jear2012 Abstract Ocimum sanctum was tested for its larvicidal and water sedimenta- tion properties; the fruit ethanol and methanol extracts of Phyllanthus emblica were tested for phytochemical, larvicidal, oviposition-deter- rent and ovicidal activities. Results emphasized that plant extracts have high toxicity against the egg and larvae of the malarial vector Anopheles stephensi and also have water sedimentation properties. LC50 of Phyllanthus emblica against Anopheles stephensi larvae ranged from 33.08 ppm to 81.26 ppm and from 23.44 to 54.19 ppm for ethanol and methanol extracts, respectively. Phyllanthus emblica also showed excellent ovipositional deterrent and ovicidal activities. The oviposi- tion activity index value of ethanol and methanol extracts of Phyllanthus emblica at 500 ppm were -0.80 and -0.92, respectively. Ocimum sanctum includes both insecticidal secondary compounds, amino acids (glycine, lysine), vitamin C and other substances, that make treated water suitable for human consumption. Water quality parameters such as color, turbidity and pH were analyzed in the water samples (pre-treatment and post-treatment of plant extracts) taken from the breeding sites of mosquitoes. Hence, the plant product can be used as both mosquitocidal and water purifier. Introduction Mosquitoes are the most single group of insects in terms of public health significance and transmitting dreaded diseases like malaria, filariasis and dengue, etc. There are approximately 460 recognized species. While over 100 can transmit human malaria, only 30-40 com- monly transmit parasites of the genus Plasmodium that causes malar- ia which affects humans in endemic areas. The known vectors of Anopheles species, which are common in India, include A. stephensi, A. culicifacies, A. fluviattis, A. minimmus, A. sudanicus and A. philip- pinensis. Anopheles stephensi Liston, 1901 (Diptera: Culicidae) is the most common human-biting malaria vector in India and many West Asian countries, and is likely responsible for 40-50% of the annual malarial incidence (Curtis, 1994). The problems of high cost, environmental risks and development of resistance in many vector mosquito species to several synthetic insec- ticides have revived interest in exploiting the pest control potential of plants (Grainage & Ahamed, 1988). Conventional water treatment relies on the addition of chemicals such as alum (aluminum sulfate) as coagulants and the addition of chlorine as a bactericide. The avail- ability of these chemicals, which depends on foreign exchange, is unreliable and unpredictable. Because of economic and political con- straints, the universal provision of piped water is not currently feasi- ble, leaving millions of people without access to safe drinking water (WHO, 2005). This led us to look for plants with water purification properties. Ocimum sanctum (holi basil), also called Tulsi in India, is ubiqui- tous in Indian tradition. Tulsi is described as a sacred medicinal plant in ancient literature (Kirtikar & Basu, 1975). It has been used to treat malarial fevers, ringworms, and other cutaneous afflictions (Butani, 1982). A variety of biologically active compounds have been isolated from the leaves, including ursolic acid, apigenin and luteolin. Some other main chemical constituents of Tulsi are Oleanolic acid, Rosmarinic acid, Eugenol, Carvacrol, Linalool, and β-caryophyllene (Merrily & Winston, 2007). This paper, therefore, describes the mos- quitocidal and water sedimentation properties of Ocimum sanctum against malarial vector, Anopheles stephensi. The plant genus Phyllanthus L. (Euphorbiaceae) is widely distrib- uted in most tropical and subtropical countries. It is a very large genus consisting of approximately 550 to 750 species and is subdivided into ten or eleven subgenera. Phyllanthus emblica L. has been used for the Journal of Entomological and Acarological Research 2012; volume 44:e17 [page 90] [Journal of Entomological and Acarological Research 2012; 44:e17] Mosquitocidal and water purification properties of Ocimum sanctum and Phyllanthus emblica K. Murugan,1 P. Madhiyazhagan,1 A. Nareshkumar,1 T. Nataraj,1 D. Dinesh,1 J.S. Hwang,2 M. Nicoletti3 1Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, India; 2Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan; 3Department of Environmental Biology, Università “La Sapienza”, Rome, Italy Correspondence: Kadarkarai Murugan, Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, India. E-mail: kmvvkg@gmail.com Key words: Anopheles stephensi, Ocimum sanctum, Phyllanthus emblica, ovipositional deterrent, ovicidal, mosquitocidal, water purification. Acknowledgements: the authors thank the Council of Scientific and Industrial Research CSIR (Sanction letter No. 37(1386) EMRII dated 02-06- 2010), Government of India, New Delhi, for providing financial assistance. Received for publication: 20 August 2012. Revision received: 28 November 2012. Accepted for publication: 28 November 2012. ©Copyright K. Murugan et al., 2012 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2012; 44:e17 doi:10.4081/jear.2012.e17 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. No n- co mm er cia l u se on ly anti-inflammatory and anti-pyretic treatments by the rural population and for the treatment of several disorders such as the Scurvy, Cancer and Heart diseases. The important constituent of plant leaves has anti- neutrophilic activity and anti-platelet properties in vitro. The extracts also have several pharmacological properties, such as anti-viral (HIV, AIDS, herpes virus, cytomegalovirus) antimutagenic, antiallergic, anti- bacterial activities (Khopde et al., 2000). Phyllanthus emblica L. con- tains a different class of secondary metabolites (Calixto et al., 1998). The present study aimed to evaluate the effect of mosquitocidal and water sedimentation properties of Ocimum sanctum on the malaria vector Anopheles stephensi and screen phytochemicals from Phyllanthus emblica L. for larvicidal, pupicidal, oviposition deterrent and ovicidal effect against the same malaria vector. Materials and methods Colonization, mosquito rearing and maintenance The eggs of Anopheles stephensi Liston, 1911 were collected from var- ious water sources (e.g., overhead tanks) in Coimbatore district, Tamil Nadu, India. The eggs were brought to the laboratory and were trans- ferred to 18×13×4 cm size enamel trays containing 500 mL of water until they hatched. After hatching, the larvae were provided with dog biscuits and yeast at a 3:1 ratio and maintained at 27+2°C, 75-85% RH, under 14 h light (L): 10 h dark (D) photoperiod cycles. Pupae were col- lected and transferred to plastic jars (12×12 cm) containing 500 mL of water, which were placed in 90×90×90 cm screened mosquito cage for adult emergence. The freshly emerged adults were maintained at 27±2°C, 75-85% RH, under 14 h L: 10 h D photoperiod cycles. The adults were provided a 20% sugar solution ad libitum, and provided with rab- bits (each exposed on the dorsal side) for 30 min two times per week (Hardstone et al., 2009). The males were provided with a 10% glucose solution on cotton wicks that was changed daily. Blood-fed females were provided filter paper-lined cups containing water for oviposition. Collection of plant material and preparation of extracts The leaves of Ocimum sanctum and Phyllanthus emblica fruits were collected from in and around Bharathiar University Campus, Coimbatore, Tamil Nadu, India. A total of 250 g of fresh, mature leaves was rinsed with distilled water and dried in a shade. The dried leaves were put in a Soxhlet apparatus (Borosil Glass Workers Ltd., Worli, Mumbai, India), and extracts were prepared using 100% ethanol (Loba Chemie Pvt. Ltd., Mumbai, India; 99 % purity) for 72 h at 30-40°C. Dried residues were obtained from 100 g of extract evaporated to dryness in a rotary vacuum evaporator. Two grams of the residues were dissolved in 100 mL of ethanol (2% stock solution), from which the following con- centrations were prepared: 0.5%, 0.1%, 2.0%, 4.0%, and 8.0%, using dis- tilled water. Amla fruits were cut into small pieces and ground into a uniform powder using a blender. The methanolic extract of amla was prepared by soaking 100 g of dried powdered samples in 250 mL of methanol for 12 h. The extracts were filtered by using Whatman n. 1 filter paper. The filtrate was used for phytochemical screening. The preliminary qualita- tive phytochemical studies were performed for testing the different chemical groups present in fruit methanol extract of Phyllanthus emblica (Trease et al., 1978; Kokate et al., 1990). Laboratory plant extract toxicity tests F1-F2 larvae/pupae from the wild adult collection were used to eval- uate larvicidal activity. Twenty-five larvae (in stages I to IV and pupae) were placed into a 500-mL glass beaker containing 249 mL of dechlori- nated water and 1 mL of desired concentrations of plant extract. Test larvae and pupae were provided with food as previously described. At each tested concentration, 2-5 trials of 3 replicates were conducted con- currently. Control groups of larvae exposed to ethanol served as control. Mortality was corrected using Abbott’s formula (Abbott, 1925). Ovicidal activity assay For ovicidal activity, the freshly laid eggs were collected by providing ovitraps in mosquito cages. Ovitraps were kept in the cages two days after the female mosquitoes had been given a blood meal. The eggs were laid on the filter paper lining provided in the ovitrap. After scoring, 100 gravids were placed in a screen cage where ten oviposition cups were introduced for oviposition 30 min before the start of the dusk period. Of these ten cups, eight were each filled with test solution of 150, 200, 250, 300, 350, 400, 450, 500 ppm, and one was filled with 100 mL of respective solvent containing water and Polysorbate 80 that served as a control. A minimum of 100 eggs were used for each treatment, and the experiment was replicated 5 times. After treatment, the eggs were sieved through muslin cloth, thoroughly rinsed with tap water, and left in plastic cubs filled with dechlorinated water for hatching assessment after counting the eggs under the microscope (Su & Mulla, 1998). The percent egg mor- tality was calculated on the basis of non-hatchability of eggs with unopened opercula (Chenniappan & Kadarkarai, 2008). The hatching rate of eggs was assessed after 98 h post-treatment according to the method of Rajkumar and Jebanesan (2009). Oviposition deterrence activity To study the ovipositional deterrence effect and the number of eggs deposited in the presence of extracts with different solvents of experi- mental plants, a multiple concentration test was carried out. For bioas- say test, 20 males and 20 females were separated in the pupal stage (by size of the pupae) and were introduced into screen cages (45×45×40 cm) in a room at 27±2°C and 75-85% relative humidity (RH) with a photoperiod of 14:10 h light and dark cycles. The pupae were allowed to emerge to adults in the test cages. Adults were provided continuously with 10% sucrose solution in a plastic cup with a cotton wick. They were blood fed (from pigeon) on Day 5 after emergence. In the multi- ple concentration test, five cups, each containing 100 mL distilled water with a 9-cm piece of white filter paper for oviposition, as well as solvent extracts at concentrations of 100, 200, 300, 400 and 500 ppm, were placed in each cage. A sixth cup without extract served as a control. The positions of the plastic cups were alternated between the different replicates so as to nullify any effect of position on oviposition. Five replicates for each concentration were run with cages placed side by side for each bioassay. After 24 h, the number of eggs laid in the treat- ed and control cups were counted under a stereomicroscope. The per- cent effective repellency for each concentration was calculated using the following formula: where ER is effective repellency, NC is number of eggs in control, and NT is number of eggs in treatment (Rajkumar & Jebanesan, 2009). The oviposition experiments were expressed as mean number of eggs and oviposition activity index, which was calculated using the follow- ing formula: Article [Journal of Entomological and Acarological Research 2012; 44:e17] [page 91] No n- co mm er cia l u se on ly where NT is total number of eggs in the test solution and NS is total number of eggs in the control solution. Oviposition active index of +0.3 and above are considered as attractants while those with −0.3 and below are considered as repellents (Kramer & Mulla, 1979). Positive values indicate that more eggs were deposited in the test cups than in the control cups and that the test solutions were attractive. Conversely, negative values indicate that more eggs were deposited in the control cups than in the test cups and that the test solutions were a deterrent. Field trials of plant extract larval toxicity Plant extracts formulations (51 g*l-1) were applied to the water sur- face with a knapsack sprayer (Ignition Products Ltd., India, 2008). Pre- treatment and post-treatment at 24, 48 and 72 h was conducted using a larval dipper. Larvicidal efficacy was carried out against late third and early fourth-instar larvae. Larvae were identified and counted to deter- mine the relative species composition of each test site. Six trials were conducted for each test site (standing water bodies) with similar weather conditions (27°C; 79% RH). The required quantity of plant extract was determined by calculating the total surface area and vol- ume (0.25 m2 and 250 L). The required concentration was prepared using 10 times the observed laboratory LC50 values (Murugan et al., 2003). Percentage reduction of the larval density was calculated using the formula: where C is the total number of mosquitoes in control and T is the total number of mosquitoes in treatment. Water quality parameters Water quality parameters such as pH, color and turbidity were inves- tigated using the methods of Clescerl et al. (2005). To prepare the coag- ulants and treatment (Schwarz, 2000), leaves of Ocimum sanctum were shade-dried, powdered using an electric blender, and mixed with a small amount of clean water to form a paste. The paste was diluted to the required strength based on raw water turbidity. Total suspended solids in raw water separated as over 50, between 50 and 150, and over 150 mg*l-1, and the final concentration used for treatment was 50, 30- 100 and over 150 mg*l- 1, respectively (Schwarz, 2000). After filtering insoluble material with a fine-mesh screen or muslin cloth, the coagu- lant was added and stirred fast for 30 s. The treated water was then cov- ered for 1 h without disturbance. Statistical analysis The SPSS (Version 9.0) software package was used to analyze data obtained from the bioassay. Lethal concentrations (LC), LC50 and LC90, Duncan Multiple Range Test) and c2 tests were used. Results Ocimum sanctum leaf extract was used to test water purification properties including water color, total suspended solids and pH, and was effective in sedimentation and purification. Before treatment, water color was 31 HU while after this was 12 HU. Total suspended solids before treatment was 40.0 mg*l-1 and this was reduced 30.0 mg*l-1, after. Similarly, pH level was 8 before treatment and 6.8 after. Significant mortality was evident after the treatment of Ocimum sanctum leaf extract (OSLE) at different concentrations against A. stephensi in laboratory (Table 1). After the treatment of OSLE at differ- ent concentration levels (0.5-8%), 38% mortality was noted at I instar larvae by the treatment of OSLE at 0.5%, whereas this increased to 90% at 8% of OSLE treatment. Mortality increased with increasing concen- tration. Lethal concentrations (LC50 and LC90) were also calculated. The LC50 and LC90 values are represented as follows: LC50 value of I instar was 1.52%, II instar was 2.22%, III instar was 3.11%, IV instar was 5.13% and pupae was 6.45%. LC90 value of I instar was 7.39%, II instar was 8.68%, III instar was 10.07%, IV instar was 14.11% and pupae was 15.24%, respectively. An. stephensi larvae were collected exclusively in overhead water tanks and mean larval count was calculated. Breeding sites treated with O. sanctum extracts, showed a reduction in An. stephensi larvae. The field experiment was carried out at drinking water tanks (0.5×0.5×1.0) with Anopheles stephensi larvae and the percentage reduction/mortali- ty was 82.5%, 87.9% and 92.4% after the 24 h, 48 h and 72 h, respective- ly (Table 2). Table 3 shows qualitative analyses of the fruit extract of Phyllanthus emblica, emphasizing the presence of proteins, tannins and ter- penoids. Steroids were absent in Phyllanthus emblica. The larvicidal and pupicidal activities of the ethanol extract of Phyllanthus emblica against Anopheles stephensi larvae under laborato- Table 1. Larvicidal activity of Ocimum sanctum against malaria vector, Anopheles stephensi. Larval Larval mortality (%)±SD LC50 95% Confidence limit Chi-square instars Concentration of Ocimum sanctum (%) (LC90) LCL UCL value and Pupa 0.5 1.0 2.0 4.0 8.0 LC50 (LC90) LC50 (LC90) I 38±0.6a 43±0.3ab 58±1.2b 75±0.5c 90±0.8d 1.52067 0.85569 6.34024 (7.38616) (2.07067) (8.99671) 2.830* II 32±0.9a 41±0.5b 50±1.0c 69±0.7d 85±1.1e 2.21933 1.57467 7.42155 (8.68104) (2.80353) (10.65323) 2.769* III 26±1.0a 35±1.2b 46±1.4c 61±0.8d 79±1.5e 3.10581 2.47306 8.56234 (10.06631) (3.76193) 12.46361 3.384* IV 19±1.5a 25±0.8ab 39±0.6b 52±1.2c 61±0.4d 5.12732 2.94904 9.02274 (14.11467) (13.82370) (56.49114) 8.278* Pupa 10±0.5a 21±1.0b 32±1.2c 46±0.8d 53±0.6e 6.44729 3.67191 9.00805 (15.23744) 570.79738 (2199.74679) 13.287* Within a column means followed by the same letter(s) are not significantly different at 5% level by Duncan Multiple Range Test; *Significant at P<0.001 (heterogeneity factor used in calculation of confidence limits). SD, standard deviation; LC50, LC90, lethal concentration; LCL, lower confidence limits; UCL, upper confidence limits. Article [page 92] [Journal of Entomological and Acarological Research 2012; 44:e17] No n- co mm er cia l u se on ly ry conditions are given in Table 4. Percentage mortality was 43% at 20 ppm concentration and increased to 82% at 100 ppm concentration against the first instar larvae. Median lethal concentrations (33.08, 48.85, 68.28, 81.26 and 86.24 ppm) for larvae and pupae were low and significant. Table 5 shows larvicidal and pupicidal activities of methanol extract of Phyllanthus emblica against the malarial vector, Anopheles stephen- si. Among the four larval stages, I Instar larvae were more susceptible than the other instars. The fruit extracts also showed considerable pupal mortality. The lowest mortality observed was 20% at 20 ppm against pupae and the highest was 98% at 100 ppm against the I instar larvae. LC50 (23.44, 33.10, 42.13, 54.19 and 64.28 ppm for four instar lar- vae and pupae, respectively) observed for larvae and pupae were very low when compared to the ethanol extract. In the oviposition deterrent assay, gravid Anopheles stephensi pre- ferred to lay eggs in the distilled water control cups than in the cups treated with solvent extracts of Phyllanthus emblica (Table 6). There was also a marked difference in the number of eggs laid. Observed results showed that the 500 ppm treated cups received a mean number of 49±1.15 and 19±1.53 eggs per cup in fruit ethanol and methanol extracts of Phyllanthus emblica treatment while the control cups received a mean number of 456±1.50 and 470±1.30 eggs per cup. The present results indicated that the oviposition deterrence was concen- tration dependent, as 500 ppm of ethanol and methanol fruit extracts of experimental plant exhibits strong deterrent effect when compared with 100 ppm against oviposition. The solvent leaf extracts strongly deterred oviposition by gravid Anopheles stephensi, with a significantly lower proportion of eggs being laid on ovitraps containing extracts in comparison with control solutions (P<0.05). The maximum percentage of effective repellency against oviposition was 96.93%, reported in 500 ppm followed by 95.95, 94.13, 87.01, 78.18 and 67.50% at 500, 400, 300, 200 and 100 ppm methanol extracts of Phyllanthus emblica, respective- ly. The percentages of egg hatchability of Anopheles stephensi with the fruit ethanol and methanol extracts of Phyllanthus emblica are pre- sented in Table 7. The ethanol and methanol extracts of Phyllanthus emblica exerted 100% mortality (no hatchability) at 400 ppm and above. Very low hatchability (19±1.20% and 0%) was observed at a 350 ppm concentration of ethanol and methanol extracts of Phyllanthus emblica, respectively against Anopheles stephensi. Almost 100% hatch- ability was obtained in the control. In the case of ovicidal activity, expo- sure to freshly laid eggs was more effective than to the older eggs. Discussion and conclusions Many approaches have been developed to control the mosquito men- ace. One such approach to prevent mosquito-borne disease is by killing mosquito at the larval stage. The current mosquito control approach is based on synthetic insecticides. Even though they are effective, they created many problems, such as insecticide resistance (Liu et al., 2005), pollution, and toxic side effects on humans (Lixin, 2006). In the present study, OSLE showed higher larvicidal activities against mosqui- to probably due to the presence of active compounds such as eugenol and (E)-6-hydroxy-4,6-dimethyl-3-heptene-2-one (Kelm & Nair, 1998). The mosquito larvicidal property of leaf and flower extracts of Ocimum sanctum L. against Aedes aegypti and Culex quinquefasciatus larvae has been previously reported (Anees, 2008). Mosquito breeding habitats vary from ponds, marshes, ditches, pools, drains, water containers and other similar water collections, and are often species-specific (Rozendaal, 1997). The increase in the mosquito vector population and the incidence of mosquito-borne diseases (e.g., malaria, dengue, and Chikungunya) is rising in India as a result of inadequate water supply systems and contamination. Storage of water, often from untreated water sources, and polluted water systems serve breeding sites of mos- quitoes that transmit mosquito-borne pathogens, in addition to water- borne pathogens (e.g., cholera, dysentery and typhoid) (Vinod, 2011). In the present study, the treatment with coagulants at the breeding sites of mosquito had not only killed mosquito larvae but it had also water purifying properties. Biopesticide spray operations had been per- formed in the past (Murugan, 2006) for the control of vectors in the tsunami affected areas of India and the plant products such as neem and other herbal combinations showed biopesticidal potency killing mosquito larvae in the contaminated water. Larvicidal effect of neem (Azadirachta indica) oil cake was studied against mosquitoes. The oil cake showed good larvicidal activity against the mosquito species test- ed (Shanmugasundaram et al., 2008). Neem is derived from the neem tree Azadirachta indica A. Juss. (Meliaceae), and its primary insectici- dal components are the tetranortriterpenoid, azadirachtin and other limonoids. The effect of neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnim- bin on insects was investigated by Senthil Nathan et al. (2006). Coagulants of Ocimum sanctum were tested for the purifying prop- erties at the laboratory by adding coagulants with waters from mosqui- to breeding sites and coagulants also had water purifying properties; treated water showed cleaning efficacy. Plant product nutrients (vita- min A and C, calcium, iron and zinc) and allelochemicals (Tannins, alkaloids, glycosides and saponins) not only removed solid contami- Table 2. Effect of Ocimum sanctum treatments of drinkng water tanks malarial vector, Anopheles stephensi. Site no. Larval density (%) Before treatment After treatment 24 h 48 h 72 h 1 79 19 15 11 2 71 14 12 8 3 65 10 8 5 4 57 7 6 3 5 47 7 2 0 6 39 4 0 0 Total 358 61 43 27 Average 59.6 10.1 7.16 4.50 % Reduction - 82.50 87.9 92.4 -Place: Vadavalli; -Habitat: drinking water; -Size: 0.5×0.5×1.0; -Depth: 1 cm; -Species: Anopheles stephensi; -Stage: larvae stage; -Calculation: 2.5×1.5 m; 3.10×1=31.00. Table 3. Phytochemical constituents present in Phyllanthus emblica. Phyto-constituents Ethanol extract Methanol extract Flavonoidds + + Tannins + + Carbohydrates + + Alkaloids + + Proteins + + Steroids – – Terpenoids + + Article [Journal of Entomological and Acarological Research 2012; 44:e17] [page 93] No n- co mm er cia l u se on ly Table 4. Toxicity evaluation of ethanol extract of Phyllanthus emblica against the malarial vector Anopheles stephensi. Larval % of larval mortality LC50 Regression 95% Confidence Chi-square stages Concentration (ppm) (LC90) equation limit value 20 40 60 80 100 LCL UCL LC50 (LC90) LC50 (LC90) I 43±1.32a 52±1.80b 68±2.59c 71±0.5cd 82±1.80d 33.08 Y=-0.44449 18.26 42.76 1.125 (128.48) +0.01343X (110.01) (162.10) II 36±0.70a 43±1.22b 59±1.14c 62±1.41cd 78±0.79d 48.85 Y=-0.66503 38.55 57.18 1.793 (142.98) +0.01361X (122.19) (180.48) III 20±0.65a 37±0.79b 48±1.58c 52±1.51cd 70±0.79d 68.28 Y=-1.04788 60.85 76.82 2.670 (151.79) +0.01535X (131.41) (186.42) IV 16±0.35a 23±0.79b 33±1.11c 44±1.81d 68±1.06e 81.26 Y=-1.44506 74.16 90.58 2.728 (153.33) +0.01778X (134.81) (183.07) Pupa 12±1.59a 21±0.65b 31±1.19c 40±1.98d 64±1.06e 86.24 Y=-1.57407 78.85 96.30 2.032 (156.45) +0.01825X (137.64) (186.67) Within a column means followed by the same letter(s) are not significantly different at 5% level by Duncan Multiple Range Test. LC50, LC90, lethal concentration; LCL, lower confidence limits; UCL, upper confidence limits. Table 5. Toxicity evaluation of methanol extract of Phyllanthus emblica against the malarial vector Anopheles stephensi. Larval % of larval mortality LC50 Regression 95% Confidence Chi-square stages Concentration (ppm) (LC90) equation limit value 20 40 60 80 100 LCL UCL LC50 (LC90) LC50 (LC90) I 49±0.70a 65±0.79b 75±0.35c 87±0.79d 98±1.27e 23.44 Y=-0.51187 12.98 30.87 3.173 (82.15) +0.02183X (74.12) (93.77) II 40±0.54a 58±0.79b 69±1.06c 82±1.08d 97±1.29e 33.10 Y=-0.74820 25.11 39.27 4.043 (89.81) +0.02260X (81.53) (101.65) III 38±0.35a 44±0.70ab 61±1.22b 76±1.41c 90±1.76d 42.13 Y=-0.82596 34.42 48.39 2.881 (107.52) +0.01960X (96.59) (123.90) IV 29±0.74a 37±0.65b 53±1.51c 62±1.29d 88±1.38e 54.19 Y=-1.06730 36.01 69.28 6.398 (119.26) +0.01969X (95.48) (187.81) Pupa 20±0.35a 29±0.70b 48±0.93c 52±1.47cd 85±1.88d 64.28 Y=-1.36664 46.70 85.52 9.100 (124.56) +0.02126X (97.90) (216.27) Within a column means followed by the same letter(s) are not significantly different at 5% level by Duncan Multiple Range Test. LC50, LC90, lethal concentration; LCL, lower confidence limits; UCL, upper confidence limits. Table 6. Oviposition deterrence activity of ethanol and methanol extracts of Phyllanthus emblica against the malarial vector Anopheles stephensi. Concentration Ethanol Methanol (ppm) Number of eggs±S.E. Number of eggs±S.E. Treatment Control ER% OAI Treatment Control ER% OAI 500 49±1.15a 456±1.50 89.25 -0.80 19±1.53a 470±1.30 95.95 -0.92 400 54±1.41b 389±1.71 86.11 -0.75 23±1.22ab 392±1.84 94.13 -0.88 300 62±1.72c 321±1.20 80.68 -0.67 37±1.84b 285±1.61 87.01 -0.77 200 86±1.01d 266±1.41 67.66 -0.51 48±1.73c 220±1.42 78.18 -0.64 100 98±1.52e 210±1.47 53.33 -0.36 52±1.32cd 160±1.32 67.50 -0.50 Within a column means followed by the same letter(s) are not significantly different at 5% level by Duncan Multiple Range Test. S.E., standard error; ER, effective repellency; OAI, oviposition active index. Table 7. Ovicidal activity of ethanol and methanol extracts of Phyllanthus emblica against eggs of Anopheles stephensi. Treatment Extract Percentage of egg hatchability±S.D. Concentration of extract (ppm) 150 200 250 300 350 400 450 500 Control Phyllanthus emblica Ethanol 96±1.24e 71±2.43d 54±1.40c 30±2.49b 19±1.20a NH NH NH 100±0.00 Methanol 74±2.17d 47±1.71c 22±2.10b 14±1.03a NH NH NH NH 98±2.95 Within a column means followed by the same letter(s) are not significantly different at 5% level by Duncan Multiple Range Test. S.D., standard deviation; NH, no hatchability (100% mortality). Article [page 94] [Journal of Entomological and Acarological Research 2012; 44:e17] No n- co mm er cia l u se on ly nants, but also greatly reduced amounts of harmful bacteria in the waste water. Earlier studies examined the antimicrobial property pres- ent in the Ocimum sanctum, and concluded that the component respon- sible was likely to be eugenol. This component has been demonstrated to have both antibacterial (Nakaruma et al., 1999) and antihelmintic activities (Pessoa et al., 2002). Nareshkumar et al. (2011) reported mosquitocidal and water purifying properties of different plant extracts (Cynodon dactylon, Aloe vera, Hemidesmus indicus and Coleus amboinicus) on various mosquito vectors (Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti) at different water samples. In the present study, we also sought to determine whether methanol and ethanol extracts from Phyllanthus emblica fruits could be used for mosquito control. We observed a functional response by all immature life stages of A. stephensi to ethanolic and methanolic extracts of Phyllanthus emblica fruits. This biological activity is attributed to the compounds present in fruits, including flavonoids, phenols, and steroids that togeth- er or independently result in morbidity and mortality in A. stephensi. Park et al. (2000) showed that the biological activity of the plant extracts might be due to the various compounds, including phenolics, terpenoids and alkaloids existing in plants. These compounds may jointly or inde- pendently contribute to produce larvicidal activity against mosquitoes. A piperidine alkaloid from Piper longum fruit was found to be active against mosquito larvae of Cx. pipiens (Lee, 2000). It was recognized that the fourth larval stage of mosquitoes was more tolerant to toxicant then early instars (Mulla, 1961; Rettich, 1976; Nareshkumar et al., 2012). Larval mortality may be due to the effect of chemicals like flavonoids, alkaloids, and terpenoids. The higher mortal- ity of mosquito larvae was due to the combined action of plant com- pounds that might be acting on the midgut epithelium cells exerting their toxic effects on mosquito. Lethal LC50 observed in the present study is very low when compared to the earlier studies. The mangrove plant Rhizophora mucronata bark and pith extract showed toxicity with LC50 values of 157.4 and 168.3 ppm, respectively, against Ae. aegypti lar- vae (Kabaru & Gichia, 2001). Exposure of A. stephensi larvae to sub-lethal doses of neem extracts in the laboratory, prolonged larval development, reduced pupal weight, caused high oviposition deterrence and high mortality (Wandscheer et al., 2004). Phyllanthus emblica L. was also effective in oviposition deter- rence and ovicidal activities. Adult female A. stephensi avoided oviposi- tion in Phyllanthus emblica-treated water, though some laid eggs, but these hatched in abnormal larvae. The ethanolic leaf extracts of Cassia obtusifolia at high concentration (400 mg*l-1) were responsible for 92.5% oviposition deterrence effect, while 300, 200 and 100 mg*l-1 were responsible for 87.2%, 83.0%, and 75.5% deterrence effect, respectively (Rajkumar & Jebanesan, 2009). The leaf extract of Solanum trilobatum reduced egg laying by gravid females of Anopheles stephensi from 18% to 99% compared with ethanol-treated controls at 0.01, 0.025, 0.05, 0.075, and 0.1% (Rajkumar & Jebanesan, 2005). Methanol extract of Phyllanthus emblica showed more deterrence and egg mortality than the ethanol extract. The crude acetone extract of Cuscuta hyaline was an effective oviposition deterrent against Culex quinquefasciatus at a concentration of 80 ppm (Mehra & Hiradhar, 2002). It has been shown that the age of the embryos at the time of treatment plays a crucial role with regard to the effectiveness of the chitin synthesis inhibitor dimilin to Culex quinquefasciatus (Miura et al., 1976). The Ovicidal effect of Solenostemma argel was low; however, concentrations of 0.05 % and 0.1 % exhibited significant effects (P<0.05), producing 65-75% and 62.9- 62.9%, respectively, on the first and second day after treatment, the 0.1% concentration reduced egg hatch by 33.7%, compared with control, and 100% mortality values were evident in concentrations as low as 0.025% at two days post-hatching against Culex pipiens (Al-Doghairi et al., 2004). The seed extract of Atriplex canescens showed complete ovicidal at 1000 ppm concentration in eggs of Culex quinquefasciatus. The bioac- tive compound Azadirachtin isolated from Azadirachta indica showed complete Ovicidal activity in eggs of Culex tarsalis and Culex quinque- fasciatus exposed to 10 ppm concentration (Murugan et al., 1996; Ouda et al., 1998). The phytochemical analysis of Phyllanthus emblica L. reveals the presence of alkaloids, tannins and saponins. These compounds are known to be biologically active. Tannins have important roles such as stable and potent antioxidants (Trease et al., 1978). Herbs that have tannins as their main component are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery (Dharmananda, 2003). That of the largest group of chemicals produced by plants are the alkaloids and their amazing effect on humans has led to the development of powerful pain killer medications (Raffauf, 1996). Fruits are an important group of foodstuffs in the diet. These compo- nents of human diet are not adequately replaceable by any other prod- ucts. Plant tissues are naturally rich in nutritive or therapeutically active products of plant secondary metabolism. The consumption of fruits has been inversely associated with morbidity and mortality from degenerative diseases (Aruoma, 1998; Özen, 2010) and is associated with low incidences and mortality rates of cancer and heart disease (Ames et al., 1993; Dragsted et al., 1993). It is not known which dietary constituents are responsible for this association, but antioxidants appear to play a major role in the protective effects of plant foods (Gey 1990; Barberousse et al., 2008; Patra & Kumar, 2010). Fruit contains considerable amounts of active components such as polyphenols, flavonoids, tannins, vitamins A, B, C and E, and carotenoids which are considered potent scavengers of free radicals and reactive oxygen species (Rice-Evans et al., 1995). The present results suggest that Ocimum sanctum can be used for mosquitocidal and water purifying purposes for promoting water sus- tainability in developing countries. Ocimum sanctum coagulum has an added advantage of having antimicrobial properties. Considering the fact that Ocimum sanctum coagulum can be locally produced, its use in water purification should be encouraged. This is likely to reduce the high cost of the current water treatment systems. Experiments on phy- tochemical screening of Phyllanthus emblica for their, larvicidal, pupi- cidal, ovipositiondeterrent and ovicidal activity have opened the possi- bility of further investigations on their efficiency, in view of the utiliza- tion of their higher biomass. However, the mechanism of action of the compounds from Phyllanthus emblica is still not clear. Studies on iso- lation, purification and the mechanism of action of individual com- pounds existing in the Phyllanthus emblica are needed and these are in progress. References ABBOTT W.S., 1925 - A method of computing the effectiveness of insec- ticides. - J. Econ. Entomol. 18: 265-267. AL-DOGHAIRI M.A., AL-REHIAYANI S., OSMAN K.A., ELHAG E.A., 2004 - Residual effects of some conventional and biorational pesticides on ladybird beetle, Adonia variegata goeze. - Pak. J. Biol. Sci. 7: 130- 133. AMES B.M., SHIGENAGA M.K., HAGEN T.M., 1993 - Oxidants, antioxi- dants and the degenerative diseases of aging. - Proc. Natl. Acad. Sci. 90: 7915-7922. ANEES A.M., 2008 - Larvicidal activity of Ocimum sanctum Linn (Labiate) against Aedes aegypti (L.) and Culex quinquefasciatus (Say). - J. Parasitol. Res. 103: 1451-1453. ARUOMA O.I., 1998 - Free radicals, oxidative stress, and antioxidants in human health and disease. - J. Am. Oil Chem. Soc. 75: 199-212. BARBEROUSSE H., ROISEUX O., ROBERT C., PAQUOT M., DEROANNE C., BLECKER C., 2008 - Analytical methodologies for quantification of ferulic acid and its oligomers. - J. Sci. Food Agric. 88: 1494-1511. Article [Journal of Entomological and Acarological Research 2012; 44:e17] [page 95] No n- co mm er cia l u se on ly BUTANI D.K., 1982 - Insect pests of Tulsi (Ocimum sanctum Linnaeus) and their controls. - Pesticides. 16: 11-12. CALIXTO J.B., ANTOS A.R.S., FILHO V.C., TUNES R.A., 1998 - A review of the plants of the genus phyllanthus : their chemistry, pharmacol- ogy, and therapeutic potential. - J. Med. Biol. 31: 225-258. CHENNIAPPAN K., KADARKARAI M., 2008 - Oviposition deterrent, ovi- cidal and gravid mortality effects of ethanolic extract of Andrographis paniculata Nees against the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). - Entomol. Res. 38: 119-125. CLESCERL LEONORE S., GREENBERG ARNOLD E., EATON ANDREW D., 2005. - Standard methods for the examination of water and wastewater (20th ed.). American Public Health Association, Washington, DC. CURTIS C.F., 1994 - The case for malaria control by genetic manipula- tion of its vectors. -Parasitol. Today. 10: 371-374. DHARMANANDA S., 2003 - Gallnuts and the uses of Tannins in Chinese medicine. - In; Proceedings of Institutes for Traditional Medicine, Portland, Oregon. Available from: http://www.itmonline.org/arts /gallnuts.htm DRAGSTED L.O., STRUBE M., LARSEN J.C., 1993 - Cancer-protective factors in fruits and vegetables: biochemical and biological back- ground. - Pharmacol. Toxicol. 72: 116-135. GEY K.F., 1990 - The antioxidant hypothesis of cardiovascular-disease - epidemiology and mechanisms. - Biochem. Soc. Trans. 18: 1041- 1045. GRAINAGE M., AHMED H., 1988 - Handbook of plants and pest control properties. John Wiley and Sons, New York, NY. HARDSTONE C.M., LAZZARO P.B., SCOTT G.J., 2009 - The effect of three environmental conditions on the fitness of cytochrome P450.monooxygenase-mediated permethrin resistance in Culex pipiens. - BMC Evol. Biol. 9: 42. KABARU J.M., GICHIA L., 2001 - Insecticidal activity of extracts derived from different parts of the mangrove tree Rhizophora mucronata (Rhizophoraceae) Lam. against the arthropods. Afr. J. Sci. Technol. 2: 44-49. PATRA K.C., KUMAR K.J., 2010 - Establishing correlation of therapeutic activity of a siddha formulation with its antioxidant activity- a com- parative study. – Int. J. Pharma Bio Sci. 1: 1-8. KELM M.A., NAIR M.G., 1998 - Mosquitocidal compounds and a triglyc- eride, 1, 3-dilinoleneoyl-2-palmitin, from Ocimum sanctum. - J. Agric. Food Chem. 46: 3092-309. KHOPDE S.M., PRIYADARSHINI K.I., GUHA S.N., SATAV J.G., VENKATESAN P., RAO M.N.A., 2000 - Inhibition of radiation- induced lipid peroxidation by tetrahydrocurcumin: Possible mechanism by pulse radiolysis. - Biosci. Biotechnol. Biochem. 64: 503-509. KIRTIKAR K.R., BASU B.D., 1975 - Indian medicinal plant. Vol 3, 2nd ed. - International Book Distributor, Dehra Dun: 2327-2328. KOKATE C.K., PUROHITH A.P., GOKHALE S.B., 1990 – Pharmacognosy. 1st ed. - Nirali Prakashan, Pune: 120. KRAMER W.L., MULLA S., 1979 - Oviposition attractants and repellents of mosquitoes: oviposition responses of Culex mosquitoes to organic infusions. - Environ. Entomol. 8: 1111-1117. LEE S.E., 2000 - Mosquito larvicidal activity of pipernonaline, a piperi- dine alkaloid derived from long pepper, Piper longum. - J. Am. Mosq. Control Assoc. 16: 245-7. LIU H., XU Q., ZHANG L., LIU N., 2005 - Chlorpyrifos resistance in Mosquito Culex aegypti and Culex pipiens pallens. - J. Med. Ent. 42: 815-820. LIXIN S., HUIQUIN D., CHONGXIA G., JIN Q., JING S., LEI M., et al., 2006 Larvicidal activity of extracts of Ginko biloba Exocarp for three different strains of Culex pipiens pallens. - J. Med. Ent. 43: 258-261. MEHRA B.K., HIRADHAR P.K., 2002 - Cuscuta hyalina Roth., an insect development inhibitor against common house mosquito Culex quinquefasciatus Say. - J. Environ. Biol. 23: 335-339. MERRILY K., WINSTON D., 2007 - Winston & Kuhn’s herbal therapy & supplements: a scientific and traditional approach. - Lippincott Williams & Wilkins, Philadelphia, PA: 260. MIURA T., SCHAFER C.H., TAKAHASHI R.M., MULLIGAN F.S., 1976 - Effects of insect growth inhibitor, dimilin on hatching of mosquito eggs. - J. Econ. Ent. 69: 655-658. MULLA M.S., 1961 - Suseptibility of various larval instars of Culex pipens, quinqufasciatus say to insecticides. - Mosq. New. 21: 320- 323. MURUGAN K., JAHANMOHINI P., BABU R., 1996 - Effect of neem kernel extract and neem oil on Nutritive and Reproductive Physiology of Heliothis armigera Hub. In: Singh R.P., Chari M.S., Raheja A.K., Kravs W., (eds.). Neem and environment, vol. 1. - Oxford and IBH Co., Pvt. Ltd. Delhi: 321-334. MURUGAN K., 2006 - Tsunami relief work - Biopesticide spray opera- tions - A case study. In: Nadim F., Pöttler R., Einstein H., Klapperich H., Kramer S., (eds.). Geohazards. - ECI Symposium Series, Volume P7. Available from: http://services.bepress.com/eci/geohaz- ards/39 MURUGAN K., VAHITHA R., BARUAH I., DAS S.C., 2003 - Integration of botanicals and microbial pesticides for the control of filarialvector, Culex quinquefasciatus-. Ann. Med. Entomol. 12: 11-23. NAKARUMA C.V., NAKARUMA T.U., BANDO E., MELO A.F.N., CORTEZ D.A.G., DIAZ FILHO B.P., 1999 - Antibacterial activity of Ocimum gratissimum L. essential Oil. - Mem. Inst. Oswardo Cruz. 94: 675-578. NARESHKUMAR A., MURUGAN K., REJEETH C., MADHIYAZHAGAN P., DONALD R. BARNARD, 2012 - Green synthesis of silver nanoparti- cles for the control of mosquito vectors of Malaria, Filariasis, and Dengue. – Vector-Borne Zoonot. 12: 262-268. NARESHKUMAR A., MURUGAN K., MADHIYAZHAGAN P., KOVENDAN K., PRASANNAKUMAR K., THANGAMANI S., et al., 2011 - Mosquitocidal and water purification properties of Cynodon dactylon, Aloe vera, Hemidesmus indicus and Coleus amboinicus leaf extracts against the mosquito vectors. - Parasitol Res. 110: 1435-1443. OUDA N.A.A., AL-CHALABI B.B.M., AL-CHARCHAFCHI F.F.M.R., MOHSEN Z.Z.H., 1998 - Extract of Atriplex canescens against Culex quinquefasciatus. - Pharm. Biol. 36: 69-71. PARK I.K., LEE H.S., LEE S.G., PARK J.D., YOUNG-JOON A.H.N., 2000 - Antifeeding activity of isoquinoline alkaloids identified in Coptis japonica roots against Hyphantria cunea (Lepidoptera: Arctiidae) and Agelastica coerulea (Coleoptera: Galerucinae). – J. Econ. Entomol. 93: 331-335. PESSOA L.M., MORAIS S.M., BEVILAQUA C.M.L., LUCIANO J.H.S., 2002 - Antihelmintic activity of essential oil of Ocimum gratissimum Linn. and Eugenol against Haemonchus contortus. - Vet. Parasitol. 109: 59-63. RAFFAUF R.F., 1996 - Plant alkaloids: a guide to their discovery and dis- tribution. - Hawkworth Press, Inc., New York, NY. RAJKUMAR S., JEBANESAN A., 2005 - Oviposition deterrent and skin repellent activities of Solanum trilobatum leaf extract against the malarial vector Anopheles stephensi. – J. Insect. Sci. 5: 15. RAJKUMAR S., JEBANESAN A., 2009 - Larvicidal and oviposition activi- ty of Cassia obtusifolia Linn (Family: Leguminosae) leaf extract against malarial vector, Anopheles stephensi Liston (Diptera: Culicidae). - Parasitol. Res. 104: 337-340. RETTICH F., 1976 - Changes in susceptibility to temphos primiphos- methyl fenitrothion and bromophos during preimaginal develop- ment of mosquitoes. - Acta. Ent. Bohemoslov. 73: 382-387. RICE-EVANS C.A., MILLER N.J., BOLWELL P.G., BRAMLEY P.M., PRID- HAM J.B., 1995 - The relative antioxidant activities of plant-derived polyphenolic flavonoids. - Free Radical Res. 22: 375-383. Article [page 96] [Journal of Entomological and Acarological Research 2012; 44:e17] No n- co mm er cia l u se on ly ROZENDAAL J.A., 1997 - Mosquitoes and other biting Diptera. In: Vector control - methods for use by individuals and communities. - World Health Organization, Geneva: 7-177. Available from: http://www.who.int/whopes/resources/vector_rozendaal/en/ SCHWARZ D., 2000 - Water clarification using Moringa oleifera. Technical Information W1e, Gate Information Service, Eschborn, Germany. - Available form: http://www.gtz.de/gate/gateid.afp Accessed: 31st October 2007. SENTHIL NATHAN S., KALAIVANI K., CHUNG P.G., MURUGAN K., 2006 - Effect of neem limonoids on lactate dehydrogenase (LDH) of the rice leaffolder, Cnaphalocrocis medinalis (Guene´e) (Insecta: Lepidoptera: Pyralidae). - Chemosphere. 62: 1388-1393. SHANMUGASUNDARAM R., JEYALAKSHMI T., DUTT M.S., MURTHY P.B., 2008 - Larvicidal activity of neem and karanja oil cakes against mosquito vectors, Culex quinquefasciatus (say), Aedes aegypti (L.) and Anopheles stephensi (L.). – J. Environ. Biol. 29: 43-5. SU T., MULLA M.S., 1998 - Ovicidal activity of neem products (Azadirachtin) against Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae). - J. Am. Mosq. Cont. Assoc. 14: 204-209. ÖZEN T., 2010 - Antioxidant activity of wild edible plants in the Black Sea Region of Turkey. - Grasas Y Aceites. 61, 86-94. TREASE G.E., EVANS W.C., 1978 - A text book of pharmacognosy, 11th ed. - Bailliere Tidall, London: 530. VINOD S., 2011 - Deforestation and water pollution impact on mosqui- toes related epidemic diseases in nanded region. – Biosci. Discov. 2: 309-316. WANDSCHEER C.B., DUQUE J.E., FUKUYAMA Y., WOHLKE J.L., FONTANA J.D., 2004 - Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti. - Toxicon. 44: 829-835. WHO (WORLD HEALTH ORGANIZATION)/UNICEF (Joint Monitoring Programme for Water Supply and Sanitation), 2005 - Water for life: making it happen. World Health Organization, Geneva. Available from: http://www.who.int/water_sanitation_health/monitoring/jmp 2005/en Article [Journal of Entomological and Acarological Research 2012; 44:e17] [page 97] No n- co mm er cia l u se on ly