Layout 1 Abstract Vector control is one of the most important components in combat- ing vector-borne diseases throughout the world. Application of insecti- cides is a widely known and popular vector control strategy. The objec- tive of the present study was to evaluate the larvicidal activity of the hexane, diethyl ether, ethyl acetate and acetone extracts of Abutilon indicum, Hyptis suaveolens and Leucas aspera against third-stage lar- vae of Anopheles culicifiacies. The results clearly suggest that all three selected plant extracts exhibited moderate larvicidal activity after 24, 48 and 72 h at 250, 500, 750 and 1000 ppm; the lethal concentrations (LC) at 50% and 90% of A. indicum, H. suaveolens against third instar larvae at 24, 48 and 72 h (hexane, diethyl ether, ethyl acetate and ace- tone) were as follows: A. indicum, LC50=1031.65, 949.18, 833.58 and 673.68 ppm; LC90=2215.87, 2234.39, 2152.97 and 2455.10 ppm; H. suaveolens, LC50=423.00, 347.50, 236.58 and 217.24 ppm; LC90=1431.91, 1292.15, 1138.49 and 1049.27 ppm and L. aspera, LC50=559.77, 401.56, 299.71 and 263.01 ppm; LC90=1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively. Overall, the highest lar- vicidal activity was observed with H. suaveolens extract followed by L. aspera and A. indicum at various concentrations at 48 and 72 h, respec- tively. The objective of this investigation was an attempt to search for a user- and eco-friendly vector control agent. The study proved that the selected plant leaf extracts could serve as potent larvicidal agents against A. culicifacies in vector control programs. Introduction Malaria is a disease that inflicts a serious negative impact on public health and socio-economic development in resource-limited settings of the world. Malaria directly or indirectly affects the health and wealth of individuals as well as nations. Indeed, malaria is identified both as a disease associated with and a cause of poverty (Karunamoorthi, 2012). Currently, malaria control is hampered by many operational and techni- cal problems. However, the development of insecticidal resistance in malaria vectors to existing conventional insecticides has made malaria vector control more challenging (Sharma & Saxena, 1996). Anopheles stephensi and Anopheles culicifacies are the two primary malarial vec- tors in India. In India, there are five sibling species reported, named A, B, C, D, and E; however, species A, C, D, and E are considered to be major vectors, while B is a minor vector (Subbarao et al., 1999). Amerasinghe et al. (1999) reported that A. culicifacies is the main vec- tor and A. subpictus is a significant secondary malarial vector in Sri Lanka. In India, A. culicifacies is a primary vector in rural as well as peri- urban areas, which constitutes nearly 65% of all malaria cases. At the moment, dichlorodiphenyltrichloroethane (DDT) (organochlorine), malathion (organophosphorus) and �d-methrin, cyfluthrin, a-cyperme- thrin and l-cyhalothrin (synthetic pyrethroids) are the most commonly applied insecticides for vector control in the public health sector. Insecticide susceptibility of A. culicifacies to DDT, malathion and �methrin was evaluated in 2009 in several districts of India by adopting the WHO standard protocol for adult susceptibility (WHO, 1998). It was found that A. culicifacies has developed resistance to all insecticides tested (Mishra et al., 2012). Currently, malaria control largely relies on a limited arsenal of materials; viz., artemisinin derivative drugs and pyrethroids. However, these products could also become ineffective due to the continuing evolution of resistance development. In this con- text, a new innovative user- and eco-friendly alternative vector control strategy is mandatory. A search for powerful contextual community- based vector control interventions is therefore warranted. Correspondence: Kalimuthu Kovendan, Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore - 641 046, India. Tel.: +91.9962447932 - Fax: +91.422.2422387. E-mail: gokulsuryah@gmail.com Key words: Abutilon indicum, Hyptis suaveolens, Leucas aspera, Anopheles culicifiacies, larvicidal activity. Funding: the authors are thankful to Science Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India, New Delhi (SR/FT/LS-156/2012) for providing financial support for the present work. Acknowledgements: the authors are grateful to Mr. M. Munirathnam, (ICMR, Madurai), Taxonomy and Field Station in Coimbatore, Tamil Nadu, for help- ing in mosquito collection and identifying mosquito species of samples for the experiment work. Received for publication: 13 June 2013. Revision received: 18 April 2014. Accepted for publication: 12 May 2014. ©Copyright K. Kovendan et al., 2014 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2014; 46:1747 doi:10.4081/jear.2014.1747 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. Larvicidal activity of indigenous plant extracts on the rural malarial vector, Anopheles culicifacies Giles. (Diptera: Culicidae) K. Kovendan,1 P. Mahesh Kumar,1 J. Subramaniam,1 K. Murugan,1 S. John William2 1Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore; 2P.G. Research & Department of Advanced Zoology and Biotechnology, Loyola College, Nungambakkam, Chennai, Tamil Nadu, India [page 90] [Journal of Entomological and Acarological Research 2014; 46:1747] Journal of Entomological and Acarological Research 2014; volume 46:1747 Jear_2014_3_Hrev_master 16/12/14 10:38 Pagina 90 No n- co mm er cia l u se on ly Abutilon indicum (Malvaceae), which is commonly known as Thuthi (the vernacular name in Tamil), is distributed throughout the driest areas in India (Chopra et al., 1992). It is well known and reputable in the Tamil traditional medicinal system called Siddha as a phytothera- peutic agent against various illnesses such as jaundice, piles, ulcers and leprosy (Yoganarasimhan, 2000). It is also reported to possess effective analgesic activity; a study by Ahmed et al. (2000) indicated that an 80% ethanol root extract of A. indicum had a potential effect against A. aegypti and guppy fish (Promsiri et al., 2006). The larvicidal activity of crude hexane, ethyl acetate, petroleum ether, acetone, and methanol extracts of A. indicum, Aegle marmelos, Euphorbia thymifo- lia, Jatropha gossypifolia and Solanum torvum were evaluated (Rahuman, 2008). Although Hyptis suaveolens is a native plant of trop- ical America, it is also widespread in tropical Africa, Asia and Australia. It grows under a wide range of soil and climatic conditions, mainly in warm areas of the country. H. suaveolens is also administered as a traditional medicine for treat- ment of various ailments, and its essential oil possesses insecticidal and larvicidal properties (Peerzada, 1997; Azevedo et al., 2001). In Nigeria, ethanol extracts of orange peel (Citrus sinensis) and bush tea leaves of H. suaveolens were compared for their toxicity against A. aegypti (Amusan et al., 2005). It has been reported that H. suaveolens extract causes notable mortality of A. aegypti larvae because the extract also con- tained insecticidal compounds such as a-tepinoline, a monoterpene that is similar in action to d-limonone, which is present in C. sinensis. Leucas aspera (Labiatae) is a small herbaceous plant. It is commonly administered as an antipyretic herb in South India. The juice of its leaves is used for psoriasis and swellings as an external application. The plant extract mixed with honey is a good remedy for stomach pain and indiges- tion. Preliminary chemical examination of L. aspera revealed the pres- ence of triterpenoids (Kamar & Singh, 1994). The whole plant is report- ed to contain oleanolic acid, ursolic acid, and 3-itosterol (Chaudhury & Ghosh, 1969). Aerial parts are reported to contain nicotine (Mangathayaru et al., 2006). The flower is reported to contain ten com- pounds, among them amyl propionate (15.2%) and isoamyl propionate (14.4%), which were dominant (Kalachaveedu et al., 2006). L. aspera leaves are used as an insecticide and mosquito repellent in rural areas (Kiritikar and Basu, 1990; Sadhu et al., 2003). The hexane crude extracts of L. aspera showed high larvicidal activity against C. quinquefasciatus and A. aegypti (Maheswaran et al., 2008; Kovendan et al., 2012b). The aim of the present investigation was to determine the effect of A. indicum, H. suaveolens and L. aspera leaf extracts against third-stage lar- vae of A. culicifacies as a target species. Materials and methods Collection and rearing of mosquitoes Larvae of A. culicifacies were collected from Kallar village, near Mettupalayam, Tamil Nadu, in different breeding habitats in using an O type brush. The mosquito larvae were fed with pedigree dog biscuits and yeast in a 3:1 ratio. Feeding was continued until the larvae trans- formed into the pupal stage. The pupae were collected from the culture trays using a dipper and transferred to plastic containers (12×12 cm) containing 500 mL of water. The plastic jars were kept in a 90×90×90- cm mosquito cage for adult emergence. Mosquito larvae were main- tained at 27+2°C, 75-85% relative humidity, under a photoperiod of 14:10 (light/dark). A 10% sugar solution was provided for a period of 3 days before blood feeding. The adult female mosquitoes were allowed to feed on the blood of a rabbit (one rabbit per day, exposed on the dor- sal side) for 2 days to ensure adequate blood feeding for 5 days. After blood feeding, enamel trays with water from the culture trays were placed in the cage as oviposition substrates. Collection of plants and preparation of plant extracts The selected medicinal plants were collected in and around Maruthamalai hills and Bharathiar University Campus, Coimbatore, Tamil Nadu. The fresh aerial part of A. indicum, and fresh leaves of H. suaveolens and L. aspera were washed thoroughly with tap water and shade dried at room temperature (28±2°C) for 5 to 12 days. The air- dried materials were powdered separately using a commercial electric blender. From each plant, 300 g of powdered material was macerated with 1.0 L of hexane, diethyl ether, ethyl acetate and acetone, sequen- tially, for a period of 72 h each and filtered. The yield of the A. indicum, H. suaveolens and L. aspera crude extracts with hexane, diethyl ether, ethyl acetate and acetone, were: A. indicum 8.94, 10.15, 9.36 and 11.55 g and H. suaveolens 8.12, 9,66, 10.47, and 9.73 g and L. aspera 11.12, 8.29, 9.34 and 10.13 g, respectively. The extracts were concentrated at a reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4°C. One gram of the plant residue was dissolved in 100 mL of acetone, which was considered as a 1% stock solution, from which concentrations were prepared ranging from 250, 500, 750 and 1000 ppm, respectively. Larval toxicity test Larvicidal activity was assessed using the procedure of WHO (1996) with slight modifications. A laboratory colony of A. culicifacies larvae was used for the larvicidal activity. Twenty-five third-instar larvae of A. culicifiacies were kept in 250-mL glass containers, containing 200 mL of dechlorinated water. Five replicates were set up for each concentra- tion (250, 500, 750, 1000 ppm) and mixed with acetone and Triton-80 (mixing solution). Larval mortality was assessed at 24, 48 and 72 h. Each experiment was replicated 3 times at room temperature (28±2°C) for three different plants. The control mortalities were corrected by using Abbott’s formula (Abbott, 1925). Corrected mortality = Observed mortality in treatment – Observed mortality in control ¥ 100 100 – Control mortality (1) Percentage mortality = Number of dead larvae ¥ 100 (2) Number of larvae introduced The lethal concentrations (LC) at 50% and 90% were calculated from toxicity data using probit analysis (Finney, 1971). Statistical analysis The average larval mortality data were subjected to probit analysis for calculating LC50 and LC90, and other statistics at a 95% upper fiduci- dal limit and lower fiducidal limit, and chi-square values were calculat- ed using SPSS 9.0 version (Statistical software package; StataCorp., College Station, TX, USA). Results at P<0.05 were considered to be sta- tistically significant. Results Preliminary screening is a good means of evaluating the potential lar- vicidal activity of crude plant extracts, which is often assessed using dif- ferent solvent extracts. Mortality from the three plants tested is present- ed in Tables 1-3. At 24 h, A. indicum demonstrated mortality levels in hexane, diethyl ether, ethyl acetate and actone extracts ranging from 22.18, 27.32, 32.55 and 41.23% at 24 h at 250 ppm to 51.65, 56.12, 61.55 and 64.35% at 1000 ppm, respectively (Table 1). For H. suaveolens, mor- tality levels at 24 h from these extracts ranged from 41.15, 46.33, 50.60 and 52.15% at 250 ppm, to 77.18, 83.14, 88.15 and 90.77% at 1000 ppm, [Journal of Entomological and Acarological Research 2014; 46:1747] [page 91] Article Jear_2014_3_Hrev_master 16/12/14 10:38 Pagina 91 No n- co mm er cia l u se on ly [page 92] [Journal of Entomological and Acarological Research 2014; 46:1747] respectively (Table 2). Finally, for L. aspera, mortality at 24 h from these same extracts ranged from 31.12, 44.50, 46.66, and 49.15% at 250 ppm, to 74.18, 75.27, 87.55 and 89.26% at 1000 ppm, respectively (Table 3). We observed correspondingly higher larval mortality from these extracts at higher concentrations after 48 and 72 h, respectively (Tables 2 and 3). The LC50 and LC90 values of hexane, diethyl ether, ethyl acetate and acetone extracts of A. indicum, H. suaveolens and L. aspera at 24 were as follows: A. indicum, LC50=1031.65, 949.18, 833.58 and 673.68 ppm; and LC90=2215.87, 2234.39, 2152.97 and 2455.10 ppm, respectively. For H. suaveolens, LC50=423.00, 347.50, 236.58 and 217.24 ppm; and LC90=1431.91, 1292.15, 1138.49 and 1049.27 ppm, respectively. Finally, for L. aspera, LC50=559.77, 401.56, 299.71 and 263.01 ppm; and LC90=1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively (Tables 1-3). Discussion A. culicifacies is one of the major malaria vectors in the Indian sub- continent and is generally regarded as intolerant to salinity, preferring to breed in newly-dug freshwater pits, domestic wells and sites used for plantings of coconuts and casurina trees in India (Russell & Rao, 1942; Sabesan et al., 1986). Ansari et al. (2000) suggested that the pepper- mint oil (Mentha piperita) showed strong repellent action against adult mosquitoes when applied on human skin. The protection obtained against A. annularis, A. culicifacies, and C. quinquefasciatus was 100, 92.3, and 84.5%, respectively. The present investigation tested three plant leaf extracts in different solvents for their potential larvicidal activity against A. culicifacies. The biological activity of the experimen- tal plant extracts varied, which may be due to the presence of various phytochemically active compounds in the plants, including phenolics, terpenoides, flavonoids and alkaloids. These active principles may have jointly or independently influenced or contributed to produce larvicidal effects against A. culicifacies. Sharma & Ansari (1994) demonstrated the protective effect of Cyperus rotundus against A. culicifacies, A. stephensi and C. quinque- fasciatus, and showed greater protection than neem oil (37.5%). Sharma et al. (1995), reported that the crude extract of Solanum nigram leaves showed significant larvicidal activity against A. culcifa- cies, C. quinquefasciatus and A. aegypti at a dose equivalent to the LC90, ranging from 0.18 to 0.21% (Singh et al., 2002). The present results concur with some of the previous findings of A. indicum against the third instar larvae of A. culicifacies, with LC50 and LC90 values of hexa- ne, diethyl ether, ethyl acetate and acetone extracts of (LC50) 1031.65, 949.18, 833.58 and 673.68 ppm, and (LC90) 2215.87, 2234.39, 2152.97 and 2455.10 ppm at 24 h, respectively. One study reported that the lethal concentration values of the aqueous extract of roots of H. abelmoschus against the larvae of A. culicifacies, A. stephensi, and C. quinquefasciatus were 52.3, 52.6, and 43.8 ppm, respec- tively (Dua et al., 2006). The LC50 and LC90 values of S. indicus, C. colli- nus and M. koenigii against third-instar larvae at 24, 48 and 72 h (in hexane, chloroform and ethyl acetate extracts) were: for S. indicus, (LC50) 544.93, 377.86 and 274.79 ppm, and (LC90) 1325.32, 1572.55 and 1081.29 ppm at 24 h; for C. collinus, (LC50 ) 375.34, 318.29 and 226.10 ppm, and (LC90) 699.65, 1577.62 and 1024.92 ppm at 24 h; and, for M. koenigii, (LC50) 963.53, 924.85 and 857.62 ppm, and (LC90) 1665.12, 1624.68 and 1564.37 ppm at 24 h, respectively (Kovendan et al., 2012a). Article Table 1. Larvicidal activity of Abutilon indicum against third instars larvae of Anopheles culicifacies. Exposure Solvents Percentage larval mortality±SD LC50 95% confidence limit x2 (time) Concentration of A. indicum (ppm) (LC90) LFL UFL (df=4) 250 500 750 1000 LC50 (LC90) LC50 (LC90) Hexane 22.18±0.86 26.31±1.12 34.56±0.98 51.65±1.50 1031.65 877.34 1360.29 1.39a (2215.87) (1733.59) (3414.23) Diethyl ether 27.32±0.42 30.40±0.65 37.19±1.32 56.12±1.25 949.18 802.18 1260.00 2.41a (2234.39) (1722.60) (3594.44) 24 h Ethyl acetate 32.55±1.23 34.12±1.45 40.66±0.44 61.55±1.18 833.58 700.82 1076.37 3.78a (2152.97) (1659.17) (3484.70) Acetone 41.23±1.65 43.65±0.68 45.22±1.36 64.35±1.33 673.68 479.50 928.05 3.52a (2455.10) (1742.72) (5435.70) 655.69 561.41 757.04 Hexane 30.63±0.54 38.50±1.69 55.60±1.74 68.92±1.95 (1586.80) (1333.98) (2079.68) 0.56a 592.82 487.55 690.49 Diethyl ether 34.12±1.55 42.26±0.72 58.77±0.95 71.23±1.27 (1556.39) (1303.21) (2061.01) 0.48a 48 h 444.90 255.91 562.52 Ethyl acetate 44.35±1.21 49.22±1.56 61.10±1.22 74.88±1.48 (1625.26) (1310.21) (2373.37) 1.10a 274.54 69.32 392.03 Acetone 50.56±1.35 63.91±1.75 71.25±1.66 85.45±1.30 (1207.15) (1026.23) (1561.63) 0.76a Hexane 48.61±1.52 65.22±1.88 84.14±1.47 91.35±1.32 273.83 140.65 362.35 0.46a (935.20) (831.15) (1100.14) Diethyl ether 51.70±0.80 70.50±1.95 91.36±1.93 96.50±1.40 245.95 136.20 321.82 0.99a (764.39) (688.68) (873.81) 72 h Ethyl acetate 58.44±1.65 79.20±1.20 93.18±1.65 98.42±1.91 230.45 130.83 299.81 0.14a (676.51) (611.13) (767.98) Acetone 61.12±1.40 80.10±1.33 96.54±1.21 100.00±0.00 179.87 66.42 253.45 1.95a (594.49) (532.79) (681.76) Control, nil mortality; SD, standard deviation; LC50, LC90, lethal concentration at 50% and 90%; LFL, lower fiducidal limit; UFL, upper fiducidal limit; x2, Chi-square value; df, degrees of freedom. Mean values of five replicates. aSignificant at P<0.05 level. Jear_2014_3_Hrev_master 16/12/14 10:38 Pagina 92 No n- co mm er cia l u se on ly [Journal of Entomological and Acarological Research 2014; 46:1747] [page 93] Article Table 2. Larvicidal activity of Hyptis suaveolens against the third instars larvae of Anopheles culicifacies. Exposure Solvents Percentage larval mortality±SD LC50 95% confidence limit x2 (time) Concentration of H. suaveolens (ppm) (LC90) LFL UFL (df=4) 250 500 750 1000 LC50 (LC90) LC50 (LC90) Hexane 41.15±0.94 54.59±1.23 65.20±1.13 77.18±1.19 423.00 262.74 527.13 0.06a (1431.91) (1195.44) (1920.40) Diethyl ether 46.33±1.89 57.16±0.88 68.15±0.92 83.14±1.93 347.50 171.86 454.55 0.72a (1292.15) (1094.04) (1682.95) 24 h Ethyl acetate 50.60±1.51 67.24±0.85 72.13±1.80 88.15±1.37 236.58 22.09 357.75 1.83a (1138.49) (973.62) (1454.65) Acetone 52.15±0.88 69.10±1.46 74.78±1.69 90.77±0.98 217.24 15.04 334.37 2.00a (1049.27) (907.98) (1305.85) 367.14 191.69 474.69 Hexane 43.18±0.56 58.14±0.84 68.93±0.92 79.50±1.25 (1343.81) (1130.97) (1772.94) 0.08a 284.56 86.93 399.26 Diethyl ether 48.50±1.16 63.20±0.91 70.18±0.79 86.00±1.77 (1207.01) (1027.61) (1555.88) 1.19a 48 h 219.38 9.19 339.44 Ethyl acetate 51.65±1.27 68.15±1.85 75.21±1.54 89.14±0.69 (1081.48) (931.45) (1360.06) 1.00a 196.53 4.99 309.95 Acetone 54.01±0.85 70.36±0.78 79.36±1.10 92.56±0.75 (962.17) (841.05) (1170.61) 0.91a Hexane 47.10±1.27 65.48±1.35 77.23±1.16 84.78±1.68 268.78 80.81 380.42 0.51a (1134.84) (977.06) (1426.91) Diethyl ether 57.03±0.78 79.85±1.18 85.55±0.77 98.32±0.88 164.33 7.30 262.41 3.56a (751.50) (668.80) (875.67) 72 h Ethyl acetate 70.01±0.96 97.36±0.89 100.0±0.00 100.0±0.00 158.80 56.706 211.84 0.04a (382.51) (339.78) (452.69) Acetone 75.13±0.70 98.56±0.00 100.00±0.00 100.00±0.00 138.30 6.89 195.95 0.01a (349.48) (307.90) (421.39) Control, nil mortality; SD, standard deviation; LC50, LC90, lethal concentration at 50% and 90%; LFL, lower fiducidal limit; UFL, upper fiducidal limit; x2, Chi-square value; df, degrees of freedom. Mean values of five replicates. aSignificant at P<0.05 level. Table 3. Larvicidal activity of Leucas aspera against third instars larvae of Anopheles culicifacies. Exposure Solvents Percentage larval mortality±SD LC50 95% confidence limit x2 (time) Concentration of L. aspera (ppm) (LC90) LFL UFL (df=4) 250 500 750 1000 LC50 (LC90) LC50 (LC90) Hexane 31.12±1.17 47.12±1.45 62.14±1.88 74.18±1.25 559.77 464.21 643.56 0.09a (1400.80) (1203.49) (1758.85) Diethyl ether 44.50±1.20 52.43±1.94 65.36±1.70 75.27±1.33 401.56 198.85 520.40 0.22a (1549.31) (1259.51) (2219.35) 24 h Ethyl acetate 46.66±1.32 65.16±1.80 69.52±1.57 87.55±0.88 299.71 126.24 405.43 2.48a (1157.96) (996.97) (1455.63) Acetone 49.15±1.50 66.98±1.57 71.55±1.36 89.26±1.44 263.01 77.46 373.42 2.56a (1108.72) (956.98) (1387.02) 426.33 279.40 524.96 Hexane 37.92±0.95 54.35±1.65 68.12±1.30 77.10±1.65 (1377.58) (1163.25) (1800.20) 0.14a 312.92 131.45 421.96 Diethyl ether 45.42±0.77 61.72±1.10 73.50±1.55 82.64±1.93 (1223.97) (1044.38) (1568.28) 0.16a 48 h 253.69 81.55 359.15 Ethyl acetate 48.16±1.13 69.41±1.72 76.92±1.42 89.18±1.18 (1038.54) (906.06) (1269.33) 0.95a 183.68 22.88 284.62 Acetone 51.75±1.95 78.56±1.23 87.30±1.22 93.48±1.35 (824.85) (732.59) (967.34) 2.14a Hexane 45.60±1.26 61.54±0.90 73.81±1.18 87.36±1.44 325.57 178.16 420.93 0.38a (1119.70) (974.76) (1374.18) Diethyl ether 52.76±1.17 79.30±1.29 91.10±1.94 94.75±1.20 183.77 39.44 276.83 2.18a (761.60) (680.09) (881.77) 72 h Ethyl acetate 63.15±0.92 86.25±1.12 96.63±1.61 100.00±0.00 144.51 14.56 225.14 0.63a (560.83) (499.23) (646.87) Acetone 88.80±1.13 93.24±1.50 100.00±0.00 100.00±0.00 138.24 11.798 211.30 2.54a (478.13) (423.46) (557.32) Control, nil mortality; SD, standard deviation; LC50, LC90, lethal concentration at 50% and 90%; LFL, lower fiducidal limit; UFL, upper fiducidal limit; x2, Chi-square value; df, degrees of freedom. Mean values of five replicates. aSignificant at P<0.05 level. Jear_2014_3_Hrev_master 16/12/14 10:38 Pagina 93 No n- co mm er cia l u se on ly [page 94] [Journal of Entomological and Acarological Research 2014; 46:1747] It is known that placing H. suaveolens branches or whole plants in and around houses is one of the most effective methods in western Kenya, for repelling the malaria vector Anopheles gambiae Giles (Seyoum et al., 2002). Kovendan et al. (2012b) reported on the effects of hexane, chloro- form, ethyl acetate and methanol extracts of J. curcas against third instar larvae of C. quinquefasciatus, with LC50 values of 230.32, 212.85, 192.07 and 113.23 ppm, respectively. For H. suaveolens, LC50 values of these extracts were 213.09, 217.64, 167.59 and 86.93 ppm, respectively. For A. indicum, the LC50 values were 204.18, 155.53, 166.32 and 111.58 ppm, respectively. Finally, for L. aspera, LC50 values were 152.18, 118.29, 111.43 and 107.73 ppm, respectively. Similarly, our results with H. suaveolens against the third-instar larvae of A. culicifacies showed LC50 and LC90 val- ues of hexane, diethyl ether, ethyl acetate and acetone extracts of (LC50) 423.00, 347.50, 236.58 and 217.24 ppm, and (LC90) 1,431.91, 1292.15, 1138.49 and 1049.27 ppm at 24 h, respectively. Previous studies have been conducted using a methanol extract of Clerodendron inerme and Acanthus ilicifolius at different concentra- tions (20, 40, 60, 80 and 100 ppm), with LC50 values against A. stephen- si first to fourth-instar larvae and pupae of 55.04, 63.33, 73.05, 80.74 and 74.33 ppm, and 52.76, 57.76, 63.36, 70.18 and 62.78 ppm, respec- tively. Corresponding LC90 values were 125.50, 137.16, 153.55, 156.93, 199.20 ppm, and 108.30, 115.83, 125.24, 131.28 and 141.03 ppm, respec- tively (Kovendan & Murugan, 2011). Mahesh Kumar et al. (2012) reported the LC50 and LC90 values of S. xanthocarpum against the first to fourth instar larvae and pupae of C. quinquefasciatus as 155.29, 198.32, 271.12, 377.44, and 448.41 ppm, and 687.14, 913.10, 1,011.89, 1,058.85, and 1,141.65 ppm, respectively. In the present results, the hexane, diethyl ether, ethyl acetate and acetone extracts of L. aspera against third-instar larvae of A. culicifacies had LC50 values of 559.77, 401.56, 299.71 and 263.01 ppm, and LC90 values of 1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively. Conclusions The larvicidal properties of crude extracts of A. indicum, H. suave- olens and L. aspera against A. culicifacies were studied under laborato- ry conditions. The results clearly demonstrated the highest mortality with H. suaveolens, followed by L. aspera and A. indicum. In addition, the study also showed that the solvents used for the extractions have some impact on the level of larval mortality. 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[Journal of Entomological and Acarological Research 2014; 46:1747] [page 95] Article Jear_2014_3_Hrev_master 16/12/14 10:38 Pagina 95 No n- co mm er cia l u se on ly