J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 305 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Screening of Methanolic Plant Extracts against Larvae of Aedes aegypti and Anopheles stephensi in Mysore *Thirumalapura Krishnaiah Mohankumar, Kumuda Sathigal Shivanna, Vijayan Valiakottukal Achuttan Department of Studies in Zoology, University of Mysore, Mansagangothri, Mysore, Karnataka, India (Received 25 Feb 2014; accepted 6 Dec 2014) Abstract Background: Mosquitoes transmit serious human diseases, causing millions of death every year. Vector control is facing a threat due to the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. Nine different locally available medicinally important plants suspected to posse larvicidal property were screened against fourth instar larvae of Aedes aegypti and Anoph- eles stephensi to a series of concentrations of the methanolic extracts. Methods: Susceptibility tests on Ae. aegypti and An. stephensi were conducted using standard WHO methods. The larvae of two mosquito species were exposed to methanolic extracts and mortality counts were made after 24 hours of exposure as per WHO method. Larvae of Ae. aegypti were more susceptible than that of An. stephensi. Results: Among the nine plant species tested, Annona reticulata leaf extract was more effective against Ae. aegypti larvae with LC50 and LC90 values of 95.24 and 262.64 ppm respectively and against An. stephensi larvae 262.71 and 636.94 ppm respectively. The least efficacy was in Cosmos bipinnatus with LC50 and LC90 values of 442.6 and 1225.93 ppm against Ae. aegypti and LC50 and LC90 values of 840.69 and 1334.01 ppm of Thespesia populnea against An. stephensi. Conclusion: The crude methanolic extract of the An. reticulata with good larvicidal efficacy could be considered for further characterization to control mosquito vectors instead of chemical insecticides. High efficacy found in An. re- ticulata extract will be considered for further studies to isolate the bioactive compound. Keywords: Ae. aegypti, An. stephensi, Annona reticulata, Larvicide, Thespesia populnea Introduction Many new and re-emerging diseases are transmitted by Arthropod vectors (Brogdon and Mc Alister 1998). “Vector borne diseases account for around 17% of the estimated glob- al burden of all infectious diseases” (WHO 2006). Over 350 species of mosquitoes are vectors of pathogens that cause diseases in humans and domesticated animals. More than fourteen mosquito genera are known to har- bour arboviruses (Mattingly 1973). “These dis- eases contribute significantly to disease bur- den, death, poverty and social debility in tropi- cal countries” (Yang et al. 2004). The prolif- eration of the diseases is not only due to the higher number of breeding places in urban agglomeration, but also due to increasing re- sistance of mosquitoes to current commercial insecticides such as organo-chlorides, organo- phosphates, carbamates, pyrethroids and also to biological insecticides (Goettel et al. 1992, Das and Amalraj 1997, Yadav et al. 1997). Thus, mosquitoes are responsible for the transmission of more diseases than any other group of arthropods and play an important role as etiologic agents of devastating malar- ia, filariasis, dengue, yellow fever, Japanese encephalitis, chikungunya and other viral dis- eases. In addition, they also cause irritation *Corresponding authors: Thirumalapura Krishnaiah Mohankumar, E-mail: mohanvbrl2012@gmail.com J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 306 http://jad.tums.ac.ir Published Online: January 06, 2016 to human by causing allergic responses that include local skin reactions as well as sys- temic reactions such as angioedema and ur- ticaria (Peng et al. 1999). Aedes aegypti, a vector of yellow fever, dengue and chikungu- nya is widely distributed in the tropical and subtropical zones. About two-third of the world’s population, live in areas infested with dengue vectors, mainly Ae. aegypti (Hahn et al. 2001). Anopheles stephensi is the primary vector of malaria in India and other west Asian countries. Every year, an esti- mated 300–500 million new infections and 600,000 cases based on world malaria report 2013 (WHO 2013) deaths result from ma- laria worldwide. In the past decade, chikungunya - a virus transmitted by Ae. spp. mosquitoes - has re- emerged in Africa, southern and southeast- ern Asia, and the Indian Ocean Islands as the cause of large outbreaks of human disease (Burt et al. 2012). Malaria is a protozoan infection of erythrocytes caused in human beings by five species of the genus Plasmo- dium (P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi). In most cases, ma- laria is transmitted via the bite of an infected female anopheline mosquito, but congenital malaria and acquisition through infected blood transfusion are well described (Falade et al. 2007). “More than 40 per cent of the world’s population-approximately 3 billion people are exposed to malaria in 108 endemic countries “(WHO 2009). About one million cases of malaria are reported in India every year. Many approaches have been developed to control mosquito menace. One such approach to prevent mosquito borne disease is by em- ploying larvicide. The current mosquito con- trol approach is based on synthetic insecti- cides. Even though their effectiveness, they created many problems like insecticide re- sistance (Liu et al. 2005), pollution and toxic side effects on human beings (Lixin et al. 2006). This has necessitated the need for re- search and development of environmentally safe, biodegradable, indigenous method for vector control. Botanicals offer great prom- ise as source of phyto-chemicals with proven potential as insecticides which can play an important role in the control of mosquitoes and in the interruption of disease transmis- sion at individual as well at community level (Mittal and Subbarao 2003). Six plant fami- lies with several representative species, Aster- acae, Cladophoraceae, Labiatae, Meliaceae, Oocystacae and Rutaceae appear to have the greatest potential for providing future mos- quito control agents. Insecticides of plant origin do not cause toxicity to human and domestic animals and are easily biodegrada- ble. In the present study leaf extract of the plant Passifloraceae, Annonaceae, Asteraceae, Lauraceae, Malvaceae, Lamiaceae was stud- ied for its insecticidal activity against ma- laria and dengue vectors. The application of botanical derivatives against mosquito has been reviewed in detailed by Sukumar et al. (1991). In this regard, of late the researchers have shown more interest on plant derivatives as botanicals offer great promise as sources of molecules for the control of both agricultural pests and medically important insect species. Now a days, the increased use of phyto- chemicals for the control of these insects may be attributed to the fact that population throughout the world are aware of the danger inherent in conventional insecticides, partic- ularly the detrimental effect on the environ- ment (Pitasawat et al. 1998). It is in this re- gard, plant derived products have received increased attention from scientists and more than 2000 plant species are already known to have insecticidal properties (Balandrin et al. 1985, Sukumar et al. 1991). Natural insecti- cides such as pyrethrum, rotenone and nico- tine among others have been extensively used until recently for insect control (Balan- drin et al. 1985). To search derivatives with insecticidal property from local plants, the present in- J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 307 http://jad.tums.ac.ir Published Online: January 06, 2016 vestigation has been made in the Vector Bi- ology Research Lab, Department of Studies in Zoology at University of Mysore. Materials and Methods Aedes aegypti and An. stephensi larvae available at the mosquito colony maintained in Vector Biology Research Lab, Department of Studies in Zoology, University of Mysore by following standard rearing techniques. The larvae were reared in large enamel or plastic trays (30x24x5 cm) containing dechlo- rinated water and fed using finely powdered dog biscuits and dry yeast in the ratio of 2: 1. Plant material and extraction Nine plant species as listed in Table 1 were collected from in and around Mysore, Karnataka, from March 2012 to Jan 2013 and the leaves were shade dried, powdered manually and subjected to methanol solvent extraction in a Soxhlet apparatus until ex- haustion, to obtain non-polar bioactive con- stituents. The pooled extract was evaporated in a rotory vacuum evaporator at 40 ºC to dryness and stored at 4 ºC in an air tight bot- tle for further analysis. This was later dis- solved in acetone and employed to prepare different concentrations. Larval bioassay Bioassays on mosquito larvae were per- formed on late third or early fourth instars, according to standard guidelines of WHO (2005). The required quantity of plant ex- tract of different concentrations was pre- pared in acetone as solvent. One ml of each of the concentration was mixed thoroughly with 249 ml of dechlorinated water in 500 ml glass beakers. Larvae were exposed in an ascending series of five concentrations ac- cording to log dose (Table 2). Parallel con- trol tests were also maintained by adding 1ml of the solvent to 249 ml of dechlorin- ated water. Twenty five early fourth instar larvae were transferred to each of the beakers. A minimum of three replicates were kept for each concentration along with the control. Observation for the dead or moribund larvae was carried out after 24 h duration at 25±2 ºC and 75±5 % of relative humidity (RH). Data analysis Larval mortality counts were adjusted for the mortality in control, if any, by employing Abbott’s formula (Abbott 1975) to give an estimate of the plant extract attributable mortality. The corrected mortality data were subjected to regression analysis of probit mortality on log dosage (Finney 1971). The significant difference in LC50 is based on the non-overlapping of 95 % Fiducial limits. Results Table 2 provides the mortality rate of mosquito larvae against different concentra- tions of methanol extracts. Table 3 and 4 provide the efficacy of methanol extracts tested against the dengue vector Ae. aegypti and malarial vector An. stephensi larvae. Fig. 1 and 2 show the log dose- probit mortality responses of all plant extracts. Though the plants exhibited larvicidal activity against the two mosquito species, out of the nine plants screened using methanol as solvent, the An- nona reticulata (Annonaceae) was found to possess better larvicidal activity against Ae. aegypti followed by An. stephensi. The high percentage of mortality with low concentra- tion was recorded of in the species against Ae. aegypti has been with LC50 and LC90 values being 95.24 and 262.64 ppm respec- tively (Table 3). Likewise LC50 and LC90 values against An. stephensi are 262.71 and 636.94 ppm respectively (Table 4). The 95% Fiducial Limits (FL) of An. reticulata for LC50 is 46.83–139.69 and 95% FL for LC90 is 172.23–931.30 and the slope is 2.90±0.55 J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 308 http://jad.tums.ac.ir Published Online: January 06, 2016 against Ae. aegypti. Similarly, the 95% FL for LC50 is 197.21–137.54 and for LC90 it is 457.41-1434.98 with the slope being 3.33±0.49 against An. stephensi. Among these two mosquito species, susceptibility of Ae. aegypti was found to be significantly more than that of An. stephensi. Further, the larvicidal efficacy of An. reticulata was found to be significant by more compared to other plants (P< 0.05). The figure depicts the log dose-probit mortality responses and slope regression lines of tested plants. 800.00600.00400.00200.000.00 Concentrtaion 100.00 80.00 60.00 40.00 20.00 0.00 M or ta lit y Thesp Passifl Laurus Hyptis Gossip Cosmos An.ret An.glab Abutilon Thesp Passifl Laurus Hyptis Gossip Cosmos An.ret An.glab Abutilon Plants R Sq Linear = 0.957 R Sq Linear = 0.963 R Sq Linear = 0.982 R Sq Linear = 0.98 R Sq Linear = 0.999 R Sq Linear = 0.976R Sq Linear = 0.93R Sq Linear = 0.999R Sq Linear = 1 Fig. 1. Regression graph showing mortality of methanolic extracts of different plant species on Aedes aegypti J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 309 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 1. List of plant screened against Aedes aegypti and Anopheles stephensi No Plants name Family Medicinal/ toxic property 1 Passiflora foetida Passifloraceae Pitta, inflammation,insomnia, depression and anxiety disorders 2 Annona glabra Annonaceae Anticancer effects, substantial antimicrobial, antifungal and moderate insec- ticidal, sporicidal and cytotoxic 3 Cosmos bipinnatus Asteraceae Jaundice, intermittent fever and splenomegaly 4 Laurus nobilis Lauraceae rheumatism and dermatitis 5 Abutilon indicum Malvaceae Febrifuge, anthelmintic, antiemetic, anti-inflammatory, and in urinary and uterine discharges, piles, and lumbago 6 Gossypium herbaceum Annonaceae Febrifuge, anthelmintic, antiemetic, anti-inflammatory. 7 Annona reticulata Annonaceae Febrifuge, anthelmintic, antiemetic, anti-inflammatory, and in urinary and uterine discharges, piles, and lumbago 8 Hyptis suoveolens Lamiaceae Gastrointestinal infections, cramps, and pain, skin infections 9 Thespesia populnea Malvaceae Antifertility, antibacterial, antiinflammatory, antioxidant, purgative and hepatoprotective activity Table 2. Concentration and Mortality of Nine plant species against Aedes aegypti and Anopheles stephensi No Plant Name Aedes aegypti Anopheles stephensi Concentration(ppm) Mortality (%) Concentration(ppm) Mortality (%) 1 Passiflora foetida 100 150 200 250 300 14 36 56 78 96 200 350 500 650 800 12 30 54 74 90 2 Annona glabra 100 150 250 400 600 16 34 60 74 90 50 200 400 550 700 10 28 48 70 92 3 Cosmos bipinnatus 150 300 450 600 750 10 28 52 64 76 50 200 350 500 750 12 30 52 78 90 4 Laurus nobilis 100 250 350 500 700 12 28 58 78 88 100 250 400 750 900 14 36 52 74 92 J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 310 http://jad.tums.ac.ir Published Online: January 06, 2016 5 Abutilon indicum 100 300 450 550 750 12 28 62 78 92 150 300 450 700 750 16 36 50 76 90 6 Gossypium herbaceum 150 200 250 300 350 10 34 62 76 90 100 250 400 550 800 08 30 48 74 88 7 Annona reticulata 30 100 150 200 250 12 40 68 84 96 100 200 300 400 500 12 30 52 70 90 8 Hyptis suoveolens 150 250 350 400 550 12 32 48 66 82 150 300 450 600 750 08 32 50 76 94 9 Thespesia populnea 100 250 400 550 700 10 28 48 68 90 500 700 900 1100 1300 10 30 52 78 92 Table 3. Efficacy of methanolic extracts of nine plant species against Aedes aegypti No Plant name LC50 (ppm) 95% FL LC90 (ppm) 95% FL Slope±SE Heterogeneity (df) Regression equation 1 Annona reticulata* 95.24 46.83-139.69 262.64 172.23-931.30 2.90±0.55 5.20(3) Y=2.90X±0.75 2 Annona glabra 114.47 54.99-191.18 320.82 191.84-3738.15 2.86±0.64 6.98(3) Y=2.86X±0.89 3 Passiflora foetida 172.31 143.20-200.36 300.99 96.24-242.06 5.29±0.70 2.73(3) Y=5.29X±6.83 4 Gossypium herbaceum 216.23 194.57-239.21 620 524.18-778.30 2.79±0.24 1(3) Y=2.79X±1.53 5 Laurus nobilis 302.66 207.85-409.02 813.94 555.81-2193.14 2.98±0.47 3.35(3) Y=2.98X±2.39 6 Hyptis suaveolens 327.18 303.55-352.76 720.37 624.61-880.33 3.73±0.35 1(3) Y=3.73X±4.40 7 Abutilon indicum 332.98 123.63-546.59 903.76 549.46-692.12 2.95±0.71 7.40(3) Y=2.95X±2.45 8 Thespesia populnea 353.07 236.81-498.69 969.48 635.18-3376.91 2.92±0.50 3.79(3) Y=2.92X±2.44 9 Cosmos bipinnatus 442.60 402.11-488.40 1225.93 1013.27-2710 2.89±0.28 1(3) Y=2.89X±2.66 Note: LC50 - median lethal concentration, FL - fiducial limits; LC90 - lethal concentration, df - degree of freedom. *Difference in LC50 from the extracts of other eight plants is significant based on non-overlapping 95% fiducial limits (P< 0.05). Table 2. Continued… J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 311 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 4. Efficacy of methanolic extracts of nine plant species against Anopheles stephensi No plant name LC50 (ppm) 95% FL LC90 (ppm) 95% FL Slope±SE Heterogeneity (df) Regression equation 1 Annona reticulata* 262.71 197.21-337.54 636.94 457.41-1434.98 3.33±0.49 2.89(3) Y=3.33X±3.06 2 Cosmos bipinnatus 258.39 105.36-471.41 1048.00 544.56-1326.18 2.10±0.45 5.74(3) Y=2.10X±0.68 3 Gossypium herbaceum 298.80 62.13-768.79 1221.72 564.60-1130.12 2.09±0.55 8.40(3) Y=2.09X±0.18 4 Laurus nobilis 336.57 222.28-475.98 1197.75 757.60-3516.32 2.32±0.33 2.82(3) Y=2.32X±0.87 5 Abutilon indicum 372.81 231.41-532.21 1000.95 655.62-4054.35 2.98±0.56 4.51(3) Y=2.98X±2.68 6 Hyptis suaveolens 391.66 361.46-422.54 843.60 748.86-986.10 3.84±0.31 1(3) Y=3.84X±4.97 7 Passiflora foetida 441.01 409.18-473.62 922.27 820.34-1077.59 3.99±0.33 1(3) Y=3.99X±5.57 8 Annona glabra 448.78 338.06-570.24 900.18 675.62-1951.54 4.23±0.69 3.94(3) Y=4.23X±6.24 9 Thespesia populnea 840.69 801.16-880.57 1344.01 1249.14-1478.27 6.28±0.50 1(3) Y=6.28X±13.39 Note: LC50 median lethal concentration, FL, fiducial limits, LC90 lethal concentration, df, degree of freedom. *Difference in LC50 from the extracts of other eight plants is significant based on non-overlapping 95 % fiducial limits (P< 0.05). 1250.001000.00750.00500.00250.000.00 Concentration 100.00 80.00 60.00 40.00 20.00 0.00 M or ta lit y Thesp Passiflo Laurus Hyptis Gossip Cosmos Annona An.glabr Abutilon Thesp Passiflo Laurus Hyptis Gossip Cosmos Annona An.glabr Abutilon Plants R Sq Linear = 0.984 R Sq Linear = 0.984 R Sq Linear = 0.999 R Sq Linear = 0.946 R Sq Linear = 0.994 R Sq Linear = 0.995R Sq Linear = 0.977R Sq Linear = 0.996R Sq Linear = 0.997 Fig. 2. Regression graph showing mortality of methanolic extracts of different plant species on Anopheles Stephensi J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 312 http://jad.tums.ac.ir Published Online: January 06, 2016 Discussion The results of the larvicidal bioassay em- ploying different plant extracts against two different mosquito species (Table 3, 4) indi- cate significant larvicidal activity (P< 0.05) with methanol extract. The biological activ- ity of these plant extract may be due to vari- ous compounds such as, phenolics, terpe- noids, flavonoids and alkaloids (Gohil et al. 2010). Such compounds may jointly or inde- pendently contribute to produce toxic activ- ity against the mosquito species. Environmental safety of an insecticide is of paramount importance while employing against pests and vectors. An insecticide need not cause high mortality on target or- ganisms in order to be acceptable (Kabaru and Gichia 2001). Resistance to insecticides dates back to the beginning of application of chemicals, since DDT was initially intro- duced for mosquito control in 1946 and just in one year, the first case of DDT resistance occurred in Ae. tritaeniorhynchus and Ae. sollicitans (Hemingway and Ranson 2000). More than 500 species of arthropods are re- ported to be resistant to various insecticides (Shelton et al. 2007). In this regard, phyto- chemicals may serve as suitable alternatives to synthetic insecticides in future, as they are relatively safe, inexpensive and are readily available throughout the world. According to Bowers et al. (1995), the screening of locally available medicinal plants for mosquito con- trol will be cost effective, reduce depend- ence on expensive imported products and stimulate local efforts to enhance public health. It is in this regard, the present study adds to our knowledge on the efficacy of the locally available medicinal plants. An earlier report points that, ethanolic extract of Annona squamosa leaf has the most promising larvicidal activity against Cx. quinquefasciatus larvae (Das et al. 2007). The larvicidal and growth regulating activi- ties of Annona squamosa and Syzygium cumini two related species have been reported against An. stephensi and other mosquitoes (Saxena et al. 1993 and Kaushik and Saini 2008). The significant activity demonstrated by extracts of A. squamosa and A. senega- lensis suggest that the two plants may have strong killing effects against insects partic- ularly mosquitoes, hence giving a promising source of larvicidal agents (Magadula et al. 2009). Previously, a collection of A. squa- mosa plant materials from Brazil indicated larvicidal effect against Ae. albopictus and Culex quinquefasciatus (Das et al. 2007) and against An. stephensi (Saxena et al. 1993). However, no reports on the larvicidal effi- cacy are available on An. reticulata. By com- paring our results with the earlier studies, it is evident that the methanolic extracts of An. reticulata leaf have promising larvicidal ac- tivity against two mosquito species. It shows the LC50 value of 95.24 against Ae. aegypti and 262.71 against An. stephensi (Tables 3, 4). Karunamoorthi and Ilango (2010) have reported that theLC50 and LC90 values of methanol leaf extracts of Croton macrostach- yus (C. macrostachyus) were 89.25 and 224.98 ppm, respectively against late third instar larvae of malaria vector, An. arabiensis (An. arabiensis). The crude leaf extract of Aza- dirachta indica with different solvents, viz. benzene, chloroform, ethyl acetate and meth- anol were tested for larvicidal activity against An. stephensi. The LC50 values were 19.25, 27.26, 23.26 and 15.03, respectively. Kamaraj et al. (2009) have reported that the peel methanol extract C. sinensis, leaf and flower ethyl acetate extracts of Ocimum canum against larvae of An. stephensi (LC50 =95.74, 101.53, 28.96, LC90=303.20, 492.43 and 168.05 ppm) respectively. The highest larval mortality was found in methanol ex- tract of O. canum against the larvae of Ae. aegypti (LC50=99.42, 94.43 and 81.56 ppm) J Arthropod-Borne Dis, September 2016, 10(3): 305–316 TK Mohankumar et al.: Screening of … 313 http://jad.tums.ac.ir Published Online: January 06, 2016 and against Cx. quinquefasciatus (LC50= 44.54, 73.40 and 38.30 ppm), respectively (Kamaraj et al. 2008). An. stephensi and Ae. aegypti. Chowdhury et al. (2009) have re- ported that the chloroform and methanol extracts of mature leaves of Solanum vil- losum showed the LC50 value for all instars between 24.20 and 33.73 mg/l after 24 h and between 23.47 and 30.63 mg/l after 48 h of exposure period against An. subpictus. Go- vindarajan (2010) evaluated larvicidal ac- tivity of crude extract of Sida acuta against three important mosquitoes with LC50 values ranging between 38 and 48 mg/l. The crude extract had strong repellent action against the three species of mosquitoes as it pro- vided 100 per cent protection against An. stephensi for 180 min followed by Ae. ae- gypti (150 min) and Cx. quinquefasciatus (120 min). Dua et al. (2009) showed that the LC50 values of the neem oil were 1.6 and 1.7 ppm while LC90 values were 3.4 and 3.7 ppm against An. stephensi and Ae. aegypti re- spectively. Shivakumar and Kataria (2011) have reported that An. showed a high sus- ceptibility to very low concentrations of Aza- dirachta indica (1–5 ppm), whereas in the case of Ae. aegypti, the diagnostic concen- tration is slightly higher at 10ppm. The lar- vicidal action on An. stephensi, at a concen- tration of 3ppm resulted in 100% mortality within 72 h and a concentration of 15 ppm produces 90% mortality within 24 h of the treatment. In the present investigation out of the nine plants screened using methanol as solvent, Annona reticulata (Annonaceae) was found to possess better larvicidal activity against early fourth instar larvae of Ae. aegypti followed by An. stephensi with LC50 and LC90 values being 95.24 and 262.64 ppm respectively (Table 3). Likewise LC50 and LC90 values against An. stephensi are 262.71 and 636.94 ppm respectively (Table 4). Annona muricata was an active larvicide with a 48-hour LC50 value of 67.4 µ g/mL. (Jacobson 1958). He has further reported similar results for the seed extracts of an- other Annona species, namely A. cherimola, A. glabra and A. squamosa, which had lethal effects on larvae of Ae. sp. Its potential as a larvicidal plant was further supported by the results of a recent study by Satoto (1993), who has found that A. squamosa seed was one of the most effective larvicides against both Culex tritaeniorhynchus and Ae. aegypti. Earlier studies further indicated that crude extracts of leaves of Annona reticulata when evaluated for in vitro anthelmintic activity on Indian adult earth worms Eisinia fetida, a dose dependent inhibition of spontaneous motility (paralysis) of the worms was no- ticed (Sonal et al. 2011). There are no re- ports available on the toxicity of Annona re- ticulata against mosquito larvae. The present studies add that, apart from all medicinal property viz, antimicrobial, antitumoric, an- thelmintic, antibacterial, cytotoxic etc, mos- quito larvicidal property also present in this plant. Therefore, the methanolic extracts of this plant inhibiting the development of lar- val growth, indicates hopes for further char- acterization of the active compound in our lab. Conclusion The results from this study indicate that phyto-products isolated from the plants are more advisable to use for control of mos- quito borne diseases. These plants are re- markably economical and ecofriendly with more larvicidal properties. Among the plants screened An. reticulata showed high larvi- cidal efficacy against two mosquito species. Hence, An. reticulata will be selected for further chemical isolation of the active in- gredient in future studies. It could be consid- ered as a potent resource for controlling mosquito larvae. 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