MATERIALS AND METHODS

Plant material and extraction: Fresh leaves of Eugenia jambolana were collected from in and around Mysore, Karnataka, India from October 2009 to May 2010 and shade dried. The extract was prepared from the fine powder by employing a soxhlet apparatus using petroleum ether, ethyl acetate, acetone and methanol as solvents. The pooled extract was evaporated in a rotary vacuum evaporator at 40°C to dryness and stored at 4°C in an air tight bottle for further analysis. After preliminary experiments, petroleum ether extract was selected for further bioassay as it exhibited maximum efficacy. The entire experiment was conducted in the Department of Studies in Zoology, University of Mysore, Manasagangothri, Mysore, Karnataka, India.

Mosquito larvae: Larvae of the three mosquito species Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi were reared in enamel or plastic trays (30x24x5 cm) containing dechlorinated water. Culex quinquefasciatus and Anopheles stephensi larvae were fed with finely powdered mixture having 2:1 parts of dog biscuits and dry yeast, whereas Aedes aegypti larvae were fed with powdered dry yeast. The rearing water was changed daily until pupation.

Larval bioassay: Bioassays on mosquito larvae were performed on late third or early fourth instars, according to the standard guidelines of WHO (2005). The required quantities of Eugenia jambolana leaf extract of different concentrations were prepared in acetone as solvent. One milliliter of each of the concentration was mixed thoroughly with 249 mL of dechlorinated water in 500 mL glass beakers. Larvae were exposed to an ascending series of five concentrations according to log dose. Parallel control tests were also maintained by adding one mL of the solvent to 249 mL of dechlorinated water. Finally, 25 early fourth instar larvae were transferred to each of the beakers. A minimum of three replicates were kept for each concentration along with the control. Observation for the dead or moribund larvae was carried out after 24 h duration at 25°C and 14 h light and 10 h dark regime.

Data analysis: Larval mortality counts were adjusted for the mortality in control, if any employing Abbott’s formula (Abbott, 1925) to give an estimate of the plant extract attributable mortality. The corrected mortality data were subjected to regression analysis of probit mortality on log dosage (Finney, 1971). The significant difference in LC₅₀ is based on the non-overlapping of 95% fiducial limits (Yang et al., 2002).

RESULTS AND DISCUSSION

The results showing the toxicity of the Eugenia jambolana leaf extracts obtained with different solvents tested against three mosquito species are presented in Table 1 along with the log dose-probit mortality responses of all extracts in Fig. 1. Out of the four organic solvent extracts petroleum ether extract was found to be highly effective against all the three mosquito species tested followed by ethyl acetate, acetone and methanol.

Table 1:	Efficacy of different solvents of Eugenia jambolana leaf extracts against larvae of three mosquito species
LC50 median lethal concentration; FL Fiducial limits; LC90 90% lethal concentration; df degree of freedom. *The difference in LC50 is significant based on the non-overlapping of 95% fiducial limits (p<0.05)

Fig. 1:	Effect of Petroleum ether leaf extract of Eugenia jambolana against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi larvae

The results show an LC₅₀ of 40.97, 53.84 and 96.00 ppm for Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi respectively by the petroleum ether extract. Similarly, LC₉₀ were found to be 83.29, 127.49 and 156.42 ppm, respectively. The larval sensitivity towards the crude extract was found in the order of Aedes aegypti>Culex quinquefasciatus>Anopheles stephensi. Further the larvicidal efficacy was found to be significantly different among all the extracts (p<0.05). The fig too depicts the log dose-probit mortality responses and slopes of regression lines of tested crude extract of Eugenia jambolana leaf extracts with different solvents.

The results of the larvicidal bioassay employing crude Eugenia jambolana extracts by different solvents employed against three different mosquito species revealed that all the organic extracts of Eugenia jambolana were bioactive (Table 1). However, significant (p<0.05) larvicidal activity was observed with petroleum ether followed by ethyl acetate, acetone and methanol extracts (p<0.05). The biological activity of this plant extract may be due to various compounds, including phenolics, terpenoids, flavonoids and alkaloids (Gohil et al., 2010). These compounds may jointly or independently contribute to produce toxic activity against the mosquito species. It was earlier reported that petroleum ether of the whole plant such Citrullus colocynthis is effective against mosquito larvae (Rahuman et al., 2008). Likewise, Latha and Ammini (2000) have studied petroleum ether extract of Curcuma raktakanda leaf against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi. The present result and the earlier report on other plants indicate that petroleum ether extract may be more effective among organic solvents. Data presented in Table 1 and Fig. 1 further show that a converse relationship exists between extract efficacy and solvent polarity, which is in line with the observation made by Mulla and Su (1999) in neem plant extracts.

Among the three species tested by the present authors, maximum effect was on Aedes aegypti compared to the other mosquito species. This finding is in agreement with that of Rahuman et al. (2000) who have tested Feronia limonia extracts against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi larvae. The varying susceptibility of the three species of mosquitoes is probably due to difference in the physiological characteristics of the three species of mosquito. This agrees with the report of Raghavendra et al. (2009), who have reported the existence of such differences among four mosquitoes assayed with Solanum nigrum extracts. This also agrees with the report of Kumar and Maneemegalai (2008), who have tested Lantana camara extract against Aedes aegypti and Culex quinquefasciatus. Maheswaran et al. (2008) too have reported variation toxicological efficacy with Aedes aegypti and Culex quinquefasciatus to the leaves of Leucas aspera. Similar observations were made by Bagavan et al. (2009) with Achyranthes aspera against Aedes aegypti and Culex quinquefasciatus. Mathew et al. (2009) also have reported variations in larvicidal efficacy from Saraca indica, Nyctanthes arbor-tristis and Clitotia ternatea extracts against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi larvae. Thus the present findings on Eugenia jambolana showed some promise for further chemical isolation of the active ingredient in future and it could be considered as a potent resource for local people for controlling mosquito larvae. Such practice would not only reduce the chemical burden on the environment but also promote sustainable utilization of locally available bioresource by rural communities.

ACKNOWLEDGMENT

The first author is grateful to UGC SAP, New Delhi for providing financial assistance. Authors are thankful to the Chairman, DOS in Zoology, University of Mysore for the facilities.