TheEffectofCaffeineonDrosophilaMelanogaster

Alec,Avery,Chiara,TeAndre
MarchApril2017

Purpose:
Thepurposeofthisexperimentwastotestandobservetheeffectsofcaffeineand
caffeineaddictiononfruitfliesandtheiroffspring.Itisimportanttoseehowanaddiction
toanysubstancecanchangebothindividualsandpopulations.

Prediction:
WepredictedthatDrosophilaexposedtocaffeinewouldbemoreinclinedtoprefer
caffeinatedfoodsources.Andbyextension,thenextgenerationthatgrewupwith
caffeineintheirfood,wouldpreferitaswell.

Introduction:
DrosophilaMelanogasterarewhatweareusingtorunourexperiment,theyrealso
knownasfruitflies.Theyareamodelorganismsbecausetheyhaveashortlifespan
andafastreproductionrate,allowingustoseetheaffectsontheiroffspring.Youcan
getlargenumberofthematatimegivingyoualargersamplesize,givingmore
accurateresultsacrosstheboard.Animalmodelsareimportantforbiologicalresearch
becausetheyallowscientiststoreproducehumandiseasesorabnormalbehavior
withouttheethicalconcernsinherentinhumanstudies.Althoughthereareobvious
differencesbetweenhumansandotheranimals,therearemanymolecularandcellular
processesthataresharedamongallspeciesthroughevolution.Forthisexperiment,we
willbeusingartificialselectiontoseetheeffectsofcaffeinepreferenceontheoffspring
offruitfliesthathaveonlybeenexposedtocaffeine,andthosewhohaveonlybeen
exposedtonormalfood,andiftheresadifference.Artificialselectionisthebreedingof
plantsandoranimalstoproducedesirabletraits.Humansshareabout60%oftheir
DNAwithfruitflies,andoverhalfoftheadultsinamericaareaddictedtocaffeine.

 Materials:

Threeplastictubes:oneforthecaffeinegroup,oneforthenoncaffeinegroup
andoneforbothoptions
CaffeinePills
Flyfood(teaspoonfood,4mLwater)
MortarandPestle
Dividerbetweenfoodoptions(cardboard)
Camera(Fortimelapse)
CO2

IndependentVariable:Food(Caffeinatedornormal)
DependantVariable:Whichfoodtheflieschoosetogoto(predisposedtocaffeineand
non).
ConfoundingVariables:D eathofflies,reproductionrates

Procedure#1

1. SerialDilution
a. Testcaffeinepillsonfliesusingserialdilutiontodetermineasafebut
effectivequantityofcaffeineforourexperiment.
b. Crushupcaffeinepillandmixwithwater.Addpartsofcaffeinewater
solutiontoregularwater,reducingthepreviouscaffeinecontentby9/10.
c. Setup4testtubeswithflyfoodmixandputeachdifferentcaffeinelevelof
thecaffeinewatersolutionineachtube.
d. Testonfliestoseewhichcaffeinelevelwillaffecttheflies,butnotkill
them.

2.Setuphabitats
a. Takethreetesttubesandplacea1inchcardboarddividerinoneofthe
tubestosplitthecaffeineandnoncaffeinefoodsources.
b. Putcottonballsonthetoptoallowthefliestobreathandtoallowusto
anesthetizetheflieseasier.

3.Makethefood
a. 1 /2teaspoondryflyfood(leveledoff)and4mlH2O.Tapandswirlvial
immediatelyafteradditionofH2O.Letsitfor10min.
 b. Makethecaffeinefoodsource.addanamount(dependingonserial
dilution,.1forourgroup)ofacrushedupcaffeinepilltotheH2Obefore
mixingwithflyfoodpowder.Repeatabovesteps.

4.AddFoodtoHabitats
a. Addcaffeinatedfoodsourcetotheonehabitatandhalfofthesurfaceof
anotherhabitat.
b. addnoncaffeinatedfoodsourcetothethirdhabitat(ourcontrolgroup)
andtotheotherhalfofthesurfaceofthesecondhabitat.*Itisimportant
tohaveacontrolgroupsoyouknowifyourfindingsaresignificant
ornot.

5.PutFliesinHabitat
a. UseCO2toanesthetizeflies.
b. Separateequallybynumberandsexintoeachexperimenthabitat.
c. Makesuretheresenoughfemalesandmalestogethertoreproduceinthe
separategroups.
d. Placetheoneflygroupintothetubewiththecaffeinatedfoodsource,and
theothergroupintothenoncaffeinatedfoodsource.

6.Reproduction
a. Waitfor23weeksforfliestoreproduce.Thiswillformanewpopulation,
selectivelybredwithexposuretocaffeine.

7.TestOffspring
a. UsingCO2,anesthetizeoffspringfromcaffeineandnoncaffeinetubes
separatelyatdifferenttimes.
b. Moveoffspringintothebothoptionstubeatseparatetimes.
c. Recordwithslowmocameraknockingthemtothebottomofthetube(10
times).
d. Seehowmanygotothecaffeineandnoncaffeinefoodsources.Record
results.

8.Comparedatafrombothgroups





 Procedure#2:

1. CreateHabitats
a. Afterrunningserialdilutionanddeterminingasafeamountofcaffeinefor
thefliestoconsume,putacaffeinatedfoodsourceinonetube.
b. Putanoncaffeinatedfoodsourceintoanothertube.

2. RecordWeight
a. weighbothtubestofindtheamount/volumeoffoodineach.
b. Writedowntheweight.

3. PlaceFlies
a. Putanequalamountoffliesintoeachhabitat.
b. Thenumberofflieswas10ineach.

4. Letthetubessitforanextendedperiodoftime,preferablyaweek.

5. TestNewWeights
a. Nowthatthefliesareaccustomedtothetubesandareeatingthefood,it
istimetoseeifthevolumeoffoodischangingatadifferentrate
dependingondifferenceofthefoodsource.
b. Anesthetizeandremoveallfliesandlarvaeinbothexhibits.
c. Measuretheweightofthetubes.
d. Recordweights.

6. CalculateResults
a. Usemathtofindouthowthevolumeoffoodischangingovertime.We
candrawconclusionsfromhowtheamountchanges.
b. Writedownanyobservationsorchanges.

Procedure#3:
1. SetupHabitats
a. Cutoutapieceofcardboardtodividethetubeintotwoequalhalves.
b. Fillonesidewithcaffeinatedfoodpredeterminedbytheserialdilutionand
filluptheothersidewithnoncaffeinatedfood.
c. Waitforfoodtodry

2. PreparetoTestFlies
a. Anesthetizefliesindividuallyandplaceintotestingvile.

3. Runexperiment.
a. Whenflywakesup,knockthetubeonthetable.
b. Recordwhichfoodsourcetheflygoestovianegativegeotaxis.

4. RecordResults
a. Createatableforthefirsttentrialsandthesecondtentrials.
b. Recordthisforeachflyandrepeatasmanytimesaspossible.

 Results
Figure1:RawDataTable&Graph
Fly1 Fly2 Fly3 Fly4

1st10 2nd10 1st10 2nd10 1st10 2nd10 1st10 2nd10

Caffeine 2 8 3 8 1 6 3 8

Normal 8 2 7 2 9 4 7 2

 Flowgram

 Conclusion:

MeaningofData
Whatweconcludedwasthatcaffeinehasastrongeffectonfruitflies.Notonly
didouroriginalexperimentfail,butallofthefliesinthecaffeinetesttubedied.Even
thoughouroriginalplanandmethodforrecordingthecaffeineaddictioninfruitflies
didntsucceedthereisstillmoretobetakenaway.Somethinginterestingthatwe
observedwasthatnotonlydidallofthefliesdie,butthevolumeoffoodwasmuch
lowerthanthenoncaffeinated.
Thiscanmeanmanythingsandwecantmakeanexactclaimonwhythis
happened,butwecameupwithtwopossibleexplanations.Eitherthefruitfliesbecame
addictedtocaffeineandthenoverate,orthecaffeineshortenedthelifespanoftheflies
andtheyhadtoconsumemorefood.Thiscouldexplainwhythefoodvolumewas
different,butnothowtheydied.Weagainhypothesizetwopossibilities.Eithertheflies
becameaddictedtothecaffeineintheirfoodanddiedviaovereating,orthecaffeine
wastoostrongandcausedmajorhealthproblemsforthedrosophila.

N extSteps
Tosalvageourexperimentandproject,wedecidedtofollowuponthese
outcomes.Werantwofollowupexperiments,thefirsttestingthedifferenceinfood
volumebetweencaffeinatedandnoncaffeinatedfoodsources.Thesecondwastesting
onindividualfliespreferencetocaffeineovertime.
Thefirstexperimentwassetupwithonetesttubewithacaffeinatedfoodsource,
andonewitharegularfoodsource.Wemeasuredthemassofeachtubebeforeweput
inflies,andthenweputthesameamountoffliesineachtube.Thisexperimentisstilla
workinprogress,butwewillmeasuretheweightofthetubesaftertheflieshavebeen
inhabitingthetubeforsometime.Toavoidconfoundingvariables,wewillremoveall
flies,eggs,andlarvaebeforeweighingthetubes.Thisexperimentistotestwhetheror
notthefliesarereallyovereatingorjustbeingaffectedbyhealthfactors.
Thesecondtestisaboutpreferenceandtoseeiffliesbecomeaddictedto
caffeineovertime.Wesplitatesttubeintotwosections,caffeinatedandnormalfood
sources.Wethenputoneflyinatatimeandmeasuredwhichsidetheywentto.We
observeanindividualflytentimes,andthentestthenextone.Whatweobservedisthat
theredoesseemtobeadevelopmentofpreferenceorevenaddictionbeginninginthe
fruitflies.
Thisisshownthroughtheaverageofthefourflieswetested.Thefirsttentimes
wetestedtheirpreferences,theflieseitherwenttothecaffeineornormalside.Thefirst
tentimestheflieschosecaffeine2.25timesonaverage,andthesecondtime,7.5
chosecaffeineonaverage.Thisshowsthepreferencegrowsovertimefromexposure
tothesubstance.

FutureImprovement
Whilethisdataissignificant,thereisstillroomforgrowthinourexperiment
sincewehaveonlytestedfourflies.Tomakethisexperimentbetterandimprovenext
time,Weshouldrunmoretestsandtrials,andcarryoutthefoodvolumetheoryfora
longerperiodoftimetobettershowthesignificance.Forouroriginalideaand
procedure,thereisclearlymoretolearn.Wewerentabletotestanyoffspringdueto
theextremeeffectsthecaffeinehasonfruitflies.Toimprovethiswemustgivetheflies
amuchsmallerconcentrationofcaffeine.Thiswouldallowourfliestohaveoffspring
thatwillsurviveandthatstillareaffectedbycaffeine.