Knockdown Resistance (kdr) Mutations in Indian Anopheles stephensi (Diptera: Culicidae) Populations.

Knockdown resistance (kdr) in insects resulting from mutation(s) in the voltage-gated sodium channel (VGSC) gene is one of the mechanisms of resistance against DDT and the pyrethroid group of insecticides. Earlier, we reported the presence of two classic kdr mutations, i.e., L1014F and L1014S in Anopheles stephensi Liston, a major Indian malaria vector affecting mainly urban areas. This report presents the distribution of these alleles in different An. stephensi populations. Seven populations of An. stephensi from six states of India were screened for the presence of two alternative kdr mutations L1014F and L1014S using allele-specific polymerase chain reaction assays. We recorded the presence of both kdr mutations in northern Indian populations (Alwar and Gurgaon), with the preponderance of L1014S, whereas only L1014F was present in Raipur (central India) and Chennai (southern India). None of the kdr mutations were found in Ranchi in eastern India and in Mangaluru and Mysuru in southern India. This study provides evidence for a focal pattern of distribution of kdr alleles in India.

Laboratory selection of carbofuran tolerant line of Culex quinquefasciatus Say, the filarial vector at Mysore.

Carbofuran is a systemic insecticide/nematicide extensively employed in modern agriculture and public health to combat various insect pests and vectors. The development of insecticide resistance due to natural selection pressure by Anopheline and Culicine mosquitoes has created serious repercussions in mosquito control and disease eradication campaigns. In this regard Culex quinquefasciatus, a widely dispersed domestic mosquito and the only vector of lymphatic filariasis in India, was selected for the present experiment. In order to assess the resistance development in this species, it was subjected to carbofuran bioassay by employing WHO method for 10 generations. The results of larval selection test up to F10 revealed a significant increase in tolerance development in every generation compared to the susceptible F1. The results revealed up to 16.225 times tolerance after 10 generations. The LC50 observed in F10 is 1.298 ppm compared to 0.08 ppm in the F1. The implications of the observations will be discussed.

Biochemical characterization of deltamethrin resistance in a laboratory-selected strain of Aedes aegypti.

A resistant strain (DR) of Aedes aegypti, generated by deltamethrin selection for 20 consecutive generations from a laboratory susceptible strain (DS) was studied for the possible resistant mechanisms. The pyrethroid resistance developed was characterized by biochemical assays and native polyacrylamide gel electrophoresis. Significant elevation in the activity of alpha- and beta-esterases, glucose-6-phosphate dehydrogenase (G6PD), CYTP450 (CYTP450), and glutathione-s-transferase (GST) were noticed in DR. The gel profiles for esterases, G6PD, and CYTP450 were different in DR as compared to DS strain. The difference was either in the form of additional bands or increased intensity of the bands or both. Gel profile variations were also evident from densitometry. Our study suggests that these enzymes play an important role in deltamethrin resistance in the DR strain.

Larvicidal activity of oak Quercus infectoria Oliv. (Fagaceae) gall extracts against Anopheles stephensi Liston.

There is a growing interest in the use of botanical insecticides to reduce the use of synthetic pesticides in order to avoid environmental side effects. Anopheles stephensi is the primary vector of urban malaria, an endemic disease in India. So, an effort to assay An. stephensi larvae with gall extracts of Quercus infectoria was made under laboratory conditions at Mysore. Ethyl-acetate extract was found to be the most effective of all the five extracts tested for larvicidal activity against the fourth instar larvae, with LC(50) of 116.92 ppm followed by gallotannin, n-butanol, acetone, and methanol with LC(50) values of 124.62, 174.76, 299.26, and 364.61 ppm, respectively. The efficacy in killing mosquito larvae may make this plant promising for the development of new botanical larvicide.

Synergist efficacy of piperonyl butoxide with deltamethrin as pyrethroid insecticide on Culex tritaeniorhynchus (Diptera: Culicidae) and other mosquitoe species.

Continuous and indiscriminate use of pesticides, especially in tropical countries for public health or agriculture purpose, has led many vector populations to become resistant to organochlorides, organophosphates, and even to carbamates and pyrethroids. Development of resistance by a vector population has been one of the reasons for the failure of the control measures in many countries. This investigation demonstrates the efficacy of piperonyl-butoxide (PBO) with deltamethrin, as pyrethroid insecticide, against the field-collected mosquitoe larvae of five species, Aedes aegypti, Anopheles culicifacies, An. stephensi, An. vagus, and Culex quinqufasciatus, and two morphological variants of Cx. tritaeniorhynchus (type A from grand pools of Mysore city and type B from rice fields of Mandya district). For testing the synergistic effect of PBO, stock solutions of deltamethrin and PBO were mixed in 1:6 ratio. The synergistic ratio and the percent suppression in deltamethrin tolerance were calculated by using LC(50) values. From the results, it is clear that, PBO is an effective synergist with deltamethrin against all of species undertaken in this investigation. So, it is suggested that PBO is a good synergist in this area for decreasing the use of pesticides in environment in vector control.

Laboratory evaluation of a juvenile hormone mimic, pyriproxyfen on Culex quinquefasciatus Say and Aedes aegypti Linn. at Mysore, India.

Insect growth regulator (IGR), pyriproxyfen is a juvenile hormone analogue used in stored product insect pest and vector control programmes. It can be considered as an alternative source to conventional insecticides because of its specific activity against immature insects, low persistence in the environment and virtually nontoxic to mammals. So, in our laboratory the effect of Pyriproxyfen was evaluated against the late 3rd instar larvae of Culex quinquefasciatus and Aedes aegypti to study the Concentration-mortality response and inhibition of adult emergence. The results showed that LC50 was 0.00084 ppm and 0.00166 ppm for Cx. quinquefasciatus and A. aegypti respectively. Toxicity of this insect compound extended till the adult emergence by inhibiting pupae formation up to 61.0% and 95.6% of adult emergence against Cx. quinquefasciatus. Likewise it affected 65.0% of pupal formation and 98.9% of adult emergence against A. aegypti. These results represent a promising strategy to use insect growth regulator, Pyriproxyfen for the instant, safe and successful improvement in the integrated vector control programme.

Efficacy of piperonyl butoxide (PBO) as a synergist with deltamethrin on five species of mosquitoes.

Development of insecticide resistance has been a challenging problem for a long time and new solutions are yet to emerge. In this regard, the use of synergist with the insecticide is thought to play a key role in reducing the resistance levels. Present study demonstrates the efficacy of PBO with deltamethrin against the field collected mosquito larvae of five species of Aedes, Anopheles and Culexfrom in and around Mysore.

Differential tolerance of two morphological variants of Culex tritaeniorhynchus (Diptera: Culicidae), a Japanese encephalitis vector, to pyrethroid insecticides in Mysore, India.

Five synthetic pyrethroids, deltamethrin, cypermethrin, permethrin, alpha-cypermethrin, and lambdacyhalothrin, were tested on the larvae of 2 morphological variants of Cx. tritaeniorhynchus collected from Mysore City (ground pools) and outside Mysore (paddy fields), in Karnataka State, India. The morphological characters, ie, length and width of siphon, siphonal index, comb scale numbers, pecten teeth numbers, length of anal gills and anal gill index of larvae of two Cx. tritaeniorhynchus populations were found to be significantly different (p<0.05). To elicit further detail of these two variants, pyrethroid bioassays were undertaken. In general, the toxicity ranking of these pyrethroids tested on the Cx. tritaeniorhynchus variants from Mysore City was deltamethrin > lambdacyhalothrin > cypermethrin > permethrin > alpha-cypermethrin. However, for the rural variety it was deltamethrin > lambdacyhalothrin > alpha-cypermethrin > permethrin > cypermethrin. Of the 5 pyrethroids tested, deltamethrin was the most effective on both variants. Alpha-cypermethrin and lambdacyhalothrin were respectively 2.17 and 2.09 times more effective on the city variety (p<0.05), based on LC50 values. The results suggest that, in addition to morphological differences, the 2 tested varieties of Cx. tritaeniorhynchus also differ in susceptibility status.

Pyrethroid susceptibility & enzyme activity in two malaria vectors, Anopheles stephensi (Liston) &. A. culicifacies (Giles) from Mysore, India.

BACKGROUND & OBJECTIVES: Anopheles stephensi and A. culicifacies are the two major vectors of malaria in Karnataka. These mosquito populations are continuously being exposed directly or indirectly to different insecticides including the most effective pyrethroids. Therefore, there is a threat of insecticide resistance development. We subjected these vectors to larval bioassay using two popular pyrethroids viz deltamethrin and permethrin. An attempt was also made to correlate the activities of certain detoxifying enzymes such as A- esterase, B-esterase, glutathione-S transferase (GST) and glucose-6-phosphate dehydrogenase (G6PD) with the tolerance levels of the two vectors. METHODS: Larval bioassay was carried out following the standard WHO procedure on field-collected larvae. The LC50 and LC90 values were calculated following Probit analysis. Biochemical estimations were done with a U V spectrophotometer and the isozyme studies employing native polyacrylamide gel electrophoresis (PAGE). RESULTS: The results of the larval bioassay revealed that A. stephensi has more tolerance to deltamethrin than A. culicifacies and vice versa for permethrin. Biochemical estimations revealed significantly (P < 0.05) higher levels of A-esterase and GST activity in A. stephensi whereas A. culicifacies showed significantly higher (P < 0.05) levels of B-esterase and G6PD activity. The total larval protein assayed was found to be more (P < 0.05) in A. stephensi. The isozyme profiles also revealed difference in mobility, intensity and the number of bands. INTERPRETATION & CONCLUSION: As these malaria vectors are exposed to different kinds of insecticides, they develop increased enzyme activities to overcome the insecticide pressure. This has enhanced the tolerance level against the pyrethroids tested. Thus, A. stephensi was found to be tolerant to deltamethrin depicting a higher activity of A-esterase and GST enzymes, whereas the higher activity of B-esterase and G6PD has resulted in the development of tolerance to permethrin in A. culicifacies.

Comparative susceptibility of two Japanese encephalitis vectors from Mysore to six insecticides.

Larval populations of Culex tritaeniorhynchus and C. fuscocephala from Mysore city were tested for their susceptibility against two pyrethroids viz., cypermethrin and deltamethrin and four organophosphorus (OP) compounds viz., fenthion, fenitrothion, temephos and malathion. The investigations revealed that, among the pesticides tested, efficacy of pyrethroids was very high as compared to OP compounds. C. tritaeniorhynchus was found to be highly tolerant to all the insecticides used. It registered 4.0, 6.5, 17.0, 6.5, 14.0 and 1.5 times more tolerance than C. fuscocephala to cypermethrin, deltamethrin, fenthion, fenitrothion, temephos and malathion respectively in terms of the LC50 value. Further studies are needed to determine the development of resistance if any, by C. tritaeniorhynchus, an important JE vector in Karnataka.

Deltamethrin tolerance & associated cross resistance in Aedes aegypti from Mysore.

BACKGROUND & OBJECTIVES: The indiscriminate use of insecticides in public health and agriculture has led to the development of resistance to these insecticides in the vector mosquitoes. To understand the development of resistance to synthetic pyrethroids, selection studies on Aedes aegypti were done at Mysore. METHODS: Ae. aegypti collected from the field were subjected to selection experiment with deltamethrin for 16 generations in the laboratory. Cross resistance test was conducted against permethrin and fenvalerate. RESULTS: Tolerance level was found to increase by 333.83 folds in terms of its LC50 values. Cross resistance of this deltamethrin selected line was tested against permethrin and fenvalerate. The results show that the selected line has developed cross resistance as much as 5.19 and 5.92 folds respectively against permethrin and fenvalerate. INTERPRETATION & CONCLUSIONS: The findings show a continuous elevation in tolerance in Ae. aegypti with increase in deltamethrin selection pressure, and development of cross resistance to other insecticides of the same class. The natural or developed tolerance has its implications in the control of these mosquitoes.

Biting density, behavior and age distribution of Culex quinquefasciatus, say in Mysore City, India.

The seasonal abundance, night indoor biting behavior and parity of Culex quinquefasciatus were investigated for a period of one year from June 1988 to May 1989 in Mysore City. Twelve-hour night-time biting catches were carried out from 1800-0600 hours at four selected urban localities. The biting cycle revealed that Cx. quinquefasciatus was active throughout the night, with peak activity at 2200-2300 hours. The biting proportion of nulliparous and parous mosquitos during different hours of the night showed that nulliparous mosquitos increased from 1800 to 0600 hours (dusk to dawn), while parous mosquitos (aged) decreased from early night to the morning. One major and two minor biting peaks were encountered here. The maximum density noticed was during March 1989 (147.5 per man night) and the lowest was during July 1988 (58.3 per man night). The biting behavior/density of this important urban mosquito vector under the existing climatic conditions are discussed in the light of the earlier reports elsewhere.

Susceptibility difference in two populations of Culex quinquefasciatus Say (Diptera: Culicidae) to three synthetic pyrethroids.

Three synthetic pyrethroids were tested for their efficacy against two populations of the filariasis vector Culex quinquefasciatus under laboratory conditions following the WHO procedure, Emulsifiable concentration and technical grade formulations of deltamethrin (2.5% wp), cypermethrin (1.0% EC) and permethrin (25% EC) were employed on the larvae of the said species collected from Mysore and Mandya. All the three pyrethroids were found to be effective. But among the three, deltamethrin was found to be the most effective and cypermethrin was more efficient than permethrin. Mysore species were found to be more susceptible than the Mandya population to all the chemicals tested though they are separated by hardly 50 km.

Adult population differences in response to six insecticides in Culex fuscocephala Theobald.

Adult bioassay was conducted in the laboratory on two populations of Culex fuscocephala, a vector of Japanese encephalitis (JE) in India, against two organochloride compounds (DDT and dieldrin), an organophosphate compound (malathion), a carbamate pesticide (propoxur) and two synthetic pyrethroids (deltamethrin and cyfluthrin) using WHO techniques. The adult populations from Mandya an irrigated agricultural area, and Mysore, a non-agricultural place in Karnataka, India, have been employed for the experiments. JE outbreaks were reported from these places at different times. Among the insecticides used malathion in 5% concentration was found to be the most effective while, cyfluthrin (0.05%) and deltamethrin (0.025%) were very effective in much lesser concentration. For both propoxur (0.1%) and DDT (4.0%) the two populations of Cx. fuscocephala expressed least susceptibility in terms of the LT50. Against dieldrin (0.4%), cyfluthrin, DDT and malathion Mysore population showed relatively more susceptibility than Mandya population. However for deltamethrin and propoxur the Mandya population registered less tolerance than the Mysore population. Thus adult population responses to different insecticides are variable in the present study.

Role of esterases and monooxygenase in the deltamethrin resistance in Anopheles stephensi Giles (1908), at Mysore.

Field collected An. stephensi larvae were colonized in the laboratory for 15 generations and acclimatized. An isofemale line was raised from this colony and the larvae were subjected to continuous deltamethrin selection pressure. LC50 and LC90 values were calculated at every generation. The values indicated that at the end of seventh generation the larvae have developed 87 fold tolerance in terms of LC50 value compared with the first generation. The reason for this kind of resistance was analyzed on the basis of differential activity of A-esterase, B-esterase, glutathione s-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD). A significant correlation (P < 0.05) was observed with B-esterase and G6PD activity with the rise in the LC50 and LC90 values. However no significant rise were observed in the other enzymes tested such as A-esterase and GST. The isozyme analysis of the A-esterase and B-esterase using polyacrylamide gel electrophoresis (PAGE) have shown differential profiles.

Indoor resting density, survival rate and host preference of Culex quinquefasciatus say (Diptera: Culicidae) in Mysore City.

Monthly indoor resting density, age distribution, survival rate and host-preference of Culex quinquefasciatus, a filariasis vector were studied from June 1988 to May 1989 in Mysore city. During this period, a total of 7522 adults were collected in 72 man-hour of search spreading to twelve spots covering the entire city. The annual per man-hour density observed was 104.5, being 76.8 for female and 24.3 for male. Nulliparous and parous (1-parous and 2-parous) mosquitoes were collected throughout the year while 3-parous and 4-parous mosquitoes were recorded during cold and rainy seasons only showing an increase in the number of gonotrophic cycles. Precipitin test of blood smears from the gut content of C. quinquefasciatus was done by taking 2536 mosquito blood smears. Of these, 94.1 per cent preferred human blood, 1.4 per cent bovine and 3 per cent avain. As the species evaluated is a major vector of filariasis, the present study on its resting density and the host preference will be helpful in vector monitoring in Mysore city. Various results on this species at Mysore have been discussed in the light of earlier studies conducted elsewhere.

Variation in morphological characters of Culex tritaeniorhynchus (Diptera: Culicidae), a Japanese encephalitis vector at Mysore, India.

Studies on Culex tritaeniorhynchus Giles, were undertaken to find deviation if any, on the mean of siphonal index, comb scales, pecten teeth, denticles on apical pecten tooth, anal gills index and length of basal hair tufts on siphon in the fourth instar larvae from ground pools of Mysore city and paddy fields outside Mysore. A total of 344 larvae including 172 samples from Mysore and 172 samples from a 26 km radius outside Mysore were collected. Analysis of variance demonstrated significant differences between the two populations on 7 parameters based on morphological characteristics such as comb scales, length of siphon, width of siphon at the middle, length of anal gills, siphonal index, anal gills index and the length of basal hair tufts. So Cx. tritaeniorhynchus from different breeding places in and around Mysore city in south India can be different varieties. One variety comparatively has shorter siphon index ratio, less comb scales, less anal gills index ratio and longer basal hair tufts on siphon than the other one. Mating behavior of these varieties show that, variety from the city pools was moderate eurygamous and the one from mainly paddy fields, moderate stenogamous.

Larvicidal, ovicidal, and oviposition-deterrent activities of four plant extracts against three mosquito species.

In mosquito control programs, insecticides of botanical origin have the potential to eliminate eggs, larvae, and adults. So, the larvicidal, ovicidal, and oviposition-deterrent activities of petroleum ether and ethyl acetate extracts of the leaves of Eugenia jambolana, Solidago canadensis, Euodia ridleyi, and Spilanthes mauritiana were assayed against the three vector mosquito species, namely Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The larval bioassay was conducted following the World Health Organization method. The maximum larval mortality was found with ethyl acetate extract of S. mauritiana against the larvae of A. stephensi, A. aegypti, and C. quinquefasciatus with LC50 values of 11.51, 28.1, 14.10 ppm, respectively. The mean percent hatchability of the ovicidal activity was observed at 48-h post-treatment. The percent hatchability was found to be inversely proportional to the concentration of the extract and directly proportional to the number of eggs. The flower head extract of S. mauritiana gave 100% mortality followed by E. ridleyi, S. canadensis, and E. jambolana against the eggs of the three mosquito vectors. For oviposition-deterrent effect, out of the five concentrations tested (20, 40, 60, 80, and 100 ppm), the concentration of 100 ppm showed a significant egg laying-deterrent capacity. The oviposition activity index value of E. jambolana, E. ridleyi, S. canadensis, and S. mauritiana against A. aegypti, A. stephensi, C. quinquefasciatus at 100 ppm were -0.71, -0.71, -0.90, -0.93, -0.85, -0.91, -1, -1, -0.71, -0.85, -1, and -1, respectively. These results suggest that the leaf/flower extracts of certain local plants have the potential to be developed as possible eco-friendly means for the control of mosquitoes.

Synergistic effect of Eugenia jambolana Linn. and Solidago canadensis Linn. leaf extracts with deltamethrin against the dengue vector Aedes aegypti Linn. at Mysore.

With the goal in mind to minimize the application of environmentally hazardous chemical insecticides, the larvicidal activity of two plant extracts along with deltamethrin was studied at University of Mysore. The extracts of Solidago canadensis and Eugenia jambolana were employed for working out the synergistic efficacy against Aedes aegypti larvae, as the extracts of both the plants exhibited high efficacy when applied individually. The deltamethrin when analyzed separately, LC50 and LC90 values were 0.00045 and 0.00148 ppm, respectively. Synergistic studies with two plant extracts on deltamethrin revealed S. canadensis as more effective with synergistic factor(SF) of 4.090 for LC50 value and 4.781 for LC90 followed by E. jambolana with SF 1.80 for LC50 and 2.467 for LC90 at 1:1 ratio of the phytoextracts and deltamethrin. Thus, S. canadensis was found to be a better larvicidal and synergistic agent. Combination of phytochemical and insecticide were found to be more effective than insecticides or phytochemicals alone which could be a good ecofriendly and cost-effective approach to reduce the dose of chemicals with high residual effect to be applied in vector control programs.