Identification of Chemical Signals that Influence the Oviposition Behaviour of Anopheles Gambiae and Exploration of their Potential in

Abstract

The African mosquito species Anopheles gambiae and An. funestus are ranked high amongthe world’s most efficient vectors of human malaria. Their juvenile stages develop inaquatic environments while adults are terrestrial. Chemical signals guide gravid females ofthese vectors to their egg-laying sites. Several attributes of a pond including presence ofother organisms influence egg hatching success and larval survival. Gravid An. gambiaefemales strongly discriminate among potential egg-laying sites to ensure viability of theiroffsprings. This study is based on the hypothesis that gravid An. gambiae females usechemical cues from microbial activity and/or those associated with competitors asinterspecific cues as well as intraspecific signals associated with their own eggs or larvae toselect suitable habitats for oviposition.The main aim of this study was to identify the chemicals that guide gravid An. gambiae totheir oviposition site and to find out their effect on oviposition behaviour. To achieve this,behavioural responses of caged gravid An. gambiae on two choice assay of test waterconsisting of Culex quinquifasciatus egg rafts and/or larvae and test water as control werecompared. We found out that An. gambiae is deterred or avoids laying eggs in the siteswhere there is C. quinquifasciatus egg rafts, larvae or both. C. quinquifasciatus larvaedeterred the oviposition by gravid An. gambiae even at low density. Moreover, when bothC. quinquifasciatus larvae and egg rafts were used with varying density of egg rafts andconstant number of larvae the deterrence was more than when the two were usedseparately.Dynamic and static trapping systems were used to collect volatiles emanating from larvae,extract from test water with C. quinquefasciatus larvae, test water extract (supernatant ofmuddy soil mixed with double-distilled water and allowed to settle for 3-7days), An.gambiae egg extract, C. quinquefasciatus egg rafts extract, soil and cultured soil bacteria.Gas chromatograph-mass spectrometry (GC-MS) was used to characterize the chemicalconstituents of the volatiles.Eleven compounds were identified from C. quinquefasciatus larval volatiles; dimethyldisulfide, dimethyl trisulfide, 3,5-dimethylbenzaldehyde, 2,4-bis(1,1-dimethylethyl)phenol,1-chlorotetradecane, isopropyl myristate, isopropyl palmitate, 4-phenylmorpholine, 3-phenyl-1-azabicyclo[2.2.2]octane, eicosane and 2,4-bis(1-methyl-1-phenylethyl)phenol.Six compounds were identified from the extract of test water with C. quinquefasciatuslarvae; 4-methylphenol, 4-(1,1,3,3-tetramethyl butyl)phenol, 4-(1,1-dimethylpropyl)phenol,2[(4-hydroxyphenyl)methyl]phenol, N,N-dimethylthiocarbamoylphenyltrithiocarbonate,2,4-bis(1-methyl-1-phenylethyl)phenol and (all-E)-2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane.The test water was extracted with dichloromethane and nine compounds were identified;2,4-bis(1,1-dimethylethyl)phenol, 4-(1,1-dimethylpropyl)phenol, 6,10,14-trimethyl-2-pentadecanone, 2-(1,1-dimethylethyl)-4-(1-methyl-1-phenylethyl)phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methyl-1-phenylethyl)phenol, phytol, 2,4-bis(1-methyl-1-phenylethyl)phenol, (all-E)-2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaeneand 4-octyl-N-(4-octylphenyl)benzenamine.Tetradecanoic acid, Z-11-hexadecenoic acid, n-hexadecanoic acid, (Z)-9-octadecenoic acid,octadecanoic acid, docosane, (all-E)-2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene were obtained from An. gambiae eggs extract. Z-11- hexadecenoic acid,n-hexadecanoic acid, (Z)-9-octadecenoic acid, octadecanoic acid, N-butyl-4,9-decadien-2-amine, arachidonic acid and 1,2,3-propanetriyl ester hexanoic acid were found in C.quinquefasciatus egg rafts extract.Volatiles trapped from the muddy soil used for preparation of test water yielded elevencompounds; d-limonene, [3aR-(3a.alpha.,4.beta.,7.alpha.)]-2,4,5,6,7,8-hexahydro-1,4,9,9-tetramethyl-3H-3a,7-methanoazulene, 2,6-bis(1-methylethyl)benzeneamine, [1S-(1.alpha.,7.alpha.,8a.alpha.)]-1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)naphthalene, (1S-cis)-1,2,3,4-tetrahydro-1,6-dimethyl-4-(1-methylethyl)naphthalene, [R-[R*,R*-(E)]]-3,7,11,15-tetramethyl-2-hexadecene, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, (1-methyldodecyl)benzene, 2[(4-hydroxyphenyl)methyl]phenol, 2-phenyl-2-(phenylmethyl)-1,3-dioxolane and 2,4-bis(1-methyl-1-phenylethyl)phenol. The same soil was cultured for bacteria and the trapped volatilesthereof yielded twelve compounds identified as: dimethyl disulfide, dimethyl trisulfide, 2-ethyl-1-hexanol, 2-phenoxyethanol, tetradecane, 2,6-bis(1,1- dimethylethyl)- 2,5-cyclohexadiene-1,4-dione, hexadecane, octadecane, isopropyl myristate, 4-hydroxy-4-methyl-2-pentanone, 1-undecene and 4-phenylmorpholine.Some of the compounds identified were evaluated for their effect on oviposition behaviouragainst gravid females of An. gambiae mosquitoes at different concentrations. Dimethyldilsulfide and 1:1 mixture of N-hexadecanoic acid and octadecanoic acid had behaviouraleffect on gravid An. gambiae. At low concentrations the compounds showed positiveoviposition response and as the concentration increased there was a negative ovipositioneffect. Erythro-6-acetoxy-5-hexadecanolide, previously isolated from C. quinquifasciatusegg rafts, showed a negative oviposition effect.This study showed that interspecific chemical signals mediate the oviposition of gravid An.gambiae to a specific site. The presence of C. quinqufasciatus larvae and/or egg rafts in apond deters gravid An. gambiae from ovipositing in that specific pond. Themicroorganisms in the soil influence to a great extent the decision of gravid An. gambiae tooviposit on a given site. The chemical cues believed to mediate oviposition behaviour byAn. gambiae have been identified and characterized. This provides the basis ofunderstanding the behavioural effect of individual and blended compounds and this may beused to develop alternative methods of controlling malaria vectors