Development of Insecticide Resistance in Culex Quinquefasciatus Mosquito (Diptera: Culicidae) in India 3

J. Pestic. Sci., 35(1), 000–000 (2010) DOI: 10.1584/jpestics.G09-36

Note

phatic filariasis with worldwide distribution. This mosquito is Development of insecticide resistance in present in high density throughout the year due to its high degree Culex quinquefasciatus mosquito of adaptability in its preference for breeding sites, host speci- ficity, and high reproductive potential.1) According to the World (Diptera: Culicidae) from different Health Organization (WHO), 1.3 billion people are at risk of locations in India lymphatic filariasis infections and about 120 million people are affected in 83 countries, of which 45.5 million people live in the Indian subcontinent and 40 million in Sub-Saharan Africa.2,3) Devi Shankar SUMAN, Sachin N. TIKAR, Marked efforts have been made to control this disease along with 4) Brahma Dutta PARASHAR* and Shri PRAKASH vector transmission at various levels ; however, the development of resistance in C. quinquefasciatus against organophosphorous Defence Research and Development Establishment, (OP), pyrethroids and microbial insecticides has been reported in Gwalior-474002, India different parts of the world.5–10) Among mosquitoes, particularly (Received August 8, 2009; Accepted October 5, 2009) Anophelines and Aedes, resistance to various insecticides has been recorded from India and neighbouring countries.11–13) In Culex quinquefasciatus is a domestic mosquito known as a pri- Jodhpur, Bikaner, Jamnagar and Bathinda cantonment areas, con- mary vector of lymphatic filariasis. We report the development of trol programmes have been operated; however, the resistance insecticide resistance in four populations of C. quinquefasciatus level to any group of insecticides has not been monitored. The Say, 1823 in Jodhpur (JD), Bikaner (BKN), Jamnagar (JMN) build up of the high population density of C. quinquefasciatus and Bathinda (BTH) in India in comparison to a susceptible has been observed in these containment areas, which may be due laboratory population against larvicides such as temephos, to the development of insecticide resistance in these areas. Thus, fenthion, Bacillus thuringiensis var. israelensis and Neemarin in consideration of this, the development of insecticide resistance (azadirachtin), and adulticides such as cypermethrin, alpha cyper- in larval and adult C. quinquefasciatus in these areas has been methrin and lambda cyhalothrin. The resistance ratio (RR) at evaluated for OP, B. thuringiensis var. israelensis, neem compound—Neemarin and pyrethroids from the point of view of LC50 between field and laboratory populations was used for resistance expression. JD strain showed 10.8-, 6.94-, 5.29- and changes in the management strategy of using insecticides to 2.82-fold resistance to temephos, fenthion, Neemarin and cyper- which C. quinquefasciatus populations are susceptible. methrin, respectively. BTH strain showed resistance to temephos, Materials and Methods fenthion, lambda cyhalothrin, alpha cypermethrin and cypermethrin (9.06-, 2.06-, 3.33-, 4.96- and 3.19-fold, respectively). RR 1. Insect collection and rearing of BKN strain to temephos, fenthion, Neemarin and alpha cyper- Larval stages of C. quinquefasciatus collected from army stations methrin was 5.17-, 4.12-, 4.33- and 3.04-fold, respectively; how- in Jodhpur (JD) and Bikaner (BKN) (Rajasthan), Bathinda (BTH) ever RR was least in the JMN strain against most insecticides ex- (Punjab), and Air Force station Jamnagar (JMN) (Gujrat) were cept alpha cypermethrin and fenthion. This study suggests that B. transported to the Defence Research and Development Establish- thuringiensis var israelensis and Neemarin may be efficient for ment, Gwalior (Fig. 1). These larvae were maintained under stan- larval control as an alternative to fenthion- and temephos-resist- dard laboratory conditions at 271°C and relative humidity ant strains of C. quinquefasciatus while lambda cyhalothrin and 755%. They were kept at a density of 100 larvae in 2 l de-chlo- cypermethrin may be used effectively as an adulticide of this rinated water in enamel bowls and given yeast tablets as larval mosquito. © Pesticide Science Society of Japan food. After pupation, pupae were transferred to adult cages. Fifty pairs of male and female mosquitoes were released into a mos- Keywords: Culex quinquefasciatus, susceptibility status, OP quito cage (750 mm650 mm650 mm). In the cage, adults insecticides, Bacillus thuringiensis var. israelensis, Neem insecti- were provided with 10% sugar solution ad libitum dispensed cide, pyrethroids. through a cotton wick in small Petri dishes. Twice a week, chicken was offered as a source for blood feeding by female mosquitoes. Glass Petri dishes with 400 ml water were kept for ovipo- Introduction sition. The egg rafts were transferred to a 2 l enamel bowl for Culex quinquefasciatus Say, 1823 is a primary vector of lym- hatching and further development of larvae to the adult stage.

* To whom correspondence should be addressed. 2. Insecticides E-mail: [email protected] The following insecticide formulations were used for susceptibil- Published online XXXXX ity studies: for larval bioassay, Abate® (Temephos 50% EC) © Pesticide Science Society of Japan (American Cynamide, NJ), Baytex® (Fenthion 82.5% EC) (Bayer 2 D. S. Suman et al. Journal of Pesticide Science

ing serially diluted insecticide concentrations were used for the study. Knockdown was recorded at 10-min intervals of 1-hr exposure and treated mosquitoes were transferred to a holding tube for recovery. Sugar solution (10%) was provided as adult diet. Final mortality was recorded after a 24-hour recovery period.

4. Data analysis Data were subjected to probit analysis (Indostat Statistical Soft-

ware, India) to determine the 50% lethal concentration (LC50)

and 90% lethal concentration (LC90) for OPs, B. thuringiensis var. israelensis, neem compound and pyrethroids.17) If control mortality ranged from 5 to 20%, the mortality of treated groups was corrected according to Abbott’s formula.18) The RR was cal-

culated as the ratio between LC50 of the ﬁeld strain and LC50 of the laboratory strain of C. quinquefasciatus.

Results 1. Larval bioassay The LAB strain of C. quinquefasciatus showed more susceptibility to temephos (LC50 0.0015 mg/l) than fenthion (LC50 0.0114 Fig. 1. Locations of collections of Culex quinquefasciatus in India. mg/l). The LC50 of JD, BKN, JMN and BTH strains of C. quin- 1, Bathinda; 2, Bikaner; 3, Jodhpur; 4, Jamnagar; 5, Gwalior. quefasciatus against temephos (Table 1) ranged from 0.0048 mg/l (JMN strain) to 0.0162 mg/l (JD strain) and the LC90 of these strains ranged between 0.0325 mg/l and 0.1125 mg/l, respectively. Ltd., Germany), Neemarin® compound (Azadirachtin 1% EC) The considerable resistance of ﬁeld strains of this mosquito to and Bacillus thuringiensis var. israelensis strain 164, serotype H- temephos was observed, ranging from 3.2- (JMN strain) to 10.8- 14 WP (Biotech International Ltd., India), and for adult bioassay, fold (JD strain) of the LAB strain. The susceptibility status of cypermethrin 10% EC, alpha cypermethrin 10% EC (Insecticide different ﬁeld strains of mosquito (Table 1) to fenthion showed

India Ltd., India) and lambdacyhalothrin 5% EC (Syngenta Crop the lowest values of LC50 and LC90 (0.0238 mg/l and 0.1314 mg/l, Protection Private Ltd., India). respectively) in the BTH strain and highest (0.0792 mg/l and 0.4343 mg/l, respectively) in JD strain. BTH (2.08-fold) and JD 3. Bioassay (6.94-fold) strains exhibited minimum and maximum levels of re- 3.1. Larval bioassay sistance in comparison to the LAB strain (Table 1).

Larval susceptibility assay was carried out according to WHO LC50 and LC90 of the LAB strain of C. quinquefasciatus guidelines.14) Late 3rd–early 4th instar of the F1 generation in 5 against B. thuringiensis israelensis serotype-14 were 0.0283 mg/l replicates (20 larvae/replicate) in 250 ml dechlorinated water in a and 0.1209 mg/l, respectively. The LC50 and LC90 activity of B. 400 ml glass beaker have been used for susceptibility evaluation. thuringiensis israelensis serotype-14 against larvae of different A 1% stock solution of various insecticidal formulations was pre- ﬁeld strains of C. quinquefasciatus ranged from 0.0351 to 0.0565 pared in distilled water and fresh stock solution was used to make mg/l and 0.1108 to 0.1467 mg/l, respectively (Table 1). subsequent 5–6 serial dilutions. All experimental beakers were The susceptibility status of larvae of different strains of this kept under a 12-hr light : dark cycle. Mortality was recorded after species to Neemarin showed a wide range of LC50 (0.0137 to

24-hr exposure to temephos, fenthion, Neemarin and B. 0.0726 mg/l) and LC90 (4.2514 to 13.67 mg/l) after 24-hr expo- thuringiensis var. israelensis insecticides. Moribund larvae were sure, showing an increase in resistance from 1.35-fold (JMN considered to be dead. strain) to 5.29-fold (JD strain) in comparison to the LAB strain (Table 1). 3.2 Adult bioassay The adult bioassay was carried out using WHO test kits accord- 2. Adult bioassay ing to WHO methods under standard laboratory conditions.15) The susceptibility status of adults of LAB, JD, BKN, JMN and Two- to four-day-old non-blood-fed 20 females of F1 generation BTH strains of C. quinquefasciatus to various pyrethroids is in 5 replicates were used for toxicity studies. Acetone was used to shown in Table 2. LC50 and LC90 of adults of these strains against prepare 1% stock solution and serial dilutions. To prepare 1% in- lambda cyhalothrin ranged from 0.0085 to 0.0293% and 0.0450 secticide-treated paper, a 12 cm15 cm Whatman ﬁlter paper no. to 0.1708%, respectively, with 0.96- to 3.33-fold higher resist-

1 is treated with 2.75 ml insecticide solution (2.42 mg/ml a.i. ance than the LAB strain of C. quinquefasciatus. The LC50 and pyrethroid) with silicon oil and control paper treated only with LC90 to alpha cypermethrin of these strains ranged from 0.0045 2.75 ml acetone and silicone oil.16) Similarly, 5–6 papers contain- to 0.0247% and 0.0214 to 0.1553%, respectively; however, vary- Vol. 35, No. 1, 000–000 (2010) Development of insecticide resistance in Culex quinquefasciatus mosquito (Diptera: Culicidae) in India 3

Table 1. Susceptibility status of different populations of late 3rd–early 4th instars Culex quinquefasciatus to various larvicides

Populations LC50 (95% FL) LC90 (95% FL) RR

Temephos

Laboratory 0.0015 (0.0013–0.0018) 0.0089 (0.0066–0.0121) — Jodhpur 0.0162 (0.0137–0.0192) 0.1125 (0.080–0.157) 10.8 Bikaner 0.0086 (0.0071–0.0104) 0.0609 (0.0415–0.0892) 5.73 Jamnagar 0.0048 (0.0040–0.0057) 0.0325 (0.0241–0.0438) 3.2 Bathinda 0.0136 (0.0113–0.0163) 0.0764 (0.0568–0.1027) 9.06

Fenthion

Laboratory 0.0114 (0.0092–0.0140) 0.0746 (0.0542–0.1027) — Jodhpur 0.0792 (0.0672–0.0932) 0.4343 (0.3149–0.5989) 6.94 Bikaner 0.0470 (0.0388–0.0568) 0.2602 (0.1960–0.3454) 4.12 Jamnagar 0.0461 (0.0384–0.0553) 0.2300 (0.1774–0.2981) 4.04 Bathinda 0.0238 (0.0196–0.0287) 0.1314 (0.0991–0.1742) 2.08

Bacillus thuringiensis var. israelensis

Laboratory 0.0283 (0.0240–0.0332) 0.1209 (0.0951–0.153) — Jodhpur 0.0389 (0.0349–0.0434) 0.1108 (0.0933–0.1315) 1.37 Bikaner 0.0565 (0.0509–0.0627) 0.1467 (0.1255–0.1714) 1.99 Jamnagar 0.0321 (0.0279–0.0367) 0.1161 (0.0940–0.1433) 1.13 Bathinda 0.0351 (0.0309–0.0398) 0.1188 (0.0967–0.1458) 1.24

Neemarin

Laboratory 0.0137 (0.0075–0.0250) 4.2514 (1.5737–11.485) — Jodhpur 0.0726 (0.0439–0.1200) 13.67 (5.0574–36.96) 5.29 Bikaner 0.0594 (0.0361–0.0978) 9.9349 (3.7809–26.105) 4.33 Jamnagar 0.0186 (0.0100–0.0342) 7.5429 (2.4685–23.048) 1.35 Bathinda 0.035 (0.0200–0.0625) 12.8763 (4.2065–39.415) 2.57

LC50 and LC90 in mg/l; FL: ﬁducial limits at 95%; RR: resistance ratio. ing degrees of resistance (2.06- to 5.48-fold) were observed in cient vector-control programs.5) different ﬁeld strains of C. quinquefasciatus in comparison to the Temephos has been found to be useful to control various in-

LAB strain. The LC50 and LC90 of adults of these strains against sects, such as Simulium, Fiddler crabs, and Aedes albopictus, in cypermethrin ranged from 0.0317 to 0.0651% and 0.1122 to various habitats.20–24) The present study showed that larvae of JD, 0.3245% with a moderate resistance level, respectively. BTH and BKN strains of C. quinquefasciatus had a considerable level of resistance against temephos in comparison to the LAB Discussion strain, but low in the JMN strain (Table 1). For fenthion, high re- The destruction of vector mosquitoes is the most efficient method sistance by larvae of C. quinquefasciatus has been observed in of controlling disease, precluding effective transmission of the JD, BKN and JMN strains in comparison to the LAB strain, but it parasite. Insecticides have been the cornerstone of controlling the was low in the BTH strain. Previously, the development of resist- vectors of tropical diseases for nearly half a century; however, in- ance to temephos, fenthion and Bti by various populations of C. discriminate use of insecticides has resulted in the development pipiens was noticed in Cyprus and Tunisia.7,8) A high level of re- of resistance in many insect vectors. The WHO Experts Commit- sistance to DDT, bendiocarb, malathion, fenitrothion as adulti- tee has suggested updating knowledge about the status of vector cides and bromophos and fenthion as larvicides was observed in resistance, evaluating current and new methodologies and dis- C. pipiens in filaria endemic areas of Egypt; however, they were cussing essential components of resistance management for effi- susceptible to temephos.25) In another study, varying levels of re- 4 D. S. Suman et al. Journal of Pesticide Science

Table 2. Susceptibility status of different populations of adult Culex quinquefasciatus to various adulticides

Populations LC50 (95% FL) LC90 (95% FL) RR

Cypermethrin

Laboratory 0.0204 (0.0166–0.0250) 0.1122 (0.0854–0.1475) — Jodhpur 0.0576 (0.0494–0.0672) 0.2500 (0.1950–0.3204) 2.82 Bikaner 0.0317 (0.0271–0.0370) 0.1489 (0.1142–0.1939) 1.55 Jamnagar 0.0328 (0.0272–0.0394) 0.2123 (0.1491–0.3022) 1.60 Bathinda 0.0651 (0.0555–0.0764) 0.3245 (0.2434–0.4325) 3.19

Alpha cypermethrin

Laboratory 0.0045 (0.0038–0.0052) 0.0214 (0.0162–0.0287) — Jodhpur 0.0093 (0.0079–0.01090) 0.0466 (0.0345–0.0630) 2.06 Bikaner 0.0137 (0.0110–0.01692) 0.1060 (0.0720–0.1561) 3.04 Jamnagar 0.0247 (0.0202–0.0302) 0.1553 (0.1129–0.2136) 5.48 Bathinda 0.0223 (0.0183–0.02727) 0.1288 (0.0961–0.1724) 4.96

Lambda cyhalothrin

Laboratory 0.0088 (0.0074–0.0104) 0.0495 (0.0356–0.0686) — Jodhpur 0.0085 (0.0072–0.0099) 0.0450 (0.0334–0.0607) 0.96 Bikaner 0.0128 (0.0107–0.0153) 0.0689 (0.0517–0.0917) 1.45 Jamnagar 0.0261 (0.0218–0.0312) 0.1384 (0.1041–0.1840) 2.96 Bathinda 0.0293 (0.0244–0.0351) 0.1708 (0.1239–0.2354) 3.33

LC50 and LC90 in % treated paper; FL: fiducial limits; RR: resistance ratio. sistance to various insecticides, including temephos (10–100- (4.33-fold), BTH (2.57-fold) and JMN (1.35-fold) strains of this fold) and fenthion (0.9–6-fold), have been recorded in Aedes ae- mosquito; however, the level of resistance is much lower than for gypti in Latin American countries.26) temephos and fenthion. B. thuringiensis has been used to control the Simulium damno- For vector control, different formulations of pyrethroids have sum vector of Onchocerciasis in 11 countries in West Africa.27) also been used, such as mosquito coils, mats, liquid formulations, Development of resistance among mosquitoes has been reported and treated bed nets with direct exposure to mosquitoes, which in B. thuringiensis var israelensis and B. sphaericus.7,10,28) The may lead to the development of resistance. Various studies present study also indicated the high efficacy of B. thuringiensis have indicated the development of pyrethroid resistance in var israelensis against all tested field populations, indicating an mosquitoes.6,8,38) In the present study, different strains of C. quin- absence of resistance in all 4 strains of C. quinquefasciatus in quefasciatus have shown low resistance to lambda cyhalothrin comparison to LAB. Other studies have shown the effectiveness (0.96–3.33-fold) and cypermethrin (1.55–3.19-fold) but high to of B. thuringiensis var israelensis for mosquito control.29–32) alpha cypermethrin (2.06–5.48-fold); however, alpha cyperme- Higher toxicity to selective organisms has resulted in its rapid use thrin (0.0093–0.0247%) and lambda cyhalothrin (0.0085– as an alternative method to control mosquitoes. The little or lack 0.0293%) have shown a lower range of LC50 than cypermethrin of B. thuringiensis var israelensis use for mosquito control in (0.0317–0.0651%), indicating their long efficacy for mosquito Jodhpur, Bikaner, Jamnagar and Bathinda areas might be respon- control. The efficacy of deltamethrin, lambda cyhalothrin and sible for the high susceptibility of C. quinquefasciatus, which is a cyfluthrin has been reported by various workers against mosqui- good indication for the further potential use of this insecticide toes,40,41–43) whereas insecticide resistance to various pyrethroids, against the development of resistance to OP compounds. such as deltamethrin (1.5–112.5-fold), lambda cyhalothrin Neem compound, having various components, such as (0.1–30-fold), beta cypermethrin (0.01–30-fold) and cyfluthrin azadirachtin, nimbin, azadiradione, salanin and vepinin etc., has (0.3–10-fold) has also been reported in A. aegypti in several shown a broad range of bioactivity against insects.33–37) In the countries of Latin America.26) present study, the susceptibility status showed the slight develop- It can be surmised from the overall results of the present study ment of resistance against Neemarin in JD (5.67-fold), BKN that larvae of C. quinquefasciatus have developed insecticide re- Vol. 35, No. 1, 000–000 (2010) Development of insecticide resistance in Culex quinquefasciatus mosquito (Diptera: Culicidae) in India 5 sistance to temephos and fenthion in Jodhpur, Bikaner, Jamnagar 16) USAEHA: Procedures for the diagnostic dose resistance test and Bathinda in India. Due to resistance development, these in- kits for mosquitoes, body lice, and beetle pests of stored prod- secticides may not be effective for larval control in these areas ucts. U.S. Army Environmental Hygiene Agency, Aberdeen and may pose a problem of toxicity in nature. The development Proving ground, Maryland. USAEHA TG No. 189, pp. 3–3 of resistance in the larval stage against Neemarin has been ob- (1992). served in Jodhpur and Bikaner, but it is still effective in Bathinda 17) D. Finney: Probit Analysis. Cambridge University Press, Cam- and Jamnagar areas, indicating its potential for use in these two bridge, England (1971). areas. To control the larval stage of this mosquito, B. thuringien- 18) W. S. Abbott: J. Econ. Entomol. 18, 265–267 (1925). 19) J. F. Reinert, R. E. Harbach and I. J. Kitching: Zoo. J. Linnean. sis var. israelensis can be used, which has shown good efficacy Soc. 142, 289–368 (2004). against all four strains. As for pyrethroids, lambda cyhalothrin 20) S. C. 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