use). Ae. aegypti BKK1 laboratory strain was used as the susceptible reference strain. The appropriate concentration of insecticide for bottle bioassay was determined empirically for Ae. aegypti BKK1 strain and found to be in the range of 800-1,050 microg/bottle. The time-mortality rate at 800 microg/bottle was 170 +/- 8.66 minutes, significantly different from the time-mortality rates in the 850, 900, 950, and 1,050 microg/bottle (p = 0.008) concentrations, which were 135 +/- 15.00, 140 +/- 8.66, 135 +/- 15.00, and 125 +/- 8.66 minutes, respectively. The cut-off concentration selected for resistance detection was 850 microg/bottle. The time-mortality rate for the Roi Et strain was 382 +/- 26.41 minutes, significantly higher than the Nonthaburi (150 +/- 25.10 minutes) and BKK1 strains (145 +/- 20.49 minutes) (p < 0.001). The temephos resistance ratio (RR100) for the Ae. aegypti Roi Et strain was 2.64-fold higher at lethal time (LT100) than for the reference Ae. aegypti BKK1 strain. The mean optical density (OD) value from the biochemical microplate assay for the non-specific esterase of the Roi Et strain was higher than the mean OD for the non-specific esterase of both the Nonthaburi and BKK1 strains. Insensitive acetylcholinesterase was not found to be responsible for the resistance in the field-collected mosquitos. This study suggests that esterase detoxification is the primary cause of resistance in the Ae. aegypti population from Roi Et. Both the bottle bioassay and the biochemical microplate assay were proven to be promising tools for initial detection and field surveillance for temephos resistance.

Laboratory bio-assay of temephos and fenthion against some vector species of public health importance.

Laboratory studies carried out using Temephos and Fenthion, two commonly used larvicides under Urban Malaria Scheme (NMEP) and NFCP for the control of mosquito larvae. The results revealed that the LC50 and LC90 values for temephos against Aedes aegypti 0.0177 and 0.0559, Anopheles stephensi 0.0148 and 0.0472, Culex quinquefasciatus 0.0157 & 0.0480 and for Culex vishnui group of mosquitoes 0.043 & 0.0118 ppm respectively. The results obtained revealed that there is a 62.8 & 94.12 times increase in the LC50 & LC90 of Cx. Quinquefasciatus which indicates that the species has developed resistance to temephos. There were 6.32 & 8.34 fold increase in Ae. aegypti and 2.27 & 2.34 fold increase in LC50 & LC90 values of An. stephensi are indicative of development of tolerance against temephos. Similarly LC50 and LC90 values estimated for fenthion against Ae. aegypti 0.0173 & 0.0392, An. stephensi 0.0274 & 0.0992 and Cx. quinquefasciatus 0.03 & 0.0469 respectively. The slope values were found to be higher in fenthion as compared to temephos. It was recorded to be 2.72 times higher in Cx. quinquefasciatus and 1.54 times against An. stephensi. However, much difference was not observed in the slope values of temephos and fenthion in Ae. aegypti.

Temephos resistance in two forms of Aedes aegypti and its significance for the resistance mechanism.

Aedes aegypti, at the larval stage, has been subjected to the temephos selection in laboratory. The level of temephos resistance was detected in a microplate by biochemical assay using WHO bioassay technique. The major enzyme-based resistance mechanisms involved in temephos resistance include elevated nonspecific esterase, oxidase and insensitive acetylcholinesterase. After 19 generations of temephos selection, the selected group showed resistance ratios of 4.64 and 16.92, when compared with a non-selected group and the WHO susceptible strain, respectively. The two seperated forms, type form and the pale form of Ae. aegypti showed low levels of resistance to temephos after 19 generations of selection, with resistance ratios of 4.82 and 4.07 for the type form and the pale form, respectively; when compared with the non-selected strain, 17.58 and 14.84, when compared with the WHO susceptible strain. This showed that the type form could develop higher level resistance than the pale form. The esterase inhibitor (S,S,S-tributyl phosphorotrithioate, DEF) or synergist implicated detoxifying esterase in all the temephos selected groups and the presence of elevated esterase were confirmed by biochemical assay. There were significant differences in elevated esterase activity between the temephos selected groups and the non-selected group. However no significant difference between the type form and the pale form was found. Besides the elevated esterase, there was no change in monooxygenase activity and no evidence of insensitive acetylcholinesterease for all temephos selected groups. These results suggest that temephos resistance could be developed in Ae. aegypti under selection pressure and that the main mechanism is based only on esterase detoxification.

Chromosomal comparisons among and within populations of Simulium (Chirostilbia) pertinax (Diptera, Simuliidae)

Chromosomal studies were carried on six larval populations of Simulium (Chirostilbia) pertinax from different locations in Brazil. Larvae were collected in the states of Paraná, Rio Grande do Sul, Rio de Janeiro and São Paulo. Polytene chromosome map comparisons within and among populations showed no differences in banding pattern, except for some limited polymorphism (secondary NOR and four band polymorphisms). There were no chromosomal variations associated with the resistance or susceptibility of the larvae to temephos. The chromosomal homosequentiality found among the six populations suggests that S. pertinax may be a monomorphic species

Field assessment of bacillus thuringiensis serotype H-14 [bactimos W.P], abatc and fuel ol against anopheles and culex in bandar abbas [Sooth of Iran]

The Optimum dosage for field application bactimos [W.P.] was determined as 0.04g/m, when efficacy of four concentration of 0.01, 0.02, 0.04 and 0.08 gim2 were compared in 1 x I m2 artificial breeding sites. This dosage caused 69.8%, 55.6% and 43.6% anopheline and 83%, 47.8% and 29.7% culicine larval mortality, in 24.48% and 72 hrs post treatment, respectively. Abate and fuel oil were significantly more effective than hactimos [W.P] as they caused 100%, 99.7% and 87.2% anopheline and 100%, 93.5% and 87% culicine lavrval mortality, in 24 hr post treatment, respectivelly. In natural breeding sites Abate, fuel oil and Bactimos [W.P.] caused 100%, 98% and 69% anopheline and 99.9%, 99% and 79.3% culicine larval mortality, in 24 hr post treatment, respectively. The wettable powder formulation of Bactimos was not regarded as a suitable formulation for anopheline control in Southern Iran

Laboratory and field tests of temephos (AbateR) on mosquito larvae and non-target organisms in rice fields in Malaysia.

The toxic effects of Abate (temephos) on mosquito larvae and non-target organisms were studied in the rice-field and in the laboratory. In the laboratory tests, Culex tritaeniorhychus larvae and cladoceran zooplanktons (predominantly Diaphanosoma and Moinodaphnia species) were found to be highly susceptible to Abate with LC50 values of 0.27 and less than 0.10 parts per billion respectively. Other non-target species in decreasing degree of susceptibility to Abate were copepods (Tropodiaptomus spp.), Aplocheilus panchax and Tubifex worms. In field study, Abate at concentrations 60, 100 and 200 gm hectare-1 is effective in maintaining the rice-fields free of Anopheles and Culex mosquitoes for at least 2 days. No mortality was observed for Aplocheilus panchax and Tubifex worms at the above concentrations; nevertheless, populations of cladoceran zooplanktons and copepods were reduced up to seven days posttreatment.