MOSQUITO-LARVICIDAL ACTIVITY OF SOME SRI LANKAN

Screening of plants of Sri Lanka for mosquito-larvicidal activity, revealed that 18 plant species out of 53 tested werelethal to Culexquinquefasciatus larvae. Methanol extracts of Camellia sinensis, methanol and petroleum ether extracts ofAcorus calamus and petroleumether extracts of Cyntbopogon nardus, Languas galanga, Canariun~ zeylanicum and Curcunza domestica displayed significant mosquito-larvicidal activity (LC,, < 10.0 mgh) against late 3rd instar Culex larvae. High mosquito-larvicidal activity was observed in the steam distillate ofAcorus calamus (3.6-7.7 mgA) against late 3rdinstarmosquito larvae of C. quinquefasciatus, Aedes aegypti, Aedes albopictus, Anopheles tessellatus and Anopheles subpictus. The essential oil of C. nardus (Ceylon citronella) too displayed a significant activity (6.3 mgh) against C. quinquefasciatus and lesser activity against A. aegypti (9.3 mgA). Petroleum ether extract of the rhizome of L. galanga was similarly effective against C. quinquefasciatus (8.3 mg/l) and A. albopictus (9.3 mgtl). The essential oil of C. donzestica had high larvicidal activity (4.5 mgA) against Anopheles culicifacies which was resistant to other plant extracts. The results suggest that potent mosquito-larvicidal compounds may be isolated from A. calamus, C. nardus and C. domestica.


INTRODUCTION
The long term use of synthetic insecticides has created a number of ecological and medicinal problems such as the development of resistant insect strains, ecological imbalance and high toxicity to mammals.' Plants are known to contain compounds of insecti~idal~-~, inse~t-repelling~*~ and insect anti-juvenile6 properties.Most of these compounds are biodegradable and less harmful to mammals than synthetic insecticides.Therefore, there is a possibility of replacing synthetic insecticides with insecticides of plant origin.
Pyrethrins obtained from Chrysanthemum cinerafoiium are still widely used as insecticides and insect-repellents in Africa and South Arneri~a.~The insecticidal activity and photostability of natural pyrethrin have been increased by chemical synthetic methods.Apart from pyrethrins, the terminal epoxy derivative. of geranylgeraniol obtained from Pterodon pubescens has been reported to be highly lethal to the trematode Schistosoma m a n ~o n i .~ The insecticidal activity ofAcorus calamus against many insects has been r e p ~r t e d .~ Steam volatile principle of the rhizomes ofA.calamus L. has been shown to be toxic to adult Culex mosq~itoes.~However, there is no recorded information on the availability of compounds of plant origin, which possess mosquito-larvicidal activity.
It was, therefore, considered important to screen the plants grown in Sri Lanka, for mosquito-larvicidal activity.The present study describes the mosquito-larvicidal activity of some local plant extracts of Sri Lanka against several species of Culex, Aedes and Anoplteles.

METHODS AND MATERIALS
Plant material: Plants for screening for mosquito-larvicidal activity (Table 1) were selected randomly and also on the basis of the information gathered from native people living in rural areas of Sri Lanka.Plant specimens were identified by comparison with reference material a t the National Herbarium, Royal Botanic Gardens, Peradeniya, Sri Lanka.Plants were washed in running water slid were separated into plant parts (leaves, stem, root, bark etc.) and wereair-dried.
Preparation ofplant extracts: For preliminary screening for mosquito-larvicidal ~ctivity, air.dried plant material (10 g) were chopped and macerated with 100 ml :;olvent for extraction (light petroleum ether: methanol or water).The extracts were transferred t o conical flasks (250 ml) and were shaken in a n orbital shaker for I h.After filtration methanol and light petroleum ether were evaporated to dryness under reduced pressure and the residues were redissolved in 5% aqueous ethanol (100 ml) and later used for the bioassay.The water extracts were directly used for the bioassay.
The plant specimens which showed considerable larvicidal activity (Table 2) were further extracted with each solvent (hexane, light petroleum (40-60%) and . .methanol) separately and sequentially (in order: hexane, light petroleum ether and methanol) by refluxing in a Soxhlet apparatus for 24 h.Extracts obtained were evaporated to dryness under reduced pressure and the residues were redissolved in 5% aqueous ethanol (100 ml) and later used for the bioassay.
Essential oils in samples were extracted by steam distillation of plant samples using a Clevanger distillation apparatus as described previously." Test organisms: Late.3rd instar larvae of six mosquito species : Culex quinquefasciatus Say, Aedes aegypti L, Aedes albopictus Skuse, Anopheles culicifacies Giles, Anopheles tessellatus Theobald and Anopheles subpictus Grassi, were used for bioassay.For preliminary screening, C. quinquefasciatus was used as the test species.Mosquito bioassay: LC,, values of plant extracts for mosquito larvae were determined by the procedure followed by The World Health Organizationlo with slight modifications.For bioassay, healthy late 3rd instar mosquito larvae were distributed in batches of 20 in beakers containing 25ml water.Test dispersions (251111) were prepared in separate beakers by adding different amounts of the extract so that a series of ten final concentrations ranging from 20 to 200ppm was produced, when the contents with larvae in beakers were added to the latter (final volume, 50ml).Mortality counts were taken .after24 h and the bioassay was carried out at 2g°C.Each bioassay was carried out with five replicates.LC,, values were estimated from a probitflog concentrationgraph..

Mosquito-larvicidal activity of essential oils and solvent extracts
Results presented in Table 2 indicate that essential oils of A. calamus, prepared by steam distillation had the highest mosquito-larvicidal activity (3.6-12.0mgA), against all mosquito species tested except A. culicifacies.C. quinquefasciatus was the most sensitive mosquito species for A. calamus oil (3.6 mgA).The hexane extract of A. calamus was as effective (4.0 mgA) as the steam distillate.Light petroleum ether and methanol extracts of A.calamus rhizome were less effective.When the A. calamus rhizome was sequentially extracted with hot hexane, light petroleum ether and methanol, mosquitolarvicidal activity was retained only in the hexane extract (Results are not presented).It has been reported that the toxicity of petroleum ether extracts of A. calamus against adult mosquitoes (C.quinquefasciatus) were almost two fold when compared to that of k e r o ~e n e .~ The essential oil of C. nardus (Ceylon citronella) also displayed a significant activity (6.3 mgA) against C.quinquefasciatus and lesser activity against A.aegypti.Citronella oil is well known to be a mosquito-repellant but its mosquito-larvicidal activity has not been tested before.
The petroleum ether extract obtained from the rhizome of L. galanga was effective against C. quinquefasciatus (8.3 mgA) and A. albopictus (9.3 mgA).
The essential oil of C. domestica had high larvicidal activity (4.5 mgA) against A. culicifacies which was resistant to other plant extracts.However, it had very low activity against other mosquito species tested.
Our results suggest that the essential oils of A. calamus, C. nardus, L.galanga and C.domestica could be used as potent mosquito-larvicides.In order to use these essential oils as mosquito-larvicides it will be necessary to carry out further studies to determine their feasibilty oflarge scale use, stability of their active compounds under field conditions, and chemical identity of active compounds.The essential oils are complex mixtures of terpenes and there could be more than one compound responsible for the mosquito-layvicidal activity.Therefore, bioassay directed isolation of active components of essential oils of A. calumus, C. nardus, L. galanga and C.domestica should be undertaken.