Environmental Science and Pollution Research

, Volume 25, Issue 11, pp 10434–10446 | Cite as

Toxicological effects of chemical constituents from Piper against the environmental burden Aedes aegypti Liston and their impact on non-target toxicity evaluation against biomonitoring aquatic insects

  • Prabhakaran Vasantha-Srinivasan
  • Annamalai Thanigaivel
  • Edward-Sam Edwin
  • Athirstam Ponsankar
  • Sengottayan Senthil-Nathan
  • Selvaraj Selin-Rani
  • Kandaswamy Kalaivani
  • Wayne B. Hunter
  • Veeramuthu Duraipandiyan
  • Naif Abdullah Al-Dhabi
Plant-borne compounds and nanoparticles: challenges for medicine, parasitology and entomology


Dengue is the most rapidly spreading mosquito-borne viral disease in the world. The mosquito, Aedes aegypti, also spreads Yellow fever, Chikungunya, and Zika virus. As the primary vector for dengue, Ae. aegypti now occurs in over 20 countries and is a serious concern with reports of increasing insecticide resistance. Developing new treatments to manage mosquitoes are needed. Formulation of crude volatile oil from Piper betle leaves (Pb-CVO) was evaluated as a potential treatment which showed larvicidal, ovipositional, and repellency effects. Gut-histology and enzyme profiles were analyzed post treatment under in-vitro conditions. The Pb-CVO from leaves of field collected plants was obtained by steam distillation and separated through rotary evaporation. The Pb-CVO were evaluated for chemical constituents through GC-MS analyses revealed 20 vital compounds. The peak area was establish to be superior in Eudesm-7(11)-en-4-ol (14.95%). Pb-CVO were determined and tested as four different concentrations (0.25, 0.5, 1.0, and 1.5 mg/L) of Pb-CVO towards Ae. aegypti. The larvicidal effects exhibited dose dependent mortality being greatest at 1.5 mg Pb-CVO/10 g leaves. The LC50 occurred at 0.63 mg Pb-CVO/L. Larva of Ae. aegypti exposed to Pb-CVO showed significantly reduced digestive enzyme actions of α- and β-carboxylesterases. In contrast, GST and CYP450 enzyme levels increased significantly as concentration increased. Correspondingly, oviposition deterrence index and egg hatch of Ae. aegypti exposed to sub-lethal doses of Pb-CVO demonstrated a strong effect suitable for population suppression. Repellency at 0.6 mg Pb-CVO applied as oil had a protection time of 15–210 min. Mid-gut histological of Ae. aegypti larvae showed severe damage when treated with 0.6 mg of Pb-CVO treatment compared to the control. Non-toxic effects against aquatic beneficial insects, such as Anisops bouvieri and Toxorhynchites splendens, were observed at the highest concentrations, exposed for 3 h. These results suggest that the Pb-CVO may contain effective constituents suitable for development of new vector control agents against Ae. aegypti.


Volatile Pb-CVO Vector Gut-histology Enzyme Beneficial insects Non-target 



The authors are grateful to the Deanship of Scientific Research, King Saud University for Partial funding through Vice Deanship of Scientific Research Chairs.

Compliance with ethical standards

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abbott WS (1925) A method for computing the effectiveness of an insecticide. J Econ Entomol 8:265–267CrossRefGoogle Scholar
  2. Adams RP (2007) Identification of essential oil components by gas chromatography/mass spectrometry, 4th edn. Allured Publishing Corporation, Carol Stream, p 804Google Scholar
  3. Agra-Neto AC, Napoleão TH, Pontual EV, de Lima Santos ND, de Andrade Luz L, Coelho LCBB, Paiva PMG (2014) Effect of Moringa oleifera lectins on survival and enzyme activities of Aedes aegypti larvae susceptible and resistant to organophosphate. Parasitol Res 113(1):175–184CrossRefGoogle Scholar
  4. Allinson G, Zhang P, Bui A, Allinson M, Rose G, Marshall S, Pettigrove V (2015) Pesticide and trace metal occurrence and aquatic benchmark exceedances in surface waters and sediments of urban wetlands and retention ponds in Melbourne, Australia. Environ Sci Poll Res 22(13):10214–10226CrossRefGoogle Scholar
  5. Arambewela LSR, Arawwawala LDAM, Ratnasooriya WD (2005) Antidiabetic activities of aqueous and ethanolic extracts of Piper betle leaves in rats. J Ethnopharmacol 102:239–245CrossRefGoogle Scholar
  6. Arokiyaraj S, Kumar VD, Elakya V, Kamala T, Park SK, Ragam M, Saravanan M, Bououdina M, Arasu MV, Kovendan K, Vincent S (2015) Biosynthesized silver nanoparticles using floral extract of Chrysanthemum indicum L.—potential for malaria vector control. Environ Sci Poll Res 22(13):9759–9765CrossRefGoogle Scholar
  7. Bilal H, Akram W, Din S, Khan IA, Hassan SA, Arshad M (2012) Larvicidal activity of selected plant extracts against Aedes albopictus Skuse (Diptera: Culicidae). Afr Entomol 20(1):8–12CrossRefGoogle Scholar
  8. Boyer S, David JP, Rey D, Lemperiere G, Ravenel P (2005) Response of Aedes aegypti (Diptera: Culicidae) larvae to three xenobiotic exposures: larval tolerance and detoxifying enzyme activities. Environ Toxicol Chem 25:470–476CrossRefGoogle Scholar
  9. Breuer M, Hoste B, Loof AD, Naqvi SNH (2003) Effect of Melia azedarach extract on the activity of NADPH-cytochrome c reductase and cholinesterase in insects. Pestic Biochem Physiol 76:99–103CrossRefGoogle Scholar
  10. Caburian AB, Osi MO (2010) Characterization and evaluation of antimicrobial activity of the essential oil from the leaves of Piper betle L. E-Int Sci Res J 2:2094–1749Google Scholar
  11. Champakaew D, Junkum A, Chaithong U, Jitpakdi A, Riyong D, Sanghong R, Intirach J, Muangmoon R, Chansang A, Tuetun B, Pitasawat B (2015) Angelica sinensis (Umbelliferae) with proven repellent properties against Aedes aegypti, the primary dengue fever vector in Thailand. Parasitol Res 114(6):2187–2198CrossRefGoogle Scholar
  12. Chellappandian M, Thanigaivel A, Vasantha-Srinivasan P, Edwin ES, Ponsankar, A et al. (2017). Toxicological effects of Sphaeranthus indicus Linn.(Asteraceae) leaf essential oil against human disease vectors, Culex quinquefasciatus Say and Aedes aegypti Linn., and impacts on a beneficial mosquito predator. Environ Sci Pollut Res, doi: 10.1007/s11356-017-8952-2
  13. Cheng SS, Lin CY, Chung MJ, Liu YH, Huang CG, Chang ST (2013) Larvicidal activities of wood and leaf essential oils and ethanolic extracts from Cunninghamia konishii Hayata against the dengue mosquitoes. Ind Crop Prod 47:310–315CrossRefGoogle Scholar
  14. Dammini-Premachandra WTS, Mampitiyarachchi H, Ebssa L (2014) Nemato-toxic potential of betel (Piper betle L.) (Piperaceae) leaf. Crop Prot 65:1–5CrossRefGoogle Scholar
  15. Edwin E, Vasantha-Srinivasan P, Senthil-Nathan S, Thanigaivel A, Ponsankar A, Pradeepa V, Selin-Rani S, Kalaivani K, Hunter WB, Abdel-Megeed A, Duraipandiyan V, Al-Dhabi NA (2016a) Anti-dengue efficacy of bioactive andrographolide from Andrographis paniculata (Lamiales: Acanthaceae) against the primary dengue vector Aedes aegypti (Diptera: Culicidae). Acta Trop 163:167–178CrossRefGoogle Scholar
  16. Edwin E, Vasantha-Srinivasan P, Senthil-Nathan S et al (2016b) Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura. Invertebr Surv J 13:153–163Google Scholar
  17. Fayemiwo KA, Adeleke MA, Okoro OP, Awojide SH, Awoniyi IO (2014) Larvicidal efficacies and chemical composition of essential oils of Pinus sylvestris and Syzygium aromaticum against mosquitoes. Asian Pac J Trop Biomed 4:30–34CrossRefGoogle Scholar
  18. Gleiser RM, Bonino MA, Zygadlo JA (2011) Repellence of essential oils of aromatic plants growing in Argentina against Aedes aegypti (Diptera: Culicidae). Parasitol Res 108:69–78CrossRefGoogle Scholar
  19. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB (2010) Dengue: a continuing global threat. Nat Rev Microbiol 8:S7–S16CrossRefGoogle Scholar
  20. Hemingway J, Ranson H (2000) Insecticide resistance in insect vectors of human disease. Annu Rev Entomol 45:371–391CrossRefGoogle Scholar
  21. Hummelbrunner LA, Isman MB (2001) Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep. Noctuidae). J Agric Food Chem 49:715–720CrossRefGoogle Scholar
  22. Hwang YS, Schultz GW, Axelord H, Krame WL, Mulla MS (1982) Ovipositional repellency of fatty acids and their derivatives against Culex and Aedes mosquitoes. Environ Entomol 11:223–226CrossRefGoogle Scholar
  23. Jukic M, Politeo O, Maksimovic M, Milos M, Milos M (2007) In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone. Phytother Res 21(3):259–261CrossRefGoogle Scholar
  24. Kalaivani K, Senthil-Nathan S, Murugesan AG (2012) Biological activity of selected Lamiaceae and Zingiberaceae plant essential oils against the dengue vector Aedes aegypti L. (Diptera: Culicidae). Parasitol Res 110:1261–1268CrossRefGoogle Scholar
  25. Khan AA, Bhatnagar SP, Sinha BN, Lal UR (2013) Pharmacognostic specifications of eight cultivars of Piper betle from eastern region of India. J Pharmacogn 5:176–183CrossRefGoogle Scholar
  26. Koodalingam A, Mullainadhan P, Arumugam M (2011) Effects of extract of soapnut Sapindus emarginatus on esterases and phosphatases of the vector mosquito, Aedes aegypti (Diptera: Culicidae). Acta Trop 118(1):27–36CrossRefGoogle Scholar
  27. Laranja AT, Manzatto AJ, Campos-Bicudo HEM (2003) Effects of caffeine and used coffee grounds on biological features of Aedes aegypti (Diptera, Culicidae) and their possible use in alternative control. Genet Mole Biol 26(4):419–429CrossRefGoogle Scholar
  28. Larson RT, Lorch JM, Pridgeon JW, Becnel JJ, Clark GG (2010) The biological activity of α-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor. J Med Entomol 47(2):249–257Google Scholar
  29. Lexmond MB, Bonmatin J, Goulson D, Noome DA (2015) Worldwide integrated assessment on systemic pesticides. Global collapse of the entomofauna: exploring the role of systemic insecticides. Environ Sci Pollut Res 22:1–4CrossRefGoogle Scholar
  30. Lija-Escaline J, Senthil-Nathan S, Thanigaivel A, Pradeepa V (2015) Physiological and biochemical effects of chemical constituents from Piper nigrum Linn (Piperaceae) against the dengue vector Aedes aegypti Liston (Diptera: Culicidae). Parasitol Res 114(11):4239–4249CrossRefGoogle Scholar
  31. Mohottalage S, Tabacchi R, Guerin PM (2007) Components from Sri Lankan Piper betle L. leaf oil and their analogues showing toxicity against the housefly, Musca domestica. Flavour Fragr J 22:130–138CrossRefGoogle Scholar
  32. Nouri L, Nafchi AM, Karim AA (2014) Phytochemical, antioxidant, antibacterial, and α-amylase inhibitory properties of different extracts from betel leaves. Ind Crop Prod 62:47–52CrossRefGoogle Scholar
  33. Park HM, Kim J, Chang KS, Kim BS, Yang YJ, Kim GH, Shin SC, Park IK (2011) Larvicidal activity of Myrtaceae essential oils and their components against Aedes aegypti, acute toxicity on Daphnia magna, and aqueous residue. J Med Entomol 48:405–410CrossRefGoogle Scholar
  34. Parthasarathy U, Asish GR, Zacharia TJ, Saji KV, Johson KG, Jayarajan K, Mathew PA, Parthasarathy VA (2008) Spatial influence on the important volite oils of Piper nigrum leaves. Cur Sci 94:1632–1635Google Scholar
  35. Pisa LW, Amaral-Rogers V, Belzunces LP, Bonmatin JM, Downs CA, Goulson D, Kreutzweiser DP, Krupke C, Liess M, McField M, Morrissey CA, Noome DA, Settele J, Simon-Delso N, Stark JD, van der Sluijs JP, van Dyck H, Wiemers M (2015) Effects of neonicotinoids and fipronil on non-target invertebrates. Environ Sci Pollut Res 22:68–102CrossRefGoogle Scholar
  36. Pitasawat B, Champakaew D, Choochote W, Jitpakdi A, Chaithong U, Kanjanapothi D, Rattanachanpichai E, Tippawangkosol P, Riyong D, Tuetun B, Chaiyasit D (2007) Aromatic plant-derived essential oil: an alternative larvicide for mosquito control. Fitoterapia 78(3):205–210CrossRefGoogle Scholar
  37. Ponsankar A, Vasantha-Srinivasan P, Senthil-Nathan S, Thanigaivel A, Edwin E et al (2016a) Target and non-target toxicity of botanical insecticide derived from Couroptia guianensis L. flower against generalist herbivore, Spodoptera litura Fab. and an earthworm, Eisenia foetida Savigny. Ecotoxicol Environ Saf 133:260–270CrossRefGoogle Scholar
  38. Ponsankar A, Vasantha-Srinivasan P, Thanigaivel A, Edwin E et al (2016b) Response of Spodoptera litura Fab. (Lepidopteran: Noctuidae) larvae to Citrullus colocynthis L. (Cucurbitales: Cucurbitales) chemical constituents: larval tolerance, food utilization and detoxifying enzyme activities. Physiol Mol Plant Pathol. doi: 10.1016/j.pmpp. 2016.12.006
  39. Pradeepa V, Sathish-Narayanan S, Kirubakaran SA, Senthil-Nathan S (2014) Antimalarial efficacy of dynamic compound of plumbagin chemical constituent from Plumbago zeylanica Lin (Plumbaginaceae) against the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Parasitol Res 113(8):3105–3109CrossRefGoogle Scholar
  40. Pradeepa V, Sathish-Narayanan S, Kirubakaran SA, Thanigaivel A, Senthil-Nathan S (2015) Toxicity of aristolochic acids isolated from Aristolochia indica Linn (Aristolochiaceae) against the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Exp Parasitol 153:8–16CrossRefGoogle Scholar
  41. Pradeepa V, Senthil-Nathan S, Sathish-Narayanan S, Selin-Rani S, Vasantha-Srinivasan P, Thanigaivel A, Ponsankar A, Edwin E et al (2016) Potential mode of action of a novel plumbagin as a mosquito repellent against the malarial vector Anopheles stephensi, (Culicidae: Diptera). Pestic Biochem Physiol 134:84–93CrossRefGoogle Scholar
  42. Pradhan D, Suri KA, Pradhan DK, Biswasroy P (2013) Golden heart of the nature: Piper betle L. J Pharm Phytochem 1:147–167Google Scholar
  43. Ray D, Pautou MP, Meyran JC (1999) Histopathological effects of tannic acid on the midgut epithelium of some aquatic Diptera larvae. J Invertebr Pathol 73(2):173–118CrossRefGoogle Scholar
  44. Rehman JU, Ali A, Khan IA (2014) Plant based products: use and development as repellents against mosquitoes: a review. Fitoterapia 95:65–74CrossRefGoogle Scholar
  45. Rodriquez A, Cera DL, Herrero P, Moreno F (2001) The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem J 355:625-631Google Scholar
  46. Rudin W, Hecker H (1989) Lectin-binding sites in the midgut of the mosquitoes Anopheles stephensi Liston and Aedes aegypti L. (Diptera: Culicidae). Parasitol Res 75:268–279CrossRefGoogle Scholar
  47. Safaei-Ghomi J, Masoomi R, Kashi FJ, Batooli H (2016) Bioactivity of the essential oil and methanol extracts of flowers and leaves of Salvia sclarea L. from central Iran. J Essent Oil Res Bear Plants 19(14):885–896CrossRefGoogle Scholar
  48. Selin-Rani S, Senthil-Nathan S, Revathi K, Chandrasekaran R, Thanigaivel A, Vasantha-Srinivasan P, Ponsankar A, Edwin E, Pradeepa V (2016a) Toxicity of Alangium salvifolium Wang chemical constituents against the tobacco cutworm Spodoptera litura Fab. Pestic Biochem Physiol 126:92–101CrossRefGoogle Scholar
  49. Selin-Rani S, Senthil-Nathan S, Thanigaivel A, Vasantha-Srinivasan P, Edwin E, Ponsankar A et al (2016b) Toxicity and physiological effect of quercetin on generalist herbivore, Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Chemosphere 165:257–267CrossRefGoogle Scholar
  50. Senthil-Nathan S (2015) A review of biopesticides and their mode of action against insect pests. In: Environmental sustainability-role of green technologies, Springer-Verlag, pp 49–63Google Scholar
  51. Senthil-Nathan S (2007) The use of Eucalyptus leaf extract as a natural larvicidal agent against malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Bioresour Technol 98(9):1856–1860CrossRefGoogle Scholar
  52. Senthil-Nathan S (2013) Physiological and biochemical effect of neem and other Meliaceae plants secondary metabolites against Lepidopteran insects. Front Physiol 359(4):1–17Google Scholar
  53. Senthil-Nathan S, Kalaivani K, Murugan K (2005a) Effects of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Trop 96:47–55CrossRefGoogle Scholar
  54. Senthil-Nathan S, Chung PG, Murugan K (2005b) Effect of biopesticides applied separately or together on nutritional indices of the rice leaffolder Cnaphalocrocismedinalis. Phytoparasitica 33:187–195CrossRefGoogle Scholar
  55. Senthil-Nathan S, Kalaivani K, Sehoon K (2006a) Effects of Dysoxylum malabaricum Bedd. (Meliaceae) extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Bioresour Technol 97:2077–2083CrossRefGoogle Scholar
  56. Senthil-Nathan S, Kalaivani K, Sehoon K, Murugan K (2006b) The toxicity and behavioural effects of neem limonoids on Cnaphalocrocis medinalis (Guene’e), the rice leaffolder. Chemosphere 62:1381–1387CrossRefGoogle Scholar
  57. Senthil-Nathan S, Hisham A, Jayakumar G (2008) Larvicidal and growth inhibition of the malaria vector Anopheles stephensi by triterpenes from Dysoxylum malabaricum and Dysoxylum beddomei. Fitoterapia 79:106–111CrossRefGoogle Scholar
  58. Sivagnaname N, Kalyanasundaram M (2004) Laboratory evaluation of methanolic extract of Atlantia monophylla (Family: Rutaceae) against immature stages of mosquitoes and non-target organisms. Mem Inst Oswaldo Cruz, Rio de Janeiro 99(1):115–118CrossRefGoogle Scholar
  59. Stark JD (2005) How closely do acute lethal concentration estimates predict effects oftoxicants on populations? Integr Environ Assess Manag 1:109–113CrossRefGoogle Scholar
  60. Stark JD, Banks JE (2001) “Selective pesticides”: are they less hazardous to the environment? Bio Sci 51:980–982Google Scholar
  61. Su T, Mulla MS (1999) Oviposition bioassay responses of Culex tarsalis and Culex quinquefasciatusto neem products containing azadirachtin. Entomol Exp Appl 91:337–345CrossRefGoogle Scholar
  62. Thanigaivel A, Chandarasekaran R, Revathi K, Nisha S, Kirubakaran SA, Sathish-Narayanan S, Senthil-Nathan S (2012) Larvicidal efficacy of Adhatoda vasica (L.) Nees against the bancroftian filariasis vector Culex quinquefasciatus Say and dengue vector Aedes aegypti L. in vitro condition. Parasitol Res 110:1993–1999CrossRefGoogle Scholar
  63. Thanigaivel A, Vasantha-Srinivasan P, Senthil-Nathan S, Edwin E, Ponsankar A, Chellappandian M, Selin-Rani S, Lija-Escaline J, Kalaivani K (2017a) Impact of Terminalia chebula Retz. against Aedes aegypti L. and non-target aquatic predatory insects. Ecotoxicol Environ Saf 137:210–217CrossRefGoogle Scholar
  64. Thanigaivel A, Senthil-Nathan S, Vasantha-Srinivasan P et al (2017b) Chemicals isolated from Justicia adhatoda Linn reduce fitness of the mosquito, Aedes aegypti L. Arch Insect Biochem Physiol 00:e21384. doi: 10.1002/arch.21384 CrossRefGoogle Scholar
  65. Van Den Dool H, Kratz PDJA (1963) A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J Chromatogr 11:463–471CrossRefGoogle Scholar
  66. Vasantha-Srinivasan P, Senthil-Nathan S, Thanigaivel A, Edwin E, Ponsankar A, Selin-Rani S, Pradeepa V, Kalaivani K, Hunter WB, Duraipandiyan V, Al-Dhabi NA (2016) Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm, Eudrilus eugeniae Kinb. Chemosphere 155:336–347CrossRefGoogle Scholar
  67. Vasantha-Srinivasan P, Senthil-Nathan S, Ponsankar A, Thanigaivel A, Edwin E, Selin-Rani S, Chellappandian M, Pradeepa V, Lija-Escaline J, Kalaivani K (2017) Comparative analysis of mosquito (Diptera: Culicidae: Aedes aegypti Liston) responses to the insecticide Temephos and plant derived essential oil derived from Piper betle L. Ecotoxicol Environ Saf 139:439–446CrossRefGoogle Scholar
  68. World Health Organisation (1981) Instruction for determining the susceptibility or resistance of mosquito larvae to insecticides. WHOVBC 81(807):1–6Google Scholar
  69. World Health Organization (2009) Guidelines for efficacy testing of mosquito repellents for human skins. WHO, Gevena, pp 4–18Google Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Prabhakaran Vasantha-Srinivasan
    • 1
  • Annamalai Thanigaivel
    • 1
  • Edward-Sam Edwin
    • 1
  • Athirstam Ponsankar
    • 1
  • Sengottayan Senthil-Nathan
    • 1
  • Selvaraj Selin-Rani
    • 1
  • Kandaswamy Kalaivani
    • 2
  • Wayne B. Hunter
    • 3
  • Veeramuthu Duraipandiyan
    • 4
  • Naif Abdullah Al-Dhabi
    • 4
  1. 1.Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental SciencesManonmaniam Sundaranar UniversityTirunelveliIndia
  2. 2.Post Graduate and Research Centre, Department of ZoologySri Parasakthi College for WomenTirunelveliIndia
  3. 3.United States Department of Agriculture, U.S. Horticultural Research LaboratoryFort PierceUSA
  4. 4.Addiriyah Research Chair for Environmental Studies, Department of Botany and Microbiology, College of ScienceKing Saud UniversityRiyadhSaudi Arabia

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