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Evaluation of larvicidal activity of medicinal plant extracts against three mosquito vectors

OBJECTIVE
To evaluate the mosquito larvicidal activity of plant extracts.


METHODS
The hexane, chloroform, ethyl acetate, acetone, and methanol leaf, flower and seed extracts of Abrus precatorius (A. precatorius), Croton bonplandianum (C. bonplandianum), Cynodon dactylon (C. dactylon), Musa paradisiaca (M. paradisiaca) and Syzygium aromaticum (S. aromaticum) were tested against fourth instar larvae of Anopheles vagus (An. vagus), Armigeres subalbatus (Ar. subalbatus) and Culex vishnui (Cx. vishnui).


RESULTS
The highest larval mortality was found in seed ethyl acetate extracts of A. precatorius and leaf extracts of C. bonplandianum, flower chloroform and methanol extracts of M. paradisiaca, and flower bud hexane extract of S. aromaticum against An. vagus with LC(50) values of 19.31, 39.96, 35.18, 79.90 and 85.90 μg/mL; leaf ethyl acetate and methanol extracts of C. dactylon, flower methanol extract of M. paradisiaca, flower bud methanol extract of S. aromaticum against Ar. subalbatus with LC(50) values of 21.67, 32.62, 48.90 and 78.28 μg/mL, and seed methanol of A. precatorius, flower methanol extract of M. paradisiaca, flower bud hexane extract of S. aromaticum against Cx. vishnui with LC(50) values of 136.84, 103.36 and 149.56 μg/mL, respectively.


CONCLUSIONS
These results suggest that the effective plant crude extracts have the potential to be used as an ideal ecofriendly approach for the control of disease vectors. This study provides the first report on the larvicidal activity of crude solvent extracts of different mosquitoes.


Introduction
Mosquitoes are responsible for the spread of more diseases than any other group of arthropods. Mosquitoborne diseases still remain a major health problem in both human and veterinary sectors. Diseases transmitted by mosquitoes include malaria, dengue hemorrhagic fever, japanese encephalitis, yellow fever and filariasis. One to two million deaths are reported annually due to malaria worldwide. Lymphatic filariasis affects at least 120 million people in 73 countries in Africa, India, Southeast Asia, and Pacific Islands. These diseases not only cause high levels of morbidity and mortality but also inflict great economic loss and social disruption on developing countries such as India, China, etc. India alone contributes around 40% of global filariasis burden and the estimated annual economic loss is about 720 corers [1] . The annual incidence and mortality estimates for Japanese encephalitis (JE) are 30 000-50 000 and 10 000, respectively [2] . The use of conventional pesticides in the water sources, however, introduces many risks to people and/or the environment. Natural products of plant origin with insecticidal properties have been tried in the recent past for control of variety of insect pests and vectors [3] . Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in water, and thus, it is easy to deal with them in this habitat. Many researchers have reported on the effectiveness of plant extracts against mosquito larvae [4,5] .
Anopheles vagus (An. vagus) is a zoophilic species, which was more often collected in human-landing captures during the rainy season. The rainy season may be important in malaria transmission due to high biting populations [6,7] . A vector species that employs a unique, robust immune response against an invading pathogen is the Armigeres subalbatus (Ar. subalbatus), a natural vector of the nematode parasites that cause lymphatic filariasis. It also serves as a Objective: To evaluate the mosquito larvicidal activity of plant extracts. Methods: The hexane, chloroform, ethyl acetate, acetone, and methanol leaf, flower and seed extracts of Abrus precatorius (A. precatorius), Croton bonplandianum (C. bonplandianum), Cynodon dactylon (C. dactylon), Musa paradisiaca (M. paradisiaca) and Syzygium aromaticum (S. aromaticum) were tested against fourth instar larvae of Anopheles vagus (An. vagus), Armigeres subalbatus (Ar. subalbatus) and Culex vishnui (Cx. vishnui). Results: The highest larval mortality was found in seed ethyl acetate extracts of A. precatorius and leaf extracts of C. bonplandianum, flower chloroform and methanol extracts of M. paradisiaca, and flower bud hexane extract of S. aromaticum against An. vagus with LC 50 values of 19 36 and 149.56 毺 g/mL, respectively. Conclusions: These results suggest that the effective plant crude extracts have the potential to be used as an ideal ecofriendly approach for the control of disease vectors. This study provides the first report on the larvicidal activity of crude solvent extracts of different mosquitoes. competent laboratory vector of Plasmodium gallinaceum (P. gallinaceum), the causative agent of avian malaria in Asia [8] and also has been implicated in the transmission of JE virus in Taiwan [9,10] . Culex vishnui (Cx. vishnui) group to monitor JE virus activity in endemic areas of Tamil Nadu, southern India [11] . The Cx. vishnui subgroup mosquitoes comprising Culex tritaeniorhynchus (Cx. tritaeniorhynchus), Cx. vishnui and Culex pseudovishnui (Cx. pseudovishnui) have been implicated as major vectors of JE in India [12] .
Many studies on plant extracts against mosquito larvae have been conducted around the world. Abrus precatorius (A. precatorius) locally known as "Kundu mani," is a slender woody vine plant.
Musa paradisiacal (M. paradisiacal) (banana), is a perennial tree like herb widely distributed in moist tropics. Due to enriched food value and versatile medicinal value, banana is one of the most important fruits and vegetable crops of India. The leaves methanol and 95% ethanol extracts of M. paradisiaca were tested against the III instar larvae of Anopheles stephensi (An. stephensi) and L4 larvae of Ae. aegypti, respectively [18,19] . Syzyium aromaticum (S. aromaticum) (clove) undiluted oils was the most effective and provided 2 h of complete repellency against Cx. quinquefasciatus and Anopheles dirus (An. dirus) [20] and the methanol and ether extracts showed complete inhibition of adult emergence at 200 and 600 ppm, respectively against Culex pipiens (Cx. pipiens) [21] .
In the light of earlier literature, it is known that larvicides play a vital role in controlling mosquitoes in their breeding sites, but still vectors resistance to them remains unanswered. In view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess in the present communication, an attempt has been made to evaluate the larvicidal efficacy of the extracts from the medicinal plant against three medically important species of Anopheles vagus (An. vagus), Armigeres subalbatus (Ar. subalbatus) and Cx. vishnui.

Preparation of plant extracts
The dried (7-15 days in the shade at the environmental temperatures, 27-37 曟 day time) leaf (500 g), flower (400 g) and seed (550 g) were powdered mechanically using commercial electrical stainless steel blender and extracted with hexane (1 500 mL, Fine), chloroform (2 000 mL, Fine), ethyl acetate (2 800 mL, Qualigens), acetone (1 800 mL, Qualigens) and methanol (4 000 mL, Qualigens) in a soxhlet apparatus (boiling point range 60-80 曟 ) for 8 h. The extract was concentrated under reduced pressure of 22-26 mmHg at 45 曟 and the residue obtained was stored at 4 曟 . One gram of crude extract was first dissolved in 100 mL of acetone (stock solution). From the stock solution, 1 000 -3.125 毺 g/ mL were prepared with dechlorinated tap water. Polysorbate 80 (Qualigens) was used as an emulsifier at the concentration of 0.05 % in the final test solution. The control was set up with acetone, dechlorinated tap water and polysorbate 80.

Mosquito culture
An. vagus larvae were collected from the rice fields, open muddy pools and ditches, Ar. subalbatus breeding in open and uncovered septic tanks and Cx. vishnui larvae were collected from rice fields stagnant water area of Melvisharam and identified Dr. V. Rajagopal, Senior Entomologist, Zonal Entomological Research Centre, Vellore, Tamil Nadu. To start the colony, the larvae were kept in plastic and enamel trays containing tap water. They were maintained at (27 依2) 曟 and 75%-85% relative humidity under 14:10 light and dark cycles. Larvae were fed a diet of Brewers yeast, dog biscuits and algae collected from ponds in a ratio of 3:1:1, respectively. Pupae were transferred from the trays to a cup containing tap water and were maintained in our insectary [(45伊45伊40) cm] where adults emerged. Adults were maintained in glass cages and continuously provided with 10% sucrose solution in a jar with a cotton wick. On day five, the adults were given a blood meal from a pigeon. Glass petridishes with 50 mL of tap water lined with filter paper were kept inside the cage for oviposition [22] .

Larvicidal bioassay
During screening with the laboratory trial, the fourth instar larvae of An. vagus, Ar. subalbatus and Cx. vishnui were collected from the insect rearing cage and identified in Zonal Entomological Research Centre, Vellore. One gram of crude extract was first dissolved in 100 mL of acetone (stock solution). From the stock solution, 1 000 毺 g/mL was prepared with dechlorinated tap water. Polysorbate 80 (Qualigens) was used as an emulsifier at the concentration of 0.05% in the final test solution. The larvicidal activity was assessed by the procedure of WHO [23] with some modification [24] . For Bioassay test, larvae were taken in five batches of twenty in 249 mL of water and 1.0 mL of the desired plant extract concentration. The control was set up with acetone and polysorbate 80. The numbers of dead larvae were counted after 24 h of exposure and the percentage mortality was reported from the average of five replicates. The control was set up with acetone, dechlorinated tap water and polysorbate 80. The experimental media, in which 100% mortality of larvae occurs alone, were selected for a dose response bioassay.

Dose -response bioassay
From the stock solution, different concentrations ranging from 3.125 to 1 000 毺 g/mL were prepared. Based on the screening results, crude different solvent leaf, flower and seed extracts of A. precatorius, C. bonplandianum, C. dactylon, M. paradisiaca, and S. aromaticum were subjected to dose response bioassay for larvicidal activity against the larvae of An. vagus, Ar. subalbatus and Cx. vishnui. The numbers of dead larvae were counted after 24 h of exposure, and the per cent mortality was reported from the average of five replicates. However, at the end of 24 h the selected test samples turned out to be equal in their toxic potential.

Results
The preliminary screening is a good mean of evaluation of the potential larvicidal activity of plants popularly used for this purpose. The effect of the leaf, flower and seed hexane, chloroform, ethyl acetate, acetone and methanol extracts of A. precatorius, C. bonplandianum, C. dactylon, M. paradisiaca, S. aromaticum were tested at 1 000 毺 g/mL and showed activity against the fourth instar larvae of An. vagus, Ar. subalbatus and Cx. vishnui (Table 1). All plant extracts showed moderate larvicidal effects after 24 h; however, the highest larval mortality was found in seed ethyl acetate,

Discussion
The vector control is facing a threat due to the emergence of resistance in vector mosquitoes to conventional synthetic insecticides, warranting either counter measures or development of newer insecticides [25] . It is evident from our results that a rise in the concentration of plant extracts was the main cause of mortality in An. vagus, Ar. subalbatus and Cx. vishnui larvae. Similar study was conducted by Mathew et al [26] and reported that the seed methanol extract of Clitoria ternatea (C. ternatea) was effective against the larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus with LC 50 values of 65.2, 154.5, and 54.4 ppm, respectively. The leaf extract of Cassia obtusifolia (C. obtusifolia) had significant larvicidal effect with LC 50 and LC 90 values were 52.2 and 108.7 mg/L, respectively against An. stephensi [27] . 100% mortality was observed in 1% concentration of petroleum ether and ethanolic extract of Caesalpinia bonduc (C. bonduc), whereas it was 55% in 2.5% of aqueous extract and 92.6% in 2.5% of fixed oil against the fourth instar larvae of Cx. quinquefasciatus [28] . Earlier authors reported that the methanolic extract of Cassia fistula (C. fistula) was tested for larvicidal activity against Cx. quinquefasciatus and An. stephensi showed LC 50 values of 17.97 and 20.57 mg/L, respectively [29] . Georges et al [30] reported that the emodin, the most abundant and active anthraquinone isolated from Cassia nigricans (C. nigricans) showed approximately 85% mortality on the larvae of Anopheles gambiaea (An. gambiaea), in 24 h. The compounds diterpenoid furans 6alpha-hydroxyvouacapan-7beta,17beta-lactone (1), 6alpha,7beta-dihydroxyvouacapan-17beta-oic acid (2) and methyl 6alpha,7beta-dihydroxyvouacapan-17betaoate (3) isolated from the ethanolic extract from seeds of Pterodon polygalaeflorus(P. polygalaeflorus) exhibited LC 50 values of 50.08, 14.69, and 21.76 毺 g /mL against fourthinstar Ae. aegypti larvae [31] . Yenesew et al [32] reported that the crude chloroform extract of Millettia dura (M. dura) showed high activity (LC 50 = 3.5 毺 g/ mL at 24 h) against second-instar larvae of Ae. aegypti. Larvicidal efficacy of leaf extract of Acacia ferruginea (A. ferruginea) showed LC 50 values of 5 362.6 ppm against late third instar larvae of Cx. quinquefasciatus [33] .  [34] . Karunamoorthi and Ilango [35] have reported that the LC 50 and LC 90 values of methanol leaf extracts of Croton macrostachyus (C. macrostachyus) were 89.25 and 224.98 ppm, respectively against late third instar larvae of malaria vector, Anopheles arabiensis (An. arabiensis). Bagavan et al [36] have reported that peel chloroform extract of  Control -Nil mortality, LC 50 -Lethal concentration that kills 50% of the exposed parasite, LC 90 -Lethal concentration that kills 90% of the exposed parasite, UCL = upper confidence Limit, LCL = lower confidence Limit, 氈 2 -Chi-square, df degree of freedom, Significant at P< 0.05 level.
In conclusion, an attempt has been made to evaluate the larvicidal activity of plant extracts against An. vagus, Ar. subalbatus and Cx. vishnui. The results reported here open the possibility of further investigations of efficacy on their larvicidal properties of natural product extracts. The isolation and purification of crude and seed methanol of A. precatorius, flower methanol of M. paradisiaca, flower bud hexane extracts of S. aromaticum are in progress.

Conflict of interest statement
We declare that we have no conflict of interest.