International Research Journal of Plant Science

Larvicidal activity of crude extracts and fractions of native plants against aedesaegyptil. and culexquinquefasciatussay. (Diptera: Culicidae)

S. Hannah Catherine Raj^* and N. Shettu Department of Zoology, Pachaiyappa’s College, Chennai, India
^*Corresponding Author:
S. Hannah Catherine Raj, Department of Zoology, Pachaiyappa’s College, Chennai, India, Email: hannah0408r@gmail.com

Received: 15-Oct-2020 Published: 04-Nov-2020, DOI: 10.14303/2141-5447.2020.0015

Introduction

Controlling all the adult mosquitoes is nearly impossible. Hence, public health programmer majorly concentrates controlling larval stage of the mosquitoes (Zahran,H.E.,Abdelgaleil,S.A., 2011). and organophosphates are the choice of chemicals in India, especially temephos is being extensively used. Nevertheless, these chemical insecticidesresulted in many unwanted effects in human and non-target organisms in the environment (Sutthanont. N et al. 2010), (Bayen, S.2012, Chen, C.D. et al.2013, Chavshin, A.R et al. 2015). Apart from this, insecticide resistance in vector mosquitoes due to the continuous application of chemical larvicides showed a more significant challenge in vector mosquito control. Because of insecticide resistance, the mosquito population also increased in the ecosystem and hence the mosquito-borne disease in human is highly prevalent, and the number of cases also shows increasing trend every year. Chemical insecticides also contaminate the environment (Ruiz-Guerrero, R. 2015)

Hence, plant extracts and plant-derived compounds will substitute to chemical insecticides to manage vector mosquitoes, and also, it is non-toxic to all the other organisms, including humans. Many researchers have studied the efficacy of several plant extracts in the recent past (Hayatie, L.2015, Pavela, R.2016, Subashini 2017)

In the present study, the leaves of Cymbopogoncitratus, Azadirachtaindica, Tageteserecta, Eucalyptus deglupta and Syzygiumaromaticumwere used for solvent extraction such as hexane, chloroform, ethyl acetate, ethanol and aqueous and screened on Cx. quinquefasciatus and Ae. aegypti mosquito larvae. Cymbopogoncitratus is reported to possess antifungal, antibacterial, antiprotozoal, antioxidant, anti-inflammatory, anti-carcinogenic anti-rheumatic and anti-protective activities (Ekpenyong C.E.2015, Chukwuocha U.M.2016, Avoseh O.2015) Cymbopogoncitratusleaves have been used in folk and ayurvedic medicine (Tarkang P.A.2012) It has also been known for insecticidal, anti-malarial, and anti-pneumonic activities (Manvitha K. 2014, Chinsembu K.C.2015) Azadirachtaindica bark extracts showed antihyperglycemic activity, hepatoprotective (Costa G.et al.2016)The different solvent extracts of Azadirachtaindica showed to possess mosquito larvicidal activity (Okumu,F.O.2007)

Tageteserecta extracts are reported to possess antibacterial, nematicidal, antioxidant, wound healing, analgesic, hepatoprotective activities (Giri RK.et al.2011, FarjanaNikkon M.et al.2011, Hussain MA.et al.2011) It has also been known for insecticidal and mosquito larvicidal activities (FarjanaNikkon M. et al.2005) Extracts and oil of Eucalyptus deglupta are used in perfumes as ingredients, disinfectants, fungicides, cleaning agents, medicines, and other medical purposes (MotiurRahman M. Et al. 2009) The different solvent extracts of Eucalyptus deglupta is also reported to possess insecticidal and larvicidal properties (Kiplang’at KP.2013, Shooshtari MB.et al.2013) Syzygiumaromaticum is reported to have anti-mutagenic, anti-inflammatory, antioxidant, anti-thrombotic, anti-carcinogenic, antiviral and anti-parasitic (Miyazawa M. 2003, Chaieb K. et al.2007, Hussein G et al.2000, Yang YC . et.al. 2000)The raw extracts obtained from the leaves of the above given plants were screened on the fourth stage larvae of Ae. aegypti and Cx. quinquefasciatus in the laboratory settings.

These plants were selected based on their broad pharmacological importance. Solvent extractions using five solvents, namely hexane, chloroform, ethyl acetate, ethanol, and aqueous were done and they were screened on the fourth stage larvae of Cx. quinquefasciatus and Ae. aegypti mosquitoes.

Materials and Methods

Collection of Plant material

Leaves of the selected five plants were brought from the fields in Chennai District, Tamil Nadu, India. A Botanist authenticated the plant species at Entomology Research Institute (ERI), Loyola College (Autonomous), Chennai. The voucher specimens (ERIL-MRG-VEC-350-355) of selected plants were preserved in the herbarium of the institution. Initially, the leave materials were shade dried in the laboratory for ten days continuously and then crushed with an electric mixer.

Preparation of Solvent extracts

The extracts were extracted from the crushed leaves of each plant by successive extraction technique using five different solvents in the order hexane-chloroform-ethyl acetate-ethanol-aqueous solvents.  So, initially, 1.5Kg powdered leaf of each plant was soaked in 3 litres of hexane for 72h with three-time shaking in a day. The Whattman No:1 filter paper was used to filter the solvent, then concentrated using rotary instruments and finally allowed to dry. Then the remainder of the plant leaves was soaked in the subsequent solvents viz., chloroform-ethyl acetate-ethanol-aqueous and crude extracts were extracted similarly.  All these crude extracts were kept open overnight at the laboratory to dry completely and then stored at 4oC in airtight glass vials in the refrigerator.

Test mosquitoes

The third instar stage larvae of Ae. aegypti and Cx. quinquefasciatus mosquito subjected in the present study were collected from Entomology Research Institute laboratory (ERI); mosquitoes did not expose to any pathogens or microorganisms, any insecticides, or repellent chemicals. The mosquito colony rearing conditions at the laboratory were: 28 ± 1oC; 70 - 75% Relative Humidity and 11 ± 0.5 hours photoperiod.

Larvicidal activity procedure

Larvicidal activities were carried out at the laboratory using the methodology prescribed by the World Health Organization (WHO 2005) with minor modifications. Test concentrations viz., 62.5ppm, 125ppm, 250 ppm, and 500ppm were prepared for each plant crude extract using DMSO and each concentration was replicated five times. In each replication, 99ml of water with twenty larvae of Ae. aegypti and 1ml of DMSO in which extract was dissolved (Total 100ml). Five controls with DMSO without plant extract was maintained along with the experiment. Similar experiments were carried out with Cx. quinquefasciatus mosquito larvae.

Similarly, the concentrations used to test the different fractions were 2.5 ppm, 5.0 ppm, 7.5 ppm and 10.0ppm produced using a dissolving agent called DMSO. Azadirachtin and temephos (positive control) were also tested with 2.5ppm, 5.0ppm, 7.5ppm and 10.0ppm concentration for comparison. Five replications of control (without any extract) were also maintained. The total dead larvaewere documented after 24 hoursof experimental time. The percentage of mortality was determined for each concentration of each plant extract using the following formula.

Percentage of Mortality = No. of Dead larvae/ No. of Larvae introduced x 100

Abbott’s formula [30] was used to get corrected percentage mortality when control mortality was below 5%:

Corrected percentage of mortality: (1 – n) in Treatment/ n in Control x 100

Fractionation of active extract

The promising ethanol extract of Eucalyptus deglupta (86 g) was initially packed in column chromatography using silica gel (100–200 sized mesh) and ethyl acetate. Then the raw extract was separated with commercial solvents from low polar to high, i.e. hexane, chloroform, ethyl acetate, ethanol, and methanol and its mixtures. All the fractions were assessed on TLC and fractions were pooled together. Finally, 8 fractions had resulted. These fractions were screened for larvicidal activity at different concentrations i.e. 2.5ppm, 5.0ppm, 7.5ppm and 10ppm. Fraction 7 eluted with ethanol: methanol (90:10) showed significant larvicidal results against both the mosquito larvae.

Statistical analysis

Dose-response curves were prepared for each derivative with larval and pupal mortality data. Further, larvicidal and pupicidal mortality data were subjected to probit analysis (US EPA probit; version 1.5) to find LC₅₀ and LC₉₀ values, and the differences were considered significant at p≤ 0.05.

Result

Larvicidal activity result of crude extracts

The larvicidal assay results are given in Tables 1 to 5, which evidenced that the ethanol extract of Eucalyptus degluptaleaves was the very active extract in killing the fourth stage larvae of Ae. aegypti and Cx. Quinquefasciatus mosquitoes (Tables 4).

Table 1. Lethal concentrations (in ppm) of crude extracts of Cymbopogoncitratus against the fourth instar larvae of Ae. aegyptiand Cx. quinquefasciatus.

Table 2. Lethal concentrations (in ppm) of crude extracts of Azadirachtaindicaagainst the fourth instar larvae of Ae. aegyptiand Cx. Quinquefasciatus

Table 3.Lethal concentrations (in ppm) of crude extracts of Tageteserecta against the fourth instar larvae of Ae. Aegypti and Cx. quinquefasciatus.

Table 4.Lethal concentrations (in ppm) of crude extracts of Eucalyptus deglupta against the fourth instar larvae of Ae. Aegypti and Cx. quinquefasciatus.

Table 5.Lethal concentrations (in ppm) of crude extracts of Syzygiumaromaticum against the fourth instar larvae of Ae. Aegypti and Cx. quinquefasciatus.

Among the 8 fractions screened, fraction 7 was identified to be a very effective fraction, which showed LC₅₀ and LC₉₀results of 5.07ppm, 12.64ppm against Cx. Quinquefasciatus larvae (Table 6) and 5.50ppm, 17.18ppm against Ae. aegypti larvae (Table 6), respectively. Following this, fraction 8 produced LC₅₀ and LC₉₀results of 10.59ppm, 23.18ppm onCx. quinquefasciatus and 10.73ppm, 21.46ppm on Ae. aegypti larvae, respectively. Remaining fractions recorded a modest or less larvicidal activity as given below.

Table 6. Lethal concentrations (in ppm) of different fractions of Eucalyptus deglupta ethanol extract against larvae of Cx. quinquefasciatus.

Fraction 1 produced LC₅₀ and LC₉₀results of 15.39ppm, 39.58ppm against Cx. quinquefasciatus and 18.42ppm, 54.96ppm against Ae. aegypti larvae, respectively. Fraction 2 produced LC₅₀ and LC₉₀results of 17.56ppm, 49.32ppm against Cx. quinquefasciatus and 21.42ppm, 69.40ppm onAe. aegypti larvae, respectively. Fraction 3 recorded LC₅₀ and LC₉₀results of 20.78ppm, 156.28ppm against Cx. quinquefasciatus and 24.64ppm, 205.76ppm on Ae. aegypti larvae, respectively. Fraction 4 recorded LC₅₀ and LC₉₀results of 11.40ppm, 23.88ppm against Cx. quinquefasciatus and 15.65ppm, 36.19ppm on Ae. aegypti larvae, respectively. Fraction 5 produced LC₅₀ and LC₉₀results of 12.42ppm, 34.97ppm against Cx. quinquefasciatus and 14.86ppm, 114.46ppm on Ae. aegypti larvae, respectively. Fraction 6 produced LC₅₀ and LC₉₀results of 25.50ppm, 128.92ppm against Cx. quinquefasciatus and 27.16ppm, 143.84ppm on Ae. aegypti larvae, respectively (Tables 6 and 7).

Table 7. Lethal concentrations (in ppm) of different fractions ofEucalyptus deglupta ethanol extract against larvae of Ae. aegypti.

Discussion

Mosquitoes are a highly risky organism because it transmits disease-causing pathogens to human. In recent years, the vector mosquito population has increased in several fold in tropical and subtropical countries, including India. On the other hand, mosquitoes have developed resistance to many available conventional chemical insecticides. Hence, mosquito control with plant extracts would be a good substitute for chemical pesticides.

In our study, the ethanol extract of Eucalyptus deglupta leaves produced the maximum larvicidal results with LC₅₀ and LC₉₀ data of 63.54ppm, 96.90ppm in killing Cx. quinquefasciatusand 72.19ppm, 137.90ppm in killing Ae. aegypti larvae, respectively. Our results were similar to the results of (RajivGandhi.et al.2016) who screened different extracts from five plants and found that the raw methanol extract of Rubiacordifolia was very active with LC₅₀ and LC₉₀ results of 95.69mg/L, 347.96mg/L in killing Cx. quinquefasciatus and 102.mg/L, 350.20mg/L in killing Ae. aegypti larvae, respectively. Similarly, (Aivazi,A.2009) reported that the ethyl acetate extract of Quercusinfectoriawas most effective with LC₅₀ and LC₉₀ results of 116.92ppm, 144.77ppm against the fourth instar larvae of An. stephensi. Likewise, (Yadav,R.2013) screened different plant extracts. They found that the methanol extracts of Euphorbia tirucalli latex and stem bark was most effective with LC₅₀ values of 177.14mg/L and 513.387mg/L against the third instar Cx. quinquefasciatuslarvae, respectively.

Besides, our study discovered that larvae of Cx. Quinquefasciatus mosquitowas more vulnerable than the larvae of Ae. aegypti. Similar to our report, numerous studies have reported earlier with wide-ranging larvicidal efficacy of plant extracts among different mosquito species. For example, the methanol extract from Solanumxanthocarpum seeds and fruits was tested on An. culicifacies, An. stephensi, Ae. Aegypti and Cx. quinquefasciatus mosquito larvae by (Bansal,Set al.2009). The results varied for fruits and seeds with LC₅₀ values of 51.6 mg/L, 52.2 mg/L, 118.3 mg/L, 157.1mg/L and 66.9 mg/L, 73.7 mg/L, 123.8 mg/L, 154.9mg/L on An. culicifacies, An. stephensi, Ae. aegypti and Cx. quinquefasciatus, respectively. In the same way, (Patil,S.V.et al.2010) reported that Ae. aegyptilarvae were more susceptible to methanolic extract of Plumbagozeylanicaroot with an LC₅₀ result of 169.61mg/l than An. stephensi larvae with an LC₅₀ result of 222.34mg/L.

In recent years, numerous studies have been conducted with plant crude extracts for its effect on mosquito larvae, and many crude extracts were reported to be very effective on various species of mosquito larvae. For instance, methanolic extracts were prepared from leaves of Moringaoleigera by (Prabhu,K.etal.2011) and tested on first to fourth-stage larvae of Anopheles stephensi. The LC₅₀ and LC₉₀results were reported to be 57.79ppm 125.93ppm for the first instar, 63.90ppm and 133.07ppm for the second instar, 72.45ppm and 139.82ppm for the third instar, and 78.93 ppm and 143.20 ppm for the fourth stage larvae, respectively (Prabhu, K.etal.2011) In another experiment, different solvent extracts were prepared from the root of Asparagus racemosus and they were studied on the larvae of Ae. aegypti, Cx. quinquefasciatus, and Anopheles stephensi (Govindarajan,M.2014)

Their study displayedLC₅₀ and LC₉₀ results of 90.97ppm, 210.96ppm and 179.92ppm, 168.82ppm and 115.13ppm, 97.71ppm against Aedesaegypti, Anopheles stephensi and Culexquinquefasciatus mosquito larvae, respectively (Govindarajan, M.2014) Similarly, different solvent extracts viz., acetone, chloroform, ethyl acetate, methanol and petroleum benzene were extracted from the leaf extracts of Clausenadentate and screened by (Manjari,M.S.et al.2014) on the fourth stage larvae of Cx. quinquefasciatus,Ae. aegyptiand An. stephensi. Their study showed that the acetone extract was most active and the LC₅₀ and LC₉₀ results were 0.045694mg/ml, 0.045684mg/ml on An. stephensi larvae and 0.150278mg/ml, 7.302613mg/ml againstCx. quinquefasciatus larvae and 0.169495mg/ml, 1.10034mg/ml against Ae. aegypti larvae (Manjari,M.S.et al.2014)

Conclusion

In this study, the different crude extracts of five plants, namely, Cymbopogoncitratus, Azadirachtaindica, Tageteserecta, Eucalyptus deglupta, and Syzygiumaromaticum were tested for their larvicidal activity against the fourth stage larvae of Ae. Aegypti and Cx. quinquefasciatus vector mosquitoes. The results undoubtedly proved that the ethanol extract of Eucalyptus deglupta was very active in killing the fourth stage larvae of Ae. aegypti and Cx. quinquefasciatus. Hence, based on these study results, the ethanol extract of Eucalyptus deglupta was further investigated to isolate the effective compound.

Acknowledgement

The authors greatly acknowledges to the Entomology Research Institute of Loyola College, Chennai for conducting the mosquito bioassay.

Competing Interests

Authors declare that there is no conflict of interest.

References

1. Aivazi,A.,Vijayan,V.A.,(2009).Larvicidal activity of oak Quercus infectoria Oliv.(Fagaceae) all extracts against AnophelesstephensiListon.Parasitol.Res.104: 1289–1293.
2. Avoseh O., O. Oyedeji, P. Rungqu, B. Nkeh-Chungag , A. Oyedeji (2015)  Cymbopogon species; ethnopharmacology, phytochemistry and the pharmacological importance, Molecules 20 7438–7453
3. Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267.
4. Bansal,S.K.,Singh,K.V.,Kumar,S.,(2009).LarvicidalactivityoftheextractsfromdifferentpartsoftheplantSolanumxanthocarpumagainst important mosquito vectors in the aridregion.J. Environ.Biol. 30(2): 221–226.
5. Bayen,S.,(2012).Occurrence, bioavailability and toxic effects of trace metals and organic contaminants in mangrove ecosystems: A review.Environ.Int.48: 84–101.
6. Chen,C.D.,NazniW.A.,Lee,H.L.,Norma-Rashid,Y.,Lardizabal,M.L.,Sofian-Azirun,M.,(2013).Temephosresistancein fieldAedes(Stegomyia)albopictus(Skuse)fromSelangor,Malaysia.Trop.Biomed. 30: 220–230.
7. Chavshin,A.R.,Dabiri,F.,Vatandoost,H.,Bavani,M.M.,(2015).SusceptibilityofAnophelesmaculipennistodifferentclassesofinsecticidesinWestAzarbaijanProvince,NorthwesternIran.Asian.Pac. J.Trop. Biomed.5 (5):403–406.
8. Chaieb K, Zmantar T, Ksouri R, Hajlaoui H, Mahdouani K, Abdelly C, Bakhrouf A. Antioxidant properties of essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species. J Mycosis 2007;50(5):403-6.
9. Chinsembu K.C., Plants as antimalarial agents in sub-Saharan Africa, Acta Trop 152 (2015) 32–48.
10. Costa G., H. Grangeia , A. Figueirinha , I.V. Figueiredo , M.T. Batista , Influence of harvest date and material quality on polyphenolic content and antioxi- dant activity of Cymbopogoncitratusinfusion, Indust. Crops Prod. 83 (2016) 738–745.
11. Chukwuocha U.M., O. Fernández- Rivera , M. Legorreta-Herrera , Exploring the antimalarial potential of whole Cymbopogon citratusplant  therapy, J. Ethnopharmacol. 193 (2016) 517–523.
12. Ekpenyong C.E., E. Akpan , A. Nyoh , Ethnopharmacology, phytochemistry, and biological activity of Cymbopogoncitratus(DC.) Stapf extracts, Chinese J. Nat. Med. 13 (2015) 321–337.
13. FarjanaNikkon M, RowshanulHabib, ZahangirAlam Saud and RezaulKarim M, (Medicinal Plants:  Conservation & Sustainable Use) Efficacy Evaluation of AzadirachtaIndica, CalotropisProcera, DaturaStramonium and TagetesErecta against Root-Knot Nematodes Meloidogyne Incognita. Pak. J. Bot, 43, 2011, 197-204. 
14. FarjanaNikkon M, RowshanulHabib M, EzaulKarim and Zennat Ferdousi.Insecticidal activity of flower of Tageteserecta against Triboliumcastaneum (Herbst). Research Journal of Agriculture and Biological Sciences, 5(5), 2009, 748-753.
15. Giri RK, Anindya Bose and Subrat Kumar Mishra. Hepatoprotective Activity of Tageteserecta against carbon tetrachloride-induced hepatic damage in rats .ActaPoloniae  Pharmaceutican Drug Research, 68(6), 2011, 999-1003.
16. Govindarajan,M.,Sivakumar,R.,(2014).Ovicidal,larvicidalandadulticidalpropertiesofAsparagusracemosus(Willd.)(Family:Asparagaceae)rootextractsagainstfilariasis(Culexquinquefasciatus),dengue(Aedesaegypti)andmalaria(Anophelesstephensi)vector mosquitoes(Diptera:Culicidae) Parasitol.Res. 113: 1435–1449.
17. Hayatie,L.,Biworo,A.,SuhartonoE.,(2015),AqueousextractsofseedandpeelofCaricaPapayaagainstAedesaegypti,J.Med.Biol.Eng.4(5):417–421.
18. Hussain MA, Tariq Mukhtar and Muhammad ZameerKayani. Combined wound healing activity of Gymnemasylvestere and Tageteserecta Linn. International Journal of Pharmaceutical Applications, 2(2), 2011, 135-140.
19. Hussein G, Miyashiro H, Nakamura N, Hattori M, Kakiuchi N, Shimotohno K. Inhibitory effects of Sudanese medical plant extracts on hepatitis C virus (HCV) protease. J Phytother Res 2000;14:510-6. 
20. Kiplang’at KP, Richard WM. Repellent activities of Ocimumbasilicum, Azadirachtaindica and Eucalyptus citriodora extracts on rabbit skin against Aedesaegypti. J EntomolZool Stud 2013; 1: 84-91.
21. Manvitha K., B. Bidya , (2014)  Review on pharmacological activity of Cymbopogoncitratus, Int. J. Herb. Med. 1 5–7.
22. MotiurRahman M, EkramulHaque M. Tageteserecta Linn and its Mosquitocidal Potency AgainstCulexquinquefasciatus. Asian Pacific Journal of Tropical Biomedicine, 2009, 186-188.
23. Manjari,M.S.,Karthi,S.,Ramkumar,G.,Muthusamy,R.,Natarajan,D.,Shivakumar,M.S.,(2014).ChemicalcompositionandlarvicidalactivityofplantextractsfromClausenadentata(Willd)(Rutaceae)againstdengue,malaria,andfilariasisvectors.Parasitol.Res.113(7):2475–24
24. Miyazawa M, Hisama M. (2003) Antimutagenic activity of phenyl propanoides from clove (Syzygiumaromaticum). J Agric Food Chem 51(22):6413-22.
25. Okumu,F.O.,Knols,B.G.J.,Fillinger,U.,(2007).Larvicidaleffectsofaneem(Azadirachtaindica) oil formulation on the malaria vectorAnophelesgambiae.MalariaJ.6: 63–68.
26. Pavela,R.,Benelli,G.,(2016).Ethnobotanical knowledge on botanical repellents employed inthe African region against mosquito vectors – a review.Exp.
27. Parasitol.167C, 103–108Ruiz-Guerrero,R.,Rodríguez-Pérez,M.A.,Norzagaray-Campos ,M. ,(2015). Toxicity of Mexican native plant extracts against larvae of Aedesaegypti (Diptera: Culicidae).AsianPac. J.Trop. Biomed.5 (4):287–291.
28. Patil,S.V.,Patil,C.D.,Salunkhe,R.B.,Salunke,B.K.,(2010).LarvicidalactivitiesofsixplantsextractsagainsttwomosquitospeciesAedesaegyptiandAnophelesstephensi.Trop.Biomed.27(3): 360–365.
29. Prabhu,K.,Murugan,K.,Nareshkumar,A.,Ramasubramanian,N.,Bragadeeswaran,S.,(2011).LarvicidalandrepellentpotentialofMoringaoleiferaagainstmalarialvector,AnophelesstephensiListon(Insecta:Diptera:Culicidae)AsianPacificJ. Trop.Biomed.124–129.
30. RajivGandhi,M.,DanielReegan,A.,Sivasankaran,K.,GabrielPaulraj,K.,Ignacimuthu,S.,(2016).Ovicidal and larvicidal activities of some plant extracts against Aedesaegypti L. and Culexquinquefasciatus Say (Diptera: Culicidae).Asian Pac. 6(6): 468–471.
31. Sutthanont,N.,Choochote,W.,Tuetun,B.,Junkum,A.,Jitpakdi,A.,Chaithong,U.,Riyong,D.,Pitasawat,B.,(2010).Chemicalcompositionandlarvicidalactivityofedibleplant-derivedessentialoilsagainstthepyrethroid-susceptible and resistant strains of Aedesaegypti(Diptera:Culicidae).J.Vector Ecol. 35: 106–115
32. Subashini,K.,Sivakami,R.,Jeyasankar,A.,(2017)LarvicidalactivityofScutellariaviolacea(Lamiaceae)leafextractsagainstthreeimportanthumanvectormosquitoes:Aedesaegypti,AnophelesstephensiandCulexquinquefasciatus(Diptera:Culicidae) from TamilNadu,India. Int. J.Mos. Res. 4(2): 108–110.
33. Shooshtari MB, Kashani HH, Heidari S, Ghalandari R. (2013)Comparative mosquito repellent efficacy of alcoholic extracts and essential oils of different plants against Anopheles Stephensi. Afr J Pharm Pharmacol; 7:310-4
34. Tarkang P.A., G.A. Agbor , N. Tsabang , R.Y. Tchokouaha , D.A. Tchamgoue , D. Kemeta, (2011) Effect of long-term oral administration of the aqueous and ethanol leaf extract of Cymbopogoncitratus(DC. ex Ness) STAPF, Annals of Biolog. Res. 3 (12) 5561–5570.
35. World Health Organization, 2005.Guidelines for laboratory and field testing of mosquito larvicides.WHO, Geneva WHO/CDS/WHOPES/GCDPP/ 13 pp.
36. Yang YC, Lee SH, Lee WJ, Choi DH, Ahn YJ.(2003) Ovicidal and adulticidal effects of Eugenia cryophyllata bud and leaf oil compounds on Pediculuscapitis. J Agric Food Chem; 51(17):4884-8.
37. Yadav,R.,Srivastava,V.K.,Chandra,R.,Singh,A,.(2002).Larvicidalactivityof latex and stem bark of Euphorbia tirucalli plant onthemosquitoCulexquinquefasciatus.J.Commun.Dis.34(4): 264–269.
38. Zahran,H.E.,Abdelgaleil,S.A.,(2011).InsecticidalanddevelopmentalinhibitorypropertiesofmonoterpenesonCulexpipiensL.(Diptera:Culicidae).J.Asia-Pac.Entomol.14(1):46–51.

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