Evaluation of toxicity of plant extracts against vector of lymphatic filariasis, Culex quinquefasciatus

The tropicopolitan mosquito, Culex quinquefasciatus is the vector of lymphatic filariasis in India. Filariasis is a major public health problem and globally 120 million people are at risk1. In India, one-third of the people are afflicted with 40 per cent of the global burden of the disease2. Mass drug administration (MDA) is now accepted as a global programme to eliminate lymphatic filariasis. There is a general consensus that the time for elimination can be drastically reduced if MDA is coupled with appropriate vector control measures. At present, control of Cx. quinquefasciatus is practiced using conventional insecticide as larvicides. This approach has been confronted with twin problems of vector resistance to insecticides and environmental hazards likely to be caused by insecticides. It is considered that many plant based compounds have potential larvicidal activity and this would be used as alternative larvicides as these are effective, ecofriendly and minimize the development of resistance in mosquitoes3. Since, India has a rich plant diversity, attempts are in progress to find potential larvicidal plants. The objective of the present study was to evaluate extracts of a few promising plants against larvae of Cx. quinquefasciatus.

Material & Methods

The study was conducted at MS Swaminathan Research Foundation and National Institute of Malaria Research - Field Unit, Chennai during the period of August 2007 to March 2009.

The leaves of five species of plants viz., Argemone mexicana (Mexican prickly poppy), Clausena dentata (Dentate), Cipadessa baccifera (Rana bili), Dodonaea angustifolia (Hop bush) and Melia dubia (Pride of India) were collected from different geographical locations of Tamil Nadu. The leaves of each plant were washed thoroughly with water and shade dried. The dried leaves were powdered separately with an electric blender and Soxhlet extracted with petroleum ether cycles for 36 h and subsequently the extract was concentrated and dried using flask evaporates34.

A stock emulsified water solution of the extract of 1000 ppm was prepared using 250 mg of the extract in a flask dissolved in 4 ml of acetone and 0.1 ml of Tween 20 (emulsifier) and made up to 250 ml using distilled water. This was sequentially diluted with water to obtain 500, 250, 125 and 62.5 ppm of the extract. Before each dilution, the solution was shaken well to ensure uniform distribution of the extract in the emulsion. Stock control solution was prepared using 4 ml of acetone and 1 ml of Tween 20 in 250 ml of water and this was appropriately diluted with water to serve as control for each treatment. Since A. mexicana showed high toxicity to larvae5, this plant extract was mixed with other four plant extracts separately in 1:1 proportion to see for synergistic effect in any of the different combinations. Larval susceptibility tests were conducted as per WHO protocol6. Susceptibility tests were performed in disposable plastic cups. In each 125 ml of the emulsion of different concentrations were taken separately and in each cup 20 larvae (late 3^rd and early 4^th instar) were introduced. Each treatment had five replicates with appropriate controls. The independent and combined toxicity observations were made on total number of larval mortality, pupal development, pupal mortality and adult emergence with five replicates at every 24 h (24, 48, 72 and 96 h) interval (Tables I & II). Pupae when present were not included for calculation of percentage mortality.

Table I Effect of petroleum ether extracts of the selected plants against late 3^rd instar larvae of Cx. quinquefasciatus

Table II Combined effect of A. mexicana with other selected plants (petroleum ether extract) against late 3^rd instar larvae of Cx. quinquefasciatus

Statistical analysis: Corrected percentage mortality was calculated using Abbott`s formula7. From the corrected mortality larval LC₅₀ and LC₉₀ values were calculated using the computation programme of Probit analysis8.

Results & Discussion

Larvicidal efficacy of the plant extracts are given in Table III. Among the five plant extracts, C. baccifera and M. dubia were almost non-toxic as 1000 ppm ensured only 12 per cent mortality. Among the other three plant species, A. mexicana showed highest toxicity having an LC₅₀ and LC₉₀ values of 48.89 and 463.94 ppm, respectively. D. angustifolia extract showed LC₅₀ value of 973 ppm and C. dentata 1435 ppm. A. mexicana extract when mixed with C. dentata (1:1 proportion) drastically reduced the LC₅₀ value to 28.6 ppm. However, similar combination with extracts of C. baccifera, D. angustifolia and M. dubia showed an increase in LC₅₀ values of A. mexicana indicating absence of any additive effect (Table III).

Table III Susceptibility of Cx. quinquefasciatus larvae to different plant extracts individually (A) and in combination of A. mexicana with other plants in 1:1 proportions (B)

The present study revealed that A. mexicana extract had high larvicidal potential among the five plant extracts tested. Further isolation and purification of the toxic principle from the extract can enhance the larval toxicity of A. mexicana. Other plants viz., C. dentata, C. baccifera, D. angustifolia and M. dubia were found to be non-toxic to the mosquito larvae. However, when the A. mexicana extract was mixed with plant extracts of C. dentata and D. angustifolia in 1:1 proportion, a reduction in their LC₅₀ values was observed. Plant based compounds are known to be ecofriendly and can be used in mosquito larval control ensuring maximum safety in any ecological condition. The present results clearly indicated that A. mexicana leaf extract had high larvicidal potency that can be exploited by further purification of the toxic principles in A. mexicana and identifying the synergistic compound in C. dentata and D. angustifolia.