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Aedes albopictus is a vector of dengue fever, which is a public health problem in Pakistan. Continuous exposure to synthetic insecticides has led to the development of resistance in mosquitoes, therefore the recent trends have been to explore eco-friendly plant extracts that can suppress mosquito populations. Investigations were made to assess the larvicidal potential of essential oils from some indigenous plants against larvae of Aedes albopictus (Diptera: Culicidae), a competent dengue vector in dengue positive areas. Results revealed that after 24, 48 and 72 h of exposure to essential oils, musambi (Citrus sinensis L. var. musambi) caused highest mortality (64.32%, 76.05% and 89.25, respectively) followed by eucalyptus (Eucalyptus camaldulensis L.) (60.93%, 72.78% and 85.49, respectively). Ae. albopictus larvae showed highest susceptibility, both in terms of LC50 and LT50, to musambi (Citrus sinensis L. var. musambi) essential oil after 24 h, but were highly and equally susceptible to essential oils from darchini (Cinnamomum cassia), musumbi (Citrus sinensis L. var. musambi), bakhra (Tribulus terrestris L.), eucalyptus (Eucalyptus camaldulensis L.) and succari (Citrus sinensis L. var. succari) after 48 and 72 h. Results are discussed with possible mode of action of plant extracts in mosquitoes.
Key Words: Plant extracts, larvicide, dengue vector, Aedes albopictus
Aedes albopictus has been one of the most potential mosquito forms that has caused of dengue and dengue hemorrhagic fever, a serious public health concerns in many developing countries including Pakistan. Although some vector borne diseases such as Japanese encephalitis and yellow fever have been reasonably brought under control by vaccination, however no effective vaccine is available yet for dengue (Pitasawat et al., 2007). Therefore, the only effective approach to minimize the incidence of these diseases is to eradicate and control mosquito vectors mainly by applying insecticides to larval habitats, destroying unwanted containers and enhancing public awareness (Corbel et al., 2004). Organophosphorus compounds such as temephos, fenthion and chlorpyrifos, are commonly used larvicides against mosquitoes and other aquatic insects. However, the mishandling of synthetic chemicals in public health programs has caused many problems to humans as well as the environment (Din et al., 2011). To lighten these problems, major consideration has been shifted steadily on the use of plant based products as larvicides or adulticides which can provide alternatives to synthetic chemicals (Junwei et al., 2006). Plants serve as one of the most important sources of several compounds which possess potential insecticidal or repellent properties and are free from harmful effects (Isman, 1995). Many plant products have been explored against mosquitoes either as larvicides and/or adulticides or as adult repellents (Sukumar et al., 1991). In this context, the present study was carried out with the aim of exploring the larvicidal potential of some indigenous plants against Ae. albopictus.
MATERIALS AND METHODS
Ae. albopictus larvae and pupae were collected round the year from all artificial containers and natural habitats of Faisalabad and reared in steel trays of 3 inch depth and were fed on fish food (Tetramin®) till adult emergence (Akram et al., 2010). Adults were maintained in transparent plastic cages (70-35-35 cm), where the males were provided with cotton wicks soaked in 10% sucrose solution while females were fed with blood of white rats every alternate day (Shaalan et al., 2006). A glass beaker with strips of moistened filter paper was provided for gravid females to lay their eggs. The population was maintained at set conditions of 27±2oC, 65% RH and L14:D10 photoperiod.
The plants with their parts selected for oil extraction are listed in Table I. These plants were collected from Faisalabad (31o30'N, 73o05'E) and identified from the Department of Agronomy, University of Agriculture, Faisalabad, Pakistan.
Extraction of oil and preparation of solution
The seeds of citrus cultivars were washed with tap water to remove the pulp and then dried in electric oven for 48 hours at 60oC and later were grounded in an electric grinder (Anex Germany). While parts of other plants like seeds, leaves and rhizomes were also dried. The grounded material was put in Soxhlet apparatus for the extraction of oil using di-ethyl ether as a solvent (Vogel, 1978). Oil was collected in small vials and the quantity was measured. Stock solutions were prepared by adding 1 ml of oil from each variety in 99 ml of acetone that was considered as 1% stock solution from which series of concentration (mg/L) were prepared (Murugan et al., 2007).
The extracted oils were used in six different concentrations including controls. Each concentration was replicated thrice having 200 ml of oil solution placed in 250ml glass beakers. Thirty late third or early fourth instar larvae of the Ae. albopictus (F1 Lab. generation) were exposed in each beaker containing oil solution (Mohtar et al.. 1999). Larvae in control were only treated with acetone. The bioassays were performed under lab conditions at 27 ± 2 oC and 65 ± 5% RH. The percentage mortality was calculated from the average of three replicates using the following formula given by Sumroiphon et al. (2006).
Percentage mortality = (Number of dead larvae / Number of larvae tested) -100
Mortality data ware recorded after 24, 48 and 72 hours of exposure to plant oils.
Abbott's formula (Abbott, 1925) was used for corrected mortality and the data so obtained was analyzed by probit analysis (Finney, 1971) using Minitab-15 statistical software (2009) for dose and time mortality regression lines. Due to the inherent variability of bioassays, pair wise comparisons of LC50 (mg/L) values were made, and if 95% fiducial limits of two treatments did not overlap at 1% level of significance, they were considered significant (Litchfield and Wilcoxon, 1949). The data on percent mortalities were analyzed with Statistix 8.1 (Analytical Software, 2005) and means were compared using Tukey's Honestly Significant Difference (HSD) test at 5% level of significance.
RESULTS AND DISCUSSION
Plant extracts exhibited strong larvicidal activities against Ae. albopictus. With respect to percent mortalities, musambi caused highest mortality (64.32%) after 24 h of exposure followed by eucalyptus (60.93%), darchini (56.48%) and succari (49.14%) (F=507; df=8,18; P<0.001). After 48 and 72 h of exposure, musambi caused highest percent mortality (76.05% for 48 h; 89.25% for 72 h) followed by eucalyptus (72.78% for 48 h; 85.49% for 72 h), darchini (68.03% for 48 h; 79.94 % for 72 h) and succari (57.78% for 48 h; 71.61% for 72 h), later both were statistically at par at both time intervals (F=460; df=8,18; P<0.001 for 48 h; F=317; df=8,18; P<0.001 for 72 h) (Fig. 1).
In terms of lethal concentration to kill 50 % population (LC50) of the subjected mosquito larvae, musambi proved to be the most effective larvicide particularly after 24 h of exposure by having least LC50 value (40.0 mg/L) while black pepper, castor, garlic and linseed proved least effective (70.3 mg/L, 71.5 mg/L, 66.5 mg/L and 75.5 mg/L, respectively) and were statistically at par (overlapping of 95% FLs). Darchini, musumbi, bakhra, eucalyptus and succari had lowest LC50 values (35.8 mg/L, 32.2 mg/L, 39.0 mg/L, 37.3 mg/L and 43.6 mg/L, respectively) after 48 h and (29.9 mg/L, 26.8 mg/L, 35.8 mg/L, 32.6 mg/L and 34.5 mg/L, respectively) after 72 h and were statistically at par (overlapping of 95% FLs) (Table 1).
With regards to lethal time to kill 50% population (LT50) of Aedes larvae, musambi took least time (13.44 h) followed by darchini and bakhra (18.91 h and 25.08 h) both having non significant difference while black pepper, castor, garlic and linseed took more than 2 days (51.22 h, 57.48 h, 55.11 h and 56.50 h respectively) to cause 50% mortality and were statistically at par (overlapping of 95% FLs).
In view of the environmental hazards and development of insecticide resistance in insects, particularly disease vectors, the recent trend is to explore eco-friendly plant extracts with the potential to suppress pest populations. Though several plant based insecticides have been reported worldwide, there is still a wide scope to investigate more potent plant products with enhanced activity, eco-friendly and could replace objectionable synthetic chemicals for mosquito control.
The ether extracts of 9 indigenous plants proved sufficiently effective against Ae. albopictus larvae. The varying results obtained in terms of percent mortalities LC50 and LT50 could probably be due to the differences in levels of toxicity among the insecticidal ingredients of each plant, and the effect of plant extract may vary with the time of collection and season as well (Tawatsin et al., 2006; Din et al., 2011). The studied plant extract caused high mortality in larval stages of Ae. albopictus, therefore, this intervention for public authorities (Silva et al., 2003) to use plant products could provide ideal approach in public health management especially mosquito control. Plant extracts are safer for non target organisms including man (Din et al., 2011), therefore, plant based formulations would be more feasible from environmental perspective than synthetic mosquitocides (Bhat and Kempraj, 2009). Essential oils against mosquitoes have been studied extensively. Senthilkumar et al. (2008) reported larvicidal effect of Blumea mollis essential oil against Culex quinquefasciatus, with LC50 of 52.2 mg/L. Similarly, Amer and Mehlhorn (2006) studied 41 plants against Aedes, Anopheles and Culex genera and found that Citrus limon was the most promising larvicide. Tiwary et al. (2004) applied Zanthoxylum armatum DC against three mosquito disease vectors and found it very effective. The mode of action of these oils on mosquito larvae is not known, however, previous studies documented that phytochemicals could interfere with the proper functioning of mitochondria particularly at the proton transferring sites (Usta et al., 2002). The constituents of plant extracts primarily affect the midgut epithelial surface and secondarily affect the gastric caeca and the malpighian tubules in mosquito larvae (Rey et al., 1999; David et al., 2000). Moreover, the plant extracts could be more effective compared to the individual active compounds, due to natural synergism that delays the development of resistance to a maximum, particularly in disease vectors (Maurya et al., 2007). However, further studies are required to confirm the exact mode of action of these essential oils.
In conclusion, the present study clearly indicates the efficacy of indigenous plant oils against Ae. albopictus and which encourages the development of alternative active ingredients as effective mosquitocides.
The funds provided by Higher Education Commission (HEC) Pakistan to carry out this project are highly acknowledged.