Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

Correlation of glutathione S-transferase and DDT dehydrochlorinase activities with DDT susceptibility in Anopheles and Culex mosquitos from northern Thailand.

Comparative DDT-susceptibility status as well as glutathione S-transferase activity and DDTase activity of Anopheles minimus (A). An. annularis and Culex quinquefasciatus were investigated to ascertain the role of these enzymes in DDT-resistance. The standard WHO susceptibility test kits was used to discriminate between resistant and susceptible populations. GST activity was measured in microtiter plates whereas DDTase activity was determined by HPLC quantitation of DDT metabolites. This is the first report of DDT-resistance in the Thai malaria vector, An. minimus species A. A positive correlation of DDT-resistance and DDTase activity was observed in this species as well as in the suspected vector, An. annularis. However, GST activity was not correlated to DDT-resistance in either species. Statistical analysis and scatter plots demonstrated the non-correlation between DDTase and GST activity in An. annularis. Studies in Culex quinquefisciatus revealed difference in GST/ DDTase and the relationship to DDT-resistance compared to the anopheline species. The Culex GST activity is correlated to DDTase activity. These results suggested that a positive correlation of GST and DDTase activity might be species dependent.

Estudio de la resistencia en una cepa de Culex quinquefasciatus, procedente de Medellín, Colombia

Para conocer el estado de la resistencia en una cepa de Culex quinquefasciatus, procedente de una localidad de la ciudad de Medellín, Colombia, se determinaron los niveles de susceptibilidad y/o resistencia a 5 insecticidas organofosforados (malatión, metil-pirimifos, clorpirifos, temefos y fentión), 4 piretroides (cipermetrina, deltamemetrina, permetrina y lambdacialotrina) y un carbamato (propoxur). Se observó resistencia a todos los insecticidas organofosforados, aunque con valores relativamente menores para metil-pirimifos y fentión. No se encontró resistencia a los piretroides lambdacialotrina y cipermetrina, ni al carbamato propoxur, insecticidas que pueden ser muy útiles para el control de mosquitos de Colombia. Se demostró mediante el uso del sinergista piperonil butóxico que las oxidasas de función múltiple desempeñaron una función importante en la resistencia a los insecticidas organofosforados y piretroides. El uso del S.S.S. tributil fosfotritiado reveló que la sobreproducción de esterasas inespecíficas constituyó un mecanismo de resistencia para los insecticidas organofosforados, excepto metil-pirimifos y para los piretroides excepto lambdacialotrina. Este resultado debe tenerse en cuenta en las estrategias que se vayan a usar para el control de Culex quinquefasciatus de Colombia. Estos 2 mecanismos de resistencia no son responsables de la resistencia al carbamato propoxur. El análisis electroforético reveló la presencia de las esterasas B1, A6 y B6, que presumimos tienen una función importante en la resistencia.

The levels of susceptibility and/or resistance to 5 organophosphate insecticides (malathion, methyl-pyrimifos, clorpirifos, temephos and fenthion), 4 pyrethroids (cypermethrin, deltamethrin, permethrin and lambda-cyhalothrin), and a carbamate (propoxur) were deternmined in order to know the state of resistance in a strain of Culex quinquefasciatus from a locality of the city of Medellín, Colombia. Resistance to all organophosphate insecticeides, though with relatively lower values for methyloirimifos and fenthion, was observed. No resistance to lambda-cyhalothrin and cypermethrin or to propoxur was found. These insecticides may be useful for the control of mosquitoes in Colombia. It was demonstrated by using the piperonil butoxide sinergist that the oxidases of multiple function played an important role in the resistance to organophosphate insecticides and pyrethroids. The utilization of S.S.S. tributyl phosphotritiate revealed that the superproduction of unspecific esterases was a mechanism of resistance to organophosphate insecticides, except methyl-pirimifos and for perythroids, exceptlambda-cyhalothrin. This result should be taken into consideration for the strategies to be used to control Culex quinquefasciatus in Colombia. These two mechanism of resistance are not responsible for the resistance to propoxur. The electrophoretic analysis showed the presence of esterases B1, A6 and B6, which seem to have an important function in resistance.

Monitoring resistance gene frequencies in Malaysian Culex quinquefasciatus Say adults using rapid non-specific esterase enzyme microassays.

The ability to identify the occurrence of different resistance genotypes in field populations of mosquito is considered important for the purpose of optimising chemical control operations. The recent development of rapid microassays of enzymes responsible for resistance has provided a means for rapidly assessing the genetic background of target mosquito populations. This concept is the topic of investigation in this study. Non-specific esterase activity, which is responsible for the resistance to organophosphates in Malaysian Culex quinquefasciatus Say adults, was determined in 3 field populations from Kuala Lumpur City using rapid enzyme assay. The optical density results were used to estimate the genotypic frequencies of the populations. Subsequently, time-dependent changes in the various frequencies were determined. Such techniques allowed rapid assessment of resistance genotypes for decision-making and its possible use in insect control merits further investigation.

Mecanismos de resistencia a organofosforados, carbamatos y piretroides en Culex quinquefasciatus (Diptera: Culicidae) / Resistance mechanisms to organophosphorous, carbamate and pyrethroid insecticides in Culex quinquefasciatus (Diptera: Culicidae)

Se estudiaron los mecanismos de resistencia en 3 poblaciones de Culex quinquefasciatus Say, 1823, de los municipios San José de Las Lajas, La Lisa y la Habana Vieja durante 1991. Se realizaron pruebas con insecticidas organofosforados, carbamatos y piretroides, se observó un mayor factor de resistencia a malatión (16,4 - 25,4) y clorpirifós (11,0 - 17,0), que a primifós metil (4,6 - 8,1) y al carbamato propoxur (1,8 - 2,7); mientras que el sinergista butoxido de piperonil suprimió la resistencia a cipermetrina en Quibú y Habana Vieja. Se observó una mayor frecuencia del mecanismos de las esterasas (0,66-0,89 que del mecanismo de la acetilcolinesterasa (0,20-0,24)

Método bioquímico para la determinación de acetil colinesterasa modificada, que confiere resistencia cruzada a insecticidas organofosforados y carbomatos en Culex quinquefasciatus Say, 1823 / Biochemical method to determine the altered acetylcholinesterase which confers cross resistance to organophosphate and carbomate insecticides in Culex quinquefasciatus Say, 1823

Se realizó el montaje de un método rápido para la determinación de la frecuencia del gen de la acetil colinesterasa modificada, que confiere resistencia cruzada a insecticidas organofosforados y carbomatos en mosquitos. Para el montaje de la técnica se utilizaron cepas de laboratorio de Culex quinquefasciatus Say, 1823. Se obtuvo una buena diferenciación en los valores de absorvancia entre los mosquitos homocigóticos susceptibles (SS), heterocigóticos (RS) y homocigóticos (RR) resistentes con valores de actividad enzimatica residual de 10-60, 70-100 y 80-120, respectivamente, lo que además se pudo diferenciar a simple vista basado en el cambio de color amarillo en la reacción. Se encontró el gen de la acetil colinesterasa modificada en las cepas de Culex quinquefasciatus estudiadas

Determination of insecticide susceptibility in Culex quinquefasciatus Say adults by rapid enzyme microassays.

Rapid enzyme microassays for the detection of resistance due to organophosphate and carbamate in individual field-collected strains of Culex quinquefasciatus adults were conducted. These tests allowed accurate differentiation by eye, on the basis of color changes of susceptible and resistant individuals. Two separate tests were conducted for the biochemical assays. In the insensitive acetylcholinesterase (AChE) test, acetylthiocholine iodide (ACTH) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were used as substrate and coupling agent respectively. The resulting yellow chromophore indicated AChE activity. Test results showed that the color intensity decreased as increasing concentrations of propoxur were added, thereby confirming the susceptibility of the enzyme to inhibitor. Assay of non-specific esterase however, indicated elevated levels which were correlated with degree of malathion resistance. Electrophoretic data revealed the presence of 2 esterase bands in all strains. It was concluded that such a pattern was not contributory to malathion resistance in adults.

Toxicological studies of insecticides on Cullex quinquefasciatus Say and Aedes aegypti (L.).

Toxicity tests carried out on the larvae of A. aegypti and C. quinquefasciatus showed the former to be more tolerant of all insecticides tested, the order of toxicity being temephos greater than DDT greater than DDVP greater than malathion greater than lindane greater than carbaryl; also the second instar larvae of A. aegypti were more susceptible than fourth instar larvae. Enzyme kinetic studies on the total non-specific esterases and CarEs of adults and larvae of both species showed the Km values for total esterases of adult A. aegypti to be 0.333 mM vs 0.233 mM for C. quinquefasciatus; for adult CarEs it was 0.250 mM vs 0.220 mM. For total larval esterases of A. aegypti it was 0.112 mM vs 0.175 mM for C. quinquefasciatus: and for larval CarES it was 0.159 mM vs 0.213 mM respectively. Although some correlation between in vivo toxicity (LD50 values) and in vitro esterase inhibition (I50 values) between species could be discerned, overall correlation could not be established.