Development, characterisation and evaluation of backhousia citriodora essential oil nanoemulsions for larvicidal activity and gut microbiome modulation in aedes aegypti (L.)

Abstract

The increasinginsecticideresistanceof Aedesaegypti hasreducedtheeffectivenessof currentvectorcontrolstrategies.Essentialoils(EOs)arepromisingnatural alternativestosyntheticinsecticides.However,theirhydrophobicityandinstability limittheirlarvicidalapplication.Thisstudyaimedtodevelopandcharacterise Backhousiacitriodora EO nanoemulsions(BCEONs)andevaluatetheireffectson larvicidalactivityandthegutmicrobiomeof Ae.aegypti larvae.Phytochemical compoundsinBCEOwereidentifiedthroughgaschromatography-massspectrometry (GC-MS).TheBCEONsweredevelopedbyultrasonicemulsificationwithfixed BCEO(10%w/w)andcornoil(5%w/w)andvaryingultrapurewater(50-80%w/w) andTween80(5-35%w/w)tooptimisetheformulation.TheoptimisedBCEONs werecharacterisedtodeterminetheirmorphology,dropletsize,zetapotential,and stability.Thelarvicidalactivityagainst Ae.aegypti wasconductedfollowingthe WHO-standardbioassayprotocol,includingmorphologicalandmorphometric analysis.Thegutmicrobiomecompositionandfunctionalpotentialoflarvaewere assessedusing16SrRNAgenesequencing.GC-MSanalysisrevealedthatBCEOwas composedof86.46%citral.TheoptimisedBCEONformulation(NE1)contained10% BCEO,5%cornoil,5%Tween80and80%w/wultrapurewater,exhibitinggood stabilityat4ºC,25ºC,and37°Cover28days,withdropletsize<123nm, polydispersityindex<0.2,zetapotentialrangingfrom-4.74to-11.55mV,and refractiveindexof1.3639-1.3652.Transmissionelectronmicroscopy(TEM) confirmedspherical-shapeddroplets.NE1exhibitedsignificantlyhigherlarvicidal activity(one-wayANOVA, P < 0.05)comparedtoBCEO,withLC50 andLC90 values of 136.78and237.71 ppmat24 hours,and118.55and198.31 ppmat48 hours, whereasBCEOshowedLC50 andLC90 valuesof242.66and344.03 ppmat24 hours, and237.51and332.37 ppmat48 hours,respectively.LarvaetreatedwithNE1 displayedseveremorphologicaldeformitiesandmorphometricchanges.Thegut bacterialprofilesoflarvaedifferedincompositionandrelativeabundanceacrossthe treatmentandcontrolgroups.TheanalysisindicatedthatNE1treatmentsignificantly (one-wayANOVA, P<0.05)reducedtherelativeabundanceofthephylum Actinobacteriota,andthegenera Microbacterium_A, whilesignificantly(one-way ANOVA, P<0.05)increasingtheabundanceof Bacillus_A. Functionalpredictions basedonKEGGpathwaysrevealedthatNE1significantly(two-sidedWelch’st-test, P<0.01)affectedpathwaysinvolvedinaminoacidmetabolism,carbohydrateand energymetabolism,geneticinformationprocessing,glycanbiosynthesisand metabolism,lipidmetabolism,metabolismofcofactorsandvitamins,metabolismof terpenoidsandpolyketides,andxenobioticbiodegradationandmetabolism.Overall, NE1demonstratedsuperiorstability,larvicidalefficacy,andpronouncedmodulation of gutmicrobiomecompositionandfunction,supportingitspotentialasasustainable larvicide.