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

Abstract

The African mosquito species Anopheles gambiae and An. funestus are ranked high among the world’s most efficient vectors of human malaria. Their juvenile stages develop in aquatic environments while adults are terrestrial. Chemical signals guide gravid females of these vectors to their egg-laying sites. Several attributes of a pond including presence of other organisms influence egg hatching success and larval survival. Gravid An. gambiae females strongly discriminate among potential egg-laying sites to ensure viability of their offsprings. This study is based on the hypothesis that gravid An. gambiae females use chemical cues from microbial activity and/or those associated with competitors as interspecific cues as well as intraspecific signals associated with their own eggs or larvae to select suitable habitats for oviposition. The main aim of this study was to identify the chemicals that guide gravid An. gambiae to their 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 water consisting of Culex quinquifasciatus egg rafts and/or larvae and test water as control were compared. We found out that An. gambiae is deterred or avoids laying eggs in the sites where there is C. quinquifasciatus egg rafts, larvae or both. C. quinquifasciatus larvae deterred the oviposition by gravid An. gambiae even at low density. Moreover, when both C. quinquifasciatus larvae and egg rafts were used with varying density of egg rafts and constant number of larvae the deterrence was more than when the two were used separately. Dynamic and static trapping systems were used to collect volatiles emanating from larvae, extract from test water with C. uinquefasciatus larvae, test water extract (supernatant of muddy 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 chemical constituents of the volatiles. Eleven compounds were identified from C. quinquefasciatus larval volatiles; dimethyl disulfide, 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. quinquefasciatus larvae; 4-methylphenol, 4-(1,1,3,3-tetramethyl butyl)phenol, 4-(1,1-dimethylpropyl)phenol, 2[(4-hydroxyphenyl)methyl]phenol, N,N-methylthiocarbamoylphenyltrithiocarbonate, 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-tetracosahexaene and 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 eleven compounds; 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-hydroxy phenyl)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 volatiles thereof 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 behaviour against gravid females of An. gambiae mosquitoes at different concentrations. Dimethyl dilsulfide and 1:1 mixture of N-hexadecanoic acid and octadecanoic acid had behavioural effect on gravid An. gambiae. At low concentrations the compounds showed positive oviposition response and as the concentration increased there was a negative oviposition effect. Erythro-6-acetoxy-5-hexadecanolide, previously isolated from C. quinquifasciatus egg 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 a pond deters gravid An. gambiae from ovipositing in that specific pond. The microorganisms in the soil influence to a great extent the decision of gravid An. gambiae to oviposit on a given site. The chemical cues believed to mediate oviposition behaviour by An. gambiae have been identified and characterized. This provides the basis of understanding the behavioural effect of individual and blended compounds and this may be used to develop alternative methods of controlling malaria vectors.