jear2012 Abstract Insecticide susceptibility tests using World Health Organization papers treated with 4% dichloro-diphenyl-trichloro-ethane (DDT), 0.05% deltamethrin, 0.05% lambda-cyhalothrin, 0.5% etofenprox, 0.15% cyfluthrin and 0.75% permethrin were conducted in Kamhororo, Masakadza and Chilonga villages, Zimbabwe. Three to 5-day old female Anopheles gambiae sensu lato adult mosquitoes were used. Deltamethrin knocked down 100% of the mosquitoes from Kamhororo, Masakadza and Chilonga at 35 min exposure. DDT did not knock down 100% of the mosquitoes from Kamhororo and Masakadza but did so in Chilonga. One hundred percent knockdown was achieved for cyfluthrin when exposed to mosquitoes from Kamhororo (60 min), Masakadza (25 min) and Chilonga (25 min). Etofenprox knocked down 100% of the mosquitoes collected from Kamhororo (30 min), Masakadza (30 min) and Chilonga (55 min). Knockdown of mosquitoes due to deltamethrin, DDT, cyfluthrin, permethrin; lambda-cyhalothrin and etofenprox were different at different observation times. One hundred percent mortality due to deltamethrin, DDT, etofenprox, lambda- cyhalothrin and cyfluthrin was recorded for mosquitoes collected from all the 3 sites. One hundred percent mortality due to pemethrin was recorded for mosquitoes collected from Kamhororo and Chilonga but mortality was 98.5% for those collected from Masakadza. No knock- down or mortality occurred in the controls from each locality. The kd50 (knockdown of 50% of the mosquitoes) values were 24.4-73.7 min (DDT), 8-13 min (pemethrin), 9.4-16.3 min (cyfluthrin), 9.4-14.4 min (etofenprox), 8.7-13 min (lambda-cyhalothrin) and 12.1-15.9 min (deltamethrin). The kd90 (knockdown of 90% of the mosquitoes) values were 45.6-199.5 min (DDT), 14.7-26.5 min (pemethrin), 16.5-34.9 min (cyfluthrin), 21.8-24.4 min (etofenprox), 16.3-31.6 min (lambda- cyhalothrin) and 21-25.3 min (deltamethrin). No insecticide resist- ance was recorded from the 3 sites. Introduction Malaria control is largely based on the use of long-lasting insecti- cide-treated nets and indoor residual spraying, but the efficacy of these control methods is endangered by the appearance of insecticide resistance in vector mosquitoes. Malaria in Zimbabwe causes signifi- cant mortality and morbidity although control efforts aimed at the main vector, Anopheles arabiensis, are instituted annually (Midzi et al., 2004, unpublished data). Anopheles gambiae sensu stricto, Anopheles arabiensis, and Anopheles funestus sensu stricto are the most important species for malaria transmission in Africa (Kawada et al., 2011). Insecticide resistance is a reduction in sensitivity of an insect pop- ulation as reflected by repeated failure of an insecticide to achieve the expected level of control when used according to recommendations (WHO, 1998). Insecticide resistance is mediated by behavioral, meta- bolic or physiological factors that result from: reduction in insecticide penetration, an increased metabolism of insecticide by metabolic enzymes and/or modification of the insecticide target site (WHO, 1998). World Health Organization (WHO, 1998) standards state that a mortality of 98-100% indicates susceptibility (no resistance); 80-97% Journal of Entomological and Acarological Research 2012; volume 44:e19 Correspondence: Nzira Lukwa, National Institute of Health Research, P.O. Box CY573, Causeway, Harare, Zimbabwe. Tel. +263.4.797052; +263.274664 - Fax: +263.4.253979. E-mail: nziraa33@yahoo.co.uk Key words: resistance, permethrin, DDT, etofenprox, deltamethrin, cyfluthrin, lambda-cyhalothrin. Authors’ contributions: NL and SS, research concept and design, data collec- tion tools, data analysis and interpretation, manuscript drafting, revision and final approval; PM, improving methodology, data collection tools, data analysis, revision and final approval; MZ, submitted manuscript revision, data analysis and manuscript final approval. Acknowledgments: the authors would like to acknowledge the following peo- ple who participated in data collection: Vimbai Chikwavaire, Clever Matiringe, Joel Mbedzi, White Soko, Tonderai Chiwade, Richard Mawoyo, Aleck Mogove Tozivepi, Letters Nyoni, Vitalis Kwashira, Darlington Mukotsi, Gumbo, Chiketa, Chin’ombe, Johane Muchenje, Cosmas Bvute, Peter Ndaima and Munjodzi Vhiriri. We also thank Dr S.L. Mutambu, Director of the National Institute of Health Research who supported this research. Funding: the authors are grateful to Mitsui Agro, Japan, for funding this data collection study through the National Malaria Control Programme. Received for publication: 7 June 2012. Revision received: 8 October 2012. Accepted for publication: 14 November 2012. ©Copyright N. Lukwa et al., 2012 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2012; 44:e19 doi:10.4081/jear.2012.e19 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Insecticide susceptibility tests conducted in Kamhororo, Masakadza and Chilonga villages in Zimbabwe during the 2011 malaria period N. Lukwa,1 S. Sande,2 P. Munosiyei,3 M. Zimba4 1National Institute of Health Research, Causeway, Harare; 2National Malaria Control Programme, Causeway, Harare; 3Bindura University of Science Education, Bindura; 4University of Zimbabwe, Biological Sciences, Mount Pleasant, Harare, Zimbabwe [Journal of Entomological and Acarological Research 2012; 44:e19] [page 107] No n- co mm er cia l u se on ly suggests the possibility of resistance that needs to be confirmed and less than 80% indicates resistance. However, when more than 100 mos- quitoes have been used per insecticide, less than 95% mortality strong- ly indicates resistance. However, no standards on knockdown times are specified to indicate resistance according to the WHO (1998). Pyrethroid insecticide resistance in An. gambiae is mainly associated with reduced target site sensitivity arising from a single point mutation in the sodium channel gene, often referred to as knock-down resist- ance (Awola et al., 2007). The susceptibility status of An. funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field collected mosquitoes; but this is not the case with An. gambiae (Morgan et al., 2010). Although insecticides have been used for a very long time in Zimbabwe, there are very few instances when resistance has been recorded (Munhenga et al., 2008). Three cases of insecticide resist- ance have been documented in Zimbabwe; one in Chiredzi involving benzene hexa-chloride (Green, 1982), one involving dichloro-diphenyl- trichloro-ethane (DDT) in Gokwe (Masendu, 2004; Masendu et al., 2005, unpublished data) and one involving DDT and permethrin in Gokwe (Munhenga et al., 2008). Munhenga et al. (2008) observed insecticide resistance to permethrin from An. arabiensis mosquitoes collected from Gwave, a locality 11 km from Kamhororo and 16 km from Masakadza. Munhenga et al., (2008) also recorded DDT resistance in Gwave (68.4% in 2006) but this reversed in 2008 (96% mortality). Insecticide susceptibility tests did not show any significant increase in the resistance status for either permethrin or DDT but an improvement in susceptibility over a 3-year period (Awola et al., 2007). Chanda et al. (2011) detected insecticide resistance to DDT, deltamethrin, lambda- cyhalothrin and permethrin in both An. gambiae ss and An. funestus ss collected in Zambia. Abilio et al., (2011) detected insecticide resistance to lambda-cyhalothrin, permethrin and bendiocarb in An. funestus col- lected in Mozambique. An. funestus mosquitoes were resistant to 0.75% permethrin and 0.05% deltamethrin (Morgan et al., 2010). There was suspected resistance to 4% DDT but these mosquitoes were fully suscep- tible to bendiocarb, malathion and dieldrin (Morgan et al., 2010). Djogbenou et al. (2011) observed full susceptibility to chlorpyrifos- methyl and very few samples displayed resistance to carbosulfan. Yewhalaw et al. (2011) observed that An. arabiensis mosquitoes were resistant to DDT, permethrin, deltamethrin and malathion, but suscep- tible to propoxur. Djogbenou et al. (2011) noted that insecticide suscep- tibility differs with geographical variation and this must be taken into account in the vector control strategies. For this reason, we conducted insecticide susceptibility tests in 3 different locations in Zimbabwe. Materials and methods Study areas Mosquito collection was performed in Midlands province, Gokwe South district, Kamhororo village (17°51’S, 28°38’E), Masakadza village (17°49’S, 28°36’E) and Masvingo province, Chiredzi district, Chilonga village (21°13’S, 31°39’E). The Kamhororo River runs through the village of the same name. It starts as an artesian well and flows for over 14 km. This is the major source of water for washing and domestic animals. There are no agricultural activities taking place along the river. However, cotton is grown extensively in the village and a lot of crop spraying takes place. Chances of pesticides getting into the river system are high when washing clothes and spraying equipment; washing facilities have been provided but the water flows back into the river. Mosquito larval collection was performed from hoof prints (a large number of cows are present and they drink this water. Masakadza vil- lage, 5 km from Kamhororo, is also on the Kamhororo River, but mos- quitoes were collected from a swamp that also started from an artesian well and flows for 1 km (this does not flow into the Kamhororo River). Water is used for washing (there are no designated washing facilities) and watering animals. Cotton growing is also widespread in the village. No agricultural activities near the swamp are conducted. Both Kamhororo and Masakadza are in dry areas where rainy water is limit- ed. Chilonga village is spanned by the expansive Runde River that flows for over 50 km. Kitchen gardening is the most common method of farm- ing in the villages although the river passes through large sugar estates in the low veldt district of Chiredzi. Mosquito collection Mosquito larvae were collected from breeding sites using larval scoops and placed in white plastic dishes (Figure 1). The collected lar- vae were morphologically identified and separated for rearing; the Kamhororo field insectary was used for Kamhororo and Masakadza mosquitoes, the Chilonga field insectary was used for Chilonga mos- quitoes. The identified An. gambiae sl mosquitoes were reared accord- ing to Awola et al. (2007) and the adults were provided with 10% sugar solution on cotton wool placed as a wick in a 50 mL glass bottle. Unfed 3-5 day old An. gambiae sl adults from the same study area were pooled together as this is the time/stage at which a sizable mosquito sample was obtained. Susceptibility tests WHO papers treated with 4% DDT, 0.05% deltamethrin, 0.05% lambda-cyhalothrin, 0.5% etofenprox, 0.15% cyfluthrin and 0.75% per- methrin were used according to the WHO (1998). The WHO (1998) states that knock-down rates should be measured every 10 min up to 60 min, but we made observations every 5 min so that we could detect even small differences. The WHO (1998) also states that in the event that 80% knockdown is not achieved after 60 min, the samples should be held for a further 20 min. We did not do this because two-thirds of the study sites had 80% of the mosquitoes knocked down within 60 min. The WHO (1998) states that 20-25 mosquitoes should be placed in each exposure tube (125 mm in length and 44 mm in diameter) but we used 15-20 mosquitoes before recording mortality after 24 h. All adult mosquitoes were removed from exposure tubes, provided with sugar water and held for 24 h. A total of 360, 240 and 236 mosquitoes from Masakadza, Kamhororo and Chilonga were posed to treated papers, respectively. The controls consisted of 50 mosquitoes in each study site. All exposure tubes were held in the vertical position. The insecticide treated papers were used once. Figure 1. Collection of mosquito larvae. Article [page 108] [Journal of Entomological and Acarological Research 2012; 44:e19] No n- co mm er cia l u se on ly Determination of kd50 and kd90 Kd50 (min required to 50% knockdown of the mosquitoes) and kd90 (min required to achieve 90% knockdown of the mosquitoes) were cal- culated using Probit Analysis. This uses the regression principle and correlates fixed time with knockdown response. In circumstances in which the data are not normally distributed or do not follow a regres- sion pattern, extrapolation is made beyond the period of observation. Data analysis Data was analyzed using analysis of variance (ANOVA) at 95% con- fidence limit. Results There was no knockdown or mortality from the 150 control mosqui- toes used in this study. Effect of deltamethrin on knocking down mosquitoes Deltamethrin knocked down 100% of the mosquitoes from Kamhororo, Masakadza and Chilonga after 35 min exposure to deltamethrin (Figure 2). One hundred percent mortality was recorded and no insecticide resistance was observed. There was no significant difference in knockdown of mosquitoes from Chilonga/Masakadza (P=0.13) and Chilonga/Kamhororo (P=0.42) after 5 min exposure to deltamethrin but a significant difference was seen in comparison with those from Kamhororo/Masakadza (P=0.03) (Table 1). There was no significant difference in knockdown of mosquitoes from Chilonga /Masakadza (P=0.22) and Chilonga/Kamhororo (P=0.09) at the 10 min observation time-point but a significant difference was seen in comparison with those from Kamhororo/Masakadza (P=0.003). There was no significant difference in knockdown of mosquitoes from Kamhororo/Masakadza (P=0.64), Chilonga/Kamhororo (P=0.47) and Chilonga/Masakadza (P=0.33) at the 15 min observation time-point. There was no significant difference in knockdown of mosquitoes from Kamhororo/Masakadza (P=0.59), Chilonga/Kamhororo (P=0.33) and Chilonga/Masakadza (P=0.301) at the 20 min observation time-point. There was no significant difference in knockdown of mosquitoes from Kamhororo/Masakadza (P=0.69), Chilonga/Kamhororo (P=0.49) and Chilonga/Masakadza (P=0.72) at the 25 min observation time-point. There was no significant difference in knockdown of mosquitoes from Kamhororo/Masakadza (P=0.27), Chilonga/Kamhororo (P=0.59) and Chilonga/Masakadza (P=0.27) at the 30 min observation time-point. There was no difference in knockdown rates from 35-60 min for mosqui- toes collected from either of the study sites. Effect of dichloro-diphenyl-trichloro-ethane on mosquito knockdown One hundred percent knockdown was not achieved for mosquitoes collected from Kamhororo and Masakadza apart from those from Chilonga when exposed to DDT (Figure 3). One hundred percent mor- tality was recorded and no insecticide resistance was observed. There was no significant difference in knockdown of mosquitoes from Chilonga/Masakadza (P=0.42), Chilonga/Kamhororo (P=0.42) and Masakadza/Kamhororo (P=0.27) after 5 min exposure to DDT (Table 2). A significant difference was found in knockdown of mosqui- toes from Chilonga/Masakadza (P=0.012) and Kamhororo/ Masakadza (P=0.048) compared with those from Chilonga/Kamhororo (P=0.42) for which no significant difference was found at the 10 min observation time-point. There was no significant difference in knockdown of mos- quitoes from Kamhororo/Masakadza (P=0.057), Chilonga/Kamhororo (P=0.52) and Chilonga/Masakadza (P=0.81) at the 15 min observation time-point. There was no significant difference in knockdown of mos- quitoes from Kamhororo/Masakadza (P=0.057), Chilonga/Kamhororo (P=0.085) and Chilonga/Masakadza (P=0.078) at the 20 min observa- tion time-point. Knockdown of mosquitoes from Kamhororo/Masakadza (P=0.06), Chilonga/Kamhororo (P=0.14) and Chilonga/Masakadza (P=0.32) were not significantly different at 25 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.16), Chilonga/Kamhororo (P=0.1) and Chilonga/Masakadza (P=0.11) were not significantly different at 30 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.01), Chilonga/Kamhororo (P=0.02) and Chilonga/ Masakadza (P=0.03) were significantly different at 35 min observation time. Knock down of mosqui- toes from Kamhororo/Masakadza (P=0.005), Chilonga/Kamhororo Figure 2. Knockdown rate of mosquitoes due to exposure to deltamethrin. Table 1. Knockdown of deltamethrin at each exposure time. Knockdown Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 2.5ab Mean 0b Mean 8.5a Range (0-5) Range (0) Range (5-10) 10 Mean 25%cd Mean 7.5d Mean 31.5c Range (20-30) Range (5-10) Range (30-35) 15 Mean 72.5e Mean 47.5e Mean 58.5e Range (65-80) Range (20-75) Range (50-65) 20 Mean 87.5f Mean 77.5f Mean 81.5f Range (85-90) Range (70-85) Range (75-85) 25 Mean 95g Mean 87.5g Mean 91.5g Range (90-100) Range (80-95) Range (80-100) 30 Mean 97.5h Mean 92.5h Mean 100h Range (95-100) Range (85-100) Range (100) 35 Mean 100 Mean 100 Mean 100 Range (100) Range (100) Range (100) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. Article [Journal of Entomological and Acarological Research 2012; 44:e19] [page 109] No n- co mm er cia l u se on ly (P=0.02) and Chilonga/Masakadza (P=0.045) were significantly different at 40 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.000) and Chilonga/Kamhororo (P=0.006) were significantly different at 45min observation time apart from Chilonga/Masakadza (P=0.07) that were not significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.001) and Chilonga/Kamhororo (P=0.01) were significantly different at 50min observation time apart from those from Chilonga/Masakadza (P=0.097) that were not significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.004), Chilonga/Masakadza (P=0.04) and Chilonga/Kamhororo (P=0.02) were significantly different at 55 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.000), Chilonga/Kamhororo (P=0.000) and Chilonga/Masakadza (P=0.03) were significantly different at 60 min observation time. Effect of cyfluthrin in knocking down mosquitoes Cyfluthrin knocked down 100% of the mosquitoes from Kamhororo (60 min), Masakadza (25 min) and Chilonga (25 min) (Figure 4). One hundred percent mortality was recorded and no insecticide resistance was observed. Knock down of mosquitoes from Chilonga/Masakadza (P=0.77) and Masakadza/Kamhororo (P=0.31) were not significantly different at 5 min exposure to cyfluthrin apart from those from Chilonga/Kamhororo (P=0.037) that were significantly different (Table 3). Knock down of mosquitoes from Chilonga/Masakadza (P=0.8), Kamhororo/ Masakadza Figure 3. Knock-down rate of mosquitoes due to dichloro- diphenyl-trichloro-ethane. Figure 4. Knockdown of mosquitoes due to exposure to cyfluthrin. Table 2. Knockdown of dichloro-diphenyl-trichloro-ethane at each exposure time. Knockdown Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 0a Mean 2.4a Mean 0a Range (0) Range (0-5) Range (0) 10 Mean 0be Mean 2.4de Mean 11.5c Range (0) Range (0-5) Range (10-15) 15 Mean 10.5f Mean 2.4f Mean 13.5f Range (0-22.2) Range (0-5) Range (10-15) 20 Mean 36.8g Mean 2.4g Mean 15g Range (27.8-50) Range (0-5) Range (10-20) 25 Mean 63.2h Mean 2.4h Mean 25h Range (38.9-94.4) Range (0-5) Range (15-35) 30 Mean 68.4i Mean 7.5i Mean 28.5i Range (50-94.4) Range (4.8-10) Range (15-45) 35 Mean 78.9j Mean 9.8k Mean 45l Range (72.2-94.4) Range (9.5-10) Range (35-50) 40 Mean 86.8m Mean 17n Mean 66.5p Range (83.3-100) Range (10-23.8) Range (60-70) 45 Mean 89.5st Mean 24.3q Mean 81.5rt Range (88.9-100) Range (23.8-25) Range (80-85) 50 Mean 89.5wx Mean 29.2u Mean 83.5vx Range (88.9-100) Range (23.8-35) Range (80-85) 55 Mean 92a Mean 36.6b Mean 86.5c Range (94.4-100) Range (28.6-45) Range (85-90) 60 Mean 100d Mean 61e Mean 91.5f Range (100) Range (60-61.9) Range (90-95) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. Table 3. Knockdown of cyfluthrin at each exposure time. Knockdown Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 15a Mean 0a Mean 15a Range (12-18) Range (0) Range (5-35) 10 Mean 32.5b Mean 21.4b Mean 33.5b Range (22.5-42.5) Range (14.3-27.2) Range (15-65) 15 Mean 92.5d Mean 45.2c Mean 58.5c Range (90-95) Range (38.1-50) Range (35-75) 20 Mean 97.5f Mean 69e Mean 86.5ef Range (95-100) Range (63.6-71.4) Range (70-100) 25 Mean 100 g Mean 78.6h Mean 100g Range (100) Range (72.7-81) Range (100) 30 Mean 100ij Mean 78.6j Mean 100i Range (100) Range (72.7-81) Range (100) 35 Mean 100km Mean 92.8k Mean 100lm Range (100) Range (90.5-100) Range (100) 40 Mean 100n Mean 95.2p Mean 100n Range (100) Range (90.9-95.3) Range (100) 45 Mean 100q Mean 95.2r Mean 100q Range (100) Range (95.3-95.5) Range (100) 50 Mean 100s Mean 95.2s Mean 100s Range (100) Range (95.3-100) Range (100) 55 Mean 100r Mean 95.2r Mean 100r Range (100) Range (95.3-100) Range (100) 60 Mean 100t Mean 100t Mean 100s Range (100) Range (100) Range (100) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. Article [page 110] [Journal of Entomological and Acarological Research 2012; 44:e19] No n- co mm er cia l u se on ly (P=0.53) and Chilonga/Kamhororo (P=0.42) were not significantly dif- ferent at 10 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.44 and Chilonga/Masakadza (P=0.11) were not significantly different at 15 min observation time apart from those from Chilonga/Kamhororo (P=0.017) that were significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.2) and Chilonga/Masakadza (P=0.41) were not significantly different at 20 min observation time apart from those from Chilonga/Kamhororo (P=0.02) that were significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.049) and Chilonga/Kamhororo (P=0.03) were significantly different apart from those from Chilonga/Masakadza at 25 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.049) and Chilonga/Kamhororo (P=0.03) were significantly different at 30min observation time apart from those from Chilonga/Masakadza. Knock down of mosquitoes from Kamhororo/Masakadza (P=1.8¥10-5) and Chilonga/Kamhororo (P=0.000) were significantly different at 35 min observation time apart from those from Chilonga/Masakadza. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.02) were signifi- cantly different apart from those from Chilonga/Kamhororo (P=0.09) and Chilonga/Masakadza at 40 min observation time. Knock down of mosqui- toes from Kamhororo/Masakadza (P=9.3¥10-6) and Chilonga/Kamhororo (P=0.000) were significantly different at 45 min observation time apart from those from Chilonga/Masakadza. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.27), Chilonga/Kamhororo (P=0.42) and Figure 5. Knockdown of mosquitoes due to etofenprox. Figure 6. Knockdown rate of mosquitoes due to permethrin. Table 4. Knockdown of etofenprox at each exposure time. Knockdown Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 2.6a Mean 0a Mean 25b Range (0-48) Range (0) Range (20-30) 10 Mean 25.6c Mean 12.8c Mean 45c Range (23.8-27.8) Range (0-26.3) Range (35-55) 15 Mean 48.7d Mean 59d Mean 66.5d Range (38-61) Range (40-78.9) Range (50-80) 20 Mean 87.2e Mean 79.5e Mean 85e Range (83.3-90.5) Range (65-94.7) Range (75-90) 25 Mean 94.2f Mean 94.9f Mean 96.5f Range (88.9-95.2) Range (95-100) Range (95-100) 30 Mean 97.4g Mean 100g Mean 100g Range (94.4-100) Range (100) Range (100) 35 Mean 97.4h Mean 100h Mean 100h Range (94.4-100) Range (100) Range (100) 40 Mean 97.4i Mean 100i Mean 100i Range (94.4-100) Range (100) Range (100) 45 Mean 100j Mean 100j Mean 100j Range (100) Range (100) Range (100) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. Table 5. Knockdown of permethrin at each exposure time. Knockdown Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 7.5a Mean 0a Mean 16.5b Range (5-10) Range (0) Range (15-20) 10 Mean 62.5c Mean 25.6c Mean 41.5c Range (60-65) Range (15.8-35) Range (30-50) 15 Mean 87.5d Mean 64d Mean 68.5d Range (80-95) Range (47.4-80) Range (55-79) 20 Mean 100e Mean 87.2e Mean 85e Range (100) Range (78.9-95) Range (80-94.7) 25 Mean 100 g Mean 94.9f Mean 96.5fg Range (100) Range (94.7-95) Range (95-100) 30 Mean 100h Mean 94.9i Mean 100h Range (100) Range (94.7-95) Range (100) 35 Mean 100h Mean 97.4i Mean 100h Range (100) Range (94.7-95) Range (100) 40 Mean 100j Mean 97.5j Mean 100j Range (100) Range (95-100) Range (100) 45 Mean 100j Mean 97.5j Mean 100j Range (100) Range (95-100) Range (100) 50 Mean 100j Mean 97.5j Mean 100j Range (100) Range (95-100) Range (100) 55 Mean 100j Mean 97.5j Mean 100j Range (100) Range (95-100) Range (100) 60 Mean 100j Mean 97.5j Mean 100j Range (100) Range (95-100) Range (100) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. Article [Journal of Entomological and Acarological Research 2012; 44:e19] [page 111] No n- co mm er cia l u se on ly Chilonga/Masakadza were not significantly different at 50 min observa- tion time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.27), Chilonga/Kamhororo (P=0.42) and Chilonga/Masakadza were not significantly different at 55 min observation time and this was the same at 60 min. Effect of etofenprox in knocking down mosquitoes One hundred percent knock down was achieved for mosquitoes col- lected from Kamhororo (30 min), Masakadza (30 min) and Chilonga (55 min) when exposed to etofenprox (Figure 5). One hundred percent mortality was recorded and no insecticide resistance was observed. Knock down of mosquitoes from Chilonga/Masakadza (P=0.01) and Masakadza/Kamhororo (P=0.006) were significantly different at 5 min exposure to etofenprox apart from those from Chilonga/Kamhororo (P=0.4) that were not significantly different (Table 4). Knock down of mosquitoes from Chilonga/Masakadza (P=0.09), Kamhororo/Masakadza (P=0.08) and Chilonga/Kamhororo (P=0.44) were not significantly differ- ent at 10 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.72), Chilonga/Masakadza (P=0.31) and Chilonga/Kamhororo (P=0.7) were not significantly different at 15 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.79), Chilonga/Masakadza (P=0.61) and Chilonga/Kamhororo (P=0.69) were not significantly different at 20 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.72), Chilonga/Kamhororo (P=0.66) and Chilonga/Masakadza (P=0.24) were not significantly different at 25 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/Masakadza (P=0.27) and Chilonga/Kamhororo (P=0.42) were not significantly different at 30 min observation time. Knock down of mos- quitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/Masakadza (P=0.27) were not significantly different at 35 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/Masakadza (P=0.27) were not significantly different at 40 min observation time. Knock down of mos- quitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/Masakadza (P=0.27) were not significantly different at 50 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/Masakadza (P=0.27) were not significantly different at 55 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza; Chilonga/Kamhororo and Chilonga/Masakadza were not significantly different at 60 min observa- tion time. Effect of permethrin in knocking down mosquitoes One hundred percent knock down was achieved for mosquitoes col- lected from Kamhororo (20 min), Masakadza (30 min) and Chilonga (20 min) when exposed to permethrin (Figure 6). One hundred percent mortality was recorded for mosquitoes collected from Kamhororo and Chilonga apart from those from Masakadza (98.5%). No insecticide resistance was recorded from the 3 sites. Knock down of mosquitoes from Chilonga/Masakadza (P=0.04) and Masakadza/Kamhororo (P=0.003) were significantly different at 5 min exposure to permethrin apart from those from Chilonga/Kamhororo (P=0.09) that were not significantly different (Table 5). Knock down of mosquitoes from Chilonga/Masakadza (P=0.09), Kamhororo/Masakadza (P=0.21) and Chilonga/Kamhororo (P=0.06) were not significantly differ- ent at 10 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.08, Chilonga/Masakadza (P=0.16) and Chilonga/Kamhororo (P=0.31) were not significantly different at 15 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.96), Chilonga/ Masakadza (P=0.09) and Chilonga/Kamhororo (P=0.24) were not significantly different at 20 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.2) and Chilonga/Masakadza (P=0.49) were not significantly different at 25 min observation time apart from those from Chilonga/Kamhororo (P=0.000) that were significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=2.25¥10-5) and Chilonga/Kamhororo (P=0.000) were significantly different at 30 min observation time apart from Chilonga/Masakadza. Knock down of mosqui- toes from Kamhororo/Masakadza (P=2.25¥10-5) and Chilonga/Kamhororo (P=0.000) were significantly different at 35 min observation time apart from those from Chilonga/Masakadza. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/ Masakadza were not significantly different at 40min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/ Kamhororo (P=0.42) and Chilonga/Masakadza were not significantly dif- Figure 7. Knockdown rate of mosquitoes due to exposure to lambda-cyhalothrin. Table 6. Knockdown of lambda-cyhalothrin at each exposure time. Knock down Chilonga Kamhororo Masakadza (min) (%) (%) (%) 0 Mean 0 Mean 0 Mean 0 Range 0 Range 0 Range (0) 5 Mean 10ab Mean 2.5b Mean 20a Range (0-20) Range (0-5) Range (15-30) 10 Mean 65c Mean 10d Mean 35cd Range (60-70) Range (10) Range (15-60) 15 Mean 82.5f Mean 35e Mean 55ef Range (80-85) Range (25-45) Range (15-80) 20 Mean 95g Mean 62.5g Mean 66.7g Range (90-100) Range (45-80) Range (25-90) 25 Mean 100h Mean 90h Mean 86.7h Range (100) Range (90) Range (60-100) 30 Mean 100i Mean 100i Mean 88.3i Range (100) Range (100) Range (65-100) 35 Mean 100j Mean 100j Mean 100j Range (100) Range (100) Range (100) Same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference Article [page 112] [Journal of Entomological and Acarological Research 2012; 44:e19] No n- co mm er cia l u se on ly ferent at 50 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/Kamhororo (P=0.42) and Chilonga/ Masakadza were not significantly different at 55 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza, Chilonga/ Kamhororo (P=0.42) and Chilonga/Masakadza were not significantly dif- ferent at 60min observation time. One hundred percent knock down was achieved for mosquitoes col- lected from Kamhororo (30 min), Masakadza (35 min) and Chilonga (25 min) when exposed to lambda-cyhalothrin (Figure 7). One hundred percent mortality was recorded for mosquitoes from all the study sites and no resistance was recorded. Knock down of mosquitoes from Chilonga/Masakadza (P=0.67) and Chilonga/ Kamhororo (P=0.54) were significantly different at 5 min exposure to lambda-cyhalothrin apart from those from Masakadza/Kamhororo (P=0.038) that were significantly different (Table 6). Knock down of mosquitoes from Chilonga/Masakadza (P=0.18) and Kamhororo/Masakadza (P=0.23) were not significantly dif- ferent at 10 min observation apart from those from Chilonga/Kamhororo (P=0.008) that were significantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.51) and Chilonga/Masakadza (P=0.37) were not significantly different at 15 min observation time apart from those from Chilonga/Kamhororo (P=0.04) that were signifi- cantly different. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.89), Chilonga/Masakadza (P=0.37) and Chilonga/Kamhororo (P=0.22) were not significantly different at 20 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.85), Chilonga/Masakadza (P=0.49) and Chilonga/Kamhororo (P=0.9) were not significantly different at 25 min observation time. Knock down of mosquitoes from Kamhororo/Masakadza (P=0.49), Chilonga/Kamhororo and Chilonga/Masakadza (P=0.49) were not significantly different at 30 min observation time. One hundred percent knockdown of mosquitoes was reported for mosquitoes collected from the 3 study areas. Determination of kd50 and kd90 The kd50 values were 24.4-73.7 min (DDT), 8-13 min (pemethrin), 9.4-16.3 min (cyfluthrin), 9.4-14.4 min (etofenprox), 8.7-13 min (lamb- da-cyhalothrin) and 12.1-15.9 min (deltamethrin) (Figure 8). The kd90 values were 45.6-199.5 min (DDT), 14.7-26.5 min (pemethrin), 16.5-34.9 min (cyfluthrin), 21.8-24.4 min (etofenprox), 16.3-31.6 min (lambda-cyhalothrin) and 21-25.3 min (deltamethrin) (Figure 9). Discussion The time required to knock-down 100% of the mosquitoes was com- pared and the results indicated that there was no difference when mos- quitoes were exposed to different sources of deltamethrin. DDT only managed to knock-down 100% of the mosquitoes collected from Chilonga and could not knock-down mosquites from either Kamhororo or Masakadza. This might be due to the great pressure being placed on the mosquitoes from Kamhororo and Masakadza through intensive application of pesticides for cotton growing while this is not the case Figure 8. Kd50 values in minutes. Dichloro-diphenyl-trichloro-ethane (DDT) values were extrapolated by Probit analysis because 100% knockdown was not achieved. Figure 9. Kd90 values. Dichloro-diphenyl-trichloro-ethane (DDT) values were extrapolated by Probit analysis because 100% knockdown was not achieved. Article [Journal of Entomological and Acarological Research 2012; 44:e19] [page 113] No n- co mm er cia l u se on ly with Chilonga (approx. 600 km from these 2 sites). It is worth noting that Gwave (a village not very far away from Kamhororo and Masakadza where insecticide resistance has been detected) serves as a potential reservoir of DDT resistant mosquitoes, as observed by Masendu (2004), Masendu et al. (2005, unpublished data) and Munhenga et al. (2008). Problems with not achieving 100% knockdown when DDT was used might be an indication of knock-down resistance, as observed by Awola et al. (2007). However, Djiegbe et al. (2011) demonstrated that a high frequency of resistant genes does not necessarily translate into resistance in An. gambiae sl mosquitoes. It is important to study this mechanism in the follow-up studies. There was no great difference between the times required to knock- down 100% of the mosquitoes due to lambda-cyhalothrin and perme- thrin from the 3 sites. This is interesting because Munhenga et al. (2008) recorded insecticide resistance from mosquitoes from Gwave but this has not been observed for mosquitoes from Kamhororo and Masakadza in terms of knock-down time. The times required for 100% knockdown of mosquitoes from Chilonga and Masakadza (for cyfluthrin) were very similar but were abnormally high for Kamhororo; the reasons for this are not known. Interestingly, the time required for 100% knockdown of mosquitoes from Chilonga (etofenprox) was high- er than that of Kamhororo and Masakadza; the reasons for this are not known. It may be linked to pest control on sugar estates since there is no sugar cane cultivation in either Kamhororo or Maskadza. In general, knock-down rates of mosquitoes appeared to be differ according to their sources and this was also time dependent. This trend was also observed for the different insecticides under study. This high- lights the need to study all the insecticide classes in order to under- stand this trend as this may provide useful information on the possibil- ity of insecticide resistance developing in some localities in Zimbabwe. It is also important to cover all geographical areas since this study was only carried out in dry areas where malaria is prevalent. Mortality rates are encouraging from all the study sites when consid- ering all the insecticides used in this study. No insecticide resistance was observed at the study sites. It is important to monitor trends in pemethrin response for mosquitoes from Masakadza (near Gwave where pemethrin resistance has been reported by Munhenga et al. 2008). Interestingly, this trend was not observed in Kamhororo that is nearer Gwave than Masakdaza. Thus, the absence of DDT and perme- thrin resistance agrees with observations of Dabire et al. (2008). The kd50 and kd90 values obtained from all the study sites (for DDT) appeared to be within the same range but these were abnormally high for mosquitoes collected from Kamhororo. This may signal future prob- lems with DDT use in Kamhororo, but according to Munhenga et al. (2008), reversal of resistance may occur. We are not sure whether this will happen for mosquitoes collected from Kamhororo. Otherwise, our results show that the tested insecticides have reasonable knock-down rates in the study areas. Use of Probit Analysis showed the difference between knock-down rates. One major observation from this program is that it extrapollates results when 100% knock down is not achieved and at times, this goes beyond the study time. These results agree with earlier observations that cases of insecti- cide resistance are very rare in Zimbabwe, in agreement with Munhenga et al. (2008), Green (1982), Masendu (2004) and Masendu et al. (2005, unpublished data). Unfortunately, Munhenga et al. (2008) did not detect any knock-down resistance or mutations from Gwave but no information is available for Kamhororo. Knock-down rates from our study agree with findings of Djogbenou et al. (2011) that insecticide susceptibility differs according to geographical variations. More stud- ies on this should be conducted across the country. References ABILIO A.P., KLEINSCHMIDT I., REHMAN A.M., CUAMBA N., RAMDEEN V., MTHEMBU D.S., et al., 2011 - The emergence of insecticide resistance in central Mozambique and potential threat to the suc- cessful indoor residual spraying malaria control programme. Malaria J. 2: 110. AWOLA T.S., ODUOLA A.O., OYEWOLE I.O., OBANSA J.B., AMAJOH C.N., KOEKEMOER L.L., COETZEE M., 2007 - Dynamics of knock- down pyrethroid insecticide resistance alleles in a field population of Anopheles gambiae s.s. in south-western Nigeria. J. Vector-Borne Dis. 44: 181-188. CHANDA E., HEMINGWAY J., KLEINSCHMIDT I., REHMAN A.M., RAMDEEN V., PHIRI F.N., et al., 2011 - Insecticide resistance and the future of malaria control in Zambia. PLoS One 6: e24336. DABIRE K.R., DIABATE A., AGOSTINHO F., ALVES F., MANGA L., FAYE O., BALDET T., 2008 - Distribution of the members of Anopheles gambi- ae and pyrethroid knock-down resistance gene (kdr) in Guinea- Bissau, West Africa. Bull. Soc. Pathol. Exotique. 101: 119-123. DJEGBE I., BOUSSARI O., SIDICK A., MARTING T., RANSON H., CHAN- DRE F., et al., 2011 - Dynamics of insecticide resistance in malaria vectors in Benin: first evidence of the presence of L1014S kdr muta- tion in Anopheles gambiae from West Africa. - Malaria J. 10: 261. DJOGBENOU L., PASTEUR N., AKOGBETO M., WEILL M., CHANDRE F., 2011 - Insecticide resistance in the Anopheles gambiae complex in Benin: a nationwide survey. Med. Vet. Entomol. 25: 256-67 GREEN C.A., 1982 - Malaria epidemiology and anopheline cytogenetics. In: Pal R., Kitzmiller J.B., Kanda T., (eds.). Cytogenetics and genet- ics of vectors. Elsevier Biomedical, Amsterdam: 21-29. KAWADA H., DIDA G.O., OHASHI K., KOMAGATA O., KASAI S., TOMITA T., et al., 2011 - Multimodal pyrethroid resistance in malaria vec- tors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya. PloS One 6: e22574. MASENDU H.T., 2004 - Vector mosquitoes and their significance in malaria epidemiology and control in Zimbabwe. PhD Thesis University of Witwatersrand, South Africa: 1-10 MORGAN J.C., IRVING H., OKEDIV L.M., STEVEN A., WONDJI C.S., 2010 - Pyrethroid resistance in an Anopheles funestus population from Uganda. PloS One 29: e11872. MUNHENGA G., MASENDU H.T., BROOKE B.D., HUNT R.H., KOEKE- MOER L.K., 2008 - Pyrethroid resistance in the major malaria vec- tor Anopheles arabiensis from Gwave, a malaria-endemic area in Zimbabwe. Malaria J. 7: 247. YEWHALAW D., WASSIE F., STEUBAUT W., SPANOGHE P., VAN BORTEL W., DENIS L., et al., 2011 - Multiple insecticide resistance: an impediment to insecticide-based malaria vector control program. PLos One 12: e16066. WHO (WORLD HEALTH ORGANIZATION), 1998 - Report of the WHO informal consultation. Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces. World Health Organization, Geneva. Available from: http://www.who.int/malaria/publications/ atoz/who_cds_cpc_ mal_98_12/en/index.html [page 114] [Journal of Entomological and Acarological Research 2012; 44:e19] Article No n- co mm er cia l u se on ly