International Journal of Research in Botany. ISSN Original Article

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1 Available online at International Journal of Research in Botany Universal Research Publications. All rights reserved ISSN Original Article Efficacy of Crude extracts of Annona reticulata and Pongamia pinnata as larvicidal for the Management of filarial vector Culex quinquefasciatus Say Diptera: Culicidae J. B. Nayak Department of Botany, Nizam College, Hyderabad, , Andhra Pradesh, India. Received 26 December 2013; accepted 02 January 2014 Abstract Objective: To evaluate the larvicidal activity of crude extracts derived from selected medicinal plants with special reference to journals and tribal knowledge, against early fourth instar larvae of Culex quinquefasciatus. Say (Diptera: Culicidae). Methods: According to WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants the fresh and healthy plant materials were collected and extracted with methanol and separated by Rotary evaporator. The efficacy was determined through bioassay method. For bioassay test, twenty numbers of fourth instars larvae were taken in six batches of twenty each for the treatment. Bowls of 100 ml capacity were kept in series, and tested for each desired plant extract concentrations 5 to 200 ppm. The control was set up with distilled water. The numbers of dead larvae were counted after every 12 hrs of treatment up to 48 hours. Results: These crude extracts have showed 100% mortality within short period. at different concentrations. And no mortality was found in control. Conclusions: Present study indicates that crude extracts of Annona reticulata and Pongamia pinnata carry huge potential as a mosquito larvicidal. This potential could be exploited for the development of safer and effective botanical mosquito larvicidal tool for the management of Cx. quinquefasciatus. Keywords: Annona reticulata, Pongamia pinnata, larvicidal activity, Crude extracts, Culex quinquefasciatus. 1. Introduction Mosquitoes are well known as annoying pests and as carriers of disease-causing agents to humans and animals. Several mosquito species belonging to genera Anopheles, Culex and Aedes are vectors for the pathogens of various diseases like malaria, filariasis, Japanese encephalitis, dengue and dengue hemorrhagic fever, yellow fever, etc. Culex quinquefasciatus is a vector of lymphatic filariasis, while this disease is widely distributed in tropical area with around 120 million people infected worldwide and 44 million people having common chronic manifestation (Bernhard et al., 2003). Tropical areas are more prone to parasitic diseases and the risk has increased due to climate change and intensifying Globalization (Karunamoorthy et al., 2010). Also, owing to poor drainage system, especially during rainy seasons, the presence of many fish ponds, irrigation ditches and the rice fields provide abundant mosquito breeding places. Mosquito vector-borne diseases contribute to the major disease burden in India (Prabhu et al., 2011). Fight against adult mosquitoes is temporary, unsatisfactory, inadequate and environmental polluting. Larval treatment is much more effective for managing this 2013 Universal Research Publications. All rights reserved notorious insect because in this stage larvae are localized and restricted to a small space due to the low mobility (Howard et al., 2007). Moreover, vector control is facing a threat due to the emergence of resistance to synthetic insecticides. In this context, essential oils have received much attention as potentially useful bioactive compounds against insects (Mathew and Thoppil, 2011; Massebo et al., 2009; Chowdhury et al., 2008). The application of easily degradable plant compounds is considered one of the safest methods in controlling insect pests and vectors (Sivagnaname et al., 2004). Plant extracts are currently studied more and more because of their role in plant protection as well as in urban entomology (Din et al., 2011). Plant extracts are safer for non-target organisms including man, therefore, plant based Formulations would be more feasible from environmental perspective than synthetic mosquitocides (Bhat et al., 2009). Herbal products with proven potential as insecticide or repellent can play an important role in the interruption of the transmission of mosquito-borne diseases at the individual as well as at the community level. Aromatic plants and their essential oils are very important sources of 1

2 many compounds that are used in different respects (Amer et al., 2006). Great deal of research have been done by number of authors around the globe and significant progress have been made in the field of management of notorious mosquito species through the utilization of botanicals (essential oils/polar and non-polar extracts) derived from aromatic medicinal plant/spices (Traboulsi et al., 2002; Prajapati et al., 2005; Lee, 2006; Pitasawata et al., 2007; Knioa et al., 2008). 2. Materials and methods 2.1. Collection of plant material: According to WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants the fresh and healthy plant materials of Annona reticulata leaf Fig (a) and Pongamia pinnata leaf Fig (b) and bark Fig (c) were collected from different places of Telangana region of Andhra Pradesh, India. In the individual cleaned mesh bags and plastic bags with the help of protective clothing gloves, which will prevent and protect from toxic and dermatitis-causing plants, poisonous animals and disease-carrying insects. The Medicinal plant parts were collected With reference to various journals and local tribal knowledge for the purpose of present study. Fig (a). Annona reticulata Fig (b). Pongamia pinnata bark Fig (c). Pongamia pinnata leaf The collected raw medicinal plant materials like leaf and bark were transferred to the Lab and immediately washed with tap water to remove soil, dust and any other microorganisms, and also to eliminate undesirable materials and contaminants. The collected medicinal plant materials were protected from insects, rodents, birds and other pests, and also from Livestock and domestic animals Plant species and their parts used for extraction. Plant Vernacular Plant Family species name part Annona reticulata Pongamia pinnata 2 Rama phalamu Leaf Annonaceae Kanuga Leaf and bark Fabaceae 2.2. Processing of plant material: The collected plant material leaf and bark were shade dried at room temperature for 7-10 days. The leaves were shade-dried, and powdered (Maheshwaran et al., 2008) (Patole and Mahajan 2009). Each sample was extracted with methanol using a soxhlet apparatus (Vogel, 1978). The dried plant material was grinded and powdered mechanically using electrical stainless steel blender (Model: Sumeet 1.5L Capacity, 6 angle blades, 2 speed w/pulse and made of Stainless Steel Blade. Libra appliances pvt. Ltd, Baroda, India). The dried and powdered plant materials were stored in plastic bags. and each sample was extracted in sample bottles with methanol by maceration method. After 5 days of the solvent extraction it was filtered with watman filter paper no.1 and then solvent extraction was obtained. Next the Solvent was separated by rotary evaporator (boiling point range º C) for 8 hrs. The pooled methanol extracts were concentrated separately by rotary vaccum evaporator at 40º C and evaporated to dryness and stored at 4º C in an air tight bottle (Jang et al., 2002). and the residue obtained was stored at 4º C in the deep freezer Preparation of stock solution: One gram of crude extract was first dissolved in100 ml of methanol and stored as stock solution. The anthelmintic assay was carried as per the method of Tandon et al., (1997). This stock solution was used to prepare the desired concentrations of the extract for the larvicidal activity on the mosquito larvae. From the stock solution 5, 10, 25, 50, 100 and 200 ppm concentrations were prepared with dechlorinated tap water. The control was set up with 100 ml tap water by adding 2ml of methanol. 3. Selection of Mosquito species: The important vector species of mosquitoes which is Culex quinquefasciatus was selected for the present study. Cx. quinquefasciatus is the vector of west Nile virus which causes Encephalitis or Meningitis which is known to affect the brain tissue, finally resulting in permanent neurological damage (Hubalek and Halouzka, 1999). 3.1 Mosquito larvae collection and culture: The fourth instar larvae were collected and transferred in to a glass beaker of 1 liter capacity containing

3 clean water, and the larvae after sorting were identified as Cx. quinquefasciatus larvae (Shyamapada Mandal 2011). Culex quinquefasciatus larvae were collected from stagnant sewage water of the River Musi in Hyderabad. The identification of mosquito larvae was done by Dr. B. Redya Naik, Entomologist, Department of Zoology, Osmania University, Hyderabad, Andhra Pradesh. The collected larvae were reared from egg to larval stage and then to adults in the laboratory itself, to avoid the species mixture. From these adults, next F1 generation larvae were used for the present study. This procedure facilitates to maintain the uniform age of larval instar (fourth instar). The larvae were reared in tap water and fed with dog biscuits and yeast tablets at the ratio of 3:1. They were maintained at 28 ± 2 C, 75 85% RH, under 14L: 10D photoperiod cycles. The larvicidal activity was assessed by the procedure of WHO (1996). The larvae were fed with dry yeast powder on the water surface (50mg/1) (Senthilnathan S. 2007). 4. Bioassay: The larvicidal activity was assessed by the standard guidelines of WHO (2005), with some modification and as per the method of Rahuman et al., (2000), Prabakar and Jebanesan (2004). A laboratory colony of C. quinquefasciatus was used for the larvicidal activity studies. Patole and Mahajan, (2009), reported that, the twenty numbers of 3 to 4 th instar larvae were taken out from culture through and transferred in to plastic bowls with the help of dropper. For bioassay test, twenty numbers of fourth instars larvae were taken in six batches of twenty each for the treatment. Bowls of 100 ml capacity were kept in series, and tested for each desired plant extract concentrations 5, 10, 25, 50, 100 and 200 ppm. The control was set up with 2 ml methanol and distilled water. The experimental media, in which 100% mortality rate of larvae occurred were selected for a dose response bioassay. Based on the screening results, crude methanol solvent extracts of leaf and bark extracts of the plant listed were subjected to dose response bioassay for larvicidal activity against the larvae of Cx. quinquefasciatus. The numbers of dead larvae were counted after every 12 hrs of treatment up to 48 hours. The percentage mortality and standard error of mean have been calculated for all the results obtained by this study. 5. Results and Discussion: Table.1. the larvicidal activity of A. reticulata leaf crude extract at different concentration is represented in Table: 2. the data shows that, 100 % mortality rate of larvae was observed at 5, 10, 25, 50, 100 and 200 ppm concentrations of crude extract. The concentration of 5 ppm showed 100 % mortality rate was recorded after 48 hrs of the treatment. And for 10, 25, 50, 100 and 200 ppm it was observed after 12 hrs of the treatment. The control did not show any mortality. The present investigation is the pioneer work on the Annona reticulata methanolic leaf extract, which showed excellent and encouraging results among all the extracts studied in this research. Even low concentrations (5 ppm) of crude extract were lethal on mosquito larvae. Table 1: Mortality rates of Culex quinquefasciatus mosquito larvae at different concentrations of crude extract of Annona reticulata leaves. Time (hours) n = Total % Mean SE (+/-) Control n = number of larvae used for bioassay test Graph 1: Mortality rates of Culex quinquefasciatus mosquito larvae at different concentrations of crude extract of Annona reticulata leaves. Table.2. The larvicidal activity of Pongamia pinnata bark crude extract at different concentrations is represented in Table: 4. the data shows that, 100 % mortality rate was observed at 5 and 10 ppm concentrations of crude extracts after 48 and 24 hrs of the treatment. At the concentration of 25, 50, 100 and 200 ppm, 100 % mortality rates were recorded after 12 hrs. The control did not show any mortality. This crude extracts has given excellent results when compared to that of others. Table 2: Mortality rates Culex quinquefasciatus mosquito larvae at different concentrations of crude extract of Pongamia pinnata bark. Time (hours) n = Total % Mean SE (+/-) Control n= number of larvae used for bioassay test. Graph 2: Mortality rates of Culex quinquefasciatus mosquito pinnata bark. 3

4 Aboli Lale and Dk Kulkarni (2010) reported the repulsion activity of Pongamia pinnata seed powder along with the leaves, pod shell and cow dung with cow urine fermented mixture, which showed 90% repulsion of mosquitoes. There is no such research on the larvicidal activity of the crude extracts of Pongamia pinnata bark. When this extract was given even at low concentrations the mosquito larvae showed 100 % mortality rate. Table.3. The larvicidal activity of Pongamia pinnata leaf crude extract at different concentration is represented in Table: 5. the data shows that, 100 % mortality rate of larvae was observed at 25 and 50 ppm of concentrations of crude extract after 48 hrs of the treatment. And at the concentration of 100 and 200 ppm 100 % mortality rate was recorded after 36 hrs. At low concentrations of crude extract i.e. 10 and 5 ppm after 48 hours of treatment the % mortality rate of mosquito larvae was decreasing to 80 and 50 % respectively, and no mortality rate was observed for control. Table 3: Mortality rates of Culex quinquefasciatus mosquito pinnata leaves. Time (hours) n= Total % Mean SE(+/-) Control o.ooo n = number of larvae used for bioassay test. Graph 3: Mortality rates of Culex quinquefasciatus mosquito pinnata leaves. Swathi et al., (2011) Reported, the Larvicidal efficacy of ethanolic extract of Pongamia pinnata leaves which were tested against late 3 rd or early 4 th instar larvae of Culex quinquefasciatus showed 90 % mortality at 796 ppm against mosquito larvae of Culex quinquefasciatus. Their results may be attributed to the solvent used for extraction which might elute the active compounds against the mosquito larvae. Comparatively better results were obtained in our study with methanol extract of Pongamia pinnata leaves. Where 100 % mortality rate of 4 th instar larvae was observed at a low concentration of 25 ppm within 48 hours. Therefore, among the 3 selected extract studied, the bark of Pongamia pinnata showed highest larvicidal activity against Culex quinquefasciatus mosquito larvae within a short time of 12 hrs. 6. Conclusions The botanical insecticides are generally safer, readily biodegradable non-toxic but active against the insect pest and lack toxicity to higher animals and they do not leave any phytotoxic residues in the environment and active against insect pest. The biological activity of this plant extract may be due to various compounds, including tannins, phenolics, terpenoids, flavonoids and alkaloids. These compounds may jointly or independently contribute to produce toxic activity against the mosquito species. The present investigation suggests that these extracts can be considered as alternative insecticide products for mosquito control in breeding sites, especially in cases where susceptibility is decreasing. These can be commercialized as botanical (Insect Growth Regulators) IGRS such as methoprene. The plant derivatives which are probable sources of biologically active ingredients for mosquito control need to be investigated for further studies. References: 1. Aboli Lale and Kulkarni DK. A Mosquito repellent Karanj kunaapa from Pongamia pinnata. Asian Agri- History 2010; 14, 2: Amer A, Mehlhorn H. Larvicidal effects of various essential oils against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae). Parasitol Res 2006; 99: Bernhard L, Bernhard P, Magnussen P. Management of patients with lymphoedema caused by filariasis in north-eastern Tanzania: alternative approaches. Physiotherapy 2003; 89: Bhat SK, Kempraj K. Biocidal potential of clove oil against Aedes albopictus - A comparative study. Afr J Biotechnol 2009; 8: Chowdhury N, Ghosh A, Chandra G. Mosquito larvicidal activities of Solanum villosum berry extract against the dengue vector Stegomyia aegypti. BMC Comp Alternative Med 2008; 3: Din S, Akram W, Khan HAA, Hussain A, Hafeez F.Citrus Waste-Derived Essential Oils: Alternative Larvicides for Dengue Fever Mosquito, Aedes albopictus (Skuse) (Culicidae: Diptera). Pakistan J Zool 2001; 43: Howard AFB, Zhou G, Omlin FX. Malaria mosquito control using edible fish in western Kenya: preliminary findings of a controlled study. BMC Public Health 2007; 7: Hubalek Z and Halouzka J. West Nile Fever A reemerging mosquito borne viraldisease in Europe. Emerging infections Diseases 1999; 2, Jang YS, Baek BR, Yang YC, Kim MK, Lee HS. Larvicidal activity of leguminous seeds and grains against Aedes aegypti and Culex pipiens pallens. J Am Mosq Control Assoc 2002; 18, Karunamoorthy K, Ilango K, Murugan K. Laboratory 4

5 evaluation of traditionally used plant-based insect repellents against the malaria vector Anopheles arabiensis Patton. Parasitol Res 2010; 106: Knioa KM, Ustab J, Daghera S, Zournajiana H, Kreydiyyeh S. Larvicidal activity of essential oils extracted from commonly used herbs in Lebanon against the seaside mosquito, Ochlerotatus caspius. Bioresource Technol 2008; 99: Lee HS. Mosquito larvicidal activity of aromatic medicinal plant oils against Aedes aegypti and Culex pipiens pallens. J Am Mosq Control Assoc 2006; 22: Maheswaran R, Sathish S and Ignacimuthu S. Larvicidal activity of Leucas aspera (Willd) against the larvae of Culex quinquefasciatus and Aedes aegypti. Int. J. Integr. Biol 2008; 2, Massebo F, Tadesse M, Bekele T, Balkew M, Michael TG. Evaluation on larvicidal effects of essential oils of some local plants against Anopheles arabiensis Patton and Aedes aegypti Linnaeus (Diptera, Culicidae) in Ethiopia. Afr J Biotechnol 2009; 8: Mathew J and Thoppil JE. Chemical composition and mosquito larvicidal activities of Salvia essential oils. Pharm Biol 2011; 49: Patole and Mahajan. (a). Efficacy of Some Plant Extracts against Mosquito Larvae. Pesticide Research Journal 2009; 21, Patole and Mahajan. (b) Efficacy of Some Plant Extracts against Mosquito Larvae. Pesticide Research Journal 2009; 21, Pitasawata B, Champakaewa D, Choochotea W, Jitpakdia A, Chaithonga U, Kanjanapothib D et al. Aromatic plant-derived essential oil: An alternative larvicide for mosquito control. Fitoterapia 2007; 78: Prabakar K and Jebanesan A. Larvicidal efficacy of some Cucurbitacious plant leaf extracts against Culex quinquefasciatus (Say). Bioresource Technology 2004; 95, Prabhu K, Murugan K, Nareshkumar A, Ramasubramanian N, Bragadeeswaran S. Larvicidal and repellent potential of Moringa oleifera against malarial vector, Anopheles stephensi Liston (Insecta: Diptera: Culicidae). Asian Pac J Trop Biomed 2011; 2: Prajapati V, Tripathi AK, Aggarwal KK, Khanuja SPS. Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Bioresource Technol 2005; 96: Rahuman AA, Gopalakrishnan G, Ghouse BS, Arumugam S, Himalayan B. Effect of Feronia limoniaon mosquito larvae. Fitoterapia 2000; 7, Senthilnathan S. the use of Eucalyptus tereticorinis SM. (Myrtaceae) oil (leaf extract) as a natural larvicidal agent against the malaria vector Anopheles stephensi Liston (Diptera Culicidae), Biosource Tech 2007; 98, Shyamapada Mandal.. Effect of Azadirachta indica A. Juss (Meliaceae) Seed Oil and Extract against Culex quinquefasciatus Say (Diptera: Culicidae) Larval Susceptibility of Indian Subcontinent. Maced J Med Sci 2011; 4, Sivagnaname N, Kalyanasundaram M. Laboratory evaluation of methanolic extract of Atlantia monophylla (Family: Rutaceae) against immature stages of mosquitoes and non-target organisms. Mem Inst Oswaldo Cruz 2004; 99: Swathi S, Murugananthan G, Ghosh SK and Pradeep AS. Larvicidal andrepellent activities of Ethanolic Extract of Pongamia pinnata leaves against Mosquitoes. RJPS 2011; 1, Tandon V, Pal P, Roy B, Rao HS and Reddy KS. In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India. Parasitol. Res 1997; 835, Traboulsi AF, Taoubi K, El-Haj S, Bessiere JM, Rammal S. Insecticidal properties of essential plant oils against the mosquito Culex pipiens molestus (Diptera: Culicidae). Pest Manag Sci 2002; 58: Vogel AI. Text Book of Practical Organic Chemistry, the English Language Book Society and Longman, London. 1978; WHO Report of the WHO informal consultation on the evaluation on the testing of insecticides CTD/WHO PES/IC/96. 1, WHO Guideline for laboratory and field testing of mosquito larvicides. Report of the Eighth WHOPES Working Group Meeting WHO/CDS/ WHOPES/ GCD Geneva, Switzerland. 13. Source of support: Nil; Conflict of interest: None declared 5