Multiple insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Boyeros municipality, Cuba and associated mechanisms.

In this study, insecticide resistance and the mechanisms responsible were characterized in Ae. aegypti of Boyeros municipality from Havana, Cuba. Boyeros represents a high epidemiological risk because it is located near the Havana International Airport, it is highly urbanized, and it has a large influx of people from endemic countries so that it qualifies as a sentinel site for surveillance. The larvae collected from five areas of this municipality showed resistance to temephos associated with metabolic enzymes. The adult mosquitoes displayed a deltamethrin resistance and less distinctly to other pyrethroids associated with a high frequency of sodium channel gene mutations (F1534C and V1016I), detected for the first time in a field population from Cuba. The presence in the field populations of two insecticide resistance mechanisms represents a limiting factor in the success of the control operations of this vector, so other strategies should be considered to preserve the effectiveness of the insecticides available in public health for vector control in Cuba.

Insecticide resistance in two Aedes aegypti (Diptera: Culicidae) strains from Costa Rica.

Dengue (family Flaviridae, genus Flavivirus, DENV) and dengue hemorrhagic fever (DHF) are presently important public health problems in Costa Rica. The primary strategy for disease control is based on reducing population densities of the main mosquito vector Aedes aegypti (L.) (Diptera: Culicidae). This is heavily dependent on use of chemical insecticides, thus the development of resistance is a frequent threat to control program effectiveness. The objective of this study was to determine the levels of insecticide resistance and the metabolic resistance mechanisms involved in two Ae. aegypti strains collected from two provinces (Puntarenas and Limon) in Costa Rica. Bioassays with larvae were performed according to World Health Organization guidelines and resistance in adults was measured through standard bottle assays. The activities of beta-esterases, cytochrome P450 monooxygenases, and glutathione S-transferases (GST), were assayed through synergists and biochemical tests, wherein the threshold criteria for each enzyme was established using the susceptible Rockefeller strain. The results showed higher resistance levels to the organophosphate (OP) temephos and the pyrethroid deltamethrin in larvae. The efficacy of commercial formulations of temephos in controlling Ae. aegypti populations was 100% mortality up to 11 and 12 d posttreatment with daily water replacements in test containers. Temephos and deltamethrin resistance in larvae were associated with high esterase activity, but not to cytochrome P450 monooxygenase or GST activities. Adult mosquitoes were resistant to deltamethrin, and susceptible to bendiocarb, chlorpyrifos, and cypermethrin. Because temephos and deltamethrin resistance are emerging at the studied sites, alternative insecticides should be considered. The insecticides chlorpyrifos and cypermethrin could be good candidates to use as alternatives for Ae. aegypti control.

Temephos resistance and esterase activity in the mosquito Aedes aegypti in Havana, Cuba increased dramatically between 2006 and 2008.

Aedes aegypti (L.) (Diptera: Culicidae) control programmes in Cuba rely on the application of the organophosphate temephos for larval control. Hence, the monitoring of resistance to this insecticide is an essential component of such programmes. Here, 15 field populations from different municipalities of Havana City were assayed for resistance to temephos. High levels of resistance were detected in all strains and resistance ratios were highly correlated with esterase activity (P = 0.00001). Populations from three municipalities were tested in both 2006 and 2008; resistance and esterase activities both significantly increased during this 2-year period. Synergist studies demonstrated that neither glutathione transferases nor monooxygenases were associated with the increase in resistance to temephos in this period. The duration of the efficacy of commercial formulations of temephos in controlling Ae. aegypti populations in Havana City was reduced by the high level of temephos resistance observed; hence these data are of clear operational significance for the dengue control programme in Cuba. New integrated strategies to avoid further increases in temephos resistance in Cuba are necessary.

Application of the pupal/demographic-survey methodology in an area of Havana, Cuba, with low densities of Aedes aegypti (L.).

Between the April and December of 2004, an Aedes aegypti (L.) pupal/demographic survey was conducted in the Playa municipality of Havana. The aims were to identify the key types of container in the production of the adult mosquitoes (and so provide a basis for targeted control) and assess transmission risk in terms of the number of pupae/person. Pupal abundance, as measured in the survey, was compared with traditional Stegomyia indices. The immature stages of Ae. aegypti were only found in 70 of the 15,153 containers that were investigated and the pupae of this species were only seen in 52 of the containers. Ground-level water-storage tanks yielded 74.1% of all the pupae, with a further 19.0% found in miscellaneous small containers. The utility of the pupal/demographic-survey methodology in the evaluation of dengue risk in the study area, and other areas of Cuba with low densities of Ae. aegypti, is discussed.

[High esterases as mechanism of resistance to organophosphate insecticides in Aedes aegypti strains]. / Esterasas elevadas como mecanismo de resistencia a insecticidas organofosforados en cepas de Aedes aegypti.

Five strains of Aedes aegypti L. one from Cuba and 4 from Venezuela were evaluated to determine their resistance to organophosphate insecticides (temephos, chlorpiriphos and pirimiphos methyl). In the Venezuelan strains only APURE showed resistance to temephos. In TACHIRA and MIRANDA moderate resistance values were noted (FR50 5 to 10x) for chlorpiriphos and high levels of resistance (FR > 10x) to this insecticide were found in ARAGUA. All the Venezuelan strains showed high levels of resistance to pirimiphos methyl. The Cuban strain from Santiago de Cuba revealed moderate resistance to temephos and pirimiphos methyl, but high resistance to chlorpiriphos. The results of the biochemical tests proved the presence of esterase and glutathione-s-transferase at high frequency in almost every strain. By the polyacrilamide gel electrophoresis, a strongly stained band was observed in all the strains with a Rf value of 0.779; it was named esterase A4 and was not seen in the susceptible reference strain. The meaning of this esterase in the resistance to organophosphate insecticides is yet to be determined. Resistance to these insecticides in Aedes aegypti is a serious problem for the control of this species therefore integrated management strategies were proposed to prevent or delay the appearance of this species in Cuba and Venezuela.

[Adaptation of microtitration plate methods for quantification of the activity of esterases and glutathione-s-transferase in Aedes aegypti]. / Adaptación de los métodos en placas de microtitulación para la cuantificación de la actividad de esterasas y glutation-s-transferasa en Aedes aegypti.

The detection techniques for the activity of non-specific esterases and glutathione-s-transferase in Culex quinquefasciatus were modified to detect such enzymes in Aedes aegypti(L). The optimal concentration values of substrate (saturating concentration) and the optimum reading time for reaction were determined for each enzyme by using 4 Aedes aegypti strains: one from Cuba 2 from Venezuela and one susceptible reference strain. The frequency of non-specific esterases turned out to be 0.76 in MIRANDA 0.42 in ARAGUA and 1 in SANTIAGO DE CUBA in which the highest frequency value of this mechanisms was reached. The frequency of glutathione-s-transferase mechanism was 0.45 in ARAGUA 0.043 in MIRANDA and 1 in SANTIAGO DE CUBA. For the first time in Cuba a set of biochemical techniques was available for detecting the resistance mechanisms of Aedes aegypti which made it possible to create sound foundations for the easy rapid detection of the resistance of this species the main dengue vector in the Americas.

[Resistance to insecticides in Blattella germanica species strains from Santiago de Cuba]. / Resistencia a insecticidas en cepas de terreno de la especie Blattella germanica procedentes de Santiago de Cuba.

A study was conducted on the level of resistance to seven insecticides, namely, 3 organophosphate compounds (malathion, chlorpyrifos and pirimiphos-methyl), one carbamate (propoxur) and 1 pyrethroid (cypermethrin, deltamethrin and lambdacialotrine) of three field-collected strains of Blattella germanica (Linnaeus, 1767) from Santiago de Cuba. These strains showed high resistance levels to malathion, cypermethrin, deltamethrin and lambdacialotrine and low resistance to pirimiphos-methyl, and also they were susceptible to chlorpyrifos and propoxur. The levels of resistance to tested organophosphate insecticides such as malathion and pirimiphos-methyl and to pyrethroid compounds like cypermethrin, deltamethrin and lambdacialotrine may be related to the increased production of esterases as a mechanism of resistance. The value of frequency of the resistant genes for enzyme acetylcholinesterase was very low, therefore, the modified acetylcholinesterase is not involved in resistance to insecticides tested in the studied strains from Santiago de Cuba.

[Course of insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae) in a region of La Habana]. / Evolución de la resistencia a insecticidas en Culex quinquefasciatus (Díptera: Culicidae) en un área de La Habana.

The use of malathion to control mosquitoes in Cuba during 7 years until 1986 selected 2 resistance mechanisms: that of elevated activity of nonspecific esterases and that of altered acetylcholinesterase (Ache) in Culex quinquefasciatus (Say). In Havana, specifically in the area under study (Quibú River), malathion was replaced by cypermethrin in 1987 and cycles of treatment with cypermethrin have been intensively used since 1987 up to now when the populations of Aedes or Culex increase. In Culex quinquefasciatus (Say) from the Quibú River the resistance levels, mainly to malathion, declined significantly from 1986 to 1997. An increase of resistance to pyrethroid was observed during that period of 11 years. The mechanism of elevated esterases rose to a frequency of 1 and there was also an increase in the frequency of the mechanism of Ache. The esterase B1, responsible for the resistance to malathion, but not to pyrethroid, was selected in this population until 1986. Starting from the use of pyrethroid for the control in this area, 2 new phenotypes of esterases named A6 and B6, apparently related to pyrethroid resistance, were selected.

Insecticide resistance studies on Blattella germanica (Dictyoptera:Blattellidae) from Cuba.

We studied the levels of resistance to seven insecticides: malathion, chlorpyrifos, pirimiphos-methyl, propoxur, cypermethrin, deltamethrin, and lambda-cyhalothrin in nine strains of Blattella germanica (Linnaeus, 1717) collected from sites in Santiago de Cuba and Havana City. The strains from Santiago de Cuba, generally had high levels of resistance to malathion, cypermethrin, deltamethrin, and lambda-cyhalothrin, but only low levels of resistance to pirimiphos-methyl, chlorpyrifos, and propoxur. In the strains from Havana City we found a moderate resistance to the organophosphate insecticides, resistance to the pyrethroids (except for three strains that showed susceptibility to lambda-cyhalothrin), and susceptibility to the carbamate insecticide (propoxur).

[Comparative analysis of resistance to insecticides in strains of Culex quinquefasciatus, from Camagüey]. / Análisis comparativo de la resistencia a insecticidas en cepas de Culex quinquefasciatus, provenientes de Camagüey.

The organophosphate, carbamate and pyrethroid resistance levels in 14 strains of Culex quinquefasciatus from Camagüey were studied by bioassays and biochemical tests in larvae with or without synergists. The use of electrophoresis in polyacrylamide gel (PAGE) allowed to identify 8 patterns of esterases, among which A3A6B6, BiB6 and B1A6B6 were the most frequent. It was observed that 6 years after the suspension of malathion, there is still resistance to this product. There was also cross resistance to propoxur. Detoxification mechanisms for deltamethryn are being developed. The most important physiological mechanisms were: elevated esterases in the resistance to malathion, modified AchE for propoxur and oxidases of multiple function for deltamethryn. In relation to the genic frequencies, the modified AchE mechanisms was more extended than that of the elevated esterases in these populations of mosquitoes of the central region of Cuba.

[Characterization of resistance to organophosphate insecticides, carbamates, and pyrethroids in Culex quinquefasciatus from the State of Miranda, Venezuela]. / Caracterización de la resistencia a insecticidas organofosforados, carbamatos y piretroides en Culex quinquefasciatus del Estado de Miranda, Venezuela.

Resistance of Culex quinquefasciatus from Miranda, Venezuela to the organophosphate insecticides malathion and chlorpirifos was higher than 30x whereas resistance to pyrethroids metylpirimifos, fention, cipametrine, deltametrine, permetrine and lambdacyalotrine and to organochlorate DDT was lower than 4x. Resistance mechanisms were analyzed with piperonyl butoxide synergist (multifunction oxidases) and S.S.S. phosphotrithiate tributyl (DEF, esterase inhibitor). Multifunction oxidases did not play a significant role in resistance to organophosphate insecticides and carbamate; however, esterases were only mechanisms of resistance to malathion and chlorpirifos. The only insecticide affected by DEF and PB was cipermetrine. Biochemical tests revealed a very low frequency of the altered acetylcholinesterase mechanism (0.13). Esterase frequencies were high (1). Electrophoresis exposed the B1, A6 and B6 esterase phenotypes.

[Levels of insecticide resistance and its mechanisms in a strain of Aedes aegypti of Santiago de Cuba]. / Niveles de resistencia a insecticidas y sus mecanismos en una cepa de Aedes aegypti de Santiago de Cuba.

As a result of the most recent dengue outbreak in Santiago de Cuba province, a strain of this vector was studied to determine the levels of sensitivity and/or resistance to organophosphate and pyrethoid insecticides. The results of bioassays showed low levels of resistance to fention, malathion and deltametrine, moderate levels of resistance to temephos, metyl-pirimifos and cipermetrine and high levels of resistance to chlorpirifios. According to the results obtained from the use of S.S.S. phosphotrithiate trybutil synergist, it was shown that esterases play an important role in resistance to temephos and chlorpirifos. Piperonyl butoxide synergist disclosed that multifunction oxidases were not involved in the resistance to any of the evaluated insecticides. Biochemical techniques were applied to detect esterase-, glutathione-S-transferase- and acetylcholineaterase-mediated resistance mechanisms of Aedes aegypti. In accordance with the high frequency values observed in each of the mechanisms, it was proved that esterases and glutathione-S-transferase were involved in the insecticide resistance but acetylcholinesterases were not. However, acetylcholinesterase gen was found in Aedes aegypti for the first time though at low frequency. The polyacrylamide-gel electrophoresis made it possible to observe a well-stained band with a relative mobility value of 0.779; this band was called A4 it was not observed in the reference strain and may be associated to organophosphate resistance which remains to be proved in future research.

[The evolution of resistance in a Culex quinquefasciatus strain starting from selection with the pyrethroid insecticide lambdacyhalothrin]. / Evolución de la resistencia en una cepa de Culex quinquefasciatus a partir de la selección con el insecticida piretroide lambdacialotrina.

The resistance change to different insecticides in Culex quinquesfasciatus strain select at the laboratory with doses of pyrethroid lambdacyhalothrin that would cause a larva mortality of 90% were studied. It was attained an increase of the resistance to this insecticide of 144.5 times compared with the original level, and it was obtained a resistant strain (287x). There was an increase of the levels of resistance to methyl-pyrimifos (2.4 times), propoxur (6 times), DDT (5.2 times), clorpirifos (22 time), cypermethrin (67.5 times), and deltamethrin (20.2 times). The frequencies of the genes that codify for the elevated esterases enzymes and for the modified acetylcholinesterase reached their maximum value. Significant changes were observed in the phenotypes for esterases in the electrophoresis in polyacrylamide gels. It was detected synergism of DEF and PB with lambdacyhalothrin. Therefore, the elevated esterases and the esterases of multiple function may contribute to resistance.

[3 combinations of esterases and their relation with the resistance to organophosphate, carbamate, and pyrethroid insecticides in Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) in Cuba]. / Tres combinaciones de esterasas y su relación con la resistencia a insecticidas organofosforados, carbamatos y piretroides en Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) de Cuba.

The possible relationship between different patterns of esterases and the resistance to various types of insecticides was analyzed by bioassays, biochemical tests, and electrophoresis carried out in a population of Culex quinquefasciatus from Camagüey and in three colonies obtained from it by family selection. The original population was heterogeneous and presented 8 combinations of esterases in polyacrylamide gel. A3A6B6, B1B6 and B1A6B6 were the most frequent of them and characterized each selected colony. Every studied colony, including the parental one, showed different levels of resistance to diverse insecticides. Only the resistance to propoxur showed less variation. In every colony it seems to exist a combination of resistance mechanisms; however, the differences found in tests with DEF synergized insecticides show that the different bands of zymograms may represent esterases contributing in a different way to resistance.

[The association of resistance to organophosphate, carbamate and pyrethroid insecticides with the mechanisms of resistance observed in Culex quinquefasciatus strains from Ciudad de La Habana province]. / Asociación de la resistencia a insecticidas organofosforados, carbamatos y piretroides con los mecanismos de resistencia observados en cepas de Culex quinquefasciatus de Ciudad de La Habana.

The mechanisms of resistance in Culex quinquefasciatus from 6 municipalities of Havana City were studied in order to determine their genetic frequency. Increased esterases and altered acetylcholinesterase are still being the major resistance mechanisms in Havana City. The mechanisms of esterases is of high frequency, followed by altered acetylcholinestenase. Resistance to cholorpirifos was found for the first time in Culex quinquefasciatus, while resistance of malathion and carbamate propoxur is maintained, and deterioration to pyrethroid susceptibility is being detected. The synergistic effect of S.S.S. trubutyl phosphotritiade (DEF) and piperonil butoxide (PB) was analysed, and it was proven that esterases enzymes and multifunction oxidases are involved in the resistance to pyrethroids. By electrophoresis in polyerylamide gel, it was determined that the combination of esterases that seems to be associated with the resistance to perythroids is B1-A6-B6, which appeared with the greatest percentage. Valves of genetic frequency in the 6 municipalities were found to be high, for both esterase genes and acetylcholinesterase genes (Ache).

[Determination of resistance mechanism in Culex quinquefasciatus Say 1823 and its operational implication in the correct use of insecticides for its control]. / Determinación de los mecanismos de resistencia en Culex quinquefasciatus Say 1823 y su implicación operacional en el uso correcto de insecticidas para el control.

Resistance to organophosphorous insecticides such as malathion, chlorpyrihos, and pyrimiphos-methyl; pyrethroids such as deltametrine and lambda cialotrine; and the propoxur carbamate was determined in 4 strain of Culex quinquefasciatus of the Eastern, Central and Western parts of Cuba. The increase of esterase enzymes was the main mechanism involved in resistance, followed by altered acetylcholinesterase (AChe). Gene frequencies were high for esterases (1), and moderate for acetylcholinesterase (0.52) in the four strains under study, as an average. Bioassays showed resistance to malathion and propoxur. The use of DEF as esterase inhibitor showed that pyrimiphos-methyl was not affected by this mechanism of resistance, and chlorpyriphos was slightly affected. After 5 years of using pyrethroids such as deltametrine and lambda cialotrine, these are still useful agents for the control of C. quinquefasciatus; the alternate use of these and pyrimiphos-methyl or chlorpyriphos could prevent the development of resistance in a short or medium term.

[Resistance to organophosphate, carbamate, and pyrethroid insecticides in Blattella germanica (Dictyoptera: Blattellidae) in 2 municipalities of the City of Havana]. / Resistencia a insecticidas organofosforados, carbamato y piretroides en Blattella germanica (Dictyoptera: Blattellidae) de 2 municipios de Ciudad de La Habana, Cuba.

We determined the resistance of 2 Blattella germanica strains collected in 2 municipalities of City of Havana--Playa (P) and Centro Habana (CH)--to six insecticides: malathion, pyrimiphos-methyl, propoxur, cipermetrine, deltametrine, and lambda cialotrine. The P strain showed resistance only to deltametrine (FR = 80); the CH strain was susceptible to propoxur and lambda cialotrine and resistant to malathion (FR = 12), pyrimiphosmethyl (FR = 13), cipermetrine (FR = 16), and deltametrine (FR = 100). Gene frequency of increased esterases and of the modified acetylcholinesterase was determined in the 2 strains. The values of increased esterases in the two collected strains were: P = 0.90; and CH = 0.91. Values of the modified acetylcholinesterase in the two strains were: P = 0.47; and CH = 0.12.

[Effect of 3 organophosphates on the reproduction of Culex quinquefasciatus Say 1823]. / Efecto de 3 organofosforados sobre la reproducción de Culex quinquefasciatus Say 1823.

An experiment was carried out with a Culex quinquefasciatus strain with resistance genes called "Quibú", this strain was pressured with 3 organophosphorous insecticides: malathion, chlorpyriphos, and pyrimiphos-methyl. The most toxic for this species was the chlorphyriphos (LC50 = 0.000009 mg/L), followed by pyrimiphos-methyl (LC50 = 0.00026 mg/L), and malathion (LC50 = 0.0035 mg/L). Larvae surviving the effects of the LC30 and LC70 of the three insecticides were selected to test their effect on fecundity, fertility and sexual index. Fecundity was not affected by the employed doses of chlorpyriphos and pyrimiphos-methyl, while a significant decrease was observed with the LC30 and LC70 of malathion. The employed doses of malathion did not affect fertility but there was a slight decrease with the LC70 selections of chlorpyriphos and pyrimiphos-methyl. The proportion of adult females showed a significant increase with the doses of chlorpyriphos and pyrimiphos-methyl; sexual index was not affected by malathion.