J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 226 http://jad.tums.ac.ir Published Online: May 27, 2017 Original Article Evaluation of Larvicidal Activity of Essential Oil from Leaves of Coccinia grandis against Three Mosquito Species Shahid Iqbal Mohammed, Kishor Sukhlal Vishwakarma, *Vijay Laxminarayan Maheshwari School of Life Sciences, North Maharashtra University, Jalgaon, India (Received 2 Mar 2015; accepted 7 May 2016) Abstract Background: To study the chemical constituents and larvicidal activity of essential oil extracted from the leaves of Coccinia grandis against three mosquito species. Methods: Essential oil was extracted by hydro distillation using clevenger apparatus and was analyzed for chemical constituents by gas chromatography-mass spectrophotometry (GC-MS). Larvicidal activity was recorded after 12 and 24h of post-exposure against three mosquito species, Anopheles stephensi, Aedes aegypti and Culex quinquefascia- tus. Dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical re - gion. The LC50 and LC90 values for three mosquito larvae were calculated by Probit analysis. Results: The GC-MS analysis revealed that essential oil contains 23 different constituents. Out of these 23 constitu- ents, major constituents identified were n-tetracosane (39.18%), n-eicosane (30.04%), tetratriacotane (2.97%), 7-oc- tadecanal (2.81%), and tricosane (2.31%). Essential oil from leaves of Coccinia grandis exhibited significant larvi- cidal activity against An. stephensi with LC50 and LC90 values 39.41ppm and 123.24ppm, respectively. This was fol- lowed by Ae. aegypti and Cx. quinquefasciatus with LC50 and LC90 values of 48.20ppm, 131.84ppm and 52.80ppm, 135.48ppm, respectively after 24h of exposure. Conclusion: The results could be useful in developing a cost effective, ecofriendly, region specific and practical strategy for the control of mosquito vectors. Keywords: Coccinia grandis, Essential oil, Mosquito, Larvicidal, GC-MS Introduction Mosquitoes are responsible for a number of human health problems causing illness and death throughout the world in both children and adults. They are vector for many diseas- es such as malaria, filariasis, dengue, Japa- nese encephalitis, chikungunya and west Nile virus infection in tropical and subtropical coun- tries (Anupam et al. 2012). Malaria is a dead- ly disease and globally about 3.3 billion peo- ples are at the risk of it. About 198 million cases of malaria and 0.58 million deaths oc- curred globally in 2013 (WHO 2014). Of the six malarial vector species, Anopheles ste- phensi is the main mosquito vector responsible for malaria in urban areas of India (Senthilku- mar et al. 2009). Dengue fever is caused by mosquito vector species, Ades aegypti in its epidemic areas affecting millions of people and thousands of deaths per year all over the world (Service 1996). Similarly, Culex quin- quefasciatus is a vector for lymphatic filariasis, commonly known as elephantiasis, in India. Lymphatic filariasis is caused by the worms Wu- chereria bancrofti, Brugia malayi and Br. timori of which, first is found to be more endemic in Indian subcontinent. According to WHO (2009) more than 1.3 billion people spread over 72 different countries worldwide are threatened by it. Collectively, these mos- quito mediated diseases are responsible for long term suffering, morbidity and high so- cio economic burden on society (Ramaiah et *Corresponding author: Prof Vijay Laxminarayan Maheshwari, E-mail: vlmaheshwari@rediffmail.com http://jad.tums.ac.ir/ http://en.wikipedia.org/wiki/Wuchereria_bancrofti http://en.wikipedia.org/wiki/Wuchereria_bancrofti http://en.wikipedia.org/wiki/Brugia_malayi http://en.wikipedia.org/wiki/Brugia_timori http://en.wikipedia.org/wiki/Brugia_timori J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 227 http://jad.tums.ac.ir Published Online: May 27, 2017 al. 2000, Intirach et al. 2012). Efficient control of these diseases would require a two-pronged strategy, (i) prompt treat- ment with effective medicines and (ii) vector control based prevention strategies. Injudi- cious use of medicines, particularly antima- larial drugs, has resulted in development of resistance by the malarial parasite and en- hanced casualties in endemic areas (Intirach et al. 2012). Mosquito management therefore, offers a better and practical alternative for con- trolling diseases mediated by them. Mosquito adulticides, causing temporary reduction in population are good but do not offer a lasting solution. Mosquito larvae being delicate, less mobile and more concentrated in their natu- ral habitat offer a much simpler and efficient point of intervention and control (Rutledge et al. 2003, Dharmagadda et al. 2005). However, there are reports of development of resistance and behavior changes in adult mosquitoes and larvae towards these chemicals (Mittal et al. 2004, WHO 2006). Moreover, they adverse- ly affect the environment, causing soil, air, water pollution and harm the beneficial non target organisms (Dharmagadda et al. 2005). Plant extract including essential oils or insec- ticides from botanical origin are attractive al- ternatives because they contain high amount of various bioactive compounds, many of which are selective and have little or no harm- ful effects on non-target organisms and envi- ronment (Rutledge et al. 2003, Dharmagadda et al. 2005). Essential oil is natural volatile substances found in many plants. Essential oils isolated from plant are generally a mix- ture of many constituents, primarily biologi- cally active monoterpenes (Govindrajan 2010). Traditionally, they have been used for flavor enhancement in food, odorants in fragrances, pharmaceuticals and confectionary industries (Zhu et al. 2001). Of late, they have received considerable attention as potentially active, human and environment friendly bio insecti- cides (Cheng et al. 2003). There are several re- ports on larvicidal activity of essential oil from neem, basil, citronella, lemon, eucalyptus, pine etc (Cheng et al. 2003, Amer and Mehlhorn 2006, Dua et al. 2009). The resistance against plant derived insecticides has not been re- ported so far (Kannathasan et al. 2011). Coccinia grandis (family Cucurbitaceae) is a unique tropical plant, commonly known as ‘little gourd’, growing abundantly and wide- ly all over the India. It is a fast growing per- ennial climbing shrub with white flowers. It grows several meters long and forms dense mat that readily cover shrubs and small trees. It is well known for its hypoglycemic activ- ity (Ajay 2009, Munasinghe et al. 2011). The present study was focused on the chemical constituents and larvicidal activity of Coccinia grandis leaf essential oil against vectors of malaria (An. stephensi), dengue (Ae. aegypti) and filariasis (Culex quinquefascia- tus). To the best of our knowledge it is the first report on the larvicidal activity of Coc- cinia grandis leaf essential oil against the three mosquito species. Materials and Methods Collection of Plant material and extraction of essential oil Plant material of Coccinia grandis was col- lected from Eklagna village Jalgaon. [20° 58ʹ 54.3ʹʹ N, 075° 27ʹ 09.5ʹ E (elevation: 199m)] Maharashtra, identified at Botanical Survey of India, Pune and a specimen voucher num- ber MSMI-1 was deposited in the School of Life Sciences, North Maharashtra University Jalgaon. The collected fresh leaves were cut in to small pieces and extraction was done using Clevenger apparatus for 6h (Singh et al. 2008). The extracted essential oil was subjected to dryness over anhydrous sodium sulfate (Na2SO4) to remove traces of mois- ture. The physical characteristics of extracted essential oil were recorded, percentage aver- age yield was calculated and it was stored at 4 ºC in amber-colored bottle in refrigerator until further analysis. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 228 http://jad.tums.ac.ir Published Online: May 27, 2017 GC and GC-MS analysis Gas chromatography mass spectroscopy (GC-MS) analysis of essential oil was per- formed using JEOL GCMS-Mate-II model gas chromatograph-mass spectrometer equipped with an AOC-20i auto injector and HP-5 ca- pillary column (30m x 0.25mm ID x 0.25μm coating thickness) column. The injector tem- perature was set at 280 °C, and the oven tem- perature was initially set at 40 °C then pro- grammed to increase up to 300 °C at the rate of 10 °C/min and finally held at 200 °C for 5 min. Helium gas was maintained at a flow rate 1.0ml/min as a carrier gas. One microliter of the sample diluted with acetone in 1:10 ratio was injected in the split mode. The percent- age of constituents in essential oil of leaves was calculated by the GC peak areas. Data handling was made through JEOL software and the compounds were identified based on the comparison of their retention time (RT) and mass spectra of WILEY, NIST library data of the GC-MS. Mosquito larvicidal assay Mosquito larvicidal activity was performed against mosquito larvae of species An. ste- phensi, Ae. aegypti and Cu. quinquefascia- tus. Larvae of An.s stephensi [21000’14.3N, 075029’39.8E (elevation: 207m)], Ae. aegypti [2101’01.2N, 075029’52.3E (elevation: 192m)] and Cu. quinquefasciatus [21000’53.5N, 0750 29’39.8E (elevation: 185m] were collected from local breeding areas of Jalgaon, India and identified using the microscopic exami- nation as per Theodore et al. (2005). The col- lected mosquito larvae were brought to la- boratory and maintained at 25–30 °C with 80–90% relative humidity and 12 h/d/night cycle in plastic trays containing dechlorinat- ed water. Mosquito larvae were fed with 10% sterile sucrose solution and pet biscuits. The mosquito larvicidal activity was performed according to standard procedure recommend- ed by WHO (1981). The extracted dried and pre weighed essential oil was dissolved in 1ml of acetone and from this different concentra- tions were made such as 3.125, 6.25, 12.50, 25, 50 and 100ppm in distilled water. Twen- ty five early fourth instar stage larvae of each of the three species of mosquito were used for larvicidal assay in 200ml beakers and three replicates were maintained for each concentration used. During the experiment, no food was given to the larvae. Statistical analysis The larval mortality rate was calculated after 12 and 24 h of exposure time. The le- thal concentrations, LC50 and LC90 and their 95% confidence limit of the lower and upper levels were calculated by probit analysis us- ing statistical software Stats Direct 2.8.0. Results The essential oil yield from fresh and finely cut leaves of Coccinia grandis was 0.14gm% (w/w). The yield was calculated after drying (removing the moisture) over anhydrous sodium sulfate (Na2SO4). The es- sential oil after dryness gave a slightly sticky clump with light yellow color and a char- acteristic odor. The GC-MS profile of the essential oil from leaves of Co. grandis is shown in Fig. 1. The various constituents of essential oil, their retention time and percent composition in order of elution from the col- umn are given in the Table 1. The GC-MS profile shows a total of 23 constituents ac- counting for 99.60% of total oil. The two major constituents of essential oil from leaves of Co. grandis were n-tetracosane (39.18) and n-eicosane (30.04%). Six constituents (peak number 4, 6, 10, 13, 18 and 19) were present between 2–3 percent were as the percentage composition of remaining ranged between 0.1–2 percent (Table 1). The essential oil extracted from leaves of Co. grandis shows promising larvicidal ac- tivity against three mosquito species An. ste- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 229 http://jad.tums.ac.ir Published Online: May 27, 2017 phensi, Ae. aegypti and Cu. quinquefasciatus, (Table 2). The LC50 and LC90 values against early fourth instar larvae of An. stephensi, after 12 and 24 h of exposure were calculat- ed to be 72.60 and 169.90 and 39.41 and 123.24ppm, respectively. Similarly, LC50 and LC90 values against early fourth instar larvae of Ae. aegypti after 12 and 24h of exposure were calculated to be 83.25 and 191.60 and 48.20 and 131.84ppm, respectively. The val- ues were marginally higher with early fourth instar larvae of Cx. quinquefasciatus than the other two species under identical conditions (Table 2). Table 1. Chemical composition of essential oil of the leaves of Coccina grandis. Peak no. Retention time (min) Chemical compounds Percentage 01. 35.95 E,E,Z-1,3, 12- Nanodecatriene-5,14-diol 0.81 02. 37.76 Heneicosane 1.34 03. 39.52 Phytol 1.35 04. 41.17 1-heptatriacotanol 2.06 05. 42.83 17-pentatriacontene 1.19 06. 44.29 Tricosane 2.31 07. 45.16 1- Dodecanol, 2-Coctyl- 1.37 08. 45.96 2,5-Furandione, 3-dodecyl 0.96 09. 47.00 Tetrapentacosane 1.98 10. 47.19 2-Dodecen-1-yl(-)sucinic anhydride 2.08 11. 48.77 n-Eicosane 30.04 12. 49.21 Octasane 1.37 13. 50.69 7-octadecanal 2.81 14. 51.06 Hexatriacontane 1.23 15. 52.77 n-tetracosane 39.18 16. 54.74 1,3 O-triacotanediol 1.09 17. 55.41 Z-14-octadecen-1-ol acetate 0.98 18. 55.96 Pentadeachal 2.09 19. 57.13 Tetratriacotane 2.97 20. 57.38 Triacotane 0.11 21. 62.62 Meissyl alcohol 0.13 22. 62.76 Palmitic acid 1.23 23. 64.63 Myristic acid 0.92 Total 99.60% http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 230 http://jad.tums.ac.ir Published Online: May 27, 2017 Table 2. Larvicidal activity of Coccinia grandis leaf essential oil after 12 and 24 h of exposure period on larvae of Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus Mosquito species Time Concentration (ppm) % of Mortal- ity±SE LC50 (LCL– UCL)a LC90 (LCL– UCL)a X2 (df=4)b An. stephensi After 12h 3.125 8.0±6.89 72.60 (43.12– 106.50) 169.90 (90.05– 265.96) 12.60 6.25 18±5.77 12.5 25±1.25 25 35±1.52 50 42±1.55 100 60±0.57 After 24h 3.125 17±0.21 39.41 (12.07– 67.619) 123.24 (43.276– 212.45) 28.581 6.25 21±0.82 12.5 41±1.00 25 55±1.52 50 66±1.20 100 75±1.85 Ae. aegypti After 12h 3.125 7.0±0.33 82.35 (25.73– 145.96) 191.60 (35.89– 370.94) 27.077 6.25 11±0.87 12.5 26±0.21 25 38±1.52 50 43±0.57 100 51±0.29 After 24h 3.125 14±1.20 48.20 (19.25– 78.66) 131.84 (49.23– 223.96) 27.862 6.25 17±0.88 12.5 33±0.86 25 51±0.68 50 61±1.15 100 71±0.26 Cx. quinquefasciatus After 12h 3.125 5.0±0.28 100.40 (36.18– 175.94) 217.39 (54.32– 413.03) 19.41 6.25 10±0.39 12.5 20±0.57 25 31±1.20 50 36±0.21 100 41±0.28 After 24h 3.125 12±0.13 52.805 (23.92– 83.50) 135.48 (55.54– 224.83) 25.756 6.25 16±0.57 12.5 29±0.39 25 47±1.20 50 59±0.92 100 69±0.96 aDegree of freedom LCL lower confidence level, UCL upper confidence level a95% confidence level http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 231 http://jad.tums.ac.ir Published Online: May 27, 2017 Fig. 1. Gas chromatography–mass spectrometry profile of essential oil obtained from leaves of Coccinia grandis Discussion Several authors have reported different compositions of essential oils obtained from different plant species (Govindrajan 2010, Senthilkumar and Venkatesalu, 2010, Zhu et http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 232 http://jad.tums.ac.ir Published Online: May 27, 2017 al. 2011, 2012, Rajkumar et al. 2011, Intirach et al. 2012, Liu et al. 2012 and Senthilkumar et al. 2013). The plants show significant var- iations, both in terms of number and percent- age composition, of different constituents in essential oil and it appears to be the charac- teristics of a particular plant. The excellent larvicidal activity of the es- sential oil of Co. grandis against three spe- cies of mosquitoes could be either due to the major components, i.e. n-tetracosane and eico- sane or synergistic action of the major and minor components present in it and is diffi- cult to pin point at this stage. Tetracosane is an alkane hydrocarbon and use of hydrocar- bons as pesticidal agents is reported (Sid- dique et al. 2004). Similarly, there are evi- dences indicating larvicidal and antimicro- bial activities of the other major component, eicosane (Akpuaka et al. 2013, Manas et al. 2014). The results show significantly im- proved bioefficacy against one of the mos- quito species, An. stephensi compared to ear- lier report which showed LC50 and LC90 val- ues of 93.3 and 192.6ppm, respectively (as against the 39.41 and 123.24ppm in present study) (Rajkumar et al. 2011). Similarly, the LC50 and LC90 values against Ae. aegypti have been found to be 47.54 and 86.54ppm for Mentha piperita, 40.50 and 85.33ppm for Zingiber officinale, 115.60 and 193.30ppm for Cu. longa and, 148.50 and 325.70ppm for Oc. basilicum, respectively (Kalaivani et al. 2012) compared to 48.20 and 131.84ppm for Co. grandis in the present study. The LC50 and LC90 values of the Co. grandis leaf essential oils against larvae of Cx. quinquefasciatus were marginally better than essential oils of Acorus calamus reported by Senthilkumar and Venkatesalu (2012). The results are also in agreement with sev- eral other previous reports where the major components of essential oils have shown ex- cellent larvicidal or insecticidal activities, eg Plectranthus amboinicus leaf essential oil (Senthilkumar and Venkatesalu 2010), Clause- na anisata leaf essential oil (Govindrajan 2011), Feronia limonia leaf essential oil (Senthilkumar et al. 2013). Similarly, Intirach et al. (2012) studied essential oils of six dif- ferent plant families and demonstrated their larvicidal activity against laboratory colonized An. cracens mosquito. A careful observation of these representative studies indicated that there was no common thread in terms of chemical constituents, in these essential oils. The composition and major and minor com- ponents of essential oil are characteristics of particular plant and, at the best may be rep- resented in the other members of same fami- ly. The composition and larvicidal activity of essential oil of a plant may vary as a function of age of plant, geographical location and sea- son. The observed variations in the efficacy of essential oils from various plants against different vectors could be due to different chemical compositions and/or synergistic ac- tion of major and minor components in them (Senthilkumar and Venkatesalu 2012). The natural diversity of essential oils in the in- digenous plants thus offer good opportunity of developing a cost effective, ecofriendly, region specific and practical strategy for the control of mosquito vectors either independent- ly or as a part of integrated vector manage- ment strategy. Though there are no reports of insecticide/larvicide resistance in the study area, the same is well documented in African countries for Anopheles species against all the approved four classes (organochlorines, pyrethroids, carbamates and organophos- phates) of insecticides (Kristan et al. 2003). Of the four classes, resistance to pyrethroids and its mechanisms in An. gambiae: the most important malarial vector in Africa has been extensively studied. It was found out that the insect develops resistance to insecticide ei- ther by altering its binding site, by point mu- tations, or by detoxifying it enzymatically before it reaches the target site (Tielong et al. 2014). Pyrethroids are the insecticides of choice for http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2017, 11(2): 226–235 SI Mohammed et al.: Evaluation of Larvicidal … 233 http://jad.tums.ac.ir Published Online: May 27, 2017 mosquito control primarily because of their superior human and environment safety rec- ords (Adedayo et al. 2012). Besides, the use of insecticide mixtures and their periodic rotation, integrated vector management, in- volving biopesticides/essential oils of plant origins could be the answer for preventing/ delaying development of resistance in mos- quitoes (Brogdon and Allister 1998). The present study is a step forward in the direction, demonstrating the larvicidal poten- tial of essential oil of a locally available plant against three most common mosquito spe- cies. The chemical analysis shows a different set of major and minor components in the essential oil than the earlier reported studies which can be gainfully utilized further. 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