jear2012 [Journal of Entomological and Acarological Research 2015; 47:1986] [page 31] Bioefficacy of Morinda tinctoria and Pongamia glabra plant extracts against the malaria vector Anopheles stephensi (Diptera: Culicidae) D. Amerasan, K. Murugan, C. Panneerselvam, N. Kanagaraju, K. Kovendan, P. Mahesh Kumar Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India Abstract Mosquito-borne diseases have an economic impact, including loss in commercial and labour outputs, particularly in countries with tropi- cal and subtropical climates; however, no part of the world is free from vector-borne diseases. The aim of the present study was to investigate the larvicidal, adulticidal and ovicidal activity of dried leaf chloroform, ethyl acetate, acetone, aqueous, and methanol extracts of Morinda tinctoria and Pongamia glabra against larvae of Anopheles stephensi (Diptera: Culicidae). Larvae were exposed to varying concentrations of plant extracts for 24 h. All extracts showed moderate larvicidal effects after 24 h of exposure; however, the highest larval mortality was found with the leaf methanol extracts of M. tinctoria and P. glabra against the larvae of A. stephensi lethal concentration (LC)50=136.24 and 141.05 ppm; LC90=342.67 and 368.89 ppm, respectively. The results of the adulticidal activity assays of chloroform, ethyl acetate, acetone, aque- ous, and methanol extracts of M. tinctoria and P. glabra showed signif- icant mortality against larvae of A. stephensi. The methanol extract showed maximum activity compared with the other extracts. The greatest effect on mean percentage hatch in the ovicidal assays was observed 48 h post-treatment. Percent hatch was inversely proportion- al to the concentration of extract, and directly proportional to the num- ber of eggs. A mortality of 100% was observed with 100-400 ppm methanol extracts and 200-400 ppm aqueous extracts of M. tinctoria, and 200-400 ppm aqueous and methanol extracts of P. glabra. This study provides the first report of the larvicidal, adulticidal and ovicidal activities of M. tinctoria and P. glabra plant extracts against the malar- ia vector, A. stephensi, representing an ideal eco-friendly approach for its control. Introduction The prevalence of mosquito-borne diseases is one of the world’s most important health problems. Mosquitoes are responsible for trans- mitting various infectious diseases; for this reason, the mosquito has been declared public enemy number one (World Health Organisation, 1996). Mosquitoes belonging to the genera Anopheles, Culex and Aedes are vectors for the pathogens of different diseases such as malaria, filariasis, Japanese encephalitis, dengue and dengue haemorrhagic fever, yellow fever and chickungunya (Rahuman et al., 2009; Borah et al., 2010). They cause allergic responses, including local skin and sys- temic reactions such as angioedema and urticaria (Peng et al., 1999). Tropical areas are more vulnerable to parasitic diseases, and the risk of contracting arthropod-borne illnesses has increased due to climate change and intensifying globalisation (Karunamoorthy et al., 2010). It is necessary to prevent mosquito-borne diseases and improve public health by controlling mosquitoes. Malaria is an infectious disease that is prevalent in tropical and some temperate areas of the world. Malaria is caused by a parasite that is transmitted from one human to another by the bite of infected Anopheles stephensi. Half of the world’s population is at risk from malaria. Each year, almost 250 million cases occur, causing 860,000 deaths (World Health Organisation, 2010). In India, 2-3 million malar- ia cases and about 1000 deaths are reported every year (Lal et al., 2010). Currently, a resistant variety of the malarial parasite is com- monly found in almost all parts of the world where malaria is endemic (Cooper et al., 2005). The increased incidence of drug resistance con- tinues to be a major issue, with ongoing problems related to drug qual- ity, availability, and cost of treating the disease (Garcia, 2010). For this reason, the transmission of malaria is best reduced by the control of the mosquito vector. Botanical and microbial insecticides have been increasingly used for mosquito control because of their efficacy and documented non-toxic effects on non-target organisms (Ascher et al., 1995). The highest number of malaria (Plasmodium falciparum) cases and malaria-related deaths is recorded from the state of Orissa, located in the eastern part of India (Sharma et al., 2010). Widely used chemical insecticides to control mosquitoes are often harmful to other beneficial organisms that prey on mosquito larvae, as well as to humans (Amer & Mehlhorn, 2006). Alternative pest control strategies, especially those that are effective and low-cost, are there- fore needed. A recent emphasis has been placed on plant materials Correspondence: Duraisamy Amerasan, Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore - 641 046, Tamil Nadu, India. Tel.: +91.9443746799. E-mail: ezhil_amar@yahoo.co.in Key words: Morinda tinctoria; Pongamia glabra; Anopheles stephensi; larvici- dal; adulticidal; ovicidal. Received for publication: 22 October 2013. Revision received: 24 November 2014. Accepted for publication: 16 January 2015. ©Copyright D. Amerasan et al., 2015 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2015; 47:1986 doi:10.4081/jear.2015.1986 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. Journal of Entomological and Acarological Research 2012; volume 44:e Journal of Entomological and Acarological Research 2015; volume 47:1986 No n- co mm er cia l u se on ly [page 32] [Journal of Entomological and Acarological Research 2015; 47:1986] that demonstrate larvicidal properties (Kovendan et al., 2012b; Mahesh Kumar et al., 2012a; Panneerselvam et al., 2013b). Morinda tinctoria, which belongs to the family Rubiaceae, grows wild and is distributed throughout Southeast Asia. Commercially known as Nunaa, it is indigenous to tropical countries and is consid- ered an important traditional medicine, where its leaves and roots are used as an astringent and emmengogue, and to relieve pain caused by gout (Thirupathy et al., 2009). There is a greater demand for fruit extracts of Morinda species in treatments for arthritis, cancer, gastric ulcers and other heart diseases (Mathivanan et al., 2006). Anti-convul- sant, analgesic, anti-inflammatory anti-oxidant activities of M. tincto- ria leaves have been reported (Jeyabalan & Palayan, 2009; Sreena et al., 2011). The major components that have been identified in the Nunaa plant include octoanicacid, potassium, vitamin C, terpenoids, scopoletin, flavones, glycosides, linoleic acid, anthraquinones, morindone, rubiadin, and alizarin (Moorthy & Reddy, 1970; Singh & Tiwari, 1976; Levand & Larson, 1979; Farine et al., 1996). Deepti et al. (2011) studied the in vitro free radical-scavenging efficacy of methano- lic, chloroform and ethyl acetate extracts of M. tinctoria, which can be recommended as potential antioxidants. Pongamia glabra Vent. (Syn. Pongamia pinnata), in the family Fabaceae, commonly known as Karanja, is a tree found all over India that bears imparipinnate leaves and pinkish-white flowers (Kirtikar & Basu, 1984). Roots, bark, leaves, flowers and seeds of this plant also have medicinal properties and are traditionally used as medicinal treat- ments. All parts of the plant have been used as a crude drug for the treatment of tumours, piles, skin diseases, wounds and ulcers (Tanaka et al., 1992). Seeds contain karanjin, ponga-mol and glabrin. Karanjin is reported to be an effective remedy for all skin diseases such as sca- bies, eczema, leprosy and ulcers (Sivarajan & Balachandran, 1999). The leaves are spicy, digestive, laxative, anthelmintic and cure piles, wounds and other inflammations. A hot infusion of leaves is used as a medicated bath for relieving rheumatic pains and for cleaning ulcers from gonorrhoea and scrofulous enlargement (Chopra, 1933; Satyavati et al., 1987). In addition, phytochemical examinations of this plant have indicated the presence of furanoflaovones, furanoflavonols, chromenoflavones, flavones, furanodiketones and flavonoid glucosides (Rangaswami et al., 1942; Murthy & Seshadri, 1944; Sharma et al., 1973; Talapatara et al., 1980; Pathak, 1983; Tanaka et al., 1992; Ahemed et al., 2004; Yin et al., 2006). The acetone, chloroform, ethyl acetate, hexane, and methanol leaf extracts of Acalypha indica, Achyranthes aspera, Leucas aspera, M. tinc- toria, and Ocimum sanctum were studied against the early fourth- instar larvae of Aedes aegypti and Culex quinquefasciatus (Bagavan et al., 2008); the chloroform fruit extract of Morinda pubescens (M. tinc- toria) showed wound-healing properties in rats (Mathivanan et al., 2006). Larvicidal effects of neem (Azadirachta indica) and karanja (P. glabra) oil cakes (separately and in combination) were studied against several mosquito species. The combination of neem and karanja oil cakes in equal proportion proved to be more effective than individual treatments against the mosquitoes C. quinquefasciatus, A. aegypti and A. stephensi, with lethal concentration (LC)95 values of 0.93, 0.54 and 0.77 ppm, respectively (Shanmugasundaram et al., 2008). Oviposition deterrent activity of the ethanolic extract of Pongamia pinnata, Coleus forskohlii, and Datura stramonium leaves against A. aegypti and C. quinquefaciatus was reported by Swathi et al. (2010). The individual and combined efficacy of Annona squamosa and P. glabra extracts against three mosquito vectors, C. quinquefasciatus, A. stephensi, and A. aegypti, compared with that of A. indica was investigated by George & Vincent (2005). Leaf extracts of some common plants such as Vitex negundo, Gliricidia maculata, Wedelia chinensis, M. tinctoria, and P. glabra, were evaluated for their acaricidal activity against the red spi- der mite, Oligonychus coffeae, in the laboratory using a leaf disc method under controlled conditions. Among them, the aqueous extract of M. tinctoria and P. glabra showed maximum ovicidal action, ovicidal deterrence and 100% adult mortality (Vasanthakumar et al., 2012). In the present study, we report for the first time on larvicidal, adulti- cidal and ovicidal activities of different solvent extracts of M. tinctoria and P. glabra against the malarial mosquito vector, A. stephensi. Materials and methods Plant collection Fully developed fresh leaves of M. tinctoria and P. glabra were collect- ed from the Maruthamalai Hills, near the Bharathiar University cam- pus in Coimbatore. Identification was authenticated by a plant taxono- mist from the Department of Botany, Bharathiar University. A voucher specimen is deposited at the herbarium of the Entomology Division, Bharathiar University. Extraction The leaves were washed with tap water, shade-dried at room temper- ature (28±2°C) for 5-10 days. The air-dried materials were powdered individually using a commercial electric blender. The finely ground plant material (1000 g/solvent) was loaded into a Soxhlet apparatus and extracted individually with five different solvents: chloroform, ethyl acetate, acetone, aqueous, and methanol. The solvent from the extract was removed using a rotary vacuum evaporator to collect the crude extract. The crude residue of these plants varies with the solvents used. The M. tinctoria and P. glabra with five different solvents yielded 58.20, 64.09, 54.11, 67.34, 88.05 g and 47.10, 53.31, 41.18, 54.30, 79.16 g of crude residue, respectively. Standard stock solutions were prepared at 1% by dissolving the residues in acetone. From this stock solution, dif- ferent concentrations were prepared, and these solutions were used for the larvicidal, adulticidal and ovicidal bioassays. Insect rearing The eggs of A. stephensi were collected from different breeding sites (overhead tanks) in Coimbatore District, Tamil Nadu, India. These were taken to the laboratory and transferred (in approximately the same aliquot numbers of eggs) to 18 cm L×13 cm W×4 cm D enamel trays containing 500 mL of water, where they were allowed to hatch. Mosquito larvae were reared (and adult mosquitoes held) at 27°C±2°C and 75%-85% relative humidity in a 14:10 (L:D) photoperiod. Larvae were fed 5 g of ground dog biscuit and brewer’s yeast daily in a 3:1 ratio. Pupae were collected and transferred to plastic containers with 500 mL of water. The container was placed inside a screened cage (90 cm L×90 cm H×90 cm W) to retain emerging adults, for which 10% sucrose in water solution (v/v) was available ad libitum. On days 5 post-emer- gence, the mosquitoes were provided access to a rabbit host for blood feeding. The shaved dorsal side of the rabbit was positioned on the top of the mosquito cage in contact with the cage screen (using a cloth sling to hold the rabbit) and held in this position overnight. Glass Petri dishes lined with filter paper and containing 50 mL of water were subsequently placed inside the cage for oviposition by female mosquitoes. Larvicidal bioassays A laboratory colony of A. stephensi larvae was used for the larvicidal activity. Twenty-five individuals of early fourth-instar larvae were kept in a 500-mL glass beaker containing 249 mL of dechlorinated water, and 1 mL of the desired concentration of plant extracts were added. Larval food was provided for the test larvae. At each tested concentra- tion, two to five trials were made and each trial consisted of five repli- cates. The control was set up by mixing 1 mL of acetone with 249 mL of dechlorinated water. The larvae exposed to dechlorinated water without Article No n- co mm er cia l u se on ly acetone served as a control. The control mortalities were corrected using Abbott’s formula (Abbott, 1925). LC50 and LC90 were calculated from toxicity data using probit analysis (Finney, 1971). Corrected mortality = Observed mortality in treatment�– Observed mortality in control ¥ 100 100 – Control mortality (1) Percentage mortality = Number of dead larvae ¥ 100 Number of larvae introduced (2) Adulticidal bioassays Sugar-fed adult female mosquitoes (5-6 days old) were used. The M. tinctoria and P. glabra leaf extracts were diluted with acetone to make different concentrations. The diluted plant extracts were impregnated on filter papers (140×120 mm). A blank paper consisting of only ethanol was used as a control. The papers were left to dry overnight at room temper- ature to let the ethanol evaporate. Impregnated papers were prepared fresh prior to testing. The bioassay was conducted in an experimental kit consisting of two cylindrical plastic tubes, both measuring 125×44 mm, following the method of World Health Organisation (1981). One tube served to expose the mosquitoes to the plant extract and the other tube was used to hold the mosquitoes before and after the exposure periods. The impregnated papers were rolled and placed in the exposure tube. Each tube was closed at one end with a 16-mesh wire screen. Sucrose- fed and blood-starved mosquitoes (20) were released into the tube, and the mortality effects of the extracts were observed every 10 min for a 3-h exposure period. At the end of 1-, 2-, and 3-h exposure periods, the mos- quitoes were placed in the holding tube. Cotton pads soaked in 10% sugar solution with vitamin B complex were placed in the tube during the holding period for 24 h. Mortality of the mosquitoes was recorded after 24 h. The above procedure was replicated three times using plant extracts of each concentration. Ovicidal activity assays Freshly laid eggs were collected by providing ovitraps in mosquito cages. Ovitraps were kept in the cages 2 days after the female mosqui- toes were given a blood meal. The eggs were laid on filter paper lining provided in the ovitrap. After scoring, 100 gravid females were placed in a screen cage where 10 oviposition cups were introduced for ovipo- sition 30 min before the start of the dusk period. Of these 10 cups, each nine were filled with test solution of 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 ppm, respectively and one was filled with 100 mL of the water and Polysorbate 80 that served as a control. The experiment was repeated trice with three replicates. A minimum of 100 eggs was used for each treatment, and the experiment was replicated five times. After treat- ment, the eggs were sieved through muslin cloth, thoroughly rinsed with tap water, and left in plastic cups filled with dechlorinated water for hatching assessment after counting the eggs under microscope (Su & Mulla, 1998). The percentage of egg mortality was calculated on the basis of non-hatch of eggs with unopened opercula (Chenniappan & Kadarkarai, 2008). The hatching rate of eggs was assessed after 98 h post-treatment, as per the method of Rajkumar & Jebanesan (2009). Statistical analysis The average adult mortality data were subjected to probit analysis for calculating LC50, LC90, and other statistics at 95% upper and lower fidu- cial limits, and Chi-square values were calculated by using the SPSS Statistical software package, ver. 16.0. Results with P<0.05 were con- sidered to be statistically significant. Results The present study explored the potential mosquitocidal properties of two plants, using different solvents for the crude extracts (Table 1). Chloroform, ethyl acetate, acetone, aqueous, and methanol leaf extracts of the plants M. tinctoria and P. glabra was studied for use as eco-friendly insecticides, as alternatives to potentially harmful synthet- ic insecticides. Results of larvicidal and adulticidal assays with these leaf extracts (Tables 2-5) confirm their potential ability to control adult and larval populations of the mosquito A. stephensi. All extracts showed moderate larvicidal effects; however, the highest larval mortality was found with the methanol extract of M. tinctoria and P. glabra against the fourth-instar larvae of A. stephensi (LC50=136.24 and 141.05 ppm; LC90=342.67 and 368.89 ppm, respectively) (Figure 1). The chi-square values are significant at the P<0.05 level. The high chi-square values in the bioassays possibly indicate the heterogeneity of the test popula- tion. The 95% confidence limits for the LC50 lower/upper fiducidal limits (LCL-UCL) and LC90 (LCL-UCL) were also calculated. No mortality was recorded in the control. The results of the larvicidal assay clearly indi- cate that the percentage of mortality was directly proportional to con- centration of the extract. After exposure to the test concentrations, the treated larvae exhibited restlessness, sluggishness, tremors, and con- vulsions, followed by paralysis. Five different solvents were tested, and the highest adulticidal activity was observed with the methanol extract of M. tinctoria followed by P. glabra, with LC50 values of 194.78 and 198.65 ppm and LC90 values of 336.27 and 357.92 ppm, respectively (Figure 2). At higher concentrations, the adults showed restless move- ment for some time, accompanied by abnormal wiggling movements, [Journal of Entomological and Acarological Research 2015; 47:1986] [page 33] Article Table 1. List of medicinal plants tested for bioactivity against eggs, larvae and adults of Anopheles stephensi. Botanical name Common name Family Medicinal property Plant parts (Tamil) tested Morinda tinctoria Roxb. Mannanunai Rubiaceae Leaves are useful as tonic, febrifuge and emmenagogue. Leaves (coffee family) It is also used for curing dyspepsia, diarrhoea, ulceration, stomatitis, digestion, wound and fever. The poultice or the paste of its leaves is applied on the wounds and swellings for relief. The green fruit and leaves are used to treat menstrual cramps, bowel irregularities and urinary tract infections Pongamia glabra Vent. Pungai Fabaceae Leaves of P. glabra have been known as a remedy for diarrhoea. Leaves or Leguminosae It is also used as a digestive and laxative and to treat inflammation and wounds. Leaf juice aids in treatment of leprosy, gonorrhoea, flatulence, coughs, and colds. The leaf infusions and extracts alleviate rheumatism and itches, respectively No n- co mm er cia l u se on ly [page 34] [Journal of Entomological and Acarological Research 2015; 47:1986] and death. The mean percentage egg hatch of A. stephensi was also tested against these solvents and leaf extracts; results are shown in Table 6. The percentage hatch was inversely proportional to the concen- tration of extract, and directly proportional to the no. of eggs. Of the extracts tested for ovicidal activity, the leaf methanol extract of M. tinc- toria resulted in 100% mortality (no hatch) at both 100 and 400 ppm. The leaf extract of M. tinctoria was more effective than P. glabra against larvae and eggs of this mosquito vector. Eggs in the control treatment had 100% hatch. Discussion and conclusions Insect pest control is often a complex, expensive task, frequently requiring the cooperative efforts of communities as well as such groups as industry, agriculture, state, and local governments. We must be con- cerned with the harmful effects of synthetic pesticides on the environ- ment and people, and reports have emerged on the resurgence of several mosquito-borne diseases in the world as a consequence of the increasing resistance of mosquitoes to commercial insecticides (Becker et al., 2003). This has necessitated the need for research and development of an environmentally safe, biodegradable and indigenous material for vec- tor control. Many herbal products were used as natural insecticides before the discovery of synthetic organic insecticides (Mittal & Subbarao, 2003). Plant allelochemicals may be quite useful in increasing the efficacy of biological control agents, because plants produce a large variety of compounds that increase their resistance to insect attack (Berenbaum, 1988; Murugan et al., 1996; Senthil Nathan et al., 2005). In this study, good larvicidal activity against A. stephensi was achieved with different solvent extracts of M. tinctoria and P. glabra. The mode of action of these leaf extracts on mosquito larvae is not known, but previous stud- ies have demonstrated that phytochemicals interfere with the proper functioning of mitochondria, more specifically at the proton transferring sites (Usta et al., 2002). Other studies by Rey et al. (1999) and David et al. (2000) found that phytochemicals primarily affect the midgut epithe- Article Figure 1. Larvicidal activity of A) Morinda tinctoria and B) Pongamia glabra leaf extracts against Anopheles stephensi with lethal concentration (LC)50 and LC90 values. Figure 2. Adulticidal activity of A) Morinda tinctoria and B) Pongamia glabra leaf extracts against Anopheles stephensi with lethal concentration (LC)50 and LC90 values. No n- co mm er cia l u se on ly [Journal of Entomological and Acarological Research 2015; 47:1986] [page 35] Article Table 2. Larvicidal activity of different solvent extracts of Morinda tinctoria against fourth instar larvar of Anopheles stephensi. Solvent Concentration (ppm) % Mortality±SD LC50, ppm LC90, ppm χ2 extracts (LFL-UFL) (LFL-UFL) Chloroform Control 0.0±0.0 80 26.36±1.62 160 45.44±1.43 191.938 (168.13-212.98) 416.188 (379.32-468.55) 3.751* 240 59.38±1.40 320 71.02±1.96 400 92.22±1.57 Ethyl acetate Control 0.0±0.0 80 29.48±1.48 160 48.34±1.59 240 62.24±1.52 177.080 (151.89-198.57) 400.763 (365.20-451.21) 3.295* 320 74.42±1.61 400 93.54±1.46 Acetone Control 0.0±0.0 80 32.82±1.04 160 49.52±1.51 165.573 (139.98-186.98) 382.190 (348.85-429.06) 4.702* 240 64.04±0.99 320 77.06±1.07 400 96.02±1.18 Aqueous Control 0.0±0.0 80 34.06±1.02 160 51.14±1.12 240 66.44±1.77 156.298 (91.6-198.70) 353.845 (297.79-470.72) 5.452* 320 81.84±1.23 400 98.34±1.31 Methanol Control 0.0±0.0 80 39.32±1.62 160 56.22±1.78 136.242 (48.50-184.70) 342.678 (282.75-480.98) 6.210* 240 70.06±1.20 320 83.24±1.64 400 99.04±1.16 SD, standard deviation; LC, lethal concentration; LFL, lower fiducidal limits; UFL, upper fiducidal limits; χ2 Chi square value. *Significant at P<0.05 level. Table 3. Larvicidal activity of different solvent extracts of Pongamia glabra against fourth instar larvar of Anopheles stephensi. Solvent Concentration (ppm) % Mortality±SD LC50, ppm LC90, ppm χ2 extracts (LFL-UFL) (LFL-UFL) Chloroform Control 0.0±0.0 80 23.34±1.47 160 44.36±1.64 240 55.06±1.13 205.973 (182.63-227.268) 436.574 (397.02-493.32) 3.103* 320 69.04±1.18 400 89.22±1.35 Ethyl acetate Control 0.0±0.0 80 28.42±1.62 160 45.12±1.69 240 60.62±1.78 188.888 (163.09-211.26) 428.639 (388.43-487.00) 2.174* 320 71.02±1.26 400 90.12±1.10 Acetone Control 0.0±0.0 80 30.06±1.10 160 47.08±1.11 240 62.32±1.53 176.264 (151.03-197.77) 399.610 (364.25-449.68) 1.206* 320 77.06±1.07 400 92.34±1.59 Aqueous Control 0.0±0.0 80 32.12±1.13 160 49.52±1.35 240 63.08±1.22 166.882 (140.53-188.85) 390.575 (355.82-439.81) 1.264* 320 79.42±1.54 400 93.04±1.06 Methanol Control 0.0±0.0 80 37.06±1.24 160 56.46±1.67 240 68.38±1.54 141.058 (110.40-165.10) 368.890 (335.10-417.11) 1.965* 320 81.66±1.83 400 95.16±1.42 SD, standard deviation; LC, lethal concentration; LFL, lower fiducidal limits; UFL, upper fiducidal limits; χ2 Chi square value. *Significant at P<0.05 level. No n- co mm er cia l u se on ly [page 36] [Journal of Entomological and Acarological Research 2015; 47:1986] lium and secondarily the gastric caeca and the malpighian tubules in mosquito larvae. Furthermore, the crude extracts may be more effective than the individual active compounds, due to a natural synergism that discourages the development of resistance in the vectors (Maurya et al., 2007). The present investigation revealed that the crude chloroform, ethyl acetate, acetone, aqueous, and methanol leaf extracts of the plants M. tinctoria and P. glabra have significant larvicidal, adulticidal as well as ovicidal activity. These results are comparable to earlier reports of Panneerselvam et al. (2012), who observed larvicidal activity of Artemisia nilagirica against A. stephensi. They reported LC50 values against the first instar of 272.50 ppm, second instar 311.40 ppm, third instar 361.51 ppm, and fourth instar 442.51 ppm; the corresponding LC90 values were: first instar, 590.07 ppm, second instar, 688.81 ppm, third instar, 789.34 ppm, and fourth instar, 901.59 ppm; the LC50 and LC90 values against the pupae were 477.23 and 959.30 ppm, respectively. The petroleum ether (PE) and methanol (MeOH) extracts of Rhinacanthus nasutus and Derris elliptica exhibited larvicidal effects against A. aegypti, C. quinquefasciatus, Anopheles dirus, and Mansonia uniformis, with LC50 values between 3.9 and 11.5 mg/L, while the MeOH extract gave LC50 values between 8.1 and 14.7 mg/L. D. elliptica PE extract showed LC50 values between 11.2 and 18.84 mg/L, and the MeOH extract exhibited LC50 values between 13.2 and 45.2 mg/L, respectively (Komalamisra et al., 2005); the n-hexane, ethyl acetate, and methanol extracts of Cassia nigricans showed 100% lar- val mortality against Ochlerotatus triseriatus (Georges et al., 2008). The leaf hexane, chloroform, ethyl acetate, acetone and methanol extracts of Acalypha alnifolia were tested for larvicidial activity against A. stephensi, A. aegypti and C. quinquefasciatus, with LC50 values for A. stephensi of 197.37, 178.75, 164.34, 149.90 and 125.73 ppm, respectively; for A. aegypti, 202.15, 182.58, 160.35, 146.07 and 128.55 ppm, respectively; and for C. quinquefasciatus, 198.79, 172.48, 151.06, 140.69 and 127.98 ppm, respec- tively (Kovendan et al., 2012c). In our results, the larvicidal activity of chloroform, ethyl acetate, acetone, aqueous, and methanol extracts of M. tinctoria and P. glabra exhibited larvicidal effects against A. stephensi with LC50 values of (with M. tinctoria) 191.93, 177.08, 165.57, 156.29 and 136.24; and (with P. glabra) 205.97, 188.88, 176.26, 166.88 and 141.05, respectively. The leaf benzene, petroleum ether, ethyl acetate, and methanol extracts of Citrullus vulgaris were previously tested for larvi- cidial activity against A. stephensi, with LC50 values of 18.56, 48.51, 49.57, and 50.32 ppm, respectively (Mullai et al., 2008b). The insecticidal activ- ity of Zingiber officinale against third-instar larval maturation and adult emergence of Anopheles pharoensis was evaluated at concentrations of 100, 70, 50, 25, 5, 2, 1, 0.9, 0.7, 0.5 and 0.3%, showing 100% larval mortal- ity and, at 0.2% and 0.1%, mortality of 66.7%. The effects of the tested extracts on adult emergence and adulticidal activity against the mosquitoes are remarkably greater than those reported for other plant extracts in the literature. For example, at the highest concentration, 50% inhibition of adult emergence was observed from the ethyl acetate fractions of Calophyllum inophyllum seed and leaf, Solanum suratense and Samadera indica leaf extracts, and the petrol ether fraction of Rhinocanthus nasutus leaf extract for C. quinquefasciatus, A. stephensi and A. aegypti (Muthukrishnan & Puspalatha, 2001). Similarly, 88% adult mortality was observed from Pelargonium citrosa leaf extracts at 2% concentration against A. stephensi (Jeyabalan et al., 2003). Adult mortality was caused by the ethanol extract of Citrus sinensis, with LC50 and LC90 values of 272.19 and 457.14 ppm, and for A. stephensi, 289.62 and 494.88 ppm, and A. aegypti, 320.38 and 524.57 ppm, respectively (Murugan et al., 2012). These findings correspond with those of Govindarajan & Sivakumar Article Table 4. Adulticidal activity of different solvent extracts of Morinda tinctoria against Anopheles stephensi. Solvent Concentration (ppm) % Mortality±SD LC50, ppm LC90, ppm χ2 extracts (LFL-UFL) (LFL-UFL) Chloroform Control 0.0±0.0 160 26.44±1.65 220 37.28±1.62 280 60.14±1.99 253.101 (235.09-269.40) 429.981 (399.62-473.73) 0.864* 340 72.44±1.46 400 86.06±1.00 Ethyl acetate Control 0.0±0.0 160 27.44±1.45 220 43.36±1.80 280 62.54±1.73 240.968 (222.22-257.28) 414.465 (386.03-455.13) 0.098* 340 76.08±1.94 400 88.06±1.11 Acetone Control 0.0±0.0 160 33.12±0.97 220 47.02±1.28 280 68.18±1.11 220.466 (201.34-236.39) 378.697 (354.98-411.62) 0.594* 340 84.52±1.39 400 92.62±1.52 Aqueous Control 0.0±0.0 160 37.36±1.47 220 50.28±1.52 280 74.54±1.74 206.379 (185.55-223.09) 363.338 (340.67-394.75) 1.082* 340 86.06±1.01 400 94.44±1.77 Methanol Control 0.0±0.0 160 41.02±1.29 220 55.56±1.57 280 76.52±1.40 194.785 (174.49-210.88) 336.270 (316.49-363.13) 1.563* 340 90.54±1.75 400 98.02±1.27 SD, standard deviation; LC, lethal concentration; LFL, lower fiducidal limits; UFL, upper fiducidal limits; χ2 Chi square value. *Significant at P<0.05 level. No n- co mm er cia l u se on ly (2012), who reported on the adulticidal activity of hexane, ethyl acetate, benzene, chloroform and methanol leaf extracts of Cardiospermum halicacabum against C. quinquefasciatus, A. aegypti and A. stephensi. The plant extracts showed moderate toxic effects on the adult mosquitoes after an exposure period of 24 h. However, when compared with other solvents, the highest mortality was found with a methanol extract of C. halicacabum against all three species. Among them, A. stephensi had the highest LC50 and LC90 values (186.00 and 346.06 ppm). Nathan et al. (2005) considered pure limonoids from neem seed, testing for biological, larvicidal, pupicidal, adulticidal and antiovipositional activity. Against A. stephensi, larval mortality was dose-dependent, with the highest dose of 1-ppm azadirachtin causing almost 100% mortality, exhibiting pupicidal and adulticidal activity and significantly decreased fecundity and longevity. In the present study, we found the methanol extract of M. tinctoria to have the highest adul- ticidal activity compared with P. glabra, with LC50 and LC90 values of 194.78, 198.65 ppm and 336.27, 357.92 ppm, respectively. Similarly, the greatest adulticidal effect was seen from Piper sarmentosum, followed by P. ribesoides and P. longum, with LD50 values of 0.14, 0.15 and 0.26 mg/female, respectively (Choochote et al., 2006). Adulticidal activity of the essential oil isolated from Mentha longifolia was screened using a [Journal of Entomological and Acarological Research 2015; 47:1986] [page 37] Article Table 5. Adulticidal activity of different solvent extracts of Pongamia glabra against Anopheles stephensi. Solvent Concentration (ppm) % Mortality±SD LC50, ppm LC90, ppm χ2 extracts (LFL-UFL) (LFL-UFL) Chloroform Control 0.0±0.0 160 23.16±1.15 220 36.32±0.81 280 58.52±1.54 262.077 (244.43-278.53) 41.527 (409.68-487.70) 0.677* 340 70.54±1.45 400 83.06±1.14 Ethyl acetate Control 0.0±0.0 160 25.48±1.70 220 42.36±1.67 280 60.42±1.44 247.164 (228.86-263.40) 421.814 (392.58-463.72) 0.082* 340 75.32±1.64 400 86.38±1.56 Acetone Control 0.0±0.0 160 30.62±1.77 220 46.38±1.48 280 65.34±1.73 227.727 (208.47-243.94) 393.580 (367.89-429.71) 0.378* 340 82.54±1.71 400 90.02±1.01 Aqueous Control 0.0±0.0 160 36.08±1.08 220 48.04±1.28 280 72.38±1.81 212.097 (190.37-229.53) 381.306 (356.05-417.02) 1.537* 340 84.76±1.35 400 91.08±1.10 Methanol Control 0.0±0.0 160 39.24±1.70 220 54.34±1.76 280 74.28±1.64 198.657 (176.28-216.19) 357.921 (335.27-389.43) 0.586* 340 88.72±1.10 400 94.26±1.78 SD, standard deviation; LC, lethal concentration; LFL, lower fiducidal limits; UFL, upper fiducidal limits; χ2 Chi square value. *Significant at P<0.05 level. Table 6. Ovicidal activity of different plant leaf extracts against eggs of Anopheles stephensi. Plant species Solvent Percent egg hatch Concentration (ppm) 12.5 25 50 100 200 400 Control M. tinctoria Chloroform 88.04±0.95 74.22±1.02 63.52±1.78 53.02±1.20 40.84±1.10 NH 100±0.0 Ethyl acetate 81.08±0.94 67.12±0.97 55.46±1.73 44.42±1.35 33.54±1.54 NH 100±0.0 Acetone 76.44±1.43 62.14±1.20 49.48±1.40 36.52±1.57 26.02±0.95 NH 100±0.0 Aqueous 70.42±1.39 56.32±1.63 40.56±1.72 27.26±1.57 NH NH 100±0.0 Methanol 63.36±1.36 51.44±1.77 38.56±1.63 NH NH NH 100±0.0 P. glabra Chloroform 80.14±1.15 71.38±1.89 59.08±1.17 49.18±1.04 34.52±1.77 NH 100±0.0 Ethyl acetate 75.36±1.57 62.44±1.52 48.98±1.86 38.52±1.07 29.38±1.42 NH 100±0.0 Acetone 70.26±1.53 57.28±1.47 43.54±1.01 31.24±1.57 22.98±0.95 NH 100±0.0 Aqueous 63.02±0.63 52.62±1.51 38.82±1.11 23.86±0.97 NH NH 100±0.0 Methanol 59.32±1.40 46.04±1.31 32.34±1.65 25.54±1.42 NH NH 100±0.0 NH, no hatch. No n- co mm er cia l u se on ly [page 38] [Journal of Entomological and Acarological Research 2015; 47:1986] fumigant toxicity assay against the house mosquito, Culex pipiens L. (Diptera: Culicidae), by Oz et al. (2007). The present study corresponds with the findings of Amerasan et al. (2012) who reported the LC50 and LC90 values of Cassia tora leaf extracts as adulticidal activity of hexane, chloroform, benzene, acetone, and methanol against C. quinquefascia- tus, A. aegypti, and A. stephensi as the following: for C. quinquefascia- tus, LC50 values were 338.81, 315.73, 296.13, 279.23, and 261.03 ppm, and LC90 values were 575.77, 539.31, 513.99, 497.06, and 476.03 ppm; for A. aegypti, LC50 values were 329.82, 307.3, and 252.03 ppm, and LC90 val- ues were 563.24, 528.33, 36 496.92, 477.61, and 448.05 ppm; and for A. stephensi, LC50 values were 317.28, 300.30, 277.51, 263.35, and 251.43 ppm, and LC90 values were 538.22, 512.90, 483.78, 461.08, and 430.70 ppm, respectively. The adulticidal activity of the essential oil of Lantana camara was evaluated against different mosquito species on 0.208 mg/cm2 impregnated papers, and the knockdown time (KDT)50 and KDT90 values of the essential oil were 20, 18, 15, 12 and 14 min and 35, 28, 25, 18 and 23 min against A. aegypti, C. quinquefasciatus, A. culici- facies, Ancylus fluvialitis and A. stephensi, with corresponding percent- age of mortalities of 93.3%, 95.2%, 100%, 100% and 100%, respectively (Dua et al., 2010). The ovicidal efficacy in the current study compared well with an earli- er report; the bioactive compound azadirachtin (A. indica) showed com- plete ovicidal activity against eggs of Culex tarsalis and C. quinquefascia- tus exposed to a 10-ppm concentration (Su & Mulla, 1998). The ovicidal activity of 21 hyphomycete fungi species against A. aegypti was reported. The tested fungi were Paecilomyces carneus, Paecilomyces marquandii, Isaria fumosorosea, Metarhizium anisopliae, Penicillium sp., Paecilomyces lilacinus, Beauveria bassiana, and Evlachovaea kintrischi- ca. These are the first results to show the effects of entomopathogenic fungi against eggs of A. aegypti, and they suggest their potential as con- trol agents of this vector (Luz et al., 2007). Assis et al. (2003) reported that egg hatching inhibition of ethyl acetate and methanol extracts of Spigelia anthelmia ranged from 97.4-100%, respectively, at 50.0 mg mL−1. The oviposition deterrent properties against A. stephensi have been observed for various plant extracts, including the methanol extract of Pelargonium citrosa, which exhibited 56% and 92% inhibition of oviposi- tion at 1 and 4 ppm, respectively (Jeyabalan et al., 2003). The benzene extracts of C. vulgaris caused 100% mortality (zero hatch) at 250 ppm, and at 200 ppm a very low hatch rate (11.8%), with complete ovicidal activity at 300 ppm. Fraction I at 80 ppm caused a very low hatch rate of 3.2%, followed by fraction II (6.9%), fraction III (4.9%), and fraction IV (5.3%) against A. stephensi (Mullai et al., 2008a). The leaf extracts of Andrographis paniculata, Cassia occidentalis, and Euphorbia hirta with different solvents (i.e., hexane, ethyl acetate, benzene, aqueous, and methanol) was studied for adulticidal, repellent and ovicidal activity against A. stephensi. Among the extracts tested for ovicidal activity against A. stephensi, the leaf methanol extract of A. paniculata caused 100% mortality (zero hatch) at 150 and 300 ppm, respectively (Panneerselvam & Murugan, 2013a). The leaf extract of Cassia fistula in different solvents (methanol, benzene, and acetone) were studied for lar- vicidal, ovicidal, and repellent activity against A. aegypti (Govindarajan, 2009). In the present work, the crude methanol and aqueous extracts of M. tinctoria resulted in zero hatch (100% mortality) at 100 and 200 ppm; followed by crude methanol. The aqueous extract of P. glabra caused zero hatch (100% mortality) at 200 ppm for A. stephensi. In the case of ovicidal activity, exposure to freshly laid eggs was more effective than with older eggs. It has been shown that the age of the embryos at the time of treat- ment plays a crucial role with regard to the effectiveness of the chitin synthesis inhibitor, dimilin, to C. quinquefasciatus (Miura et al., 1976). Malarvanan et al. (2009) reported that exposure of Cipadessa baccifera, Melia dubia, Clausena dentata and Dodonaea angustifolia to petroleum ether, hexane, chloroform, acetone and water extracts exhibited ovicidal activity against Helicoverpa armigera, and maximum activity was observed with the hexane extract of Clausena dentate. The leaf extract of Solanum trilobatum reduced egg laying by gravid females of A. 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). Recently, ovicidal, repellent, adulticidal and field evaluations of plant extracts were reported against dengue, malaria and filarial vectors (Kovendan et al., 2012a). Findings of the present investigation reveal that the leaf extracts of M. tinctoria and P. glabra possess remarkable lar- vicidal, adulticidal and ovicidal activity against this malarial vector. 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