International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 4(10), 16-21, October (2015) Int. Res. J. Environment Sci. International Science Congress Association 16 Efficacy of Phytochemicals as Sustainable Sources of Larvicidal Formulations for the Control of Culex sitiensRathy M.C., Sajith, U. and Harilal C.C.Division of Environmental Science, Department of Botany, University of Calicut, Malappuram District, Kerala, 673635, INDIAAvailable online at: www.isca.in, www.isca.me Received 15th July 2015, revised 24th August 2015, accepted 30th September 2015 AbstractMosquitoes are significant group of arthropods in terms of public health, as they spread serious human diseases, causing millions of deaths every year and the development of resistance to chemical insecticides ensuing in rebounding vectorial competence. Controlling mosquitoes at the larval stage is easy as target specificity of the larvicides used can be ensured. Phytochemicals derived from the vast diversity of plant species are important sources of environmentally safe and biodegradable chemicals, which can aid in mosquito control. The present study has been outlined to assess the feasibility of using phytochemicals, as a larvicidal agent against third instar larvae of Culex sitiens. Twelve species of plants belonging to Zingiberaceae (2), Asclepiadaceae, Caricaceae, Myrtaceae (2), Rutaceae (2), Calophyllaceae, Annonaceae, Euphorbiaceae, Bignoniaceae families were screened for this purpose. Aqueous leaf extracts of selected plants were prepared (0.5, 1.0, 2.0, 4.0, and 8.0 ml) and tested against mosquito larvae reared from eggs under laboratory conditions, for a period of 6 hours.Mortality percentages and LC50 were calculated as per WHO protocols and standards. Of twelve plants studied, all the plant extracts infatuated significant larvicidal potential with LC50 values ranging from 1.31 to 4.79 mg/ml against the third instar larvae of Culex sitiens. Extracts from plants like Calotropis gigantea, Pimenta dioica, Curcuma longa, Polialthia longifolia, Saritaea magnifica, Ricinus communis, Alpinia galanga, Carica papaya, Murraya koenigii and Eucalyptus globulus have the highest potential to be used as an effective larvicidal agent, signifying an ecofriendly approach for the control of mosquito vectors. Further investigations would be desired to separate and make out the constituents responsible for larvicidal properties. Keywords: Aqueous extracts, culex sitiens, mortality percentages, LC50. Introduction Mosquitoes are the most imperious group of insects, adversely influencing the health status of human beings. They have a worldwide distribution and are involved in the transmission of many dreadful diseases affecting millions of people every year1,2. They do not only transmit parasites and pathogens but they also source of allergic reaction that includes local skin and systemic sensitivity3,4. Several mosquito species belonging to the genera Anopheles, Culex and Aedes are acting as vectors for many pathogenic organisms causing diseases like Malaria, Filariasis, and Japanese Encephalitis, Dengue fever, yellow fever etc. These diseases spread globally, causing high levels of human mortality and thereby acting as factors impedimenting the economic development of most of the developing countries across the world5-8. Two million people primarily in tropical countries are being at the risk of mosquito-borne diseases9,10. To prevent mosquito-borne diseases and reduce fatality, it is necessary to stumble on a method to control the mosquito population. For this substantiation many chemical insecticides were developed and used against the vector and with significant success, but it contains some distressing drawbacks like selectiveness and non-biodegradability which leads to toxic hazards to ecosystem, environment and human health11. So an alternative strategy needs to be overcome this problem. The plant based herbal insecticides are found to more efficient, safe and best substitute for chemical insecticides. Natural products of plant origin are safe to use than synthetic insecticides. Therefore biological and ecofriendly natural resources are broad search area for the control of mosquito vectors12. In this light, the present endeavor is designed to determine the efficiency of aqueous leaf extracts of plants belonging to varied taxonomic groups on Culex sitiens competent vector of Ross River Virus and Japanese encephalitis. Material and Methods Plant collection and processing: The selection of plants were carried out based on their local availability and reported medicinal properties. The materials were collected from the plants in field located in Botanical garden, University of Calicut. The materials were taken from healthy plants free from dust, dirt and other impurities and were fetched to the laboratory for subsequent procedure. Preparation of extracts: The washed plant materials were chopped properly and kept in clean trays. For the preparation of extracts, approximately twenty grams (20gms) of plant material (Leaves) was taken and ground in a homogenizer using distilled water. The extract was filtered and the filtrate was made upto International Research Journal of Environment Sciences _____________________________________________ ISSN 2319–1414Vol. 4(10), 16-21, October (2015) Int. Res. J. Environment Sci. International Science Congress Association 17 1000 ml with distilled water and retained as stock solution for further experimentation. Serial dilutions of the stock solutions were prepared for assessing treatment efficiencies.Collection and culture of mosquito species: Vector species Culex sitiens was selected for the present study. Culex sitienscommonly breeds in all impoundments, including pools,puddles, wells, ditches; as well as cement tanks, jars and cans. It is a competent vector of Ross River Virus and Japanese encephalitis13. This species is also involved in the transmission of filariasis (Brugia malayi), although only in a secondary role. Mosquito larvae, collected from controlled breeding sites maintained with coconut shells, jars and cans kept at varying distances round households were used in the present study. Collected larvae were pooled in the laboratory and subjected to species level identification using standard manual14. In the laboratory, the larvae were transferred to enamel tray until adult emergence. After emergence, the mosquitoes were identified and species confirmed before rearing. Cyclic generations (two generations) of Culex sitiens were maintained andkept separately in mosquito cages in an insectary. For general rearing; mosquitoes were maintained at 26°C, 84% relative humidity, under a 16hr light and 8hr dark cycle with 1hr crepuscular period at the beginning and end of each light cycle based on the procedure of Kamaraj et al15 with slight modification. The adult mosquitoes were fed on five percent glucose solution. For continuous maintenance of mosquito colony, the adult female mosquitoes were blood feud with artificial blood feeding membrane16. Ovitraps (Petridishes with tap water lined with filter paper) was placed inside the cages for egg laying17. The eggs laid were then transferred to enamel trays maintained in the larval rearing chamber. The larvae were fed with larval food (dog biscuits and yeast in the ratio 3:1)18. The larvae on becoming pupae were collected, transferred to plastic bowls and kept inside mosquito cages for adult emergence19 Larvicidal Bioassay: Bioassay for the larvicidal activity was carried out using WHO20 procedure with minor modifications. Twenty numbers of early third instar larvae were introduced into treatment trays containing 250 ml of their natural growth media. To the treatment set, varying concentrations of the plant extracts (ie.0.5, 1.0, 2.0, 4.0 and 8.0 ml) were added from the stock solution; maintaining a relative concentration of the plant extracts as 10,20,40,80 and 160 mg/ml respectively. A control was also maintained for the treatment set. Mortality counts of larvae were monitored at regular intervals i.e.6, 12,24,48,72 and 96 hours after treatment and the control mortality as corrected using Abbott’s21 formula when the control mortality ranged between 5-20 percent10,22-24 Percentage mortality = Number of dead larvae X 100 Number of larvae introduced Statistical analysis: The mortality percentage was corrected using Abbott’s21 formula during the scrutiny of the larvicidal potentiality of the plant extracts. The experimental data was executed with MS Excel 2007 to find the Standard deviation and LC50 using probit analysis25. Results and Discussion The toxicity of aqueous leaf extracts of twelve species of plants belonging to various families were experienced against third instar larvae of Culex sitiens. Details of plants used for the present study and mortality percentage of mosquito larvae noticed are depicted in table-1. The efficacy of various leaf extracts on Culex sitiens exposed to 96 hours, for confirming lethality as per WHO19 standards are given Table 2.The data pertaining to LC50 are depicted in figure 1 and 2.Figure-1 LC50 value of promising plant extract against Culex sitiens 1012 0.5ml 1ml 2ml 4ml 8ml LC50 Calotropis gigantea Curcuma longa Pimenta dioica Ricinus communis Saritaea magnifica International Research Journal of Environment Sciences _____________________________________________ ISSN 2319–1414Vol. 4(10), 16-21, October (2015) Int. Res. J. Environment Sci. International Science Congress Association 18 Table-1 List of plant species used for the preparation of aqueous leaf extracts and their impact on Culex sitiens larvae Name of plant Family Condition at which 100% larval mortality noticed Alpinia galanga Zingiberaceae 12hours in 80mg/ml Calotropis gigantea Apocynaceae 12hours in 10mg/ml Carica papaya Caricaceae 12 hours in 80mg/ml Curcuma longa Zingiberaceae 48 hours in 10 and 24 hours in20mg/ml Eucalyptus globulus Myrtaceae 96 hours in 40and72 hours in 80mg/ml Glycosmis pentaphylla Rutaceae 50% mortality at 96hrs 8ml Mesua ferrea Calophyllaceae 72 hours in 80mg/ml Murraya koenigii Rutaceae 12 hours in 80mg/ml Pimenta dioica Myrtaceae 12 hours in 20mg/ml Polialthia longifolia Annonaceae 12hours 20mg/ml Ricinus communis, Euphorbiaceae 24hours 20mg/ml Saritaea magnifica Bignoniaceae 12hours 160mg/ml and 24hours 20mg/ml Table-2 The Efficacy of leaf extracts to the third instar larvae of Culex sitiens Plants Concentration of the extract (ml in 250 ml of growth medium) Mean ± S.D Control 0.5 1 2 4 8 Alpinia galanga 0 95 98.33 86.67 100 100 29.33±16.89 Calotropis gigantea 0 100 100 100 100 100 50±0 Carica papaya 0 26.67 55 100 100 100 27.83±18.25 Curcuma longa 0 100 100 100 100 100 43.33±8.17 Eucalyptus globulus 0 0 95 100 100 100 22.25±2.77 Glycosmis pentaphylla 0 0 0 0 0 56.67 13.64±11.82 Mesua ferrea 0 75 85 75 100 100 20.15±4.13 Murraya koenigii 0 0 0 46.67 100 100 18±15.14 Pimenta dioica 0 100 100 100 100 100 45.67±8.67 Polialthia longifolia 0 33.33 95 100 100 100 37.13±15.78 Ricinus communis, 0 70 100 100 100 100 31.33±4 Saritaea magnifica 0 91.67 100 100 100 100 36.11±7.03 International Research Journal of Environment Sciences Vol. 4(10), 16-21, October (2015) International Science Congress Association LC 50 Discussion: Mosquitoes are vectors for the pathogens of various diseases like malaria, Filariasis, Japanese encephalitis, Dengue, yellow fever, Chikungunya etc that causes high levels of morbidity and mortality19. So, one of the approaches for control of these mosquito- borne diseases is the interruption of disease transmission by killing or averting mosquitoes from biting human beings. The wide spread use of synthetic organic insecticides during the last five decades has upshoted in environmental hazards and developmen t of resistance in the major vector species26,27 . This has demanded the search for herbal preparations that do not produce any adverse effects in the nontarget organisms and are easily biodegradable remains a top research issue for scientists associated wi vector control6,28,29. Phytochemicals may serve as suitable alternatives to synthetic insecticides in future as they are relatively safe, inexpensive and are readily available throughout of the world26 . In fact, many researchers have reporte effectiveness of plant extracts and isolated components tested against different species of mosquito28,30,31. The larvicidal activity of different crude solvent (hexane, ethyl acetate, and methanol) extracts of plants like indica, Cass ia angustifolia, Diospyros melanoxylon, Dolichos biflorus, Gymnema sylvestre, Justicia procumbens, Mimosa pudica, Zingiber zerumbet against adult and early fourth instar larvae of Culex quinquefasciatus and Culex gelidus experimented by Kamaraj etal32 . Vahitha et al the larvicidal efficacy of methanol leaf extracts of zeylanica and Acacia ferruginea were tested against the late third instar larvae of Culex quinquefasciatus of 2,214.7 and 5,362.6 ppm, respectively. The toxicity of crude extracts from the Ricinus communis against fourth instar larvae Anopheles arabiensis quinquefasciatus. The LC50 values of 2nd , 3rd and 4 1012 LC50 Environment Sciences __________________________________ ___________ Association Figure-2 50 value of plant extract against Culex sitiens Mosquitoes are vectors for the pathogens of various diseases like malaria, Filariasis, Japanese encephalitis, Dengue, yellow fever, Chikungunya etc that causes high levels of So, one of the approaches for control borne diseases is the interruption of disease transmission by killing or averting mosquitoes from biting human beings. The wide spread use of synthetic organic insecticides during the last five decades has upshoted in t of resistance in the . This has demanded the search for herbal preparations that do not produce any adverse effects in the nontarget organisms and are easily biodegradable remains a top research issue for scientists associated wi th alternative Phytochemicals may serve as suitable alternatives to synthetic insecticides in future as they are relatively safe, inexpensive and are readily available throughout . In fact, many researchers have reporte d the effectiveness of plant extracts and isolated components tested The larvicidal activity of different crude solvent (hexane, ethyl acetate, and methanol) extracts of plants like Aristolochia ia angustifolia, Diospyros melanoxylon, Dolichos biflorus, Gymnema sylvestre, Justicia procumbens, Mimosa against adult and early fourth instar Culex gelidus were . Vahitha et al 33,34 worked out the larvicidal efficacy of methanol leaf extracts of Pavonia were tested against the late with LC50 values Ricinus communis Anopheles arabiensis and Culex , 3rd and 4 th instar larvae of Anopheles arabiensis and 403.65, 445. 66 and 498.88ppm, 1091.44, 1364.58 and 1445.44ppm respectively were reported by Elimam et al Singh et al36,37 reported that the LC and hexane extracts of Momordica charantia stephensi, Culex quinquefasciatus larvae are 0.50, 1.29, and 1.45% (aqueous extracts) and 66.05, 96.11, and 122.45ppm (hexane extracts), respectively. The monotonous observations carried out by various researchers in the field of mosquito control by plants. In the present endeavor the effect of the leaf extracts of galanga, Calotropis gigantea, Carica Papaya Eucalyptus globulus Glycosmis pentaphylla Murraya koenigii Pimenta dioica communis and Saritaea magnifica instar larvae of Culex sitiens at varying concentrations. All plant extracts showed utmost larvicidal effects after 96 However, the highest larval mortality was found with leaf extracts of Calotro pis gigantea (43.33±8.17), Pimenta dioica (45.67±8.67) (36.11±7.03) and Ricinus communis instar larvae of Culex sitiens (table - The present investigation revealed that the aqueous e Calotropis gigantea (Leaf) was the most efficient, divulging a substantial LC50 value of 10.8 mg/ml. But the leaf extract of Glycosmis pentaphylla was least efficient with an LC 5.66 mg/ml (figure-1 and 2). It was also noticed that the extracts made from Calotropis gigantea , dioica, Ricinus communis and Saritaea important larvicidal potential with LC 1.01 to 10.8 mg/ml (Figure 1).Other extracts ( Carica papaya, Mesua ferrea, Murraya koenigii, Polialthia longifolia, and Eucalyptus globulus plants ___________ ISSN 2319–1414 Int. Res. J. Environment Sci. 19 and Culex quinquefasciatus are 66 and 498.88ppm, 1091.44, 1364.58 and 1445.44ppm respectively were reported by Elimam et al 35. reported that the LC 50 values of crude aqueous Momordica charantia against Anopheles and Aedes aegypti mosquito larvae are 0.50, 1.29, and 1.45% (aqueous extracts) and 66.05, 96.11, and 122.45ppm (hexane extracts), respectively. The monotonous observations carried out by various researchers in the field of mosquito control by plants. the present endeavor the effect of the leaf extracts of Alpinia Carica Papaya Curcumalonga Glycosmis pentaphylla Mesua ferrea Polialthialongifolia Ricinus were tested against the third at varying concentrations. All plant extracts showed utmost larvicidal effects after 96 hours. However, the highest larval mortality was found with leaf pis gigantea (50±0), Curcuma longa (45.67±8.67) , Saritaea magnifica and Ricinus communis (31.33±4) against the third - 2). The present investigation revealed that the aqueous e xtract of (Leaf) was the most efficient, divulging a value of 10.8 mg/ml. But the leaf extract of was least efficient with an LC 50 value of It was also noticed that the extracts , Curcuma longa, Pimenta Saritaea magnifica obsessed important larvicidal potential with LC 50 values ranging from 1.01 to 10.8 mg/ml (Figure 1).Other extracts ( Alpinia galanga, Carica papaya, Mesua ferrea, Murraya koenigii, Polialthia Eucalyptus globulus ) showed only moderate 0.5ml1ml2ml4ml8ml International Research Journal of Environment Sciences _____________________________________________ ISSN 2319–1414Vol. 4(10), 16-21, October (2015) Int. Res. J. Environment Sci. International Science Congress Association 20 toxicity against Culex sitiens larvae (figure-2). Conclusion A Study has been undertaken to assess the efficiency of phytochemicals derived from selected plants as potential and sustainable sources of larvicidal agents for the management of mosquito vectors. Twelve plant species belonging to Zingiberaceae (2), Asclepiadaceae, Caricaceae, Myrtaceae (2), Rutaceae (2), Calophyllaceae, Annonaceae, Euphorbiaceae, Bignoniaceae,familieswere screened for this purpose. Aqueous leaf extracts of selected plants were prepared (0.5, 1.0, 2.0, 4.0 and 8.0 ml) and tested against mosquito larvae reared from eggs under laboratory conditions, for a period of 96hours. Mortality percentages and LC50 were calculated as per WHO protocols and standards. Extracts from plants like Calotropis gigantea, Pimenta dioica, Curcuma longa, Polialthia longifolia, Saritaea magnifica, Ricinus communis, Alpinia galanga, Carica papaya, Murraya koenigii, and Eucalyptus globulus were noted to be lethal to the larvae. 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