International Journal of Entomology Research International Journal of Entomology Research ISSN: 2455-4758 Impact Factor: RJIF 5.24 www.entomologyjournals.com Volume 3; Issue 1; January 2018; Page No. 95-100 Mosquitocidal potential of Rhodomyrtus tomentosa leaf extracts against Dengue and Zika virus vector, Aedes aegypti Muthu Kasinathan, Jayapal Subramaniam, C Elanchezhiyan, Samivel Kanthammal, Mayakrishnan Vijay Department of Zoology, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu, India Abstract Dengue is a mosquito-borne flavivirus found in tropical and sub-tropical regions of the world, mostly in urban and semi-urban areas. Aedes aegypti is primary vector and its day-biting mosquitoes spread disease, 40% of the world’s population living in an area at risk for dengue. The present study explored the effects of Rhodomyrtus tomentosa acetone and methanol leaf extract of R. tomentosa on larvicidal and pupicidal toxicity against dengue and zika virus vector, Aedes aegypti. The R. tomentosa dried plant materials (powder), 300 g of the plant materials was extracted with 1 L of organic solvents of acetone and methanol for using a Soxhlet apparatus boiling point range 60–80°C for 8 h at room temperature (27±2°C). The extracts were concentrated at reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4 °C. The yields of the R. tomentosa acetone and methanol crude extracts were 12.86g and 14.11g, respectively. The larvicidal and pupicidal lethal toxicity was observed after 24 h of exposure; No mortality was observed in the control group. The LC50 and LC90 of R. tomentosa leaf acetone extract (RTAE) against the first to fourth instar larvae and pupae A. aegypti were; the LC50 value of first instar was 312.17, 352.65, 407.21, 471.60 and 523.91 ppm and 722.67, 739.19, 848.42, 944.81 and 1083.29 ppm, respectively. The LC50 and LC90 values of R. tomentosa leaf methanolic extract (RTME) against the first to fourth instar larvae and pupae of A. aegypti were; the LC50 value of LC90 values were; 263.82 ppm (I), 299.43 ppm (II), 331.64 ppm (III), 386.16 ppm (IV) and 419.56 ppm (pupae), respectively. The LC 90 value of 1st instar was 609.83 ppm, 2nd instar was 677.22 ppm, 3rd instar was 746.13 ppm, 4th instar was 858.83 ppm, and pupa was 908.72 ppm (pupae), respectively. Overall, highlighted the larvicidal and pupicidal evaluation of acetone and methanolic leaf extract of Rhodomyrtus tomentosa against Dengue and Zika virus vector, Aedes aegypti. This is a new eco-friendly approach for the control of dengue and zika virus vector, Ae. aegypti mosquito as target species. The natural products of biopesticides are eco- friendly for the dengue vector control management programs. Keywords: larvicidal, pupicidal, eco-friendly, biopesticides and vector borne diseases Introduction daily life and is presently at the forefront among mosquito- Dengue is a mosquito-borne flavivirus found in tropical and controlling agents. However, the environmental threat that sub-tropical regions of the world, mostly in urban and semi- these chemicals pose, effects on non-target organisms, and the urban regions. Dengue virus is transmitted by female resistance of mosquitoes to insecticides have all increased mosquitoes mainly of the species Aedes aegypti and transmits during the last five decades [2, 5]. Biopesticides provide an chikungunya, yellow fever and Zika infection [1]. It is the alternative to synthetic pesticides because of their generally fastest spreading vector-borne viral disease and is now low environmental pollution, low toxicity to humans, and endemic in over 100 countries, resulting in 40% of the world’s other advantages [6]. Many herbal products have been used as population living in an area at risk for dengue. Dengue natural insecticides before the discovery of synthetic organic fever/dengue hemorrhagic fever (DHF) continues to be of insecticides [7]. major public health importance in countries of the Western Rhodomyrtus tomentosa (Aiton) Hassk. (Family Myrtaceae) is Pacific and Southeast Asia. It is caused by one of four distinct an ornamental, evergreen shrub grows up to four meters tall. serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) [1]. This plant species is native to southern and south-eastern Asia The chemical based insecticides are play a very important role [8]. It has been often used in traditional medicine to treat colic in controlling mosquito vector in their breeding sites, but this diarrhoea [9], dysentery, abscesses, haemorrhage, and kind of control methods showed a negative impact in areas of gynecopathy10. In addition, it is used to formulate skin- beneficial and nontarget organisms and still mosquito vectors whitening, antiaging, and skinbeautifying agents11. The extract resistance to synthetic insecticides. In view of the recently from this plant possess strong inhibitory activity against gram- increased interest in developing plant origin insecticides as an positive bacteria [12, 13], antimicrobial properties [14, 15, 16, 17]. The alternative to synthetic insecticides [2, 3]. The environmental R. tomentosa plant part has been employed in traditional safety and low cost insecticide is considered to be of medicine to treat colic diarrhoea, haemorrhage, dysentery, paramount importance and should not cause mortality on non- abscesses, and gynaecopathy [18, 19, 20, 21]. No data regarding its target organism in order to be acceptable [4]. mosquitocidal properties are available. Hence, the present Chemical control is an effective strategy used extensively in investigation was aimed to explore the mosquitocidal potential 95 International Journal of Entomology Research of the extracts (Ethanol and Methanol) from Rhodomyrtus rhodomyrtus tomentosa leaves against dengue and zika virus vector, Aedes Species - tomentosa (Aiton) Hasaaic rose aegypti under the laboratory condition. myrtus Material and Methods Preparation of plant extracts Collection of eggs Maintenance of larvae Aedes aegypti The Rhodomyrtus tomentosa plant was collected in and The eggs of Aedes aegypti were collected from Vector Control around Mettuppalayam, Coimbatore, India. R. tomentosa plant Research Centre (VCRC-ICMR, Pondicherry), India without was washed with tap water and shade dried at room expose to any insecticide and in and around Coimbatore temperature (27±2°C). An electrical blender powdered the district, India at different breeding habitats with the help of dried plant materials (leaves). From the powder, 300 g of the ‘O’ type brush. These eggs were brought to the laboratory and plant materials was extracted with 1 L of organic solvents of were transferred to 18 X 13 X 4 cm size enamel trays Acetone and Methanol for using a Soxhlet apparatus22 boiling containing 500 ml of water and kept for larval hatching. The point range 60–80°C for 8 h. The extracts were filtered mosquito larval culture was maintained in our laboratory at through a Buchner funnel with Whatman number 1 filter 27±2°C, 75-85% RH, under14L: 10D photoperiod cycles. The paper. The crude plant extracts were evaporated to dryness in mosquito larvae were fed with dog biscuits and yeast at 3:1 rotary vacuum evaporator. One gram of the plant residue was ratio. The feeding was continued till the larvae were dissolved in 100 ml of acetone (stock solution) and considered transformed into pupae. as 1% stock solution. From this stock solution, different concentrations were prepared ranging from 120 to 600 ppm, Maintenance of pupae and adult Aedes aegypti respectively. The pupae were collected from the culture trays and were transferred to plastic containers (12 X 12 cm) containing 500 Larvicidal and pupicidal bioassay (WHO 2005) ml of water. The plastic jars were kept in 90 X 90 X 90 cm The larvicidal bioassay was assessed by using slandered WHO sized mosquito cage for adult emergence. The freshly emerged Protocols23 for experimental treatment, one ml of desired adults were maintained 27±2°C, 75-85% RH, under 14L: 10D concentration obtained from stock solution of different photoperiod cycles. The adults were fed with 10% sugar products of Rhodomyrtus tomentosa extracts. Laboratory solution for a period of three days before they were provided colonies of mosquito larvae/pupae were used for the an animal for blood feeding. larvicidal/pupicidal activity. Twenty-five numbers of I to IV instar larvae and puape were introduce into 500 ml glass Blood feeding of adult Mosquitoes beaker containing 249 ml of de-chlorinated water and 1ml of The female mosquitoes were allowed to feed on the blood of a desired concentrations of biopesticide. Larval food was given rabbit (exposed on the dorsal side) for two days. The males for the test larvae. At each tested concentration 2 to 5 trials were provided with 10% glucose solution on cotton wicks. were made and each trial consisted of three replicates. The cotton was always kept moist with the solution and Experiments were conducted at 27±1°C, 85% RH with changed every day. An egg trap (cup) lined with filter paper photoperiod of 12L:12D. Symptoms of the treated larvae were containing water was placed at a corner of the cage egg observed and recorded immediately and at timed intervals and collection. no foods offer to the larvae, mortality and survival was registered after 24 hours of the exposure period. dead larvae Collection of plant materials were identified when they failed to move after probing with a The tribal medicinal plant, Rhodomyrtus tomentosa were needle in the siphon or cervical region, moribund larvae were collected from Nilgiri hills, Western Ghats, Tamil Nadu, those incapable of rishing to the surface (within a reasonable India. The plants were identified (Taxonomy) at BSI period of time) or showing the characteristic diving reaction (Botanical Survey of India, Coimbatore, Tamil Nadu, India.) when the water was disturbed. The larvae showed and the specimens were deposited at Zoology Department, discoloration, unnatural positions, tremors, unco-ordianation Annamalai University, Annamalai Nagar, Chidambaram, or rigor were also counted.