Microsoft Word - 1-Dr. Sallahodin .doc Iranian J Arthropod-Borne Dis, 2007, 1(2): 1-6 S Sulaiman et al.: Evaluation of Pyrethrin … 1 Original Article Evaluation of Pyrethrin Formulations on Dengue/Dengue Haemorrhagic Fever Vectors in the Laboratory and Sublethal Effects *S Sulaiman, K Fadhlina, O Hidayatulfathi Department of Biomedical Science, Faculty of Allied Health Sciences, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia (Received 3 May 2007; accepted 2 Oct 2007) Abstract In Southeast Asia, Aedes aegypti (L.) has been incriminated as principal vector of dengue viruses and Ae. albopictus as the secondary vector of dengue fever. Therefore, the aim of this study was to investigate the effectiveness of three formula- tions of pyrethrin derived from Tanacetum cinerariaefolium against the dengue/dengue haemorrhagic fever vectors Aedes aegypti and Ae. albopictus in the laboratory. The testings employed 2 methodologies: the WHO Larval Bioassay and WHO Adult Bioassay. The results showed that all the three pyrethrin formulations had larvicidal and adulticidal activities. The impact of the sublethal doses of pyrethrin formulations on Aedes spp. larvae resulted in 4-6% of alive adult emergence compared to 90% of Ae. aegypti emerging adults and 96% Ae. albopictus alive adult emergence in the control. The impact of sublethal doses of the pyrethrin formulations caused very low fecundity on both Aedes spp. compared to the control (P< 0.05). Keywords: Pyrethrin, Dengue vectors, Sublethal effects Introduction Pyrethroids have been widely used for den- gue vector control. Lambda-cyhalothrin had been shown to be effective against dengue vectors under laboratory and field conditions in Malay- sia (Lim and Visalingam 1990, Lim and Lee 1991, Sulaiman et al. 1991, 1993). Alphacyperme- thrin had been shown to be effective against dengue vectors in the field in Malaysia, demon- strating both adulticidal and larvicidal effects (Sulaiman et al. 1995). Pyrethrin derived from pyrethrum daisy Tanacetum cinerariaefolium is a highly effective insecticide for controlling insect pests. Pyrethrum had shown repellent activity against Mansonia mosquitoes (Hadis et al. 2003), sampling indoor resting African malaria vectors is done traditionally by hand catches with oral or mechanical aspirators and pyrethrum catches (Harbison et al. 2006) and entomological evalua- tion of malaria vectors at different altitudes (Kul- karni et al. 2006). Pyrethrum is being used for impregnating of bednets and curtains, made of polypropylene fibre (Curtis et al. 1992). The objective of this study was to evaluate the efficacy of various pyrethrin formulations on the larval and adult stages of the dengue vectors, and the effect of sublethal dose on the larval stage of the dengue vectors on adult emer- gence and fecundity in the laboratory. Materials and Methods The pyrethrin formulations used were pyre- thrin 50%, pyrethrin 0.4g/l+ PBO 1.5g/l, pyrethrin 44g/l+ PBO 160g/l and insecticides Abate® and Malathion. The pyrethrin formulations were sup- *Corresponding author: Prof Dr S Sulaiman, Tel: +03 40405416, Fax: +603 26929032, E-mail: salsul@medic.ukm.my Iranian J Arthropod-Borne Dis, 2007, 1(2): 1-6 S Sulaiman et al.: Evaluation of Pyrethrin … 2 plied by Botanical Resources Australia Pty Ltd, Tasmania, Australia. Larval Bioassay The larval bioassay was conducted on Aedes aegypti and Aedes albopictus, based on WHO instruction (1981a). The experiment was conducted at 25± 1 ºC and relative humidity 80± 5%. Twenty five Ae. aegypti and Ae. albopic- tus 4th instar larvae were exposed to 250 ml of prepared pyrethrin concentrations and Abate® in 600 ml beakers. Four replicates were con- ducted and mortality was recorded after 24 h. The LC50 and LC90 levels, regression slopes and associated 95% fiducial limits were determined by computer using probit analysis (Raymond 1985). Adult Bioassay The adult bioassay was conducted on Ae. aegypti and Ae. albopictus using WHO test kit (1981b). Fifteen 2-5 day old female mosquitoes were exposed for 1h to the filter papers in the WHO test kit, impregnated with various concen- trations of pyrethrin formulations and malathion as positive control, then transferred to holding tubes. Sugar solution soaked in cotton was pro- vided as food. Four replicates were conducted and the number of adult mortality was recorded after 24 h. LC50 and LC90 levels, regression slopes and associated 95% fiducial limits were determined by Probit analysis (Raymond 1985) Studies on sublethal effects were based on Loh and Yap study (1989) with some modifica- tions. The concentration used was the LC50 of each pyrethrin formulation from the larval bio- assay. Fifty 4th instar larvae of Ae. aegypti and Ae. albopictus were used for each sublethal dose of pyrethrin formulation. Mortality was recorded after 24 h exposure and the surviving larvae were transferred into beakers containing distilled water. The number of surviving lar- vae, pupae and emerging adults was recorded. The emerging adults were blood fed and al- lowed to oviposit and the eggs were counted. The eggs were allowed to hatch into larvae, pu- pae and adults and recorded. The control in- cludes non insecticide treated mosquitoes. Data were analyzed using Mann-Whitney U test. Results Table 1 showed that both Ae. aegypti and Ae. albopictus 4th instar larvae were more sus- ceptible to pyrethrin 44g/l+ PBO 160g/l than pyrethrin 0.4g/l + PBO 1.5g/l or pyrethrin 50%. The pyrethrin 44g/l+ PBO 160g/l LC50 and LC90 for Ae. aegypti 4th instar larvae were 0.002 ppm and 0.007 ppm. The LC50 and LC90 for Ae. albopictus 4th instar larvae were 0.004 ppm and 0.012 ppm, respectively. The LC50 and LC90 values for pyrethrin 50% were higher than LC50 and LC90 for pyrethrin 0.4g/l+ PBO 1. 5g/l for Ae. aegypti 4th instar larvae (0.038 ppm and 0.135 ppm:0.028 ppm and 0.080 ppm). Similarly, the LC50 and LC90 for pyrethrin 50% were higher than LC50 and LC90 for pyrethrin 0.4g/l+ PBO 1.5g/l for Ae. albopictus 4th instar larvae (0.069 and 0.336: 0.056 and 0.166), re- spectively. Using Mann-Whitney U test values of LC50 and LC90 for each insecticide tested against 4th instar larvae of both Aedes spp. showed significant difference (P< 0.05). However, Abate® showed a much lower susceptibility to both Aedes spp. 4th instar larvae compared to the three pyrethrin formulations. Although the pyrethrin formulations have larvicidal effects against both Aedes spp. but Abate still remains the insecti- cide of choice as larvicide. Iranian J Arthropod-Borne Dis, 2007, 1(2): 1-6 S Sulaiman et al.: Evaluation of Pyrethrin … 3 Table 1. Mortality response of pyrethrin formulations to Aedes aegypti and Aedes albopictus 4th-instar larvae in the laboratory Aedes aegypti Aedes albopictus Insecticides LC50 (95%CI) (ppm) LC90 (95%CI) (ppm) Slope±SE LC50 (95%CI) (ppm) LC90 (95%CI) (ppm) Slope ± SE Pyrethrin 50% 0.038 (0.029-0.052) 0.135 (0.090-0.280) 2.334±0.371 0.069 (0.039-0.083) 0.336 (0.268-0.487) 2.276±0.674 Pyrethrin 0.4g/l + PBO 1.5g/l 0.028 (0.022-0.036) 0.080 (0.058-0.131) 2.842±0.413 0.056 (0.044-0.071) 0.166 (0.120-0.281) 3.226±0.464 Pyrethrin 44g/l+PBO 160g/l 0.002 (0.002-0.003) 0.007 (0.005-0.011) 2.973±0.428 0.004 (0.003-0.006) 0.012 (0.008-0.021) 2.974±0.603 Abate® (Control) 0.00003 (0.00001-0.00007) 0.0016 (0.00045-0.01382) 0.909±0.136 0.00001 (0.0000-0.00002) 0.004 (0.0006-0.1409) 0.594±0.112 Table 2 indicates that both Ae. aegypti and Ae. albopictus adults are more susceptible to pyrethrin 44 g/l + PBO 160 g/l than pyrethrin 0.4 g/l + PBO 1.5 g/l and pyrethrin 50%. The LC50 and LC90 of pyrethrin 44 g/l + PBO 160 g/l for Ae. aegypti were 0.209 µg/cm² and 0.469 µg/cm². The LC50 and LC90 for Ae. albopictus were 0.197 µg/cm² and 0.492 µg/cm², respec- tively. The LC50 and LC90 for pyrethrin 50% were higher than that of pyrethrin 0.4g/l + PBO 1.5g/l for Ae. albopictus adults. However, the LC50 for pyrethrin 50% was higher than that of pyrethrin 0.4g/l + PBO 1.5g/l for Ae. aegypti but the regression slope of pyrethrin 0.4g/l + PBO 1. 5g/l indicated that a slight increase in its concentration would cause a higher mortality of Ae. aegypti adults, compared to pyrethrin 50%. Using Mann Whitney U test the value of LC90 for each insecticide formulation tested against adults of both Aedes spp. were sig- nificantly different (P< 0.05). However, malathion showed a much lower susceptibility values to both Ae. aegypti and Ae. albopictus adults, compared to the three pyrethrin formulations. Table 2. Mortality response of pyrethrin formulations to Aedes aegypti and Aedes albopictus adults in the laboratory Aedes aegypti Aedes albopictus Insecticides LC50 (95%CI) (µg/cm²) LC90 (95%CI) (µg/cm²) Slope ± SE LC50 (95%CI) (µg/cm²) LC90 (95%CI) (µg/cm² ) Slope ± SE Pyrethrin 50% 1.331 (0.901-2.128) 3.982 (2.080-4.013) 2.745±0.604 1.980 (1.007-2.202) 5.244 (4.896-5.993) 3.036±0.573 Pyrethrin 0.4g/l + PBO 1.5g/l 1.701 (0.967-1.996) 3.743 (2.765-4. 010) 3.636±0.699 1.671 (1.010-2.100) 4.721 (3.787-5.220) 3.104±0.564 Pyrethrin 44g/l+PBO 160g/l 0.209 (0.160-0.277) 0.469 (0.339-0.875) 3.713±0.753 0.197 (0.091-0.298) 0.492 (0.287-0.992) 3.227±0.622 Malathion (Control) 0.00006 (0.00003-0.00007) 0.00050 (0.00048-0.00098) 1.303±0.266 0.0008 (0.0006-0.0013) 0.0071 (0.0058-0.0071) 1.364±0.249 Iranian J Arthropod-Borne Dis, 2007, 1(2): 1-6 S Sulaiman et al.: Evaluation of Pyrethrin … 4 Table 3 shows the impact of Aedes spp. larvae which was treated with sublethal doses (LD50) of pyrethrin formulations. All the three pyrethrin formulations resulted in 4-6% of emerg- ing adults of Ae. aegypti and Ae. albopictus alive, compared to 90% of Ae. aegypti adults and 96% of Ae. albopictus alive adults in the control, respec- tively. Thus, all the pyrethrin formulations at LD50 when treated on both Aedes spp. larvae caused low production of emerging adults. Table 3. Impact of treatment of mosquito larvae with sublethal doses of pyrethrin formulations Pyrethrin 50% Pyrethrin 0.4g/l + PBO 1. 5g/l Pyrethrin 44g/l + PBO 160g/l Control Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus No. of larvae tested 50 50 50 50 50 50 50 50 Mortality of larvae after 24 hr (%) 44 48 50 52 52 48 2 2 Mortality of pupae (%) 2 0 8 4 10 12 0 0 Mortality of emerging adults (%) 6 8 16 16 20 14 0 0 Emerging adults alive (%) 6 6 4 4 4 4 90 96 Table 4 indicated that when Aedes spp. larvae were treated with sublethal doses of pyrethrin formulations, the fecundity of subse- quent adults produced very low number of eggs for both Aedes spp. in the range of 0-4 eggs, compared to 198-227 eggs produced in the control. None of the eggs derived from the treated lar- vae hatched. However, 99% of the control eggs of both Aedes spp. became adults. It was ob- served that the female mosquitoes derived from different formulations of pyrethrin treated lar- vae were not feeding well on Argus spp. This would affect the number of eggs laid. Table 4. Impact of mosquito fecundity after the larval exposure to sublethal doses of pyrethrin formulations Pyrethrin 50% Pyrethrin 0.4 g/l + PBO 1. 5 g/l Pyrethrin 44 g/l + PBO 160 g/l Control Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus No. of eggs produced 2 4 0 2 0 3 1189 2270 % of eggs hatched 0 0 0 0 0 0 99 99 % of larvae becoming pupae 0 0 0 0 0 0 99 99 % of pupae becoming adults 0 0 0 0 0 0 99 99 Discussion According to Rayman (2006), a 2% pyre- thrum solution, a naturally occurring pyrethroids found in Chrysanthemum spp. flowers is the recommended agent for aircraft disinsection because they are extremely effective insecticide and pose minimal health risks. According to Gerry et al. (2005) the aerial application of ULV pyrethrin insecticide for control of adult mos- quitoes did not result in undue exposure to the pilot. Jensen et al. (1999) conducted ULV applica- tion of pyrethrin, malathion and permethrin on non-target invertebrates, sentinel mosquitoes Iranian J Arthropod-Borne Dis, 2007, 1(2): 1-6 S Sulaiman et al.: Evaluation of Pyrethrin … 2 and mosquitofish in California seasonal wetlands, USA. All the insecticides tested including pyre- thrin were able to control adult mosquitoes without substantial effects on the aquatic insects or fish in the seasonal wetlands. Victor et al. (2002) in their application of temephos and fogging with pyrethrum 2% extract in villages at Tamil Nadu, India, were effective against immatures and adults Aedes aegypti. Thus, the pyrethrin formulations can be widely used in controlling dengue vectors. Mohapatra et al. (1999) evaluated cyfluthrin and fenfluthrin on their activity against differ- ent developmental stages of three vector species viz., Anopheles stephensi, Ae. aegypti and Culex quinquefasciatus. Both compounds were more active against the fourth larval instars of all mosquito species tested, cyfluthrin in culicines and fenfluthrin in anophelines brought about maximum inhibition of adult emergence. Mohapatra et al. (1999) also found that py- rethroids cyfluthrin and fenfluthrin significantly reduced the fertility rates (P< 0.001) of Anopheles stephensi, Aedes aegypti, and Culex quinque- fasciatus. Loh and Yap (1989) who studied the efficacy and sublethal effects of pyriproxyfen on Ae. aegypti, found that the eggs hatchability was reduced by 36.8%. According to Xue et al. (2005) application of deet forced egg retention time reduced the number of eggs laid per female Aedes albopic- tus. The rate of egg hatched was considerably reduced after three weeks of retention. The fe- cundity and fertility of gravid female Ae. al- bopictus were affected by the time duration of forced egg-retention. Ali et al. (2006) found that fecundity and fertility based on number of laid eggs per female and percentage of egg hatch in Stegomyia albopicta when exposed to 0. 1% boric acid sugar bait were significantly reduced and ovarian development retarded. Focks et al. (1991) studied Aedes aegypti which were fed on rabbits subcutaneously injected with ivermectin exhibited reduced survival and egg production compared to females fed on the control rabbits. Eggs from these rabbits were also less likely to hatch and subsequent larval survival was lower than the controls. Similarly, our studies also indicated that pyrethrin formulations affected the produced eggs and then a few produced eggs could not hatch into larvae. To conclude, all the pyrethrin formulations had larvicidal and adulticidal effects on Aedes aegypti and Aedes albopictus. Furthermore, treat- ment at sublethal doses at the larval stage pro- duced very low emerging adults and fecundity. Acknowledgements We wish to thank Faculty of Allied Health Sciences, University Kebangsaan Malaysia for providing research facilities and Botanical Re- sources Australia Pty Ltd for providing the py- rethrin formulations. References Ali A, Xue RD, Barnard DR (2006) Effects of sublethal exposure to boric acid sugar bait on adult survival, host-seeking bloodfeeding behavior, and reproduction of Stegomyia al- bopicta. J Am Mosq Control Assoc. 22: 464-468. Curtis CF, Myamba J, Wilkes TJ (1992) Various pyrethroids on bednets and curtains. Mem Inst Oswaldo Cruz. 87 suppl. 3: 363-370. Focks DA, Mc Laughlin RE, Linda SB (1991) Effects of ivermectin (MK-933) on the repro- ductive rate of Aedes aegypti (Diptera: Culicidae). J Med Entomol. 28: 501-505. 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