Crude extracts of three plants Citrullus colocynthis, Leucas aspera, and Strychnos nux-vomica were screened for their larvicidal activities against the third instar of Culex quinquefasciatus. The mortality rate of the mosquito larvae was evaluated against the three botanicals at different periods of exposure (24h, 48h, and 72h). Hexane, Chloroform and Ethyl acetate were used as solvents for the crude extracts. C. colocynthis showed the maximum larvicidal effect (100% mortality) in 750 ppm and 1000 ppm in hexane extract at 24h period of exposure. L. aspera also showed the same activity in hexane extract at 24 h, while S. nux-vomica showed slightly lower effect.
The vector-borne diseases caused by mosquitoes are one of the major health problems in many countries. Dengue, malaria, yellow fever, filariasis and chickungunya are some of the deadly diseases spread by mosquitoes. Lymphatic filariasis, is a widely distributed tropical disease with around 120 million people infected worldwide and 44 million people having common chronic manifestation3. It is caused by Wucherria bancrofti, and this pathogen is transmitted by Culex quinquefaciatus2. Culex quinquefasciatus differs itself from the rest by laying their eggs in the form of rafts. They hatch within 2-3 days found in water. Four larval instars, which take 3-4 days to pupate, it lasts for 1-4 days adult emerges and matures sexually within a day.
The most efficient approach to control the vector is to target the immature stages of the life cycle. Synthetic chemical larvicides continue to be applied for controlling mosquitoes in most parts of the world. But many of these chemicals are toxic to human, plant, and animal life and resistance can be problematic in maintaining control, especially with organophosphate and pyrethroid larvicides. There has been serious concern about the use of chemical based mosquito larvicides and repellents in recent past. As a result, researchers are currently investigating natural substances to use as insecticides for controlling larval mosquitoes. Plants may be a source of alternative agents for control of vectors because they are rich in bioactive chemicals.
Das et al.,5 have evaluated the larvicidal efficacy of botanicals against Aedes albopictus and Culex quinquefasciatus. Chowdhury et al.,4 have tested the efficacy of Solanum villosum berry extract against the dengue vector Stegomyia aegypti. In the present study, the larvicidal efficacy of three botanicals Citrullus colocynthis, Leucas aspera, and Strychnos nux-vomica were examined against the thrid instar larvae of Culex quinquefasciatus.
Materials and Methods
Citrullus colocynthis, Leucas aspera and Strychnos nux-vomica were chosen based on their abundance, availability and medicinal properties. Citrullus colocynthis and Leucas aspera were collected from the field and Strychnos nux-vomica from the hilly areas of Vazhakulam, Eranakulam district, Kerala. Chosen plant samples were confirmed of their taxonomic position following the descriptions of Longman10.
The egg rafts of Culex quinquefasciatus were collected from the open drains of river Coovum, near Choolaimedu, Chennai, Tamil Nadu, India. The egg rafts of Culex quinquefasciatus were placed in Petri dishes (10.5 cm diameter), containing aged tap water. Larvae were fed with finely ground mixture of yeast and dog biscuits in the ratio of 3:1. They were maintained at 28± 2o C. They first developed into pupae through four instars, in 8-10 days. Pupae were transferred into beakers containing aged tap water and kept in mosquito cage for emergence. The adult mosquito came out in 2-3 days. Blood meal from a chick was given to adult mosquitoes after three days of emergence. After 3-4 days of blood feeding of adult mosquitoes, the petri dishes filled with tap water were placed inside the cage for oviposition. The egg rafts were separated and placed in glass petri dishes for hatching15.
The whole plant of C. colocynthis, the leaves of S. nux-vomica and the leaves and inflorescence of L. aspera were dried at room temperature (28 ±2o) for one week. The air dried plant materials were powdered separately using electrical blender. 100 gm of powder was mixed with 300 ml of hexane and kept for 24 hrs and then filtered and it was stored in reagent bottle. Again, 100 ml of hexane is added to the sample powder and kept for another 24 hrs with periodical mixing with a glass rod. It is then filtered and the extract solution was stored in the same reagent bottle. The powder was allowed to dry for 2 hrs before pouring the other solvents of chloroform and ethyl acetate, respectively and also for other two plants.
The crude extracts thus obtained were filtered through Whatman’s filter paper No.1 and were evaporated to dryness in a rotary vacuum evaporator at the room temperature (40oC) and low pressure (25-30 mm hg)13. Such dried and semisolid crude extracts were stored in the refrigerator until they were used for bioassay test against Culex larvae. Larvicidal activity was assessed using the standard method16. Twenty five larvae of early third instar were released in a 250 ml glass beaker containing 100 ml of dechlorinated water.
From the stock solution (1, 00,000 ppm), 250, 500, 750, 1000 ppm concentrations were prepared. The plant extract was introduced into the beaker containing the larvae is respective concentration (250, 500, 750, 1000 ppm), which was mixed with acetone and triton-80 (mixing solution). Data pertaining to the larval mortality was recorded at the end of 24, 48 and 72 hrs. Simultaneously positive and negative controls were also maintained. Each experiment was replicated 5 times at room temperature (28±2oC) for three different plants. Percent larval mortality of Culex quinquefasciatus was calculated following to the procedure of Abbott1.
Results and Discussion
Maximum mortality was recorded in 1000 ppm concentration in all the solvents. Minimum larvicidal activity was noted in 250 ppm concentration. In 500 and 750 ppm showed moderate larvicidal activity in different period of exposures. Within the solvent extracts hexane crude extracts of all the three plant extracts showed maximum mortality of the larvae and was observed to be 100 ± 0 at 24 hr of exposure in 750 ppm and 1000 ppm respectively. The least mortality was observed in ethyl acetate and chloroform crude extracts (Table 1 & 2).
Results revealed high mortality at higher concentrations. The maximum mortality was exhibited in longer duration. The mortality was found to be higher in hexane crude extracts in all plants compared to the other solvents. Mortality counts were made using Abbott’s formula1 after 24hr of the treatment LC50 , LC95, slope and chi-square values were calculated using probit analysis7.
In recent times biologically active plant materials have attracted considerable interest in mosquito control programmes because of their biodegradable nature. Some popularly used plants are screened for antihelminthic, antiparasitic, mosquitocidal and repellent activity and are reported89. These plant materials are used as larvicides, adulticides and repellents to control mosquitoes.
Sathish Kumar and Maneemegalai,12 have tested the larvicidal effect of Lantana camara against Aedes aegypti and Culex quinquefasciatus. Dua et al.,6 have elucidated the larvicidal activity of neem oil (Azadirachta indica) formulation against various species of mosquitoes. Singha and Chandra,14 have tested the mosquito larvicidal activity of some common spices and vegetable waste on Culex quinquefasciatus and Anopheles stephensi.
Since the vectors develop resistance against chemicals in the environment due to gene mutation. Bioactive principles of botanical extracts would provide effective control over mosquito larvae. A detailed assessment by isolation and purification of components from the crude extracts of these potential plant extracts will throw light on their relevance in the management of vector mosquitoes at the larval stages.
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- Ansari MA, Mittal PK, Razdan RK, Sreehari U. 2005. Larvicidal and mosquito repellent activities of pine (Pinus longifolia, Family: Pinaceae) oil. J Vect Borne Dis., 42:95-99.
- Bernhard L, Bernhard P, Magnussen P. 2003. Management of patients with lymphoedema caused by filariasis in north-eastern Tanzania: alternative approaches. Physiotherapy 89:743-749.
- Chowdhury N, Anupam Ghosh and Goutam Chandra. 2008. Mosquito larvicidal activities of Solanum villosum berry extract against the dengue vector Stegomyia aegypti. BMC Complementary and Alternative Medicine 8:10.
- Das NG, Goswami D and Rabha B. 2007. Preliminary evaluation of mosquito larvicidal efficacy of plant extracts. J Vect Borne Dis. 44:145-148.
- Dua VK, Akhilesh C Pandey, Kamaraju Raghavendra, Ashish Gupta, Trilochan Sharma and Aditya P Dash. 2009. Larvicidal activity of neem oil (Azadirachta indica) formulation against mosquitoes. Malaria Journal 8:124
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- Geerts SK, Blerk V and Triest L. 1994. Effect of Ambrosia martimia on Anopheles stephensi and Aedes aegypti J. Ethnopharmacology 42:7-11.
- Kelm MA, Nair MG and Schutzki RA. 1997. Mosquitosidal compounds from Magnolia salicifolia. Int. J. Pharmacolognosty 35(2):84-90.
- Longman O. 1994. Indian Medicinal Plants – a compendium of 500 species Orient Longman Ltd, Madras. 318.
- Sathish Kumar M and Maneemegalai S. 2008. Evaluation of larvicidal effect of Lantana camara Linn against mosquito species Aedes aegypti and Culex quinquefasciatus. Advances in Biological Research 2(3-4):39-43.
- Saxena RC, Harshan V, Saxena A, Padma S, Sharma MC and Laxman kumar M. 1993. Larvicidal Chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi (Diptera: Culucidae) J. Am. Mosq. Cont. Assoc., 9(1):84-87.
- Singha S and Chandra G. 2011. Mosquito larvicidal activity of some common spices and vegetable waste on Culex quinquefasciatus and Anopheles stephensi. Asian Pac J Trop Med., 4(4):288-93.
- Verma KVS and Rahman SJ. 1986. Development of Knockdown resistance (KDR) against fenvelarate in a DDT-resistant strains of Culex quinquefasciatus Curr. Sci., 55:914-916.
- W.H.O. 1996. Report of the WHO informal consulation on the evaluation and testing of insecticides Control of Tropical Disease Division WHO publications, CTD/WHOPES/IC/96.1, Geneva. 69.