Mosquito larvicidal potential of four common medicinal plants of India

Vector control is an essential requirement in control of epidemic diseases such as malaria, filariasis, dengue, etc. that are transmitted by mosquitoes. Excessive use of synthetic pesticides causes emergence of pesticide resistance and harmful effect on non-target organisms. This has necessitated an urgent search for development of new and improved mosquito control methods that are economical and effective as well as safe for non-target organisms and the environment. Herbal insecticides of plant origin become a priority in this search. Several laboratory and field based studies have already been carried out in this area and some potentially active larvicides of plant origin like octacosane, falcarinol, geranial, azadirachtin, pipernonaline plumbagin, β-sitosterol, etc. have been isolated so far1.

The objective of present study was to evaluate the larvicidal activity of the crude extracts of four plants viz. Alternanthera sessilis L. (Amaranthaceae), Trema orientalis L. (Cannabaceae), Gardenia carinata Smith. (Rubiaceae) and Ruellia tuberosa L. (Acanthaceae) on Culex quinquefasciatus as target species. Their medicinal properties are presented in Table I2345678. Qualitative analysis of the crude plant extracts was also done to have an idea about the chemical profile of active ingredient (s).

Table I Botanical description and medicinal properties of plants used in the study

Material & Methods

The present study was conducted in the department of Zoology, University of Burdwan, at Burdwan, West Bengal, India during April-June 2011. Mosquito larvae available in the cemented drains surrounding the University campus were collected by 250 ml dipper9. Majority of the larvae were of the species Cx. quinquefasciatus and a few were Armigeres subalbatus. Larvae were reared to adult stage and identified101112. Collected larvae were kept in the plastic bucket (20 l) with the addition of artificial food (powdered mixture of dog biscuits and dried yeast powder in the ratio of 3:1), and aquatic weeds kept free from exposure to pathogens, insecticides, or repellants and maintained at 31-33°C temperature and 85 per cent relative humidity (RH). Some of the previously identified larvae were also reared upto the adult stages to confirm their species identity.

Preparation of crude extract: Fresh, mature, green leaves of all the selected plants were collected during April – June, 2011, from plants growing on the outskirts of Burdwan and authenticated by Dr Ambarish Mukherjee, Professor in Botany, University of Burdwan, West Bengal, India. After collection, all the leaves were initially rinsed with distilled water and dried on paper towel. Wet weight quantities (50 g) of plant leaves were crushed with a mixer-grinder machine, and the juice was filtered by Whatman No. 1 filter paper. The clear filtrate was used as a stock solution (100% concentration of crude extract) for bioassays experiments. Required concentrations (0.5, 1 and 1.5%) were prepared using sterilized distilled water.

Dose-response larvicidal bioassay: The larvicidal bioassay was done following the World Health Organization13 standard protocols with suitable modifications. Each of the earlier prepared concentrations of crude extract was transferred into the sterile glass Petri-dishes (9 cm diameter/150 ml capacity). The first-, second-, third-, and fourth-instar larval forms of Cx. quinquefasciatus (20 each) were separately introduced into different Petri-dishes containing appropriate concentrations, 20 mg of larval food (dried yeast powder) was added per Petri dish. Mortality rates were recorded after 24, 48, and 72 h post-exposure. The experiments were replicated thrice at three different days and conducted at 25-30°C and 80-90 per cent RH. A set of control experiment (without having the test solution) using tap water was also run parallel.

Phytochemical analysis of the plant extracts: Crude extracts of all four plants were subjected to qualitative phytochemical analysis141516. The phytochemicals analysed were saponins, terpenoids, alkaloid, steroids, tannin, flavonoids and cardiac glycosides.

Effect on non-target organisms: The crude extracts of all the plant leaves were tested against two non-target invertebrates, Diplonychus annulatum (5^th instar larval forms) and Chironomus circumdatus present in the same habitat of the mosquito larvae. As per the procedure used by Suwannee et al17, the non-targets were exposed to the sub lethal dose, LC₅₀ (at 24 h for 3^rd instar larvae of Cx. quinquefasciatus) of the crude extract. Ten early 4^th instar chironomid larvae were placed in a plastic tray containing 200 ml pond water with mud. Ten 3^rd instar nymph of D. annulaum were kept in pond water in a plastic tray (12.6 × 10 × 6 inches). Numbers of dead were recorded after 24 h, 48 h and 72 h of exposure and percentage mortality was recorded. Each experiment was replicated thrice. A set of control (without having the test solution) for each organism was run parallel.

Statistical analysis: The percentage mortality observed was corrected by Abbott's formula18. Statistical analysis of the experimental data was performed using the computer software “STAT PLUS 2007 (Trial version: http://statplus.en.softonic.com)” and “MS EXCEL 2007” to find out the (Log probit analysis (at 95% confidence level), regression equations and mean percentage mortality. Comparison of mean percentage mortality, standard error and their upper and lower bound at 95 per cent confidence level are estimated by Tukey and Duncae test.

Results

Highest mortality was seen at 1.5 per cent concentration of crude extract, tested against all larval instar and was significantly higher (P < 0.05) than 0.5 and 1 per cent concentrations at 24, 48 and 72 h of exposure (Table II). The relative efficacy of the plant extracts against target species was as follows: A. sessilis > T. orientalis > G. carinata > R. tuberosa leaf extract. The result of regression analysis of crude extract of all the plants showed that the mortality rate (Y) was positively correlated with concentration of exposure (X). The result of log probit analysis (95% confidence level) showed that LC₅₀ values gradually decreased with exposure periods and a lowest value at 72 h exposure to II^nd instar followed by III^rd, IV^th and I^st instar larvae (Table III). Comparison of mean percentage mortality of 1^st–IV^th instars of Cx. quinquefasciatus larvae at different test concentration of tested plants, standard error and their upper and lower bound at 95 % confidence level are presented in Table IV. No changes in the survival rate and swimming activity of the non target organisms were observed within 72 h post exposure. The results of preliminary qualitative phytochemical analysis of tested plant revealed the presence of some secondary metabolite that may be responsible for their biocontrol potentiality (Table V).

Table II Results of larvicidal bioassay of the tested plants against Cx. quinquefasciatus larva

Table III LC₅₀ values and corresponding regression equations of four plant leaf extracts against different instars of Cx. quinquefasciatus

Table IV Mean percentage mortality of 1^st -4^th instars of Cx. quinquefasciatus larvae at different concentrations of tested plants

Table 5 Result of qualitative phytochemical analysis of the crude leaf extract of the tested plants

Discussion

The mosquito borne parasites are continually developing resistance to the available insecticides and at present, there is no vaccine to prevent infections transmitted by mosquitoes. Vector control in the larval condition is the best available option as the larvae are confined to water bodies which are mainly man made and can be easily located. Furthermore, for the protection of environment and other non-target organisms which share same habitat with the mosquito larvae, plant based insecticides are in demand for mosquito control as synthetic insecticides are non-biodegradable, toxic to environment and also responsible for resistance19. Testing the plant crude extracts against mosquito can lead to identify potential bioactive compounds that can be used as larvicides to control mosquito. Botanical derivatives have drawn attention as potential insect control agents targeting only larval stages in the mosquito control programme in the last three decades202122.

The findings of the present investigation indicated that 1.5 per cent crude extract of mature leaves of A. sessilis showed highest mortality against II^nd instar larvae of Cx. quinquefasciatus. T. orientalis showed 53.3 per cent mortality against III^rd instar, G. carinata showed 50 per cent mortality against III^rd instar and R. tuberosa showed 50 per cent mortality in II^nd and III^rd instar larvae after 72 h, respectively. The phytochemical analysis of the plant extracts revealed the presence of several bioactive secondary metabolites that singly or in combinations may be responsible for the larval toxicity. As no mortality occured in the non-target organisms, it was assumed that tested plant extracts were safe to use in the aquatic ecosystem.

Green synthesis of pesticides of biological origin may serve as suitable alternatives to synthetic or chemical insecticides in future as these are relatively safe, inexpensive, and are readily available in many areas of the world. Plant shows a vast range of phytochemicals which may be used in place of chemical pesticides due to their ecofriendly nature. Further investigations are needed to elucidate this activity against a wide range of mosquito species and also the active ingredient(s) of the extract responsible for the mosquitocidal activity.