TheEffectsofBisphenolSontheBehavior

Patternsof
Drosophilamelanogaster
with
FMR1GeneInsertions

AroobaLodhi
NilesNorthHighSchool
2016

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TableofContents

Acknowledgements..3

PurposeandHypothesis.......4

Variables......5

ReviewofLiterature........7

Materials........19

Procedure.......20

ObservationKey....24

Results
DataTables....29
Graphs....37
DataAnalysis.....42
Discussion......45
ExperimentalError.........47

Conclusion.........49

ReferenceList........51

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Acknowledgements
Aspecialthanksgoestomysupervisor,Ms.ChristineCamel.Thesupportandguidance
sheprovidedmewithinordertoprogressmyprojectwastrulyappreciated.Anotherthanksis
alsoextendedtoMr.RichardThielsenwhoseassistancewascrucialtobegintheexperimental
process.
MyutmostgratitudeisalsogiventoDr.AmyMarieYuofTuftsUniversitySchoolof
Medicinewithoutwhomthisprojectwouldnotbepossible.TheideasandgeneralknowledgeDr.
Yuwaswillingtosharewithmeledtothesuccessofthisprojectwellbeforethe
experimentationprocesshadevenbegun.
MyfinalthanksextendstothecontributionsIreceivedfrommyclassmateswhentwo
handsandonebrainwerenotenough.LilyShearer,JulianaTichota,andSumonaBanerjee:
thankyouforalwayslisteningintheplanningprocessandthanksforallthesolutionsyou
providedmewithinordertostartmyexperimentation.Duringmytestingtrials,Iappreciatethe
greatinterestyoualldisplayedinmyprojectandyourwillingnesstoassistmeinthe
experimentalprocess.Allotherclassmates,teachers,andfamilymemberswhosupportedmein
thisendeavoralsohavemygreatestgratitudeandappreciation.

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Purpose

TheultimatepurposeofthisstudyistoobservetheeffectsofBPSonthebehaviorpatternsof
Drosophilamelanogaster
withFMR1geneinsertionstodetermineifexposuretothechemical
intensifiesFragileXSyndromesymptomsmodeledbythespecimen.Ifthisexperimentis
successful,itcouldpromptfurtherresearchattemptingtofindmorehazardslinkedwiththe
manufacturingofplasticitemslacedwiththeBPSchemical.Iffurthersafetyhazardsare
correlatedwithBPSproduction,newalternativechemicalscouldbestudiedinordertocreate
safercommonplaceproducts.

Hypothesis

If
Drosophilamelanogaster
modelingFragileXSyndromewithinsertedFMR1genesare
exposedtothechemicalBisphenolS,thenthegeneticallymodifiedfruitflieswithexposurewill
displaydrasticchangesintheirlocomotion,reactionstostimuli,andclumpingpatterns.

Rationale

Previousstudieshavedeterminedthatexposuretothenowbanned,harmful,plasticproducing
chemicalBisphenolA(BPA)hasaffectedlocomotionpatternsandreducedresponsesto
environmentalchangesingeneticallymodifiedautisticrats,zebrafish,andmonkeys(Smit,
2015).FragileXSyndromeisaninheritedgeneticconditioncharacterizedbyanabnormalX
chromosomewhichisprimarilyplacedunderthebroadercategoryofAutisticSpectrum
Disorders.BPSreplacedtheharmfulBPAchemicalinproductionofplastics,butsincethenew

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chemicalisamodificationoftheformer,highexposuretoitmaystillaffectvariousbehavior
patternsof
Drosophilamelanogaster
modelingFragileXSyndromesymptomswiththeinsertion
ofanFMR1gene.

Variables

IndependentVariable:

ExposureoftheFMR1
Drosophilamelanogaster
totheBPS
chemical

DependentVariable:
ThebehaviorchangesofFMR1
Drosophilamelanogaster
intermsof
locomotion,clumpingpatterns,andreactionstonewstimuliintheenvironment
Thebehavioralpatternsofeachexperimentalandcontrolgroupwillbedeterminedby
utilizingthekeyprovidedrankingpossiblebehavioralchangesinthespecimen.Thekey
providedrankslocomotion,clumpingpatterns,reactiontolight,andreactiontoanadditivefood
sourceonascalefrom1to3with1describingregular
Drosophilamelanogaster
behavior
patternsand3describingirregularbehavior.Inordertorecordgroupbehavior,eachofthenine
specimentubeswillbeassignedanumberspecifictothebehaviorpatternsbeingobservedeach
weekin10minuteintervals,daily,foratestingperiodofthreeweeks.

ControlGroup:

ThenormalWildType
Drosophilamelanogaster
specimenwithno
exposuretotheBPSchemicalwillestablishabaselinefornormalbehaviorpatterns.The
behaviorpatternsofFMR1specimenwithnoBPSexposureandFMR1specimenwithBPSwill
becomparedtotheestablishedbaseline.

ControlledVariables:
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Typeoffoodmediasource
Amountoffoodsourcegiventoeachgroup
AmountofaddedBSP
Weeklyintervalsfortransferringallspecimen
Methodforanesthetizing
Environmentalconditions(roomtemperature,atmosphere,lighting)
Typeanddistanceoflighting(Week2Trials)
Typeandamountofadditivebananas(Week3Trials)
Typeofglassculturetubes(includingthespongesandcaps)

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ReviewofLiterature

Indevelopingcountriesaroundtheworld,reportsofchildrenwiththeinabilitytothink,
reason,andlearnhavebeenemerging.Separationofchildrenwhodisplayaggressive,anxious,
andawkwardsocialbehaviorhasformedincountlessschools,fuelingthelevelofostracism
facedbythementallydisabled.Inresponsetothis,thebehavioralsciencefieldhasbecome
heavilyfocusedonresearchingcausesandfactorslinkedtoarangeofdisorderdiagnoses.Dueto
theincreasingawarenessofdisorderssuchasAutisticSpectrumDisorder(ASD)within
communityboundaries,thehopeofmanyresearchersistodevelopanunderstandingonhow
differentmanmadestressessuchasmanyplasticproducingchemicalsaffectvariousstrainsof
Drosophilamelanogaster
(fruitfly)genes.Withsimilargeneticcodesfornormalcognitive
functionsandadisplayofcomplexbehaviorsrelevanttohumans,fruitfliesareaprimemodelto
observeforgeneticandbehavioralpurposes.Additionally,becauseASDisagrowing
developmentaldisorderinmanyyouthstoday,informationfromthistypeofresearchcanbe
appliedtohumanbeingsintermsofunderstandinghowcommonchemicalscanpotentiallyaffect
humanautismlinkedgenes.Studiessuchasthiswillcreateabetterunderstandingofhow

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environmentalandmanmadestressescanaffectvariousdevelopmentaldisorders,furtheringthe
searchforproducingsaferitemsandenvironmentalconditions.

AutismSpectrumDisorderandFragileXSyndrome
AutismSpectrumDisorder(ASD)isarangeofcomplexneurodevelopmentdisorders
thatischaracterizedbysocialimpairments,communicationdifficulties,andrepetitivepatternsof
behavior(AutismFactSheet,2015).Peoplediagnosedwithautismremainindifferenttosocial
interactionandhavedifficultylearninghowtospeakinaccordancewithsocialcues(Autism
Factsheet,2015).Withinthisbroadcategoryofdisorders,FragileXSyndromeisaninherited
geneticconditioncharacterizedbyanabnormalXchromosomeprimarilycausinglearning
disabilitiesandcognitiveimpairmentmorecommonlyassociatedwithASD(FragileX
Syndrome,2014).AlthoughthereisnocorrelationbetweenindividualsdiagnosedwithFragileX
Syndromeandearlydeath,symptomsoftheconditionallowforlittleindependencedueto
dependabilityonapersonwithsufficientsocialandintellectualabilitiesneededforeveryday
activities.IndividualsdiagnosedwiththegeneticconditiondisplaysymptomsofADD,ADHD,
socialanxiety,handbiting,pooreyecontact,sensorydisordersandincreasedriskfor
aggression(FragileXSyndrome,2014).Althoughmanypatientsareabletoadapttothese
symptomsandlivenormalliveswiththeassistanceofacaretaker,FragileXsyndromeremains
thefocusofmanystudiesbecauseitisthemostcommonformofASDinapproximately46%of
malesand16%offemalesdiagnosednationally(LearningAboutFragileX,2013).
FragileXSyndromeisinheritedinanXlinkeddominantpatternbecausetheFMR1
mutatedgeneislocatedontheXchromosomeandasinglecopyofthealteredgeneinanycellis
enoughtocausethedisorder(LearningAboutFragileX,2013).Thegeneticconditioniscaused

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byamutationintheFMR1genewhereaspecificCGGtripletDNAsegmentisexpandedand
repeatedthroughoutthegenomesequence,asdisplayedbelowin
Figure1:NormalGenevs.
FMR1Mutation
(LearningAboutFragileX,2013).Inpeoplewithoutthecondition,theCGG
tripletsegmentisonlyrepeatedfrom5timestoabout50timeswhereasinpeoplediagnosed,the
DNAsegmentisrepeatedover200times(LearningAboutFragileX,2013).Becauseofthe
abnormalrepetitionoftheCGGsegment,theFMR1geneissilencedorinactivatedthus
preventingthegenefromproducingavital
fragilexmentalretardationprotein
(LearningAbout
FragileX,2013).Lossofthisnecessaryproteinresultsinthesignsandsymptomsoffragilex
syndromeinbothmalesandfemales.

Figure1:NormalGenevs.FMR1Mutation

(LearningAboutFragileX,2013)
Inregardstotreatmentfortheinheritedcondition,researchershavenotbeenableto
produceadefinitecureortreatmentmethodtopreventthedisorderorsignificantlyreduce
symptoms(FragileXSyndrome,2014).Toremedyspecificbehaviorsymptoms,special
educationandanticipatorymanagementfocusonavoidingstressfulstimulationofpatients
sensestodecreaseaggressivebehavior(LearningAboutFragileX,2013).Someprofessionals
recommendtheuseofmedicationtomanageparticularbehaviorsymptoms,butnospecific

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medicinehasbeenshowntobenefitthepatientduetointensifyingsideeffectsthatmayincrease
somesymptomswhilereducingothers.SincesymptomsofFragileXSyndromevaryonacaseto
casebasis,medicationisnormallyprescribedforindividualsymptomswithLithiumasthemost
commonformoodinstabilityandProzacforaggression.Intermsofphysicalsymptoms,doctoral
proceduresaretypicallyusedtotreatvision,degradingtissue,hearing,andheartproblems
presentinthepatient(LearningAboutFragileX,2013).Althoughnotherapyorbehavior
interventionhassignificantlyreducedspecificbehaviorproblems,earlyintervention,special
educationcourses,andvocationaltrainingarehighlysuggestedtohelpthepatientadapttoliving
withthecommonsymptoms(AutismFactSheet,2015).

Drosophilamelanogaster
Inordertoresearchanddeterminehowcommonchemicalsandenvironmentalconditions
arelinkedtothesymptomsofFragileXSyndrome,manyresearchersutilizelargesamplesof
Drosophilamelanogaster
specimen.Thecommonlyreferredfruitflyorvinegarflyremainsone
themostfrequentlyobservedmodelsforgeneticresearchduetoitssmallsizeandabilityto
reproduceinmassamounts(Kimball,2014).ThesefruitfliesarepartoftheInsectaclassandfall
undertheDrosophilidaefamily(Miller,2000).Theyinhabitmoistareasinbothruralandurban
areaswheretheycanbefoundnearrottingfruits,hencetheirnametheyreceivetheiressential
nutrientsfromdecayingvegetables,fruits,andothersugarysubstancesthroughamouthdesigned
toconsumeliquids(Kimball,2014).Althoughfruitfliesareconsideredtohaveapositiveimpact

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onhumanlifeduetotheirscientificcontribution,theyarealsoconsideredapestduetotheir
occupationandconsumptionoffruitinhomes,especiallyduringthewinter(Miller,2000).
Thecommonfruitflyisnormallyatancolor
andonly3mminlengthwitharoundedhead,two
largecompoundeyes,threesmallersimpleeyes,and
shortantenna(Miller,2000).Asshownin
Figure2:
DrosophilamelanogasterDevelopment
,maleand
femalespecimencanbedifferentiatedbecause
femalesareslightlylargerwhilemaleshavemoreblackpigmentationonthelowerendoftheir
abdomen(Kimball,2014).Asin
Figure3:DrosophilaPhysicalStructure(TopView)andFigure
4:DrosophilaPhysicalStructure(SideView),
thephysiologicalstructureoftheflyconsistsofa
exoskeletonwiththreemainbodysegmentsandthreepairsofsegmentedlegs(Miller,2000).
Thiswelldevelopedexoskeletonofthefruitflyhelpstoprotecttheinternalorgansthatallowthe
organismtoreproduce.Reproductioninallspeciesoffruitfliesisrapidandproduceshundreds
oflarvaeeggswithinacoupleweeks(Miller,2000).Asinallinsectspecies,maturefruitflieslay
eggsplacedonfruitwhichthenhatchintoflylarvae(maggots).Themaggotstheninstantlystart
consumingthefruittheywerelaidonandbecomesexuallyactiveafterjustoneweek,
reproducingrelativelyquickly(Miller,2000).Essentially,becausethefruitflyhasashort
lifespan,asimplegenome,andiseasilymadetoreproduceincaptivity,itisaprimecandidate
forgeneticresearch(Miller,2000).

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Inregardstothebehavioroffruitfly,malesandfemalessharethesamesimplisticurges
andreactionstoenvironmentalstimuli.Thelocomotionpatternsofnormalfruitfliestendtobe
quickasthespecimenmovesinstraightlinesfromonepointtothenextunlikethesluggish
movementofthosediagnosedwithFragileXSyndrome(Smit,2015).AlsounlikeFragileX
Syndromepatients,becausemostinsectshaveasenseofpersonalspace,fruitfliestendto
distancethemselvesfromoneanotherbyatleastacoupleofcentimeterswhenkeptincontained
areassuchasculturetubes(Smit,2015).Additionally,regularfruitflyspecimenareeasily
attractedbythesmellofanyfoodsourceandwillusuallymatewithanyindividualofthe
oppositesexwhenplacedtogether(Miller,2000).Additionally,thefruitfliesaresensitiveto
lightintensityandwillusuallymigratetowardsourcesofbrightlights(Miller,2000).Although
thisbehaviorexistswithinnormalfruitflies,researchconductedonfruitflyspecimenwith
autisticgeneticmutationshasfoundthatanaddedbrightlightsourcedoesnottriggerthequick
impulseofthealteredspecimentochangedirectionandtraveltowardsthelight(Smit,2015).
Instead,thespecimenappeartobedazedandconfusedastheirflyingpatternsdonot
demonstratetheabilityoftheflytoprocessandrespondtochangesintheenvironment(Smit,
2015).Thisdiscoverypromptedfurtherquestioningtodeterminehowotherenvironmental
changesaffectedthebehaviorofautisticmodifiedfruitfliestohelpstudythegeneticcomponent
behindthesuddenreactiontooutsidestimulitriggeredwithinFragileXSyndromepatients.
BisphenolA(BPA)andBisphenolS(BPS)
Inordertostudytheeffectsofenvironmentalfactorsand
manmadestressesonthebehaviorofgeneticallymodifiedfruit
flies,researchersdevelopedaninterestinfocusingoneveryday

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productsthatcouldintensifyandalterspecificbehaviorsofFragileXSyndromepatients.
BisphenolA(BPA)wasonecommonplasticproducingchemicalfoundinmanyAmerican
householditemssuchasfoodcontainers,babybottles,andsippycupsforkids(FDAsBPABan,
2013).In2013,theU.SFoodandDrugAdministrationhadbannedtheuseofBPAinmass
productionduetofindingslinkinglowdosesofthechemicaltochronicdiseasesincluding
cancer,autisticdisorders,impairedimmunefunction,earlyonsetofpuberty,obesity,diabetes
andhyperactivity(FDAsBPABan,2013).Afterthisban,anothervariationoftheBPA
chemicalknownasBisphenolS(BPS)wasafavoredreplacementbecauseitwasthoughttobe
moreresistanttoleaching(Bilbrey,2014).Althoughproductioncompaniesclaimedthatthe
BPSreplacementwasasaferchemicalduetoitsinabilitytocontaminatefoods,recentresearch
hasfoundthatBPSstillleaksintofoodasNearly81percentofAmericanshavedetectable
levelsofBPSintheirurineandonceitentersthebody,itcanaffectcellsinwaysthatparallel
BPA(Bilbrey,2014).Asshownin
Figure5:ChemicalStructuresofBPAandBPS
,thephysical
makeupofbothchemicalsaresignificantlysimilar,promptingresearcherstoquestionthe
parallelsthatcanbedrawnbetweenthehazardousBPAanditsnewerBPSreplacement(FDAs
BPABan,2013).SinceBPSisnowthemostcommonchemicalusedtoproduceplastic
householditems,researchershavedevelopedaninterestinobservinganylinksbetweenthe
chemicalandFragileXSyndromeashadbeendonetodeterminethedangersofthechemicalon
cellalterationinyoungchildrenandadults.
PriorResearchStudies
StudiesconductedtodeterminenewparallelsbetweenBPAandBPSconsistof
observingtheeffectsbothchemicalshaveonproteins,hormones,andvitalstructureswithinthe

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body.AfterexposinglowconcentrationsofBPAtoseparategroupsofmicespecimen,
researchersfoundthattheenzymeactivityofthemicewasdisruptedinawaythatdecreasedthe
rateofmetabolism(Richteretal,.2008).Inaddition,otherformsofdisruptiontothespecimens
endocrinesystemsincludedthealterationofhormonereceptorgeneswhichultimatelydecreased
thethebodyweightofthespecimen,makingthemweaker(Richteretal,.2008).Becausethe
internalfunctionalstructuresofthemicespecimenwereobservedtobephysicallyaltered,new
studiesdesignedtoobserveanychangesthatexposuretotheBPSchemicalcanhaveonthe
internalsystemsofvariousanimalspeciesshouldbedonetodetermineanyshorttermor
longtermhealthhazardsofthechemicaltohumansandotheranimals.
Inadditiontothephysicalchangeswithintheinternalsystemsofthemiceexposedto
BPAatcriticalperiodsofdevelopment,thespecimenalsoexhibitederraticchangesinbehavior
(Richteretal.,2008).ThestudyledbyresearcherCatherineRichterandherteamalsofound
evidencelinkingtheexposureofBPAtoincreasedaggressionwithinthemiceusingacomposite
aggressionscoretoquantifytheresults(2008).OtherbehavioralchangeswithexposuretoBPA
plasticproducingmonomerincludedanincreaseinhyperactivity,fearofpainfulstimuli,and
anxiousmotoractivitywhichdeterminedthatthechemicalshavetheabilitytointensifyspecific,
strongimpulses(Richteretal.,2008).Byconductingthisstudy,theresearcherswereableto
confirmthat
lowdosesofBPAduringdevelopmenthavepersistenteffectsonbrainstructure,
functionandbehaviorinratsandmice(
Richteretal.,2008
).Sincethisstudyprimarilyfocused
ontheeffectsofBPAonbehavioralchangesinmice,itpromptstheneedtostudythehazardous
effectsofthenewerBPSmonomeronsimilarchangesinordertodetermineifthelatterchemical
withanalmostidenticalchemicalmakeuplendstointensifyingspecificanimalbehaviors.

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Usingfruitflyspecimenasamodelforhumangenetics,otherstudieshavedetermined
thatexposuretobothBPAandBPSislinkedtoanincreaseofaggressivebehavior,ultimately
correlatingexposuretothechemicalsandtheonsetofAutismSpectrumDisorder(Keisukeetal.,
2003).AdistinguishablesymptomofASDconsistsofthediagnoseddisplayingerraticbehavior
andviolenttendencieswhenstimulatedbyanoutsideforce(AutismFactSheet,2015).Interms
oftheexperiment,fruitflieswereintroducedtobothchemicalsthroughtheirfoodmediasource
andoverthecourseof12weekstheaggressivetendenciesofthespecimenweremonitoredby
observingtheamountoftimesonefruitflyphysicallyengagedincontactwithanotherina
specificamountoftime(Keisukeetal.,2003).Theresultsfoundthatfruitflyspecimenexposed
tolowlevelsofBPAandBPSdisplayedrelativelythesameamountofaggressiveness,butin
comparisontothosefruitfliesthatwerenotexposed,thephysicalcontacttendencieswere
increased(Keisukeetal.,2003).AfterconductingmoretestingtrialsonspecificallyBPA,the
researchersdeterminedthatinsomecases,constantexposuretothechemicalpromptedtheonset
ofaggressivesymptomsfoundinhumanchildrendiagnosedwithASD(Keisukeetal.,2003).
However,thisresearchalonedeterminedalinkbetweenaggressivebehaviorandexposuretothe
harmfulchemicalsitdidnotdetermineifexposurewasthecauseofthesymptoms.
Furthermore,otherstudiesconductedtodeterminelinksbetweenBPAexposureand
geneticconditionssuchasFragileXSyndromeattempttocreateacontrolgrouptowhich
parallelsinvolvingBPSexposureandgeneticconditionscanbecomparedwith.Inastudydone
todeterminetheeffectsBPAhasontheoffspringofrodentspecimen,thedigestionoflowlevels
ofBPAduringprenataldevelopmentbythefemaleledtothecreationofoffspringwithvaried
geneticconditions(Palanzaetal.,2008).Afterobservingthebehavioroftheexposedoffspring,

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theresultsdisplayedthatmaternalexposuretoBPAreducedtheintensityofreactiontostimuli
beforepubertyandasadults(Palanza,2008).Additionally,theexposedoffspringalsodisplayed
adecreaseinbehavioralactivityinregardstoexplorationandactivityinafreeexploratoryopen
fieldandexplorationintheelevatedplusmaze(Palanzaetal.,2008).Basedonthesefindings,
theresearchersconcludedthatexposuretoBPAwasalsolinkedtotheonsetofvarious
developmentaldisordersasthespecimendisplayedsymptomsofreducedreactionsto
environmentalchangesandhadhyperactivepatternsofmovement(Palanzaetal.,2008).
Drawingfromthisconclusion,leadresearcherPalanzaandherteamsupportedthebanofBPA
productsandbegantoconductstudiestodetermineifthereplacementBPShadanysimilar
effectsondevelopmentalorgeneticdisordersymptoms.
InadditiontolinkingthecontaminationofBPAtothecausesofspecificconditions,
researchersdevelopedaninterestisdetermininghowthehazardouschemicalcouldintensify
symptomsofpreexistingAutismSpectrumDisorders.Inordertodoso,astudywasconducted
inwhichthefruitflywasusedasamodeltoevaluatetheeffectsofBPAonautismrelated
impairmentstodisplayacorrelationbetweenthechemicalandtheonsetofdevelopmental
disorders(Kaur,Simon,A.Chauhan&V.Chauhan,2015).Acontrolgroupofautisticfruitflies
wascomparedwithanexperimentalgroupofautisticfruitfliesexposedtoBPAduringearly
stagesoflife,andthemovementsofthespecimenweretrackedusingavisionscanner(Kauret
al.,2015).TheresultsgathereddisplayedfruitfliesthatwereexposedtoBPAearlyoninlife
displayeddisturbancesinlocomotionpatternsthatmayrelatetothedecisionmakingand
motivationalstateoftheanimal,confirmingthebeliefthatexposuretoBPAhadintensified
incoordinationsymptomsofautisticanimals(Kauretal.,2015).Studiessuchasthisledtothe

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investigationofBPAanditspotentialhazardouseffectsoncausingorintensifyingbehavior
symptomswhicheventuallyresultedintheterminationofmassproduceditemswithBPA.
Shortlyafter,BPSwasbroughtupasaviablereplacementandwasquicklyputintoproduction
makinghouseholdplasticitemswithoutbeingproperlytestedtoseetheparallelsthenew
chemicalhadwithitsformer(Bilbrey,2014).
MaintenanceandMethods
Sincefruitfliesareoneofthemostcommonmodelsusedtoobservebehavioralchanges,
theprotocolsformaintainingthespecimeninalabarerelativelysimpleduetotheirsmallsize.
Fruitflyspecimenarekeptinsmallculturetubesingroupsofabout1030specimeninorderto
beabletoobservesmallergroupsatatime(Service,n.d.).Additionally,ineachculturetube,a
spongesoakedinfoodmediarestsatthebottomtoallowthespecimentogainallnutrientswhen
necessary(Service,n.d.).Ifnecessary,thespongemayalsocontainlow,concentrateddosesof
chemicalsaswhenaresearchstudyistestingtheeffectsofthechemicalonthebehaviorofthe
exposedspecimen.Inordertoreplenishthefoodmediasource,thefruitfliesmustbetransferred
toanewculturetubewithanewfoodsourceatweeklyintervals(Service,n.d.).Before
transferringthespecimen,theymustberenderedunconsciousbyusingananesthetickitthatwill
temporarilydrugthefruitflies(Service,n.d.).Afterconfirmingthatallthespecimenare
unconscious,carefullytakethethefruitfliesoutandgentlyplacethemintotheirreplacement
tubewiththenewfoodsourcethisprocessmustbedonerelativelyquickbecausemost
anestheticsonlyrenderthespecimenunconsciousfor12minutes(Service,n.d.).
Theculturetubesshouldbekeptatroomtemperatureandshouldpreferablybeplacedin
welllightedareas(Service,n.d.).

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Inmanystudies,thebehaviorofthefruitfliesisobservedandanalyzedusingvideo
footagethatdetailsthemovementofthespecimenwithspecialattentiontotheirphysical
structures(Smit,2015).Inothercases,akeyusingarangeofnumberstorankthespecimens
movementaswellasotherpatternsofbehaviorisdevelopedthroughcarefulobservationof
regularfruitflybehavior(Smit,2015).Often,thesekeysareutilizedtocomparethebehaviorof
geneticallymodifiedfruitfliesorfruitfliesexposedtocertainenvironmentalandmanmade
stressestoregularbehavioralpatterns.Inmoststudies,thekeysdevelopedtorankcertain
behaviorsareusedincaseswherecomparisonsaretobemadebetweenvariousspecimengroups
whereasmostmovementtrackingvideodevicesareusedtostrictlydetermineonespecimen
groupsbehaviorpattern.
Inconclusion,the
Drosophilamelanogaster
specieshasincreasinglybeenutilizedasan
experimentalmodelforhumangeneticresearchbyobservingtheeffectsofmanmadeand
environmentalstressesontheirbehaviorandgenes.Byfocusingonphysicalandbehavioral
changesinparticular,researchershopetolearnmoreaboutthehazardsassociatedwithcertain
commonmaterialsinordertocreatesaferproducts.Insocietiestoday,developmentaldisorder
diagnoses,particularlyFragileXSyndromeintheASDcategory,areincreasinginresponseto
changingenvironmentsanddailyexposuretopotentiallyharmfulchemicals.Notably,dailyBPS
chemicalexposureisbecominganissuethatisundercarefulobservationtodetermineifany
hazardsparallelthoseoftheformerBPAchemical.Inordertogainmoreinsightintohow
commonexposuretotheBPSchemicalspecificallyaffectsfruitflybehaviors,theideabehind
variousstudiesistoobserveif
Drosophilamelanogaster
withinsertedFMR1FragileX
SyndromegenesexposedtotheBPSchemicalwilldisplaydrasticchangesintheirlocomotion,

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reactionstostimuli,andclumpingpatterns.Inthismodernage,itiscriticaltotakemeasurements
reducingtherisksassociatedwithhazardouschemicalstopreventthesubstantialeffects
intensifyingdisordersymptomscanhaveonvariouspopulationsaroundthecountry.

Materials

WildTypeDrosophilamelanogaster(30specimen)
FMR1GeneInsertionGeneticallyModifiedDrosophilamelanogaster(60specimen)
4,4SulfonyldiphenolBisphenolS(35grams)
Formula424DrosophilaMedium,Plain(1Liter)
Freezer
DrosophilaGlassCultureTubes(1030)
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CylinderSponges(1030)
IncandescentLampswithLightBulbs(3)
DarkChamberLabArea
Bananas(15)
Balance
CrucibleHoldingDish
TapWater
10mLBeakers(24)
DisposablePipettes
StirringRod
Scoopula
GraduatedCylinder
Ruler
MaskingTape
GlovesandFaceMask

Procedure:

PreparingtheFoodMediumandBPSSource:
1. ObtainFormula424DrosophilaPlainMediumandsixculturetubes.Addapproximately
4gramsofdrymediaandapproximately10mLofwatertothebottomofeachculture
tube.
2. Makesuretoaddenoughwatertothemediaflakesuntilanaccuratesolidconsistencyis
reached.Thesixculturetubesshouldbefilledofthewaywiththemediafoodsource.

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3. ObtainaBisphenolS(BPS)chemicalsampleandusingwater,concentratethechemical
toasafedosageina10mLbeakerbyadding2gramsofthechemicalpowderwith5mL
ofwater.
4. Addapproximately23mLoftheconcentratedBPSchemicaltotwooftheculturetubes
preparedwiththemedia.ThesetwoculturetubesshouldbelabeledBPS.

PreparingtheDrosophilaHabitats:
5. Obtainapproximately30geneticallynormalWildType
Drosophila
specimenand
approximately60FMR1geneticallymodified
Drosophila
specimenthatwillbeobserved
forthedurationofthethreeweektestingperiod.
6. Makesuretohavepreviouslypreparedthesixglasstubeswiththefood(andBPS)
sourcesthatwillserveasthehabitatsforthethreedifferent
Drosophilamelanogaster
groups.
7. LabelthefirsttwotubesControlwithnumbers12andplaceapproximately10
geneticallynormalflieswithnoBPSexposureineachtubetoserveasacontrolbaseline
fornormalbehavior.
8. LabelthenexttwotubesNoBPSwithnumbers34andplaceapproximately10FMR1
fruitflieswithnoBPSexposureineachtubetoestablishabaselineforautisticlike
behavior.
9. WiththelasttwotubeslabeledBPSwithnumbers56,placeapproximately10FMR1
fruitflieswithBPSexposureineachtubetoserveastheexperimentalgrouptoobserve
theeffectsofBPSonautisticlikebehaviors.
10. Placeallsixlabeledtubesinastable,dryarea.Makesuretoplugthetopsofthetubes
withfoamspongestopreventescape.

MaintenanceoftheFruitFlies:
11. Keepthefruitflyspecimenintheirassignedtubesatroomtemperatureforthethreeweek
testingperiod.
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12. TheFormula424willneedtobereplacedeveryweekbytransferringthefruitfly
specimenintoanewtubewithanewfoodmediumsource.
13. Repeatsteps111inordertocreatethenewfoodmediumtobeplacedintosixnewtubes
forallthreegroups.Makesuretokeeptrackofeachindividualcontrolandexperimental
specimenastheyarebeingtransferred.
14. Beforetransferringthefruitfliesintotheirnewassignedtubes,theymustberendered
unconsciousbyplacingeachtubeonahorizontalslantinsideafreezerforapproximately
12minutes.
15. Foreachoftheindividualgroups,monitortheamountoftimespentinsidethefreezerand
after12minutes,takethetubesoutandlaythemdownontheirside.Donotleavethe
tubesstandingverticallytopreventthespecimenfromdrowninginthemediawhile
unconscious.
16. Whilethefruitfliesareunconscious,transferallthespecimenintotheirnewlyassigned
tubepayingcloseattentiontothelabelsoneachtube.
17. Transferthefruitfliesbyholdingthefrozentubeataslantandslowlypouringthe
unconsciousspecimenintothenewlypreparedtubealsoheldataslant.Afterthetransfer
iscomplete,immediatelycapthenewtubestopreventescapeandplacethemona
horizontalslant.
18. After12minutes,thespecimenshouldregainconsciousnessandthenewtubescanbe
placedverticallyupright.
19. Rinsetheoldtubeswithwaterandsoapandallowthemtodry.

WeekOneTesting:
20. Forweekoneofthetestingperiod,maintainallsixfruitflygroupsataconstantroom
temperatureinaspecificareaofthelaboratory.
21. Onadailybasisfor5days,atadesignatedtime,utilizethekeyprovidedtoobserveand
recordthelocomotionandclumpingpatternsofthefruitfliesinallsixtubes.

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22. Assigneachcontrolandexperimentalgroupalocomotionnumberandaclumping
patternnumberbasedontheprovidedkeyatintervalsof10minutesforatotalof30
minutes.
23. Remembertoassignnumbersbasedonthekeytoeachgroupasawhole,notas
individualspecimen.
24. Attheendoftheweek,followsteps1219totransferthefruitfliesintoanewassigned
culturetubewithafreshfoodmediumsource.

WeekTwoTesting:
25. Forweektwoofthetestingperiod,maintainallsixfruitflygroupsataconstantroom
temperatureinadarkenedareaofthelaboratory.
26. Obtainthreelampseachwithanincandescentlightbulb.Assignonelamptoshineonthe
sideofthetwoControltubes.AssignonelamptoshineonthesideofthetwoNo
BPStubes,andassignthelastlamptoshineonthesideofthetwoBPStubes.Make
suretodistancethelampsfromtheculturetubesatapproximatelya0.3meterdistance.
27. Onadailybasisfor5days,atadesignatedtime,utilizethekeyprovidedtoobserveand
recordthelocomotion,clumpingpatterns,andreactiontolightwithinthefruitfliesinall
ninegroups.
28. Assigneachgroupalocomotionnumber,aclumpingnumber,andareactionto
lightnumberbasedontheprovidedkeyatintervalsof10minutesforatotalof30
minutes.
29. Remembertoassignnumbersbasedonthekeytoeachgroupasawhole,notas
individualspecimen.
30. Attheendoftheweek,followsteps1219totransferthefruitfliesintoanewassigned
culturetubewithafreshfoodmediumsource.Thereshouldnolongerbeany
incandescentlightsshiningonthespecimentubes.

WeekThreeTesting:

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31. Forweekthreeofthetestingperiod,maintainallthreefruitflygroupsataconstantroom
temperatureinaspecificareaofthelaboratory.
32. Obtainabananaandsliceoutsixpiecesoftherawfruit.Placeoneoftheseslicesnearthe
topoftheeachvialandsealthecaptopreventescapeorcontamination.Thisonefruit
slicewillremainintheindividualtubesforthedurationofthetestingday.
33. Onadailybasisfor3daysatadesignatedtime,utilizethekeyprovidedtoobserveand
recordthelocomotion,clumpingpatterns,andreactionstoanadditionalfoodsource
withinthefruitfliesinallgroups.
34. Assignthecontrolandeachexperimentalgroupalocomotionnumber,aclumping
patternnumber,andareactiontoadditionalfoodsourcenumberbasedonthe
providedkeyatintervalsof10minutesforatotalof30minutes.
35. Remembertoassignnumbersbasedonthekeytoeachgroupasawhole,notas
individualspecimen.
36. OnadailybasisforWeekThreeTesting,makesuretoswitchoutthesixbananaslices
forafreshoneattheendoftheobservationalperiodforthatday.

DisposalandCleanUp
37. Inthecaseofdeadfruitflyspecimen,disposeofthespecimenbyplacingthemina
plasticbagtothenthrowawayintothegarbage.
38. Disposeoftheoldfoodmediumsourcesbyrinsingtheculturetubeswithwaterandsoap.
39. MakesuretodisposeofthethefruitslicesfromWeekThreestrialsinagarbagecan.
40. Washoutthetubesandanyequipmentusedtotouchthefruitflyspecimenduringthe
durationoftheexperiment.

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ObservationalKey

LocomotionPatterns

NumberRange

Description

Over80%ofthefruitfliesintheculturetubesdisplaypatternsofquick
movementsinastraightlinefromonepointtothenext.Thespecimentravel
longerdistancesinonestrideandmovefromareatoareafrequently.

About50%ofthefruitfliesintheculturetubesdisplaypatternsofquick
movementsinastraightlinefromonepointtothenext.Thespecimentravel
longerdistancesinonestrideandmovefromareatoareafrequently.

Theother50%ofthefruitfliesintheculturetubesdisplaypatternsof
sluggishnessandmoveslowly,ifatall.Theytravellessdistanceswith
circularmotionsandtendtonotmovefromanareafrequently.

Over80%ofthefruitfliesintheculturetubesdisplaypatternsof
sluggishnessandmoveslowly,ifatall.Theytravellessdistanceswith
circularmotionsandtendtonotmovefromanareafrequently.

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ClumpingPatterns

Number
Range

Description

VisualReference

Over80%ofthefruitfliesintheculture
tubesdisplaypatternsofdistancingfrom
eachindividualspecimenby
approximately12cm.Thespecimendo
notfrequentlycomeintocontactwith
oneanother.

(Servicen.d.)

About50%ofthefruitfliesintheculture
tubesdisplaypatternsofdistancingfrom
eachindividualspecimenby
approximately12cm.Thespecimendo
notfrequentlycomeintocontactwith
oneanother.

Theother50%ofthefruitfliesinthe
culturetubesdisplaypatternsofclose
clumpingcategorizedbyfrequently
contactingoneanother.Theindividual
specimendonothaveadistanceof
approximately12cmbetweeneach
other.Manyofthespecimenalsodisplay
patternsofpilingupononeanother.
26

(HarvardMedicalSchool,2010)

Lodhi

Over80%ofthefruitfliesintheculture
tubesdisplaypatternsofcloseclumping
categorizedbyfrequentlycontactingone
another.Theindividualspecimendonot
haveadistanceofapproximately12cm
betweeneachother.Manyofthe
specimenalsodisplaypatternsofpiling
upononeanother.

(Service,n.d.)

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ReactiontoLightPatterns

Number
Range

Description

Over80%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
towardtheincandescentlightsource.Thespecimenalsodevelopatendencyto
stationthemselvesonthesideoftheculturetubewheretheadditionallight
sourceshines.

Over80%ofthefruitfliesintheculturetubesdisplaypatternsofmovingaway
fromtheincandescentlightsource.Thespecimenalsodevelopatendencyto
stationthemselvesonthedarkersideoftheculturetubeswithoutadditional
light.

About50%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
towardtheincandescentlightsource.Thespecimenalsodevelopatendencyto
stationthemselvesonthesideoftheculturetubeswheretheadditionallight
sourceshines.

Theother50%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
awayfromtheincandescentlightsource.Thespecimenalsodevelopa
tendencytostationthemselvesonthedarkersideoftheculturetubeswithout
additionallight.

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ReactiontoAdditionalFoodSourcePatterns

Number
Range

Description

Over80%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
towardtheadditionalfoodsource.Thespecimenmorefrequentlygather
nutrientsfromtheaddedfoodsourcethanthefoodmediaatthebottomofthe
culturetubes.

About50%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
towardtheadditionalfoodsource.Thespecimenmorefrequentlygather
nutrientsfromtheaddedfoodsourcethanthefoodmediaatthebottomofthe
culturetubes.

Theother50%ofthefruitfliesintheculturetubesdisplaypatternsofmoving
awaytheadditionalfoodsourceornotreactingtothefoodatall.Thespecimen
continuethepreviouspatternofgatheringnutritionsfromthefoodmediasource
atthebottomoftheculturetubes.

Over80%ofthefruitfliesintheculturetubesdisplaypatternsofmovingaway
theadditionalfoodsourceornotreactingtothefoodatall.Thespecimen
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continuethepreviouspatternofgatheringnutritionsfromthefoodmediasource
atthebottomoftheculturetubes.

Results:DataTables

Week1Trials

Day1:LocomotionPatterns

Day2:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day1:ClumpingPatterns

Day2:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:LocomotionPatterns

Day4:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

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Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:ClumpingPatterns

Day4:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day5:LocomotionPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

Day5:ClumpingPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

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BPS5

BPS6

Week2Trials
Day1:LocomotionPatterns

Day2:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day1:ClumpingPatterns

Day2:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day1:ReactiontoLightPatterns

Day2:ReactiontoLightPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

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Day3:LocomotionPatterns

Day4:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:ClumpingPatterns

Day4:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:ReactiontoLightPatterns

Day4:ReactiontoLightPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

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Day5:LocomotionPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

Day5:ClumpingPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

Day5:ReactiontoLightPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

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Week3Trials
Day1:LocomotionPatterns

Day2:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day1:ClumpingPatterns

Day2:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day1:ReactiontoAdditionalFoodSourcePatterns

Day2:ReactiontoAdditionalFoodSourcePatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

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Day3:LocomotionPatterns

Day4:LocomotionPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:ClumpingPatterns

Day4:ClumpingPatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

Day3:ReactiontoAdditionalFoodSourcePatterns

Day4:ReactiontoAdditionalFoodSourcePatterns

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control1

Control2

Control2

NoBPS3

NoBPS3

NoBPS4

NoBPS4

BPS5

BPS5

BPS6

BPS6

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Lodhi

Day5:LocomotionPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

Day5:ClumpingPatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

Day5:ReactiontoAdditionalFoodSourcePatterns
FruitFly
Groups

10Mins
Ranking(#)

20Mins
Ranking(#)

30Mins
Ranking(#)

Control1

Control2

NoBPS3

NoBPS4

BPS5

BPS6

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Results:Graphs

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Results:DataAnalysis

Accordingtothe
Control1AverageBehaviorPatterns
graph,thespecimenreceiveda
rankingof1forthethreeweektestingperiodsignifyingthatlocomotion,clumping,reactionto
light,andreactiontoadditionalfoodsourcepatternsremainedconsistentandnormal.Aswith
Controlgroup1,the
Control2AverageBehaviorPatterns
graphalsoexhibitsarankingof1for
thethreeweektestingperiodsignifyingthatover80%offruitfliesinthesecondcontrolgroup
alsodisplayednormallocomotion,clumping,reactiontolight,andreactiontoadditionalfood
sourcepatterns.
Accordingtothe
NoBPS3BehaviorPatterns
graph,thespecimenreceivedarankingof
1forweeksoneandtwointermsofnormallocomotionandclumpingpatternsforthefirstweek
andnormallocomotion,clumping,andreactiontolightpatternsforthesecondweek.Forweek
3,theNoBPS3groupaveragedarankingof1.2forlocomotionandclumpingpatterns.
Additionally,withanaverageofrankingof1.8forreactiontoadditionalfoodsourcepatterns,a
smallamountofspecimenbegantoshiftfromthenormalbehaviorranking.Accordingtothe
No
BPS4BehaviorPatterns
graph,thespecimeninthisgroupdisplayedsimilarpatternstogroup3
witharankingof1forlocomotionandclumpingpatternsforweek1.Week2displayed1.2for
locomotionpatterns,2forclumpingpatterns,and1.4forreactiontolightpatternssignifyingthat
themajorityofspecimendisplayednormalbehaviorpatternsinthosecategorieswhileasmall
amountshiftedfromthenormalrankingof1.Week3displayedarankingof1fornormal
locomotionpatternsandarankingof1fornormalreactiontoadditionalfoodsourcepatterns
whilereceivingarankingof1.9forcrowdedclumpingpatterns.

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Lodhi
Accordingtothe
BPS5BehaviorPatternsGraph,
thelocomotionpatternsofthe
specimenprogressedfromarankingof1.6forweekone,toarankingof2forweektwo,anda
rankingof2.7forweek3signifyingalargeshiftfromthenormalmovementpatterns.For
clumpingpatterns,thespecimendisplayedalargeshiftfromtheregulardistancedbehavior
progressingfromarankingof1forweekonetoarankingof2.4forweektwo,and2.1forweek
three.Group5alsodisplayedirregularreactionstolightandadditionalfoodsourcebyranking
averagesof2.1and1.6forweekstwoandthreerespectively.Accordingtothe
BPS6Behavior
Patterns
Graph,locomotionpatternsprogressedfromanormalrankingof1forweekonetoa
rankingof2forweektwoandarankingof2.6forweekthree.Theclumpingpatternsalso
progressedfromarelativelynormalrankingof1.4forweek1toarankingof2forweektwoand
arankingof2.6forweekthree.Group6alsodisplayedirregularreactionstolightandadditional
foodsourcebyrankingaveragesof2.1forweeksoneandtwoforbothbehaviortypes.
Accordingtothe
AverageWeek1
Patterns,allControlandNoBPSgroupsdisplayeda
normalrankingof1forlocomotionandclumpingpatterns.BPSGroup5displayedirregular
locomotionpatternsbutdisplayednormalclumpingpatterns.Additionally,BPSGroup6
displayedirregularclumpingpatternsbutdisplayednormallocomotionpatterns.Accordingto
AverageWeek2Patterns
,bothControlgroupsandNoBPSgroup2displayedregular
locomotion,clumping,andreactiontolightpatternswitharankingof1.NoBPSgroup4
displayedslightlyirregularbehavioraveragingrankingsbetween1and2forallthreebehavior
categorieswithclumpingpatternshavingthegreatestshiftwitharankingof2.BPSgroups5and
6bothdisplayedstrongerirregularbehaviorpatternswithrankingshigherthantheothergroups
averaging2forlocomotionpatternsforgroup5and6andclumpingpatternsforgroup6.

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Additionally,reactiontolightpatternsreceivedrankingsover2signifyingirregularbehavior
patterns.Accordingto
AverageWeek3Patterns
,bothControlgroupsdisplayedregularbehavior
patternswitharankingof1whereastheNoBPSgroupsdisplayedirregularlocomotion,
clumping,andreactiontoadditionalfoodsourcepatternsreceivingrankingsbetween1and2.
BPSgroups5and6displayedevenstrongerirregularbehaviorpatternswithrankingsbetween2
and3forlocomotion,clumping,andreactiontoadditionalfoodsourcepatterns.
Regardingthecomprehensivedataaccumulatedinthe
AverageBehaviorPatterns
graph,
Controlgroups1and2displayedregularbehaviorwitharankingof1throughouttheentire
testingperiod.NoBPSgroups3and4displayedregularbehaviorforlocomotionandclumping
patternswhiledisplayingirregularbehaviorforthereactiontolightandadditionalfoodsource
patternswithrankingsbetween1and2.BPSgroups5and6displayedthemostirregular
behaviorintermsofallbehaviorpatternsreceivingaveragerankingsabove1.5foralltypesof
behavior.Additionally,therankingsforBPSgroups5and6weremuchcloserinnumbertoa
rankingof2forallfourcategoriesthanallothergroups.Inrelationtoeachother,theNoBPS
groupsremainedcloserinrankingtotheControlgroupswhereastheBPSgroupsreceived
rankingsgreaterinnumberthantheNoBPSgroups.Essentially,theaveragerankingsforthe
Controlgroupsdisplayedconstant,normalbehaviorwhiletherankingsfortheBPSgroups
displayedthemostirregularbehaviorpatternshiftsshownthroughthehighestrankingsasthe
trialsprogressed.

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Results:Discussion

Accordingtothecomprehensivedataforallthreeweeks,theresultsdisplaythatthefruit
flieswithFMR1mutationsheldintubeswithBPSexhibitedthemostintenseautisticbehaviors
basedontheirhigherrankings.TheControlgroupswithnogeneticmutationsorBPSexposure
exhibitedregularbehaviorbyexhibitingquick,erraticmovements,distancingthemselveswithin
thetubes,andbeingattractedtothelightsourcetotheadditionalfoodsourcebecausetheywere
accustomedtotheirenvironmentandwerenotexposedtooutsidechemicals.TheNoBPS
groupswithFMRImutationsbutnoexposuretoBPSdisplayedbehaviorthatexhibitedslight
shiftsfromtheregularpatternsbutnottotheextentoftheBPSgroupsbecausetheywere
geneticallymodifiedbutwerestillaccustomedtoanenvironmentexemptfromtheadditionof
unnaturalchemicals.Essentially,asshownthroughthehigherrankingsoftheBPSgroupsin
comparisontotheNoBPSgroups,exposureofBPSisshowntointensifyparticularautism
symptomsbecausetheadditionofanunnaturalBPSchemicalcreatedanewenvironmentthat
affectedthealreadyalteredgeneticmakeupofthespecimen.
Additionally,therankingsgiventotheControlgroupsandtheNoBPSgroupswere
similarinnumbersinlargepartduetosurvivinginanenvironmentthatthespecimenwere
accustomedtofreefromtheadditionofanunnaturalchemical.AlthoughtheNoBPSgroupsdid
47

Lodhi
haveslightshiftsfromtheregularbehaviorpatterns,theirchangesinlocomotionandclumping
remainedrelativelysimilartothoseoftheControlgroupswhilechangesinthereactiontolight
andadditionalfoodsourceweremoresignificantthiscanbeattributedtotheNoBPSgroup
specimenalreadydisplayingirregularbehaviorfromweekoneandintensifyingthesepatternsin
thelatertwoweeks.ThegreatestshiftinbehavioroccurredfromtheControlgroupstotheBPS
groupswherethelattergroupsdisplayedirregularbehaviorattributedtosurvivinginan
unnaturalenvironmentwithalteredgenesinsteadofonlyhavingamutatedgeneticmakeup.The
locomotionandclumpingpatternsoftheBPSgroupsdifferedthemostfromtheControlandNo
BPSgroupswhereasthereactiontolightandadditionalfoodsourcepatternsremainedcloserto
theNoBPSgroupthiscanonceagainbeattributedtotheBPSspecimenalreadydisplaying
extremelyirregularbehaviorfromweekoneandintensifyingthesepatternsinthelatertwo
weeks.AlthoughtheNoBPSgroupsalsodisplayedslightchangesinbehaviorpatterns,thedata
suggeststhatexposuretoBPSmayindeedintensifyparticularautisticbehaviorsymptoms
becausetherankingsoftheBPSgroupsremainedlargerthanthoseattributedtotheNoBPS
groups.
Itisinferredthatthisintensifyingofautisticsymptomsoccurredduetotheexposureof
BPStotheFMR1mutatedfruitfliesjustaspreviousstudiesdeterminedthatthenowbanned
BPAplasticproducingchemicalhadbeenlinkedtocancer,autisticdisorders,andotherimmune
disorders(FDAsBPABan,2013).BecauseBPSisacommonchemicalthatissimilarin
structuretoBPA,theadverseeffectsonfruitflieswithFMR1mutationsappearedtobesimilar
topreviousstudiesdeterminingthatautisticfruitflieschangedlocomotionandclumpingpatterns
withexposuretotheBPAchemical(Smit,2015).Additionally,althoughthereisnowidely

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acceptedreasonastohowBPAdirectlyaffectsgenes,itisinferredinthisexperimentthat
exposuretoBPSaffectedtheFMR1mutationsinthefruitfliesbylimitingtheirreactionsto
outsidestimuliasaresultofalteredcognitiveandphysicalmakeupthiscomparisonissimilarto
thatofpreviousresearchwhichdeterminedthatexposuretoBPAwaslinkedtospecimen
exhibitingsymptomsofreducedreactionstoenvironmentalchangesandhyperactivemovement
patterns(Palanzaetal.,2008).Essentially,thisexperimentsupportsfindingspreviouslynoted
throughevidenceofthereducedbehaviorpatternsinrelationtoBPAexposurebydisplaying
evidencethatthesimilarlystructuredBPSmayshareadverseeffectswiththeformerchemicalby
affectingthegeneticmakeupandcognitivefunctionsofthefruitflies.

Results:ExperimentalError

Inthisexperiment,errorscouldhaveoccurredmainlyduetotheunpredictivemannersof
thefruitflies.First,sincetherewereapproximately10fruitflieswithineachtube,therewasno
definitewayofaccountingfortheamountofmedia(orBPS)eachindividualspecimen
consumedduringthethreeweektestingperiod.AlthoughaspecificamountofmediaandBPS
wasgiventotheassignedgroups,somespecimeninthegroupcouldhaveconsumedmorethan
another.Thiscouldhaveledtosomespecimenconsumingmoreorlessmedia(orBPS)during
thetestingperiodduetolackorexcessoffoodfromthepastdays.ConsumingmoreorlessBPS
couldhavealteredthebehaviorpatternsofthespecimeneitherbyintensifyingparticular
symptomsorreducingothers.Inordertohelpavoidthechanceofthiserror,thetubeswere
monitoredtomakesureapproximatelythesameamountofspecimenremainedineachgroupfor
thetestingweek.Additionally,eachgroupwasgivenapproximately4gramsofdrymediaand
forgroups5and6,approximately2mLoftheconcentratedBPSchemical.

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Anothererrorcouldhavebeenthedeathofsomespecimenalongwiththequick
reproductiveabilitiesofthefruitflies.Assomespecimendiedduringthetestingperiod,the
reproductivecapabilitiesofthefruitfliesreplacedthedeadbutcouldhavealteredthedata
becausenewspecimenwerenowbeingobserved.Sincefruitfliesareminisculeinsizeandlarge
innumber,therewasnodefinitewaytokeeptrackofthedeadandthenewbornsoallthe
specimeninonetubeweregroupedtogether.Thisgroupingwaseffectivebecausethespecimen
inonetubewerekeptinthatconstantenvironmentforthetestingperiodhowever,havingno
distinctionbetweenthenewlybornandtheolderfruitfliescouldhavealteredthebehaviorbased
onnaturalpatternsoftheyoungcomparedtothoseoftheolderspecimen.Additionally,some
tubesmayhaveheldmoreorlessspecimenduetorandomdeathsandfasterreproductive
capabilitiesinfluencingthedatabycreatingalargerorsmallersamplesizeforthatparticular
group.Inordertoavoidhavingnodistinctionoftheyoungandtheold,fruitfliesalteredtonot
reproducecouldbeobservedtocreatemorestablesamplegroupswiththesamespecimenbeing
observedfortheentiretestingperiod.
Moreover,thetransferringofthefruitfliesmayhavealsoaffectedthedataonaminiscule
levelbecausethespecimenineachgroupwereplacedindifferenttubesattheendofeachweek.
Althoughtheenvironmentsinsidethetubewerekeptthesameintermsofdrymediaand/orBPS
exposure,transferringthespecimencouldhaveslightlyshiftedthedatabecausethefruitflies
wereplacedinamorefreshenvironmentattheendofeachweek.Becausethefoodsource(s)
werechangedattheendofeachweek,thelessfreshmediaand/orBPSchemicalcouldhavehad
lessofaneffectonthebehaviorofthefruitfliesattheendoftheweekincomparisontothe
moreintenseexposuretothefreshmediaand/orBPSchemicalatthestartoftheweek.Toavoid

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thisweeklypatternofbehaviorchangesinresponsetoafreshorspoiledfoodsource,eachtube
shouldbegivenafreshmediaand/orBPSsourceattheendofeachday.
Additionally,experimenter'sbiasmayhavealteredtherankinggiventoeachcontroland
experimentalgroupbecausetheexperimenteralreadyhadanideaofwhattheexpecteddata
shouldlooklike.Inordertoavoidhavingexperimenter'sbiasaffecttherankinggiventoeach
group,anoutsidepersonunawareofthestudyssetupwasgiventheobservationkeyandasked
torankeachgroupatparticularintervals.Topreventtheoutsidepersonsrankingfromalso
developingabias,23peopleweregiventhekeyandaskedtorankthebehaviorofeachgroup
ondifferentdaystogatheraccuratedata.

Conclusion:

Inthisexperiment,thepurposewastodeterminetheimpactofBPSonthebehavior
patternsof
Drosophilamelanogaster
withFMR1geneinsertionstodetermineifexposuretothe
chemicalintensifiesFragileXSyndromesymptomsmodeledbythespecimen.Thehypothesis
thataccompaniesthispurposestatesthatif
Drosophilamelanogaster
modelingFragileX
SyndromewithinsertedFMR1genesareexposedtoBPS,thenthegeneticallymodifiedfruit
flieswithexposurewilldisplaydrasticchangesintheirlocomotion,reactionstostimuli,and
clumpingpatterns.Essentially,exposuretothenewerBPSchemicalshouldintensifyparticular
autisticbehaviorsofthefruitfliessinceFragileXSyndromeisplacedunderthebroader
categoryofAutismSpectrumDisordersandsinceBPSissimilarinstructuretothepreviously
harmfulBPAchemicalusedtoproduceplastics.

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Inordertoconductthisexperiment,acontrolgroupofgeneticallynormalflieswithno
BPSexposure,agroupofFMR1fruitflieswithnoBPSexposure,andagroupofFMR1fruit
flieswithBPSexposurewereobservedandtheirbehaviorchangeswerecompared.Usingan
observationalkeyoveraperiodofthreeweeks,thelocomotion,clumpingpatterns,reactionto
light,andreactiontoadditionalfoodsourceswererankedandusedtodetermineifBSPexposure
hadanintensifyingeffectonfruitfliesmodelingautismsymptoms.TheControlgroups
establishedabaselinefornormalbehaviorpatternsconsistingofquickmovements,creating
distancesbetweenotherspecimenwithinthetube,andshowingattractiontoadditionallightand
foodsources.Usingtheobservationalkey,theNoBPSgroupsandBPSgroupswereobservedto
determineiftheydisplayedintensifiedautisticsymptomsofsluggishmovements,crowded
clumping,andretractionfromadditionallightandfoodsources.
Accordingtothedata,itcanbeconcludedthatthehypothesiswasindeedsupported
becausethefruitflieswithFMR1mutationsexposedtoBPSdisplayedsignificantintensified
particularautisticsymptomswhentestedduringthethreeweekperiod.Evenmoreso,forall
threeweeks,therankingsofthebehaviorcategoriesdisplaythattheBPSgroupsexhibited
strongerautisticsymptompatternsthantheNoBPSgroupwhichshowedslightshiftsfromthe
regularbehaviorpatternsestablishedbytheControlgroups.Uponfurtheranalyzation,itis
inferredthatthefruitflieswithFMR1mutationsandexposuretoBPSdisplayedintensified
behaviorpatternsbecausetheadditionofaharmfulchemicaltotheirsurroundingsreactedwith
thealreadyalteredgeneticmakeupofthespecimentointensifybehaviorduetochanged
cognitiveandphysicalabilities.Overall,becauseautismisagrowingdevelopmentaldisorderin
manyyouthstoday,informationfromthisresearchcanbeappliedtohumanbeingsintermsof

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understandinghowcommonchemicalscanpotentiallyaffecthumanautismlinkedgenessimilar
tothoseoffruitflies.Researchsuchasthiswillcreateabetterunderstandingofhow
environmentalandmanmadestressescanaffectvariousdevelopmentaldisorders,therefore
furtheringthesearchforproducingsaferitemsandenvironmentalconditions.

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