Insecticide resistance and, efficacy of space spraying and larviciding in the control of dengue vectors Aedes aegypti and Aedes albopictus in Sri Lanka

Pesticide Biochemistry and Physiology 107 (2013) 98–105

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Insecticide resistance and, efficacy of space spraying and larviciding in the control of dengue vectors Aedes aegypti and Aedes albopictus in Sri Lanka S.H.P.P. Karunaratne a,⇑, T.C. Weeraratne a, M.D.B. Perera b, S.N. Surendran c a b c

Department of Zoology, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka Regional Office, Anti-Malaria Campaign, Kurunegala, Sri Lanka Department of Zoology, Faculty of Science, University of Jaffna, Jaffna, Sri Lanka

a r t i c l e

i n f o

Article history: Received 27 September 2012 Accepted 14 May 2013 Available online 30 May 2013 Keywords: Dengue vectors Insecticide resistance Fogging Temephos

a b s t r a c t Unprecedented incidence of dengue has been recorded in Sri Lanka in recent times. Source reduction and use of insecticides in space spraying/fogging and larviciding, are the primary means of controlling the vector mosquitoes Aedes aegypti and Ae. albopictus in the island nation. A study was carried out to understand insecticide cross-resistance spectra and mechanisms of insecticide resistance of both these vectors from six administrative districts, i.e. Kandy, Kurunegala, Puttalam, Gampaha, Ratnapura and Jaffna, of Sri Lanka. Efficacy of the recommended dosages of frequently used insecticides in space spraying and larviciding in dengue vector control programmes was also tested. Insecticide bioassay results revealed that, in general, both mosquito species were highly resistant to DDT but susceptible to propoxur and malathion except Jaffna Ae. aegypti population. Moderate resistance to malathion shown by Jaffna Ae. aegypti population correlated with esterase and malathion carboxylesterase activities of the population. High levels of acetylcholinesterase (AChE) insensitivity in the absence of malathion and propoxur resistance may be due to non-synaptic forms of AChE proteins. Moderate pyrethroid resistance in the absence of high monooxygenase levels indicated the possible involvement of ‘kdr’ type resistance mechanism in Sri Lankan dengue vectors. Results of the space spraying experiments revealed that 100% mortality at a 10 m distance and >50% mortality at a 50 m distance can be achieved with malathion, pesguard and deltacide even in a ground with dense vegetation. Pesguard and deltacide spraying gave 100% mortality up to 50 m distance in open area and areas with little vegetation. Both species gave >50% mortalities for deltacide at a distance of 75 m in a dense vegetation area. Larval bioassays conducted in the laboratory showed that a 1 ppm temephos solution can maintain a larval mortality rate of 100% for ten months, and the mortality rate declined to 0% in the eleventh month. In the field, where 1 ppm concentration is gradually decreased with water usage, 100% mortality was observed only for the first four months, <50% mortality for the next two months, and 0% mortality was observed eight months after the application of temephos. Deltacide can be effectively used for space spraying programmes in Sri Lanka. Larval control can be successfully achieved through temephos with public participation. Ó 2013 Published by Elsevier Inc.

1. Introduction Aedes aegypti (Linnaeus) and Ae. albopictus Skuse are the major vectors of arboviral diseases such as dengue fever, yellow fever and chickungunya [1]. Dengue fever (DF) and dengue hemorrhagic fever (DHF) are of major public health concern in Sri Lanka. During year 2009, over 32,000 cases and 315 deaths were reported across the country [2]. Sri Lanka also experienced an outbreak of chikungunya during 2006/2007 period [3]. In the absence of a licensed ⇑ Corresponding author. Fax: +94 81 2388018 E-mail address: [email protected] (S.H.P.P. Karunaratne). 0048-3575/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.pestbp.2013.05.011

vaccine, the major focus in dengue disease control programmes of the island is vector control through elimination of breeding sites and application of insecticides. Spraying/fogging of insecticides has been widely used for several years in Sri Lanka to control dengue vectors, especially during disease outbreaks. Four major groups of synthetic insecticides i.e. organochlorines, organophosphates, carbamates and pyrethroids, are commonly used in insect pest control programmes. Target site for organophosphates and carbamates is insect acetylcholinesterases. For pyrethroids and a group of organochlorines (DDT + its analogues) the target site is Na+ channel regulatory proteins of the nerve membrane. For the rest of the organochlorines (cyclodienes), it is

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c-aminobutyric acid (GABA) receptors [4]. Continuous exposure to insecticides leads to development of insecticide resistance in vector populations. Mosquitoes develop resistance through increased activity of insecticide detoxifying enzymes and/or insensitive trget sites [4,5]. Chemical control with insecticide residual spraying (IRS) is not recommended for dengue vectors as these mosquitoes do not usually rest on walls. Space spraying of insecticides and application of larvicides are the major methods of chemical control used in rapid control of vector populations, especially during the outbreaks. Malathion, pesguard FG161 and deltacide are commonly used for space spraying in Sri Lanka while temephos is used as the main larvicide. It is important to assess the efficacy of these insecticidebased control methods in order to strengthen the present vector control programmes. Since the resistant individuals have a selective advantage over susceptible individuals, resistance genes spread in the population rapidly and once they become dominant, control failures occur. Understanding mechanisms of insecticide resistance in vector populations is essential to delay or prevent/revert the development of resistance and also to design new insecticides to combat resistant strains. Present study was designed and conducted to identify insecticide resistance status and also to identify some of the underlying resistance mechanisms of Ae. aegypti and Ae. albopictus populations in selected localities of Sri Lanka. The efficacy of space spraying of the recommended adulticides and application of the recommended larvicide were also studied.

1 - Peradeniya

6

2 - Teliyagonna 3 - Dehiowita 4 - Pasyala 5 - Chilaw 6 - Thiruelvely

55

2

4

1

3

2. Material and methods 2.1. Study sites and mosquito collection Mosquito eggs and larvae were collected from selected localities in six administrative districts in Sri Lanka i.e. Peradeniya in Kandy district (7° 150 N, 80° 360 E), Theliyagonna in Kurunegala district (7° 280 N, 80° 230 E), Chilaw in Puttalam district (7° 35’ N, 79° 48’ E), Pasyala in Gampaha district (7° 10’ N, 80° 07’ E) and Dehiowita in Ratnapura district (6° 58’ N, 80° 15’ E) from August 2007 to August 2008, and Thirunelvely in Jaffna district (9° 68’ N, 80° 03’ E) from November 2009 to January 2010 (late collection from Jaffna was due to security reasons) (Fig. 1). Both Ae. aegypti and Ae. albopictus eggs and larvae were collected from all the five districts except from Jaffna where only Ae. aegypti eggs could be collected. All the laboratory experiments were carried out in the Department of Zoology, Faculty of Science, University of Peradeniya, Sri Lanka. Mosquito eggs were collected using ovitraps prepared using black plastic cups and filter paper strips were used as oviposition substrates. Collection of filter paper strips with eggs from the ovitraps placed in field sites was done on weekly basis. Carefully removed filter papers were brought to the laboratory and immersed in plastic basins containing dechlorinated tap water allowing the eggs to hatch. Ae. aegypti and Ae. albopictus larvae were identified using standard keys [6,7] and reared in separate basins. Powdered fish meal pellets were given as larval food. Once the adults were emerged, they were transferred to adult mosquito cages and fed on cotton pads soaked in 10% glucose solution (25 ± 2° C, 80% RH). In addition to the egg collections, larvae were directly collected from different breeding habitats in the field sites and reared under the same laboratory conditions to obtain adults. Three day old adults were used for bioassay experiments. For biochemical assays, the adult mosquitoes (2–3 days old) were frozen and stored at 20° C and used for assays within two weeks upon storage.

Fig. 1. Map of Sri Lanka showing the study sites (1–6) where the collection of Ae. aegypti and Ae. albopictus eggs/larvae were carried out.

2.2. Chemicals and equipment Chemicals were purchased form Sigma chemicals U.K. unless otherwise stated. All the technical grade insecticides (DDT, malathion, carbamates and pyrethroids) (97–99% pure) used to prepare insecticide impregnated papers were a gift from Prof. Janet Hemingway, Liverpool School of Tropical Medicine, UK. Pesguard FG 161Ò (d-tetramethrin + cyphenothrin) and deltacideÒ (deltamethrin + s-bioallethrin + piperonyl butoxide) were from Bayer, Thailand, and malathion (97–99% pure) used for fogging was from United Phosphorus Ltd., India. Temephos (1% sand granules) was from Zagro Chemicals, Malaysia. UVmaxELx 800™ absorbance microtitre plate reader was from molecular devices, Bio-Tek. USA. Miniprotean II gel electrophoresis apparatus and protein assay kit were from BIO-RAD, U.K. 2.3. Preparation of insecticide impregnated papers Insecticide-impregnated papers were prepared according to the standard World Health Organization (WHO) method [8]. WHO recommended discriminating dosages of insecticides for Aedes mosquitoes i.e. 4% DDT (an organochlorine), 0.75% malathion (an organophosphate), 0.1% propoxur (a carbamate) and 0.25% permethrin (a pyrethroid) [9] were prepared by mixing the technical grade insecticide with the spreading agent. DDT, malathion and propoxur solutions were prepared in olive oil. Permethrin solutions were made in Dow-Corning 556 silicone fluid. Rectangles of

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Whatman no. 1 filter papers (12 cm 15 cm) were used for insecticide impregnation. Insecticide/oil solutions (0.7 ml) were mixed with an equal volume of acetone and the mixture was spread uniformly on the filter paper. Acetone was used to ensure an even distribution of the solution on the paper. Insecticide impregnated papers were left overnight at room temperature to allow complete evaporation of acetone. Papers were then foil wrapped and stored at 20° C until use. 2.4. Adult bioassay experiments 2.4.1. Tarsal contact bioassays The standard World Health Organization (WHO) procedures were followed to determine susceptibility/resistance status of adult mosquitoes [10]. Bioassays were conducted by means of tarsal contact exposure of mosquitoes to insecticide impregnated papers using WHO test kits. Adult female mosquitoes (2–3 day old) were tested against the WHO discriminating dosages of DDT, malathion, propoxur and permethrin. Batches of 10–20 mosquitoes (depending on the availability) were exposed to insecticide impregnated papers for 1 h at 25 ± 2° C and 80% relative humidity (RH). Mortalities were recorded after a recovery period of 24 h. At least five replicates (a minimum of 100 mosquitoes) were carried out per insecticide/species/district. Control experiments were carried out by exposing the mosquitoes to papers impregnated with carrier oil alone. Data were considered only if the control mortalities were less than 20%. If there were mortalities in controls, actual mortalities were calculated using Abbott’s formula [11]. 2.4.2. Cage bioassay Efficacy of space spraying of the standard concentrations of malathion (250 ml malathion in 5 L kerosene oil), pesguard FG 161 (d-tetramethrin + cyphenothrin) (30 ml in 5 L kerosene oil) and deltacide (deltamethrin + s-bioallethrin + piperonyl butoxcide) (100 ml in 5 L kerosene oil) was tested against dengue vectors in Kurunegala municipal area using standard WHO cage bioassays [12]. Cylindrical cages (20 cm length 5 cm diameter) prepared using 16-mesh nylon netting were hung at 10, 25, 50, 75 and 100 m distance away from the place of insecticide fogging in downwind direction. Three replicates were arranged for each distance. All cages were hung 1.5 m above the ground level. Twenty adult mosquitoes (2–3 day old) were released to each cage and fogging was carried out by delivering standard dosages of insecticides. The experiments were repeated in three different habitat types e.g. open ground area, an area with little vegetation and an area with dense vegetation. After a spray round of 8 min, cages were removed and the mosquitoes were transferred into clean plastic cups with 10% glucose soaked cotton pads. Mosquitoes were examined after 1 h to determine the initial knockdowns and the mortalities were recorded after a 24 h recovery period. 2.5. Larval bioassays 2.5.1. Laboratory trials Efficacy of 1% temephos sand granules was tested against Ae. aegypti and Ae. albopictus larvae under laboratory conditions. Each of 25 cement tanks (20 cm 20 cm) was filled with 10 L of dechlorinated water and 1 g of temephos was added to give the WHO recommended concentration of 1 ppm. Bioassays were carried out using three randomly selected tanks as replicates for each time point. Twenty late third instar larvae were introduced to each of tank and grounded fish food was given as larval food. Mortalities were recorded after a 24 h exposure period. Control experiments were done in 10 L dechlorinated water alone. Initially the bioassays were conducted weekly. Once the larval survival was initiated, bioassays were conducted daily.

2.5.2. Field trials A locality, where residents store water in cement tanks for bathing and washing purposes, was selected from the Kurunegala municipal area. These cement tanks (180 x 120 x 90 cm) have been reported as good breeding sites for Aedes species (13). Temephos (1%) sand granules were applied into 148 cement tanks in 148 households to give approximately 1 ppm concentration. Required amount of insecticide was wrapped in a piece of cloth and allowed to float on the water surface of each tank. Bioassays were conducted at weekly intervals using ten randomly selected tanks out of 148 tanks. Larval cages, made from 5 L capacity buckets, were used to hold and expose larvae to the water in tanks. Bottom of each bucket was covered with 100-mesh nylon strainer cloth to allow water circulation and the top of the bucket was covered with a net. The bucket was fixed to a hole in a polystyrene board enabling the larval cage to float in the water of the cement tank (one cage per tank). Twenty late 3rd instar larvae of both Ae. aegypti and Ae. albopictus were introduced separately into each of larval cages placed in 10 randomly selected tanks. After a 24 h exposure the buckets were removed from the tank and dead/live larvae were counted. Bioassays were repeated so that a minimum of 200 larvae from each species were tested against 1 ppm temephos. Control experiments were done using the tanks filled with water alone. 2.6. Biochemical assays 2.6.1. Microtitre plate assays Unfed adult females (200 per assay) of each of the species Ae.aegypti and Ae. albopictus from each district were subjected individually to carboxylesterase, glutathione S-transferase (GST), monooxygenase, acetylcholinesterase and protein assays. All the biochemical experiments were carried out according to the procedures outlined by WHO [14]. Mosquitoes were individually homogenized in 150 lL of ice-cold distilled water. Fifty microliter of each crude homogenate was taken for AChE assays. Remaining homogenates were centrifuged at 13,000g for 2 min and the supernatants were used for other assays. 2.6.2. Malathion metabolism Batches of 25 mosquitoes (about 40–60 mg wet weight) were homogenized in 0.5 ml of 25 mMTris–HCl buffer (pH 7.5) and centrifuged at 13,000g for 5 min. Supernatant was incubated with 300 lM malathion for 2 h at room temperature. The mixture was then extracted twice with 0.5 mL acidified chloroform. The chloroform extract was dried under a current of air, re-dissolved in 30 lL acidified chloroform and loaded onto a thin layer chromatography plate. After running with n-hexane: diethyl ether (1:3) the plate was sprayed with 0.5% (w/v) 2,6-dibromoquinone 4-chloromide in cyclohexane and left at 100 °C for 2 h to visualize malathion and its metabolic products. Tris buffer (0.5 mL), incubated with 1 lL of malathion and NaOH solution (10 lL of 300 lM Malathion + 10 lL of 300 lM NaOH and heated at 80 °C) was run as a positive control. Tris buffer (0.5 mL), incubated with 1 lL of 300 lM malathion, was run as a negative control. 2.6.3. Polyacrylamide gel electrophoresis (native PAGE) Native polyacrylamide gel electrophoresis (PAGE) was used to visualize elevated esterase isozymes [15]. Gels were stained for esterases activity with the substrates a- and b–naphthyl acetate and esterase bands were identified with their relative mobility (Rf) i.e. ratio of the migratory distances of the esterase band and xylene cyanol dye front. For insecticide inhibition studies, gels were separately incubated with 0.1 mM paraoxon, propoxur and permethrin in 50 mM phosphate buffer (pH 7.2) after the electrophoresis for 10 min and then exposed to the substrate solutions. Control experiments were done by incubating with buffer without

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insecticides. The degree of inhibition of each band was recorded visually according to their colour intensity compared to the control.

Table 1 Percentage mortalities of Ae. aegypti and Ae. albopictus populations to the discriminating dosages of DDT, malathion, propoxur and permethrin (n = 100 for each insecticide/species/district). Results are grouped as susceptible (98–100% mortality), possibly resistant (80–97% mortality) and resistant (<80% mortality) [16].

2.7. Analysis of results Results of the tarsal contact bioassays were interpreted as susceptible (98–100% mortality), possibly resistant (80–97% mortality) and resistant (<80% mortality) according to WHO recommendations [16]. Percentage mortalities obtained from cage bioassay (space spraying) experiments were statistically analyzed according to the insecticide, habitat type, distance and mosquito species using two-way analysis of variance (ANOVA minitab 16 version). Multiple comparisons between the significant levels of interactions of the variables were done by Tokey’s method.

District

DDT (4%)

Malathion (0.8%)

Propoxur (0.1%)

Permethrin (0.25%)

Kandy Kurunegala Ratnapura Gampaha Puttalam Jaffna

Ae. ae. 9a 30a 31a 10a 18a 60a

Ae. ae. 97b 100c 100c 90b 92b 52a

Ae. ae. 82b 100c 100c 100c 100c 100c

Ae. ae. 51a 54a 60a 42a 38a 92b

Ae.al. 8a 28a 30a 13a 20a ⁄

Ae.al. 97b 100c 100c 83b 88b ⁄

Ae.al. 98c 100c 100c 100c 100c ⁄

Ae.al. 25a 45a 54a 39a 38a ⁄

⁄

Not tested (no Ae. albopictus could be collected from Jaffna collection sites). Ae. ae.- Aedes aegypti, Ae.al.- Aedes albopictus. a Resistant. b Possibly resistant. c Susceptible.

3. Results

tive insecticide- distance interaction group consisted of pesguard-10 m, malathion-10 m, deltacide-10 m and deltacide25 m. The most effective distance- habitat interaction group consisted of 10 m- open ground, 10 m- little vegetation and 10 m- dense vegetation. The most effective insecticide- habitat group comprised of deltacide- open ground, deltacide- little vegetation and deltacide- dense vegetation.

3.1. Insecticide bioassays Results of the adult insecticide bioassays conducted with the discriminating dosages of DDT, malathion, permethrin and propoxur for Ae. aegypti and Ae. albopictus populations from different districts are presented in Table 1. Populations of both species were resistant to DDT in all the districts. However the lowest DDT resistance (40% resistance) was from Ae. aegypti from Jaffna district and the highest resistance was from Kandy and Gampaha districts (about 87–91% survivals). All the populations were more or less susceptible to malathion except for Jaffna Ae. aegypti population (48% survivals). A possible resistance to propoxur was shown only by Kandy Ae. aegypti population while all the others were susceptible to propoxur. Resistance to permethrin (40–75%) was shown by all the populations except Jaffna Ae. aegypti, which showed only 8% permethrin resistance (Table 1).

3.3. Larva bioassays Laboratory larval bioassays with 1 ppm temephos for both species resulted in 100% mortality for the first ten months. Survival of larvae was observed after ten month (Fig. 2). In the field where 1 ppm concentration was gradually decreased with usage of water, 100% mortality was observed only for the first four months, >50% mortality for next two months and 0% mortality was observed eight months after the application of temephos (Fig. 3).

3.2. Space spraying 3.4. Biochemical assays Results of the space spraying experiments are given in Table 2. Statistical analysis of cage bioassay results showed that the efficacies of the three insecticides were significantly different from each other (P < 0.05). Deltacide showed the highest efficacy followed by pesguard and malathion. Mortalities were significantly higher in open ground followed by little vegetation and dense vegetation habitat types (P < 0.05). At 10 m distance, all the cage bioassays gave 100% mortalities. With increased distance, percentage mortalities were significantly decreased (P < 0.05) and at 100 m distance all bioassays gave 0% mortality (Table 2). There was no significant mortality difference between the two species Ae. aegypti and Ae. albopictus for different variables tested. There were significant interaction effects between insecticide- distance, distance- habitat and insecticide- habitat (F = 43.62, 5.64, 3.83 respectively). According to multiple comparisons (at 95% confidence level) most effec-

Enzyme activity/quantity profiles of insecticide detoxifying enzymes i.e. carboxylesterases, GSTs and monooxygenases for all Ae. aegypti and Ae. albopictus populations are shown in Fig. 4. Mean values for the populations are given in the Table 3. Ratnapura, Gampaha and Puttalam populations of Ae. aegypti showed high carboxylesterase and GST activity profiles. Gampaha Ae. albopictus population had a high carboxylesterase profile where as Puttalam and Kandy populations had high GST profiles. Monooxygenase quantity profiles were high in Kurunegala for Ae. albopictus. Elevation of esterases was evident from gel electrophoresis studies as well. Native PAGE resolved one elevated esterase band from Ae. aegypti from Jaffna district (Rf = 0.67 with xylene cyanol marker) and the elevated activity was inhibited only by malathion (Figures not shown).

Table 2 Percentage mortalities of Aedes aegypti and Ae. albopictus when exposed to malathion, pesguard and deltacide fog at five different distances in three different habitats (n = 100; a = Ae. aegypti, b = Ae. albopictus). Open ground

Malathion Pesguard Deltacide

a b a b a b

With little vegetation

With dense vegetation

10 m

25 m

50 m

75 m

100 m

10 m

25 m

50 m

75 m

100 m

10 m

25 m

50 m

75 m

100 m

100 100 100 100 100 100

76 80 100 100 100 100

60 68 78 64 88 85

0 0 0 0 45 50

0 0 0 0 0 0

100 100 100 100 100 100

70 72 100 100 100 100

56 60 80 82 80 78

0 0 0 0 32 38

0 0 0 0 0 0

100 100 100 100 100 100

67 60 72 85 100 100

50 52 48 58 76 74

0 0 0 0 30 40

0 0 0 0 0 0

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110

100

90

80

% Mortality

70

60

50

40

30

20

Ae. albopictus

10

Ae. aegypti

0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

No. of days Fig. 2. Percentage mortalities of Ae .aegypti and Ae. albopictus larvae with ten month old 1 ppm temephos. Cement tanks (25) with 1 ppm temephos in dechlorinated water (10 L) were prepared in the laboratory. Larval bioassays were conducted by exposing 20 late third instar larvae/tank for 24 h using three randomly selected replicates, initially on weekly basis. Survival of larvae was observed only during the eleventh month after application of temephose where the bioassays were conducted on daily basis.

4. Discussion In Sri Lanka, DDT was the insecticide used in mosquito control programmes in 1960s and early 1970s. It was subsequently banned in 1975/1977 due to development of insecticide resistance in malaria vectors and also due to environmental concerns. DDT was replaced by malathion and other organophosphates, which are still being used in mosquito control programmes in the country. In Sri Lanka, use of carbamates has been restricted only to agricultural sector. Development of resistance to malathion and other organophosphates by malaria vectors demanded the introduction of pyrethroids in early 1990s [17]. Due to the increased incidence of dengue at an alarming rate, health authorities instigated to use insecticides, in addition to use of biological agents and removal/destroying Aedes breeding sites, to control dengue vector mosquitoes.

High resistance to DDT in both Ae. aegypti and Ae. albopictus shows that even after three decades of cessation of DDT usage, resistance to DDT still prevails among Sri Lankan mosquitoes. This phenomenon has already been reported for malaria vectors and other mosquito species in Sri Lanka [17–19]. It has been suggested that the GST based resistance mechanism in malaria mosquitoes was originally selected in Sri Lanka with high usage of DDT prior to 1975/77, and was favored and maintained by the subsequently used organophosphate insecticides [4,20]. Despite having a very low activity level of GSTs to metabolize DDT, Kandy Ae. aegypti

120

100

80

% Mortality

Malathion metabolism studies were undertaken on samples of both Ae. aegypti and Ae. albopictus from all districts to determine whether a malathion carboxylesterase resistance mechanism was present in these populations. Metabolism of malathion into monoand di-acid products within the standard time period was detected in homogenates from Gampaha and Jaffna Ae. aegypti populations, and Gampaha and Puttalam Ae. albopictus populations. Other populations did not show this mechanism. Biochemical assays were undertaken to determine the involvement of insensitive AChE enzymes in organophosphate and/or carbamate resistance. Percentage remaining activities of AChEs, when inhibited by propoxur, are shown in Table 4. Except for Jaffna and Kandy, all Ae. aegypti populations showed higher AChE insensitivity levels. Highest level of insensitivity among Ae. albopictus populations was shown by Ratnapura population.

60

40

20

Ae. aegypti Ae. albopictus

0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32

No. of weeks Fig. 3. Changes in the percentage mortalities of Ae.aegypti and Ae. albopictus larvae after exposing to temephose for a 24 h period in water storage cement tanks in households in the field (temephose concentration was initially adjusted to 1 ppm but later diluted with usage).

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Aedes aegypti

(A) 3.0 2.5

2.5

2.0

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0 Gampaha

Jaffna

Kandy

Kurunegalla

Puttalam

Ratnapura

(B) 1.6

1.6

1.4

1.4

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

Gampaha

Jaffna

Kandy

Kurunegala

Aedes albopictus

3.0

Puttalam

Ratnapura

Gampaha

Kandy

Kurunegala

Puttalam

Ratnapura

Gampaha

Kandy

Kurnegala

Puttalam

Ratnapura

Kurunegala

Puttalam

(C) 0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

0.00

Gampaha

Jaffna

Kandy

Kurunegala

Puttalam

Ratnapura

0.00

Gampaha

Kandy

Ratnapura

Fig. 4. Profiles of (A) carboxylesterase activity (lmol min1 mg1), (B) glutathione S-transferase activity (lmol min1 mg1), and (C) monooxygenase quantity (equivalent units of cytocrome P450) in different populations of Ae. aegypti and Ae. albopictus.

population showed a high DDT resistance indicating possible involvement of a kdr type resistance mechanism. Dengue vector susceptibility to malathion may be due to their inadequate exposure to malathion and/or proper management of

malathion usage. Heavy and indiscriminating usage of malathion has been reported from 1983–2010 during the civil war in Jaffna district and this may have selected malathion resistance genes in mosquito populations [21]. Increased enzyme activities to

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Table 3 Mean carboxylesterase activity (lmol min1 mg1), mean glutathione S- transferase (GST) activity (lmol min1 mg1) and mean monooxygenase quantity (equivalent units of cytocrome P450) of Ae. aegypti and Ae. albopictus populations (n = 100 for each assay/species/district). Carboxylesterase assays (kinetic) were done by mixing mosquito homogenates individually with q-nitrophenyl acetate (qNPA) and GST assays (kinetic) with 1-chloro-2,4,-dinitro benzene (CDNB) + reduced glutathione (GSH). Monooxygenase quantities were estimated by titrating the amount of bound haem in the mosquito homogenate with tetramethyl benzidine (TMBZ) against a standard curve of cytochrome C [13]. Enzyme

Kandy

Carboxylesterases GSTs Monooxygenases

*

Mean SE Mean SE Mean SE

Kurunegala

Ratnapura

Gampaha

Puttalam

Jaffna

Ae. aegy.

Ae. albo.

Ae. aegy.

Ae. albo.

Ae. aegy.

Ae. albo.

Ae. aegy.

Ae. albo.

Ae. aegy.

Ae. albo.

Ae. aegy.

Ae. albo.

0.116 0.009 0.116 0.009 0.0055 0.0003

0.106 0.012 0.546 0.055 0.0048 0.0005

0.113 0.012 0.169 0.014 0.0068 0.0005

0.061 0.006 0.312 0.043 0.0120 0.0011

0.164 0.020 0.741 0.052 0.0012 0.0007

0.335 0.036 0.264 0.048 0.0009 0.0001

0.448 0.186 0.662 0.104 0.0031 0.0003

0.862 0.255 0.303 0.079 0.0008 0.0001

0.114 0.046 0.516 0.075 0.0077 0.0001

0.378 0.058 0.490 0.082 0.0023 0.0004

0.125 0.042 0.170 0.017 0.0015 0.0002

* * * * * *

Not tested (no Ae. albopictus could be collected from Jaffna collection sites).

Table 4 Inhibition of acetylcholinesterases of Ae. aegypti and Ae. albopictus. Each crude homogenate from individual mosquito was tested for AChE activity (using dithiobis- 2 nitrobenzoic acid and acetylthiocholine iodide) with and without propoxur. Mean percentage remaining activities were calculated by taking percentage remaining activity in the inhibited fraction (with propoxur) compared to uninhibited control fraction (without propoxur) for each homogenate (n = 200 per species per district). Ae. aegypti

Kandy Kurunegala Ratnapura Gampaha Puttalam Jaffna

Ae. albopictus

Mean% remaining activity

SE

Mean% remaining activity

SE

9.17 59.04 73.14 68.07 80.69 6.76

1.67 2.59 5.92 6.13 6.41 2.62

17.80 15.30 76.81 31.66 29.98

1.73 2.53 7.41 4.08 4.00

*

*

SE-standard error. * Not tested.

metabolize malathion could be detected in this population by malathion carboxylesterase and gel electrophoresis studies. Malathion carboxylesterases mechanism, which could be detected in three other populations also, has not been reported previously from Sri Lankan Ae. aegypti and Ae. albopictus [22]. Resistance to carbamates cannot be expected as Sri Lanka has never used carbamates to control mosquitoes. Altered AChE mechanism may be responsible for the possible resistance seen in Kandy Ae. aegypti population. Resistance to permethrin was detected in all the populations of Ae. aegypti and Ae. albopictus except from Jaffna where the usage of pyrethroids was not significant [21]. Involvement of kdr type mechanism in pyrethroid resistance can also be anticipated in the absence of high monooxygenase levels. However, the higher rate of mortality observed with the spraying of deltacide, which includes the monooxygenase inhibitor piperonyl butoxide, may indicate some involvement of monooxygenases in pyrethroid resistance. Involvement of a number of mutations in the voltage gated sodium channel of nerve membranes have been reported in pyrethroid resistant strains of Ae. aegypti from other geographical areas (23). Screening of Sri Lankan Ae. aegypti and Ae. albopictus populations for the presence of these mutations is important for effective dengue vector control strategies. Although AChEs were less sensitive to propoxur inhibition, resistance to malathion and propoxur was not detected as it could have been anticipated. Two ace genes for acetylcholinesterases have been reported from Ae. aegypti [24] Mutations in ace-1 have been associated with resistance to insecticides in other mosquitoes. Although the presence of altered AChE- based resistance mechanism in Ae. aegypti have been indicated, mutations involved have not been reported [23,25,26]. In cotton whitefly, Bemisia tabaci, it has been shown that the resistance against organophos-

phates and carbamates vary regardless of high level of AChE insensitivity in individuals of a population. Two forms of AChEs have been identified; a protein responsible for the synaptic transmission and a protein not involved in the nerve impulse transmission. Sensitivity level of these two forms towards organophosphates and carbamates is different and the presence of these two proteins, synaptic and non-synaptic, can be identified using specified substrates and kinetic parameters [27,28]. In Sri Lanka, technical malathion, pesguard and deltacide are used as insecticides for space spraying to reduce the vector density of Aedes mosquitoes. The results of the present study suggest that the most effective insecticide for space spraying is deltacide, followed by pesguard and technical malathion. Although all insecticides could kill 100% of both species of Aedes adults at 10 m distance in all three habitat types, killing effect was negligible when the mosquitoes were 75 m away from the origin of fogging except for deltacide. Space spraying operations have been evaluated in several previous trials in other countries. In Malaysia, it was reported that combination of pesguard-PS 102 and Vectobac 12 AS (Bti) applied by vehicle mounted ULV machine against Ae. aegypti produced >90% mortality in adults which were placed at a distance of 100 m away from the spraying machine [29]. In India, it was shown that indoor thermal fogging using deltacide has a strong adulticidal effect against Ae. aegypti [30]. The formulation of deltacide may be more effective as it contains deltamethrin (a killing agent), S-bioallethrin (a knock-down agent) and piperonyl butoxide (a synergist). The results show that even with hand operated thermal fogging machines, the effects of deltacide is relatively high even for a habitat with dense vegetation. Temephos (1%) S.G. at the concentration of 1 ppm is recommended by WHO as a larvicide for Aedes larvae [31]. In Sri Lanka, use of temephos is promoted in household water storage tanks and containers that could not be covered with lids. Although the initial temephos concentration dilutes over the time, the results of the present study indicate that its effectiveness stands for more than three months. Temephos is not an expensive insecticide and 1 ppm temephos S.G. can be highly recommended to use as a larvicide for Aedes breeding habitats including water storage tanks. However, further studies are needed to investigate the public perception, water use practices and container types in different socio-ecological settings in order to use temephos extensively with community participation. In other geographical areas, insecticide resistance in Ae. aegypti is known to be widespread and Ae. albopictus is considered as a much more susceptible species although the published reports of bioassays on the latter is very limited [32]. In general, Ae. aegypti is considered as a urban domestic species and Ae. albopictus as a vector found in rural and semi-urban areas. However, it has been shown that Ae. albopictus has dominated over the major vector

S.H.P.P. Karunaratne et al. / Pesticide Biochemistry and Physiology 107 (2013) 98–105

Ae. aegypti over the time [33,34]. This may be due to higher adaptability of Ae. albopictus to unfavorable weather conditions gaining the ability to replace the primary vector [35]. In the present study, comparable cross-resistance spectra were seen in both species indicating that loss of habitat boundaries has given both species a similar exposure to insecticides. Development of resistance is dengue vectors in Sri Lanka is mainly due to the selection pressure through indoor residual spraying activity to control malaria vectors for many years, outdoor fogging, larviciding and domestic use of insecticides. Present study also reveals that space spraying with deltacide would be more effective than other chemicals used by the island for adult control. Larval control can be done by the effective usage of temephos and this has to be achieved through public participation. It is expected that the results of the present study would help health authorities to use appropriate insecticides in each administrative district to delay the onset of resistance and to use appropriate insecticides to get the maximum effect through space spraying and larviciding. Acknowledgments This work was funded by the National Science Foundation of Sri Lanka (Research Grant No. RG/2006/HS/02). References [1] S.C. Weaver, W.K. Reisen, Present and future arboviral threats, Antiviral Res. 85 (2010) 328. [2] Ministry of Healthcare and Nutrition, Sri Lanka, Survey on Dengue Epidemic 2009 (Part 1), Weekly Epidemiological, Report. 36(50), 2009, pp. 143–152. [3] World Health Organization, Prevention and Control of Chikungunya in SouthEast Asia: Report of the Expert Group Meeting Aurangabad, India, Regional Office for South-East Asia, (2008) Available from: (accessed 26 09 2012). [4] S.H.P.P. Karunaratne, Insecticide resistance in insets: a review, Ceylon J. Sci. (Biological Sciences). 25 (1998) 72–99. [5] J. Hemingway, N.J. Hawkes, L. McCarrol, H. Ranson, The molecular basis of insecticide resistance in mosquitoes, Insect Biochem. Mol. Biol. 34 (2004) 325– 327. [6] H. D. Pratt, C.J. Stojanovich, N.J. Magennis, Work book on identification of Aedes aegypti larvae, U.S. Department of health, education and welfare public health service 57 (1964). Available from: http: //www.eric.ed.Gov/ERICDocs/ data/ericdocs2sql/content_storage01/0000019b/80/36/7e/b7.pdf (accessed 5 04 2010). [7] K. Tanaka, K. Mizusawa, E.S. Saugstad, A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawa island) and Korea (Diptera: Culicidae), Contrib. Am. Entomol. Inst. 19 (1979) 987. [8] World Health Organization, Insecticide resistance and vector control: 13th report of the WHO expert committee. 265 1963, pp. 106. [9] World Health Organization, WHO Pesticide Evaluation Scheme (WHOPES) Available from: (accessed 5 12 2006). [10] World Health Organization, Insecticide resistance and vector control, Tech. Rep. Ser. 265 (1963) 41–47. [11] W.S. Abbott, A method of computing the effectiveness of an insecticide, J. Econ. Entomol. 18 (1925) 265–267. [12] World Health Organization, Guidelines for assessing the efficacy of insecticidal space sprays for control of dengue vector Aedes aegypti, WHO/CDS/CPE/PVC/ 2001.1. (2001). [13] T. C. Weeraratne, M. D. B. Perera, M. A. C. M. Mansoor, S.H.P.P. Karunaratne, Prevalence and the breeding habitats of dengue vectors Aedes aegypti and Aedes albopictus in semi-urban areas in two different climatic zones in Sri Lanka, International J. Trop. Insect Sci. (accepted). [14] World Health Organization, Techniques to detect insecticide resistance mechanisms (field and laboratory manual), WHO/CDS/CPC/MAL/98.6. (1998) 1–35.

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