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Behavioral choice assays and alcohol preference in Drosophila melanogaster
(USC Thesis Other)
Behavioral choice assays and alcohol preference in Drosophila melanogaster
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Content
!
!
Behavioral!Choice!Assays!and!Alcohol!
Preference!in!Drosophila*melanogaster*
By!
Zachary!Marshel!Polonus!
!
*
*
A!Thesis!Presented!to!the!!
FACULTY!OF!THE!USC!GRADUATE!SCHOOL!
UNIVERSITY!OF!SOUTHERN!CALIFORNIA!!
In!partial!Fulfilment!of!the!
Requirements!for!the!Degree!
MASTER!OF!SCIENCE!
(Biochemistry!and!Molecular!Biology)!
August!2016!
!
!
Copyright!2016!! ! ! ! ! !Zachary!Marshel!Polonus!
! ! ! !
!
!
!
ACKNOWLEDGMENTS.
! I!would!like!to!show!my!gratitude!to!my!advisor!Dr.!Sergey!Nuzhdin!for!your!
encouragement!in!my!research!over!the!last!several!years.!!Your!advice!has!been!
invaluable!to!me.!I!would!also!like!to!thank!the!other!members!of!my!thesis!
committee!Dr.!Zoltan!Tokes!and!Dr.!Joseph!Hacia.!
! I!would!like!to!thank!the!entire!Nuzhdin!Lab!for!always!being!helpful!and!
supportive.!!I!would!like!to!express!special!thanks!to!Dr.!Sarah!Signor!who!has!
given!me!constant!guidance!and!help!in!both!the!conduction!of!my!research!and!
the!writing!of!this!thesis.!!Lastly,!I!would!like!to!thank!Dr.!Brad!Foley!for!assisting!
me!with!the!statistical!analysis!on!this!thesis.!!!
!
Thank.you.all.for.your.help.and.support. .
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Table.of.Contents.
1.! Introduction..!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!1.
a.! Drosophila*as!A!Model!OrganismYBackground!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!1.
b.! Raleigh!Inbred!Lines!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!!5.
c.! !Alcohol!and!Drosophila!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!!5.
d.! Ethanol!Metabolism!in!Drosophila*.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!6.
e.! Behavioral!Changes!Associated!to!Ethanol!in!Drosophila!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!!7.
f.! Mode!of!Ethanol!Intoxication!and!Response!Regulation!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!7.
g.! YYMaze!Use!in!Drosophila*Behavioral!Assays!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!!8.
h.! Olfaction!in!Drosophila.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!*.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!..!10.
i.! Objectives!of!the!Project!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!11.
2.! Results..!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!12.
a.! Behavioral!Choice!Assay!–!Ethanol!Intoxication!Phase!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!12!
b.! Behavioral!Choice!Assay!Data!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!12!
c.! Ethanol!Preference!for!Full!Data!Set!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!14.
d.! Ethanol!Preference!Phase!II!of!Experiments!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!!15.
3.! Discussion..!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!17.
4.! Materials.and.Methods..!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!20.
a.! Drosophila*melanogaster*Rearing!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!20.
b.! Genetic!Cross!Creation!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!21.
c.! Behavioral!Choice!Assay!Assembly!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!22.
d.! Behavioral!Choice!Assay!Procedure!Phase!One!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!24.
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e.! Behavioral!Choice!Assay!Procedure!Phase!Two!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.27.
5.! References!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!.!29!
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Abstract.
Excessive!alcohol!consumption!is!a!common!cause!of!preventable!death!in!most!countries,!
and!imposes!a!large!socioeconomic!burden!in!terms!of!health!care!and!violent!crime.!Not!all!
individuals!who!consume!alcohol!will!become!alcoholics,!so!to!study!variation!in!susceptibility!to!
its!effects!and!differences!in!inclination!for!chronic!use,!we!need!to!look!into!our!genetic!
makeup.!!Using!Drosophila*melanogaster!as!a!model!organism,!we!are!able!to!study!the!effects!
of!alcohol!and!how!it!is!related!to!genetic!differences.!!Drosophila!can!be!used!as!the!model!
organism!not!only!because!we!have!the!tools!decode!their!genomes!and!sequence!individual!
genotypes!in!large!reproducible!quantities,!but!also!because!they!have!similar!reward!pathways!
and!physical!symptoms!of!alcohol!use!as!humans.!!In!this!study,!D.*melanogaster*are!given!the!
choice!between!an!odor!for!which!they!have!a!known!preference,!ethyl!acetate!(EA)!and!ethanol!
(ETOH).! ! D.* melanogaster! are! given! this! choice! with! and! without! a! previous! exposure! to!
ETOH.!!We!have!found!two!significant!pieces!of!evidence!to!support!the!hypothesis!that!D.*
melanogaster!is!a!good!model!of!ethanol!abuse.!!First,!we!found!that!there!was!a!significant!
interaction!between!preYexposure!to!ETOH!and!preference!for!ETOH.!!After!further!analysis!of!
selected!genotypes,!we!found!that!there!was!a!significant!interaction!between!the!change!in!
preference!for!ETOH!and!the!genotype.!!This!demonstrates!potential!variation!in!susceptibility!to!
the!rewarding!effects!of!ethanol!in!naturally!occurring!genotypes.!!In!the!coming!months,!mRNA!
will!be!extracted!and!sequenced!to!give!further!insight!as!to!what!transcriptomic!expression!
might!be!linked!to!this!varying!preference.!!With!this!information!relating!gene!expression!to!
preference!and!susceptibility!toward!ETOH,!we!will!move!one!step!closer!to!understanding!how!
some!people!are!more!susceptible!to!the!negative!aspects!of!alcohol!use.
!
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Page!|!1!
1.!Introduction!
!
As!early!as!7,000!BCE!evidence!of!purposeful!fermentation!and!creation!of!consumable!
alcohol!was!achieved!in!Northern!China.!!Since!that!time,!alcohol!has!become!ubiquitous!to!all!
nations!and!cultures.!!While!its!production!can!be!considered!an!art!form,!its!role!in!both!religious!
and!cultural!settings!has!become!inseparable,!and!globally,!the!repercussions!of!alcohol!use!on!
health!and!society!is!undeniable.!!Available!to!all!over!the!legal!age,!alcoholic!beverages!have!
great!propensity!for!causing!addiction,!injury,!death,!and!myriad!of!health!issues.!In!the!US!alone,!
nearly!88,000!deaths!are!caused!by!alcohol!related!issues,!making!it!the!third!most!preventable!
cause!of!death.!!In!addition!to!this,!alcohol!misuse!problems!cost!the!United!States!223.5!billion!
dollars!as!seen!in!a!2006!study!(NIH,!2016).!!While!the!cost!is!great,!not!all!who!drink!alcohol!
misuse!it,!become!addicted,!or!contribute!to!the!statistics!above.!!The!difference!between!those!
who!use!alcohol!responsibly!and!those!who!do!not!cannot!is!not!just!attributed!to!a!person’s!
nurture,!but!also!to!their!nature.!It!is!generally!believed!that!a!person’s!genetic!makeup!can!make!
them!more!susceptible!to!the!negative!affects!of!alcohol!consumption!and!addiction.!!To!
understand!how!the!genetics!of!an!individual!makes!them!more!or!less!likely!to!succumb!to!ill!
effects!of!alcohol,!the!model!organism,!Drosophila,!was!used!to!begin!to!elucidate!that.!!This!
section!will!describe!why!Drosophila*were!used!for!this!project!and!how!it!will!help!us!move!
forward!in!trying!to!understand!the!genetic!basis!of!alcohol!abuse.!
Drosophila*as.A.Model.Organism.–.Background.
! In!the!order!Diptera!and!family!Drosophiladae,!Drosophila,!more!commonly!known!as!the!
fruit!fly,!has!become!a!critical!part!in!much!of!biological!exploration!and!creativity.!!The!most!well!
!
!
Page!|!2!
understood!and!widely!used!subgenus,!Drosophila*melanogaster,!which!was!used!to!in!this!
project,!was!popularized!in!1910!when!it!was!used!to!confirm!the!Chromosomal!Theory!of!
Inheritance!(Morgan,!2010).!!Originating!in!SubYSaharan!Africa,!D.*melanogaster!has!migrated!to!
all!continents!that!are!occupied!by!humans.!!D.*melanogaster!is!a!human!commensal!organism!
that!migrated!via!the!trade!of!fruit,!upon!which!they!are!commonly!found.!!Part!of!what!makes!
the!Drosophila!such!an!effective!model!organism!is!its!ability!to!reproduce!quickly,!consistently,!
and!in!large!numbers!in!a!laboratory!setting.!This!alone!would!not!make!Drosophila!a!great!
model,!but!the!they!also!contain!compact!genomes!that!are!easy!to!manipulate!with!many!
orthologous! genes! to! humans.! ! Many! of! these! orthologous! genes! are! also! thought! to! be!
associated!with!human!disease!(Hale,!Korey,!Larracuente!&!Roberts,!2015).!!These!characteristics!
alongside!the!recent!development!of!powerful!technology!for!genetic!analysis!have!allowed!
Drosophila!to!become!a!mainstay!around!the!world!for!behavioral,!evolutionary,!and!genetic!
research.!!!
! The!short!Drosophila*life!cycle!is!an!integral!part!of!their!utility!in!research.!In!a!9Y10!day!
period,!D.*melanogaster,!can!develop!from!a!fertilized!egg!into!adult!form!at!25!degrees!Celsius.!!
The!time!of!maturation!from!egg!to!adult!can!easily!be!affected!by!changes!in!temperature.!!At!
lower!temperatures!this!process!can!take!up!to!19!days!to!occur!(Hale,!Korey,!Larracuente!&!
Roberts,!2015).!!Following!fertilization,!the!process!of!embryogenesis!takes!24!hours!on!average!
to!complete!and!leads!to!three!larval!stages.!!The!first,!second,!and!third!larval!stages!are!referred!
to!as!instars!and!are!easily!differentiated!by!molting!events!that!occur!between!each!of!the!
stages.!!The!first!and!second!instar!takes!an!average!of!1!day!while!the!third!instar!usually!lasts!2!
days.!!Thus,!it!typically!takes!5!days!for!completion!of!larval!development.!!At!this!point!the!
!
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Page!|!3!
Drosophila!goes!through!metamorphosis!in!a!hard!chitin!pupal!case!that!acts!as!protection!for!4Y
5!days!(Hale,!Korey,!Larracuente!&!Roberts,!2015).!!During!this!time,!the!adult!Drosophila!is!
formed!as!the!larval!tissues!are!broken!down.!!This!process!is!mediated!by!the!steroid!hormone!
ecdysone,!which!is!instrumental!in!regulating!gene!expression!changes!from!larval!to!adult!
pattern!(Yamanaka,!Rewitz!&!O’Connor,!2013).!!Once!the!adult!Drosophila*springs!from!its!pupal!
case,!or!ecloses,!it!will!then!become!sexually!mature!within!8!hours!permitting!the!lifecycle!to!
repeat.!!!
! The!Drosophila*genome!contains!both!intra!and!interYspecies!variation!much!like!other!
eukaryotic!organisms,!and!it!has!become!one!of!the!most!well!studied!genomes.!!With!a!genome!
size!around!180!Mb,!in!comparison!to!the!human’s!3000!Mb,!the!Drosophila!has!lent!itself!to!
being!heavily!sequenced!as!technology!has!progressed!(Bosco,!Campbell,!LeivaYNeto,!Markow,!
2007).!!Of!the!180!Mb!in!the!Drosophila*genome,!120!Mb!are!comprised!of!euchromatin.!!!!
Euchromatin!accounts!for!the!DNA!that!is!loosely!bound!around!histones!which!allows!for!active!
transcription.!Drosophila*also!have!only!four!chromosomes!pairs.!The!Drosophila*genome!was!
the!second!multicellular!organism!to!be!sequence,!after!the!C.*Elegans,!using!whole!genome!
shotgun!sequencing!(Adams!et*al,*2000).!!By!2002,!13,600!genes!had!been!identified!in!D.*
melanogaster!(Mirsa!et*al.*2002).!!Currently,!over!13,920!protein!coding!genes!have!been!
elucidated!using!sequenced!transcripts!from!cDNA!and!RNAseq!data.!!The!D.*Melanogaster*genes!
are!organized!by!giving!an!annotation!of!CG,!for!proteinYcoding,!and!CR!for!nonproteinYcoding!
genes.!!In!2007,!with!the!aim!to!make!the!D.*melanogaster!genome!more!available!for!scientific!
use!and!collaboration,!the!modENCODE!project!was!initiated.!!This!project!sought!to!define!and!
understand!all!of!the!functional!elements!in!the!both!the!C.*elegans*and!the!D.*melanogaster*
!
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Page!|!4!
(modENCODE!Consortium,!2010).*Functional!genomics!is!a!subset!of!genomic!studies!that!
focuses!on!understanding!the!relation!between!a!gene,!its!functions,!and!interactions.!!Due!to!
the!large!consortiums!and!individual!labs,!scientists!now!have!access!to!enormous!data!sets!that!
describe! the! Drosophila* genome.! ! The! scientific! communities’! continued! interest! in! the!
understanding!of!Drosophila*continues!to!make!it!a!powerful!genetic!tool.!
! The!use!of!Drosophila!can!be!a!powerful!tool!to!uncover!mechanisms!of!neurobiology!and!
behavior!as!it!relates!to!genetics.!!This!type!of!study,!called!neurogenics,!has!helped!uncover!how!
genetics!effects!sensory!biology,!sexual!behavior,!levels!of!aggression,!circadian!rhythms!and!
more!(Voshall,!2007).!!Beginning!with!experiments!in!the!1960’s,!The!Benzer!Lab!identified!the!
bacteriophages!genetic!structure!and!hypothesized!that!single!genes!could!control!specific!
behaviors.!A!prime!example!of!neurogenenics!came!in!the!form!of!the!gene!called!fruitless*(fru)*
discovered!by!two!of!Benzer’s!trainees,!Hall!and!Yoshiki!Hotta.!!The!fruitless!gene!was!found!to!
govern!heterosexual!behavior!in*Drosophila.**This!is!significant!because!Drosophila*have!complex!
and! specific! courtship! rituals! that! go! through! a! number! of! different! stages.! ! When! the!
transcription!factor!encoded!by!the!fruitless!gene!was!mutated,!groups!of!male!Drosophila!
became!homosexual!and!performed!the!courtship!rituals!upon!each!other!creating!a!large!chains!
of!males!all!in!concert!!(Voshall,!2007).!!In!today’s!world!of!advanced!genetic!techniques!such!as!
RNAYSeq,!scientists!are!able!to!combine!genetic!screens!with!simple!behavioral!assays!to!help!
elucidate!the!genetic!basis!of!behaviors.!!This!is!meaningful!because!of!evolutionary!genetic!
conservation.! ! Evolutionary! genetic! conservation! relates! gene! function! in! less! complex!
organisms,!like!Drosophila,!to!more!complex!organisms!like!mice!or!humans.!!Thus,!behavioral!
genetic!studies!of!Drosophila*can!give!us!insight!to!those!same!types!of!behaviors!in!humans!
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(Bellen!et*al.*2010).*In!addition,!examples!of!gene!mutations!in!Drosophila!giving!rise!to!similar!
phenotypic!changes!in!humans!that!relate!to!disease!have!laid!the!ground!work!for!continued!
study!and!understanding!of!neurogenics.!!
!Raleigh.Inbred.Lines.
The!best!understood!North!American!population!of!Drosophila*originates!from!Raleigh!
North!Carolina!and!are!denoted!as!Raleigh,!Drosophila.!!From!this!original!location,!192!different!
Drosophila*lineages!have!been!inbred!to!a!point!close!to!complete!homozygosity!and!have!also!
been!fully!sequenced.!!All!of!the!192!lineages!are!derived!from!a!single!outbred!lineage.!!The!
work!done!to!establish!this!collective!of!public!genomic!information!is!referred!to!as!the!
Drosophila*melanogaster*Genetic!Reference!Panel!or!the!DGRP!(Mackay!et*al.*2012).!!The!DGRP!
is!important!because!it!allows!future!research!and!use!of!Raleigh,!(R),!Drosophila*melanogaster*
to!compare!genomic!information.!(Mackay!et*al.*2012).!!In!this!experiment!14!of!the!192!
sequenced!Raleigh!Drosophila*were!used!in!hopes!of!eventual!genomic!comparison!to!the!
existing!DGRP!available.!!!
Alcohol.and.Drosophila**
Drosophila*melanogaster,*commonly!known!as!the!fruit!fly,!is!known!as!such!because!of!
its!tendency!to!be!found!on!rotting!or!fermenting!fruit.!!In!the!natural!environment!of!the!
fermenting!fruit,!ethanol!is!produced!and!thus!Drosophila*have!become*accustomed!to!ethanol!
exposure!in!varying!concentrations.!!This!consistent!exposure!over!time!has!possibly!lead!to!the!
genetic!evolutionary!changes!in!the!Drosophila’s*ability!to!regulate!responses!to!ethanol!via!
natural! selection! (Guarnieri! and! Heberlein,! 2003).! ! This! example! of! natural! selection! has!
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conceivably!lead!Drosophila*to!a!point!of!being!resistant!to!the!effects!of!ethanol!intoxication!
and!to!have!the!ability!to!metabolize!ethanol!into!a!usable!source!of!energy.!!!
Ethanol.Metabolism.in.Drosophila*
Much!like!in!the!case!of!mammals,!Drosophila*convert!ethanol!in!a!three!step!process!that!
allows!it!to!enter!the!citric!acid!cycle.!!Ethanol!is!first!oxidized!into!acetaldehyde!by!the!enzyme!
alcohol!dehydrogenase,!ADH.!!Following!this!reaction,!the!acetaldehyde!is!converted!into!acetate!
using!the!enzyme!aldehyde!dehydrogenase,!ALDH.!!Lastly,!the!acetate!is!ligated!to!a!CoYA!and!
produces!acetyl!CoYA,!which!can!enter!in!the!TCA!cycle!and!be!used!in!the!synthesis!of!fatty!acids.!!
It!has!been!determined!that!both!ADH!and!ALDH!are!critical!for!ethanol!metabolism!in!adult!
Drosophila*by!testing!the!effects!of!ethanol!on!Drosophila*with!deficiencies!in!one!or!both!of!the!
enzymes.!!It!was!found!that!without!either!enzyme!there!is!a!resulting!increase!in!sensitivity!to!
ethanol!(Guarnieri!and!Heberlein,!2003).!!!One!major!difference!between!the!ADH!enzyme!in!
Drosophila*and!mammals!is!that!the!mammalian!ADH!enzyme!is!a!longer!zinc!metalloenzyme,!
where! as! the! ADH! enzyme! in! drosophila! is! shorter! and! more! analogous! to! a! shortYchain!
dehydrogenase!(MckinleyYMcKee,!Winberg,!Petterson,!1991).!!The!significance!of!the!shortYchain!
dehydrogenase!is!that!is!it!conserved!in!many!eukaryotic!organisms!ranging!from!yeast!to!
humans.!!This!indicates!the!possibility!that!Drosophila*may!have!evolved!from!organism!in!the!
same!family!that!were!not!able!to!survive!in!environments!containing!ethanol.!!
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Behavioral.Changes.Associated.to.Ethanol.in. Drosophila!
Coinciding! with! the! similarities! of! the! mechanisms! by! which! Drosophila* metabolize!
ethanol!to!mammals,!there!too!are!similarities!in!the!ways!Drosophila*physically!respond!to!
ethanol!exposure.!!It!was!found!using!3!different!types!of!behavioral!assays;!narrow!tubes!assays,!
locomotor!chambers,!and!inebriometers,!that!low!to!moderate!concentrations!ethanol!can!cause!
Drosophila*to!have!hyperactive!locomotion!(Singh!and!Heberlein,!2000)!(Kaun!et*al.!2011).!!At!
higher!concentrations!of!ethanol,!it!was!found!that!the!result!was!complete!sedation!after!a!
period!loss!of!coordination!(Devineni!et*al.*2011).!Many!experiments!have!also!been!performed!
in!order!to!elucidate!specific!mutations!in!Drosophila*cause!variation!in!sensitivity!and!tolerance!
to!ethanol.!!
Mode.of.Ethanol.Intoxication.and.Response.Regulation.
! Unlike! other! drugsYofYabuse! such! as! heroin! or! cocaine! which! bind! to! very! specific!
receptors!in!the!brain!to!potentiate!the!effects!of!reward!pathways!in!the!brain,!alcohol!has!a!
much!less!specific!mechanism!of!action!that!leads!to!a!myriad!of!effects!on!mammals!and!
Drosophila.!!As!is!directly!related!to!the!metabolism!of!ethanol,!ADH!plays!a!crucial!role!in!
adjusting!the!Drosophila*sensitivity!to!ethanol.!!Defective!adh!genes!and!decreased!expression!
of!ADH!have!been!shown!to!cause!an!increase!in!sensitivity!to!ethanol!and!decreased!preference!
for!ethanol!(Ogueta!et*al.*2010).!!
!One!main!pathway!affected!by!ethanol!ingestion!is!the!dopamine!reward!pathway.!It!was!
found!that!dopamine!was!necessary!for!the!expression!of!conditioned!preference,!or!creation!of!
the!reward!pathway!in!response!to!ethanol.!The!dopaminergic!pathway!is!expressed!through!the!
!
!
Page!|!8!
neurons!mushroom!body!of!the!Drosophila*brain!(Kaun!et*al.!2011).!!!This!finding!implicates!the!
dopaminergic!pathway!as!a!potential!site!of!addiction!studies.!Neuropeptide!F,!NPF,!which!has!
the!homolog!neuropeptide!Y!in!humans,!has!also!been!implicated!to!effect!the!circuit!in!which!
acute!ethanol!sensitivity!is!modulated.!!It!was!found!that!increased!expression!of!the!NPF!
correlated!with!higher!sensitivity!to!ethanol!and!the!opposite!was!found!for!lower!expression!
and!knockout!of!NPF!(Wen!et*al.*2005).!!One!theory!as!to!how!ethanol!interacts!with!cells!to!
cause!intoxication!is!through!the!disruption!of!lipid!membranes.!!This!disruption!also!leads!to!
perturbation!of!transmembrane!protein!function!that!are!a!part!of!the!lipid!membranes.!One!
type!of!transmembrane!protein,!the!NMDA!receptor,!is!particularly!important!because!it!is!part!
of!the!glutamate!receptor!family!that!responds!to!crucial!excitatory!neurotransmitters.!!These!
excitatory!neurotransmitters!are!partially!responsible!for!a!number!of!neural!functions!including!
cognitive! function! and! motor! coordination.! ! It! was! also! determined! that! the! intoxicating!
properties!of!ethanol!intoxication!correlated!to!increased!solubility!in!lipid!membranes!(Peoples,!
Weight,!1995).!!The!various!genes!and!pathways!associated!with!ethanol!intoxication!and!
sensitivity!may!play!a!combined!role!in!the!overall!development!preference!for!ethanol.!!We!
eventually!aim!to!understand!what!that!role!is!by!understanding!the!expression!of!the!proteins!
associated!with!each!important!factor.!
YPMaze.Assay.Use.in.Drosophila*Behavioral.Assays!
! During!our!research,!we!implemented!the!use!of!a!YYmaze!behavioral!assay!in!order!to!
evaluate!the!preference!for!ethanol!in!naturally!occurring!Raleigh!inbred!lines.!The!YYmaze!
behavioral!assay!is!simple!and!efficient!because!it!gives!the!Drosophila*an!option!between!two!
choices,!and!each!of!the!choices!can!be!controlled!conditions!manipulated!and!controlled.!!Due!
!
!
Page!|!9!
to!the!simplicity!and!easily!replicable!nature!of!the!YYmaze!behavioral!assay,!it!has!come!into!use!
for!a!number!of!different!studies!covering!topics!from!olfaction!and!habituation!to!modeling!
addiction.!!YYmaze!assays!have!also!been!widely!used!in!other!animal!models!like!the!mouse!
(Silverman!et*al.*2010).!
! In!one!recent!study,!a!simple!YYmaze!behavioral!assay!was!used!to!test!the!chemosensory!
response!of!Drosophila*based!on!olfaction.!!Here,!they!tested!the!Drosophila*level!of!avoidance!
of!two!different!aversive!smells!emitted!by!piece!of!filter!paper!soaked!in!either!acetic!acid!or!
phenylacetic!acid.!!In!a!controlled!condition!of!total!darkness,!they!were!able!to!generate!a!
consistent!index!of!olfactory!preference!using!the!YYmaze!apparatus!(Simonet!et*al.*2014).!!The!
YYmaze!assay!model!in!our!study!was!closely!based!on!the!one!in!this!study!which!employs!an!
environment!that!allows!the!Drosophila!to!make!a!choice!based!solely!on!olfaction.!
In!a!more!complex!study!a!YYmaze!apparatus!was!used!to!determine!if!Drosophila*
experience!the!rewarding!effects!of!ethanol!intoxication!and!what!neurons!were!responsible!for!
this!pathway.!!Here,!the!YYmaze!was!connected!to!a!series!of!mechanisms!that!controlled!the!
flow!of!vaporized!ethanol!and!other!cues!using!different!tube!systems.!!By!giving!two!neutral!
olfactory!cues!and!having!one!associated!with!an!intoxicating!dose!of!ethanol!they!were!able!to!
generate!a!conditioned!preference!index!for!ethanol!preference.!!One!conclusion!made!was!that!
while!ethanol!was!initially!aversive,!the!Drosophila*showed!preference!when!tested!24!hours!
after!initial!exposure.!!It!was!also!discovered!that!Drosophila!would!overcome!an!aversive!
stimulus!in!the!form!of!an!electric!shock!to!attain!ethanol!(Kaun!et*al.!2011).!!This!kind!of!
conditioned!preferential!behavior!is!hypothesized!to!be!a!process!much!like!addiction!or!at!least!
the!mimicry!of!the!mammalian!reward!pathway.!!It!is!important!to!note!that!they!did!not!explore!
!
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Page!|!10!
variation!in!the!development!of!preference,!as!the!main!goal!of!the!paper!was!to!establish!
Drosophila!as!a!model!system!for!alcohol!research,!demonstrating!that!it!meets!the!DSMYIV!
guidelines!for!addiction.!!In!this!study!we!intend!to!understand!the!development!and!variation!of!
preference!for!ethanol!as!a!paradigm!for!addiction.!!While!it!is!controversial!that!Drosophila!can!
be!used!to!represent!potential!behavioral,!biochemical!and!genetic!patterns!akin!to!ethanol!
addiction!in!humans,!we!postulate!that!this!possibility!is!one!worth!exploring.!
Olfaction.in.Drosophila*
. The! Drosophila! olfactory! system! is! well! understood! because! of! its! simplicity,! the!
possibility!to!evaluate!single!neuronal!connectivity,!and!its!relation!to!the!vertebrate!olfactory!
system.! Odor! is! conveyed! through! olfactory! receptors! neurons,! ORNS,! located! on! small!
projections!called!sensilla!that!are!attached!to!the!antennae!and!maxillary!palps.!!There!are!50Y
60!different!types!of!ORNs!that!respond!to!different!odorants!(Wilson,!2013).!!Located!on!the!
sensilla!are!odorant!receptors!and!odorant!binding!proteins,!(OBP).!!OBPs!are!apart!of!large!
family!of!genes!expressed!in!the!sensilla!and!are!essential!for!managing!much!of!the!intake!of!
Drosophila*olfaction!(Swarup,!Williams,!Anholt,!2011).!!OBPs!include!both!pheromoneYbinding!
proteins,!PBP,!and!general!odorant–binding!proteins,!GOBP,!and!they!serve!to!relay!information!
to!the!ORNs.!!LUSH,!an!OBP,!was!determined!to!have!a!sterically!defined!ethanolYbinding!pocket!
and!without!that!lush*gene!expression!there!was!an!abnormal!response!to!ethanol.!This!implies!
that!an!ethanolYLUSH!complex!is!necessary!to!activate!olfactory!neurons.!!It!was!also!found!that!
there!is!conservation!in!the!ethanol!binding!motif!in!LUSH!in!mammalian!alcohol!sensitive!
receptors!(Kruse,!2003).!
!
!
Page!|!11!
! For!this!experiment,!one!of!the!odors!used!in!the!YYmaze!assay!was!an!ethyl!acetate!
mixture!with!paraffin!oil.!!Ethyl!acetate!was!used!because!it!is!an!attractive!odor!that!reacts!
strongly!with!the!Drosophila*olfactory!system.!!It!is!also!a!compound!that!can!be!found!naturally!
in!the!wild!on!fruit!during!fermentation!which!suggests!that!it!may!be!an!important!attractant!
for!Drosophila*(Grewal!et*al.**2014).!
!! Olfaction!plays!an!important!role!in!this!experiment!because!it!is!the!only!stimulus!given!
to!the!Drosophila*in!the!YYmaze.!!Because!ethanol!has!a!specific!OBP!and!ethyl!acetate!is!a!natural!
attractant!for!Drosophila,!our!behavioral!choice!assay!represents!a!strong!model!to!understand!
preference!as!a!potential!indicator!of!addiction.!
Objectives.of.the.Project.
. During!this!project,!our!main!objectives!are:!!1.!To!confirm!conditioned!preference!for!
ethanol!in!naturally!occurring!inbred!Drosophila*genotypes,!and!2.!To!study!the!interaction!in!the!
variation!in!the!preference!for!ethanol!with!the!chosen!genotypes.!
! Accomplishing!these!objectives!will!then!make!it!possible!to!conduct!further!research!
using!!Drosophila*to!elucidate!the!transcriptional!variation!using!mRNA!next!gen!sequencing.!!!
!
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Page!|!12!
2.!Results.
Behavioral.Choice.Assay.Observations.–.Ethanol.Intoxication.Phase.
Upon!exposure!to!ethanol,!the!classic!symptoms!of!were!evident.!The!Drosophila!went!
through!an!evident,!active!hedonic!phase,!and!by!20!minutes,!in!all!replicates,!every!fly!was!
unconscious.!!
After!the!4Yhour!recovery!phase,!the!Drosophila*appeared!to!be!fully!recovered,!at!least!
qualitatively,!and!they!were!responsive!to!movement!of!the!loading!vials.*
Behavioral.Choice.Assay.Data:.
! For!the!full!panel!of!14!genotypes,!3!assays!were!completed!under!both!preYexposure!and!
nonYpreYexposure!conditions,!using!20!Drosophila*in!the!first!round!of!experiments.!The!more!
intensive!screening!for!the!panel!subsample!involved!50!Drosophila!per!assay.!!The!data!includes!
both!phases!of!the!experiment.!!The!raw!data!included!the!genotype,!date,!a!distinction!between!
preYexposure!to!ethanol!vs!no!preYexposure,!choice!of!ethanol,!choice!of!ethyl!acetate,!or!no!
choice.!!!
! We!were!most!interested!in!testing!whether!preference!for!ethanol,!in!contrast!to!ethyl!
acetate,! changes! after! preYexposure.! We! measured! preference! of! ethanol,! P
Et
,! as! the! log!
transformed!ratio!between!the!number!of!Drosophila!that!chose!ethanol,!Et
n,
!and!the!Drosophila!
that!chose!ethyl!acetate,!Ea
n
.!To!avoid!division!by!zero,!we!added!a!nominal!value,!0.5/n
tot
,!to!
the!numerator!and!denominator,!where!n
tot
!is!the!total!number!of!Drosophila!in!the!experiment.!
!
"#
=%&
'(
)
+0. 5 &
#.#
'/
)
+0. 5 &
#.#
!
!
!
Page!|!13!
This!is!equivalent!to!a!logit!transformation!of!the!proportion!of!Drosophila!choosing!
ethanol.!!It!is!important!to!use!a!logit!transformation!of!proportional!data!because!it!removes!
the!boundaries!and!makes!the!distribution!symmetrical!around!zero.!!Using!proportions!would!
not!allow!us!to!perform!power!transformations!and!regression!analysis.!
!In!each!experiment!there!were!Drosophila!that!chose!neither!the!ethanol!nor!the!ethyl!
acetate.!!Therefore,!we!also!tested!whether!there!was!an!effect!of!ethanol!preYexposure!on!the!
number!of!Drosophila!that!made!any!choice!C
n
,!vs!no!choice,!NC
n
.!!We!transformed!this!ratio!as!
above!to!obtain!the!metric!for!preference!to!choose,!P
C
:!!
!
0
=%&
1
)
+0. 5 &
#.#
21
)
+0. 5 &
#.#
!
! We!analyzed!the!data!using!standard!regression!models!in!R!(lm{R}).!We!were!interested!
in!testing!for!the!effect!of!genotype!and!ethanol!exposure!on!the!P
Et
.!To!see!if!there!was!an!
interaction!between!genotype!and!the!response!to!preYexposure,!we!tested!for!the!significance!
of!these!terms!using!model!reduction.!!By!doing!so!we!were!able!to!evaluate!the!significance!of!
interactions,!removing!one!variable!at!a!time!while!still!preserving!the!system!response.!!This!
allowed!us!to!discover!which!interactions!were!and!were!not!significant.!!!We!also!included!date!
as!a!covariate,!to!account!for!block!effects.!!!
!
!
!
!
!
!
!
Page!|!14!
Ethanol.Preference.for.Full.Data.Set.
!
Figure.1...The!YYaxis!shows!the!Drosophila*preference!for!ethanol,!P
Et
!.!!The!XYaxis!shows!the!
exposure!treatment,!where!0!is!no!preYexposure!and!1!is!ethanol!preYexposure.!
!
!! !We!tested!for!an!effect!of!preYexposure!on!overall!preference,!P
Et
,!for!the!full!dataYset,!
using!linear!models.!The!effect!of!preYexposure!on!preference!was!significant!(est=1.48,!r
2
=0.79,!
df=1,!F=420.2,!p<2EY16).!
!
!
Page!|!15!
Ethanol.Preference.for.Phase.Two.of.Experiments.
.
Figure.2..The!YYaxis!shows!the!Drosophila*preference!for!ethanol,!P
Et.
!The!XYaxis!shows!the!
genotypes!represented!as!integers.!!For!each!genotype!an,!N,!denotes!no!preYexposure!condition!
and!a,!P,!denotes!preYexposure!condition.!
!
! This!plot!portrays!the!variation!in!the!change!for!ethanol!preference!for!each!genotype!
over!the!two!conditions.!To!further!understand!this!concept,!we!studied!the!interaction!
between!the!different!variables!in!this!second!phase!of!experiments.!
!
!
!
!
!
!
!
Page!|!16!
!
Degrees!of!
Freedom!
Sum!Squared! Mean!
Squared!
F!Value! Pr(>F)!
Date! 1! 10.466! 10.466! 216.0794! 1.685eY13!
PreYExposure! 1! 4.751! 4.751! 98.090! 5.932eY10!
Genotype! 5! 1.193! 0.239! 4.924! 0.00305!
PreY
Exposure:!
Genotype!
5! 0.6572! 0.1314! 2.7134! 0.0442!
Residuals! 24! 1.163! 0.0484! ! !
Figure.3...This!table!describes!the!interaction!between!date,!preYexposure,!genotype,!and!preY
exposure!and!genotype,!with!ethanol!preference.!!!
! !
! This!table!demonstrates!that!within!the!subsample!involving!50!Drosophila*per!assay,!
there! is! a! significant! interaction! between! the! preYexposure! condition! and! genotype! with!
preference!for!ethanol.!!The!P!value!of!.0442!conclusively!shows!that!the!ethanol!preference!is!
significantly!linked!to!the!interaction!between!genotype!and!preYexposure!condition.!!!We!also!
included!date!as!a!covariate,!to!account!for!block!effects.!!
!
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3.!Discussion.
.
. Significant!studies!have!been!conducted!suggest!that!Drosophila*is!a!good!model!for!
ethanol!abuse!and!addiction.!!In!this!study!we!have!found!evidence!that!support!this!idea.!!!
The!first!piece!of!significant!evidence!was!found!upon!comparing!the!preference!for!
ethanol!with!the!conditions!of!preYexposure!and!without!preYexposure!to!ethanol.!!We!
concluded!that!there!is!a!significant!interaction!between!preYexposure!condition!and!the!
preference!for!ethanol!as!was!seen!in!figure!2.!!With!consistency,!the!Drosophila*had!a!
statistically!significant!preference!for!ethanol!in!the!behavioral!assay!when!they!had!been!
previously!exposed!to!ethanol.!!This!evidence!served!in!part!to!confirm!the!theorized!idea!that!
Drosophila*can!develop!a!preference!for!ethanol!following!exposure.!!It!also!served!to!validate!
the!experimental!design.!!This!finding!is!still!important!because!it!suggests!that!a!high!dose!of!
ethanol,!enough!to!cause!full!sedation,!with!an!ample!recovery!period!may!cause!dopaminergic!
reward!pathways!to!be!activated.!!!!!
After!concluding!that!there!was!a!significant!interaction!between!ethanol!preference!
and!preYexposure!condition,!we!then!thought!it!was!viable!to!do!another!phase!of!experiments!
with!a!larger!sample!size,!50!Drosophila,!to!understand!more!interactions.!!The!interaction!of!
direct!interest!was!the!potential!variation!of!change!in!ethanol!preference!from!the!two!
exposure!conditions!compared!between!genotypes.!In!has!been!determined!via!many!different!
behavioral!studies!using!Drosophila!with!altered!or!knocked!out!genes!involved!in!ethanol!
absorption!and!processing!that!large!changes!in!tolerance!and!preference!occur,!but!it!has!not!
been!explored!in!naturally!occurring!genotypes.!!In!this!research,!we!determined!that!there!is!a!
significant!interaction!between!the!genotype!and!the!change!in!preference!from!the!two!
!
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Page!|!18!
exposure!conditions.!!This!is!important!because!it!represents!a!model!that!postulates!that!
Drosophila,*like!humans,!have!a!different!innate!susceptibility!to!the!intoxicating!effects!of!
ethanol!regardless!of!initial!preference.!!Innate!susceptibility!is!crucial!to!the!understanding!of!
the!development!of!alcoholism!in!humans.!!It!has!been!found!that!in!vertebrates!higher!initial!
sensitivity!to!alcohol!correlates!to!increased!instances!of!alcoholism!(Davies,!2004).!This!new!
finding!helps!continue!previous!studies!that!have!been!conducted!using!Drosophila!as!a!model!
of!addiction!and!alcoholism!specifically!because!it!is!similar!to!the!variation!found!in!humans.!!
While!humans!do!not!get!placed!in!YYmaze!assays!and!have!to!decide!to!consume!alcohol,!they!
do!so!in!varying!ways!partly!based!on!their!initial!reaction!to!alcohol,!and!that!idea!is!what!we!
have!tested!in!this!study.!
In! the! future,! we! plan! to! continue! this! study! by! uncovering! some! of! the! genetic!
mechanisms!behind!the!variation!in!change!for!preference.!By!extracting!and!sequencing!the!
mRNA!from!all!of!54!samples!collected!in!the!phase!two!of!experiment,!we!hope!to!observe!some!
of!the!genotypic!differences!that!are!expressed!in!proteins!that!could!relate!ethanol!preference.!!
Recently,!247!candidate!genes,!that!are!associated!with!the!development!of!the!nervous!system!
and!catecholamine!synthesis,!were!identified!as!being!associated!to!alcoholYrelated!phenotypes.!!
TwoYthirds!of!these!gene!had!previously!been!associated!with!alcoholYrelated!phenotypes!in!
humans,!mice,!and!Drosophila*(Mozrova,!2015).!!By!studying!these!candidate!genes!and!their!
interacting!networks!we!may!be!able!to!understand!some!of!what!transcriptional!differences!
account!for!phenotypic!variation!between!genotypes.!!!
Like!all!studies,!this!study!has!limitations!that!must!be!considered.!!One!limitation!is!the!
number!of!replicates!for!each!genotype.!!Behavioral!assays!are!extremely!delicate!and!time!
!
!
Page!|!19!
intensive,!so!it!was!only!feasible!to!conduct!three!replicates!for!each!of!the!genotypes!in!each!
condition!for!phase!one!and!two.!!More!trials!would!have!given!more!power!to!detect!more!true!
significant!differences.!!A!difficulty!with!the!experimental!design!was!the!dispersal!of!the!ethanol!
during!the!preYexposure!condition.!!During!preYexposure!it!was!not!uncommon!for!there!to!be!
complications!with!ethanol!dispersion!that!would!invalidate!the!assay.!!Due!to!the!breeding!
design!necessary!for!the!experiment!this!often!resulted!in!additional!delays!of!up!to!30!days.!
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4.!Materials.and.Methods.
Drosophila*melanogaster!Rearing:!!!
1.! Genotypes:!!14!genotypes!of!Drosophila*melanogaster,!R59,!R136,!R153,!R176,!R208,!
R228,!R273,!R303,!R318,!R340!R387,!R441,!R756,!and!R852!were!used!in!the!first!phase!
of!the!experiment.!!For!the!second!phase!we!used!the!6!genotypes,!R59,!R303,!R387,!
R441,!R765,!and!R852.!!W1118!tester!strain!was!used!in!both!phases!for!making!genetic!
crosses.!
2.! Drosophila*Food:!Each!vial!contains!food!from!a!mixture!of!44L!water,!3.0L!of!light!corn!
syrup,!4200!mL!of!corn!meal,!1200!mL!nonYactive!yeast,!1200!mL!of!soy!flour,!500!ml!of!
Agar,!2950!mL!of!malt!extract,!450!ml!of!Acid!Mix!A,!and!260!mL!of!Tegocept!Acid!Mix.!!
Acid!Mix!A!contain!1!L!Propionic!acid,!1196!mL!of!water,!and!167!mL!of!Phophoric!acid!
(85%).!!The!Tegocept!Acid!Mix!contains!456!g!Tegocept!and!1560!mL!of!Ethanol!(100%).!
3.! !Drosophila*Maintenance:!During!the!expansion!process,!increasing!the!number!of!vials!
of!each!genotype,!each!vial!was!transferred!every!4!days.!!By!transferring!every!4!days!it!
gives!Drosophila*the!opportunity!to!lay!eggs!and!then!do!so!again!in!new!vials!while!the!
larva!grown!in!the!original!vial.!!Transferring!is!the!process!by!which!a!vial!with!food!that!
already!contains!Drosophila!of!both!sexes,!usually!more!than!20!per!vial,!is!transferred!to!
a!new!vial!with!food.!!Transfers!allow!you!to!either!increase!the!number!of!vials!for!a!given!
genotype!or!maintain!the!number!of!vials!for!a!given!genotype.!!If!the!vials!are!not!
transferred!for!extended!periods!of!time,!greater!than!3Y4!weeks,!the!Drosophila*will!die!
because!the!food!will!become!dry!or!mold.!!To!maintain!population!size!and!number!of!
!
!
Page!|!21!
vials!we!transferred!each!vial!once!every!two!weeks.!!Each!vial!was!labelled!with!the!
genotype!and!date!of!transfer!after!every!transfer.!!!
Genetic!Cross!Creation:!
1.! In!order!to!create!standardized!lines!that!will!be!useful!for!mRNA!sequencing!crosses!were!
made!with!each!of!the!14!genotypes!and!a!tester!genotype!w1118.!!w1118!tester!
genotype!was!used!to!create!a!control!cohort!for!genetic!analysis!(Tower,!2014).!!
2.! We!used!male!w1118!Drosophila*and!virgin!female!Drosophila*from!each!genotype.!!To!
generate!virgin!female!Drosophila*for!crosses,!the!vials!were!cleared!after!a!10Y14!day!
period.!!Clearing!is!the!process!by!which!all!of!the!live!adult!Drosophila!in!a!vial!are!
disposed!of,!leaving!only!the!larva!that!adhere!to!the!sides!of!the!vial!left.!!This!is!
important!because!when!checking!the!vials!in!a!4Y8!hour!period!after!clearing!it!all!of!the!
adult!flies!will!be!virgins.!!The!time!elapsed!from!the!date!transferring!to!clearing!can!vary!
based!on!visual!confirmation!of!the!developmental!stage.!!Visual!confirmation!occurs!
when!you!can!see!in!the!vials!that!there!are!a!large!number!of!larva!that!are!in!the!end!of!
the!hardYchitin!stage!and!resemble!black!spots.!!!
3.! Once!female!virgins!were!collected,!10!Drosophila*per!food!vial,!they!were!stored!in!an!
incubator!at!25!degrees!Celsius!regulated!by!a!12Yhour!day!and!night!cycle.!!Collection!is!
the!process!by!which!females!and!male!Drosophila!can!be!differentiated!and!sorted.!!This!
is!done!by!emptying!the!vial!contents!onto!a!plate!that!has!a!low!flow!of!CO2!gas!which!
renders!the!Drosophila*unconscious!and!using!a!small!paint!brush!to!separate!them!by!
gender!and!age.!The!different!sexes!can!be!distinguished!by!different!patterning!on!
genital!and!abdomen!regions.!!Virgin!females!Drosophila!can!be!distinguished!because!
!
!
Page!|!22!
they!appear!as!puffy,!white,!larger!Drosophila!with!a!large!dark!spot!on!the!abdomen.!!
The!process!of!collection!always!took!place!under!an!8Yminute!window!to!minimize!the!
chances!of!brain!damage!occurring!due!to!oxygen!deprivation.!
4.! To!ensure!the!virginity!of!the!female,!they!are!incubated!for!a!day!in!a!vial!before!cross!
creation.!!The!old!vials!are!kept!and!verification!is!made!via!the!lack!of!larvae!in!the!vial.!!!!
If!larva!do!appear!in!those!vials!the!corresponding!crosses!are!discarded.!!
5.! After!the!oneYday!waiting!period,!10!w1118!male!Drosophila*were!collected!and!placed!
into!a!food!vial.!!The!female!virgins!were!then!added!to!the!vial!containing!the!males.!
After! completion! of! the! crosses! and! correct! labeling! denoting! the! date! and! which!
genotype!was!crossed!with!w1118,!the!vials!were!placed!back!into!the!incubator.!!!
6.! After!a!period!of!10Y14!days,!depending!on!the!appearance!of!dark!larvae!spots,!the!vials!
were!cleared.!!After!clearing,!F1!males!were!collected!from!the!vials!and!placed!into!new!
food!vials!in!groups!of!20!or!50!Drosophila*depending!on!whether!they!were!to!be!used!
in!phase!one!or!two!of!the!experiment!respectively.!!These!new!male!Drosophila,!were!
then!stored!for!3Y5!days!in!the!incubator!before!use!in!the!behavioral!habituation!assay.!!
Only!males!were!used!in!this!assay!to!eliminate!changes!in!behavior!based!on!social!
interaction!with!females.!!!
Behavioral!Choice!Assay!Assembly:!!
1.! Assay!Environment:!!When!designing!this!assay!which!aimed!to!test!preference!based!on!
olfaction!alone,!it!was!important!to!use!an!environment!that!eliminated!confounding!
variables!such!as!temperature,!light!exposure!and!time!of!day.!!This!environment!was!
created!by!making!a!dark!room!that!prohibited!light!entry.!!While!a!full!dark!room!was!
!
!
Page!|!23!
not!assessable,!one!was!created!using!heavy!curtains!affixed!with!adhesives.!Within!the!
dark!room!was!another!light!proof!container,!a!dark!box,!created!to!cover!the!assay!
apparatus! and! allow! entry! and! exit! from! the! dark! room! without! disturbing! the!
environment.!!Temperature!was!kept!constant!at!22!degrees!Celsius.!The!assay!was!
conducted!at!10:30!am!directly!after!the!Drosophila*day!cycle!began.!!This!is!important!
because!Drosophila*are!most!active!at!dawn!and!dusk.!!
2.! !FarYred!Light:!!In!order!to!perform!the!assay!in!equivalent!to!pitch!black!conditions,!we!
used!a!farYred!light.!!The!farYred!spectrum!is!between!red!and!infraYred!light!from!710Y
850!nm.!!Drosophila*have!very!limited!vision!over!600!nm!(Heisenberg!and!Wolf,!1984).!
Using!this!farYred!light!is!important!because!it!allowed!us!to!see!during!the!procedure!
while!rendering!the!Drosophila*blind!because!they!are!unable!to!see!the!farYred!light!
spectrum.!!!
3.! Assay!Apparatus:!Each!apparatus!required!the!use!of!three!1!ml!pipette!tips,!a!small!
plastic!YYshaped!hollow!joint!large!enough!for!the!Drosophila*to!crawl!through,!a!razor!
blade,!two!Drosophila*vials!without!food,!two!synthetic!vial!plugs,!cotton!balls,!tape,!and!
a!1.5!ml!test!tube!rack!(4X12).!!!
4.! Apparatus!Construction:!!To!construct!the!apparatus,!two!of!the!1!ml!pipette!tips!were!
used!cut!41!mm!from!the!wider!end!and!7!mm!from!the!narrower!end!using!a!razor!blade.!!
The!smaller!opening!was!cut!just!enough!to!let!Drosophila!easily!go!through!but!not!allow!
them!to!easily!pass!back!through!the!opening.!It!also!limited!the!odor!flow!into!the!loading!
chamber!(Simonet!et*al.*2014).!!The!large!end!was!cut!in!order!to!make!it!fit!snuggly!on!
the!YYshaped!joint.!!The!YYshaped!joint!has!two!smaller!ends!and!one!slightly!larger!end.!!
!
!
Page!|!24!
The!two!smaller!ends!connect!to!the!first!two!pipette!tips.!!Next,!the!two!synthetic!vial!
plugs!were!pierced!through!the!middle!with!the!razor!blade!and!with!a!clockwise!twisting!
action!a!small!hole!was!made.!This!hole!was!then!penetrated!by!the!preYcut!1!mL!pipette!
tip.!!Once!this!connection!was!made,!the!YYshape!joint!could!be!effectively!attached!to!
the!empty*vials!that!will!act!as!choice!chambers.!!Before!attaching!the!choice!chambers!
to!the!apparatus,!a!pinch!of!cotton!ball!was!placed!in!and!pushed!to!the!bottom!of!each!
of!the!food!vials.!One!was!labelled!with!ETOH!standing!for!ethanol!and!the!other!EA!
standing!for!ethyl!acetate.!The!loading!chamber!was!made!with!last!1!mL!pipette!tip,!cut!
44!mm!from!wider!end!as!to!fit!in!the!large!end!of!the!YYshaped!joint.!!Once!connected,!
the!apparatus!was!completed.!
!
Figure.4...This!figure!depicts!the!assembly!of!the!YYmaze!behavioral!assay!apparatus.!!On!the!
left,!the!key!components!of!the!apparatus!are!separated.!!On!the!right,!the!apparatus!is!
complete!and!erected!to!demonstrate!how!it!would!look!during!an!assay.!
!
Behavioral!Choice!Assay!Procedure!Phase!One:!!
1.! Intoxication!Stage:!In!this!stage,!each!group!of!F1!males!were!given!high!doses!of!ethanol!
for!20!minutes.!!For!each!group!of!F1!males,!an!intoxication!chamber!was!made!using!
empty!vial!that!was!labelled!with!the!date,!number!of!F1!males,!and!the!genotype.!!It!was!
è
!
!
Page!|!25!
then!placed!next!to!the!food!vial!containing!the!F1!males.!!Using!a!100Y1000!microliter!
pipette,!300!microliters!of!100%!ethanol!was!dispersed!on!to!a!new!synthetic!vial!plug.!
The!dispersal!of!the!ethanol!was!done!in!the!center!of!the!vial!plug,!within!a!2!millimeters!
of!the!plug!to!ensure!consistent!dosage!of!ethanol!for!each!trial.!!When!designing!the!
assay,!it!was!discovered!that!dispersing!the!ethanol!unevenly!on!the!plug!caused!variation!
in!the!intoxication!time.!As!soon!as!the!ethanol!was!dispersed,!the!F1!males*were!
transferred!to!the!empty!vial!and!the!plug!with!ethanol!was!then!put!into!the!vial!with!
the!F1!males.!!The!plug!is!pushed!in!until!top!of!the!plug!meets!the!top!of!the!vial!as!to!
maintain!a!consistent!volume!for!each!of!the!trials.!!Each!intoxication!stage!lasted!20!
minutes!and!the!plug!was!then!removed.!!If!multiple!groups!were!undergoing!the!assay!
at!the!same!time,!20!second!intervals!were!used!to!stagger!the!intoxication!to!maintain!
consistent!time!for!each!group.!!At!the!conclusion!of!this!stage,!the!F1!males!were!then!
transferred!into!a!loading!chamber!using!a!funnel.!!The!loading!vial!was!then!covered!with!
a!piece!of!tape!to!prevent!the!F1!males!from!escaping!and!labelled!according!to!genotype!
and!condition.!!A!small!incision!was!made!in!the!tape!to!allow!oxygen!flow!into!the!loading!
chamber.!!The!loading!chamber!was!then!placed!into!a!vial!rack!in!the!dark!room!and!
covered!by!the!container.!!Neither!cold!nor!CO2!were!used!within!24!hours!of!this!
procedure.!!!This!type!of!sedation!has!been!shown!to!have!negative!effects!on!Drosophila!
behavior!(Baron,!2000).!
2.! Recovery!Stage:!!The!F1!males!then!were!let!to!recover!for!4!hours!in!the!dark!room.!
3.! Assay!Conduction:!!After!3!hours!and!50!minutes!had!passed!we!entered!the!dark!room,!
with!the!F1!males!still!in!the!dark!box,!and!set!up!the!assay!apparatus.!!Each!apparatus!
!
!
Page!|!26!
was!constructed!prior!to!the!assay!and!brought!into!the!dark!room.!!In!the!dark,!the!farY
red!light!was!turned!on!and!a!5Yminute!period!was!necessary!to!adjust!to!seeing!at!that!
light!spectrum.!!Prior!to!entering!the!dark!room,!a!50!ml!vial!of!ethanol!at!100%,!ETOH,!
and!a!50!ml!vial!with!a!mixture!of!ethyl!acetate!and!paraffin!oil,!EA,!were!set!aside.!!The!
mixture!of!ethyl!acetate!and!paraffin!oil!was!a!1:36!ratio!(Simonet!et*al.*2014).!!Just!before!
4!hours!had!passed,!the!apparatus!was!completed!by!dispersing!55!microliters!of!the!
ETOH!and!EA!onto!the!cotton!balls!that!were!in!each!of!the!ends!of!the!choice!chambers!
using!a!20Y200!microliter!pipette.!The!ETOH!choice!chamber!was!filled!first!and!replaced!
back!onto!the!apparatus!followed!by!the!EA!choice!chamber.!!Unlike!ethanol,!the!EA!has!
a! high! viscosity! thus! it! must! be! withdrawn! slowly! in! order! to! ensure! that! no!
inconsistencies!in!volume!are!drawn.!!The!apparatus!was!then!ready!to!be!connected!to!
the!loading!vials!with!the!F1!males.!!The!loading!chamber!was!placed!onto!the!open!end!
of!the!YYshaped!apparatus.!!This!was!done!by!gently!tapping!the!loading!vial!down!while!
simultaneously!removing!the!tape!from!the!top!and!connecting!it!with!the!the!open!end!
of!the!apparatus.!!The!completed!apparatus!was!then!placed!back!into!the!rack!with!the!
loading!vial!at!the!bottom.!!This!created!a!YYshape!as!seen!in!figure!4.!!The!loading!vial!at!
the!bottom!takes!advantage!of!Drosophilas’*natural!negative!geotaxis,!the!tendency!to!
walk!against!gravity!(Orr!and!Sohal,!1994).!!If!more!than!one!trial!was!run!simultaneously,!
the!ETOH!choice!chamber!would!alternate!from!left!to!right!to!eliminate!directional!bias.!
The!dark!box!was!then!replaced!over!the!active!behavioral!choice!assays.!!The!assay!was!
let!to!run!for!1!hour.!!!
!
!
Page!|!27!
4.! Recording!Results!and!Sample!Storage:!Before!1!hour!had!elapsed,!two!1.5!mL!microtubes!
were!labelled!with!the!genotype,!date,!and!either!ETOH!or!EA.!!Liquid!nitrogen!was!also!
acquired!as!it!would!be!used!in!the!last!step.!!At!55!minutes,!the!dark!room!was!entered!
to!allow!for!time!for!the!researchers’!sight!to!adjust.!!At!1!hour,!the!dark!box!was!removed!
and!the!results!were!recorded.!!The!number!of!F1!males*that!choose!either!the!ETOH!or!
EA!choice!chambers!were!counted!then!written!on!the!respective!microtube!and!on!a!
recording!sheet.!!!Then!the!F1!males*would!be!transferred!into!the!respective!microtube!
using!the!funnel.!!After!this!was!completed!the!microtubes!would!be!flash!frozen!using!
the!liquid!nitrogen!and!then!placed!into!the!Y80!degrees’!Celsius!freezer.!
5.! Assay!Totals!and!Controls:!!For!each!of!the!14!genotypes,!3!assays!replicates!were!run.!!!
Each!assay!contained!20!F1!males.!In!order!to!prevent!potential!bias!by!day,!a!single!
genotype!was!tested!over!multiple!days.!!To!set!controls!for!the!experiment,!another!3!
assays!replicates!were!run!with!each!of!the!14!genotypes!without!the!preYexposure!
condition.!!To!carry!out!the!control!assays,!the!exact!same!procedure!was!used,!but!with!
the!exception!of!the!initial!intoxication!stage!which!was!done.!!!
Behavioral!Choice!Assay!Procedure!Phase!Two:!!
1.! Choosing!Genotypes:!!After!completing!phase!one,!the!6!genotypes,!R59,!R303,!R387,!
R441,!R765,!and!R852!were!chosen!for!more!extensive!study.!!!
2.! Assay!Conduction:!!The!same!procedure!was!used!for!phase!two!as!was!done!in!phase!
one.!!The!only!change!was!that!each!trial!contained!50!F1!males!instead!of!20!F1!males.!!
This!is!important!because!it!would!allow!for!easier!generation!of!mRNA!from!larger!
!
!
Page!|!28!
sample!size!and!it!will!be!more!statically!significant.!!For!each!of!the!6!genotypes,!3!assays!
with!preYexposure!to!ethanol!and!3!control!assays!were!run.!!!
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Abstract (if available)
Abstract
Excessive alcohol consumption is a common cause of preventable death in most countries, and imposes a large socioeconomic burden in terms of health care and violent crime. Not all individuals who consume alcohol will become alcoholics, so to study variation in susceptibility to its effects and differences in inclination for chronic use, we need to look into our genetic makeup. Using Drosophila melanogaster as a model organism, we are able to study the effects of alcohol and how it is related to genetic differences. Drosophila can be used as the model organism not only because we have the tools decode their genomes and sequence individual genotypes in large reproducible quantities, but also because they have similar reward pathways and physical symptoms of alcohol use as humans. In this study, D. melanogaster are given the choice between an odor for which they have a known preference, ethyl acetate (EA) and ethanol (ETOH). D. melanogaster are given this choice with and without a previous exposure to ETOH. We have found two significant pieces of evidence to support the hypothesis that D. melanogaster is a good model of ethanol abuse. First, we found that there was a significant interaction between pre-exposure to ETOH and preference for ETOH. After further analysis of selected genotypes, we found that there was a significant interaction between the change in preference for ETOH and the genotype. This demonstrates potential variation in susceptibility to the rewarding effects of ethanol in naturally occurring genotypes. In the coming months, mRNA will be extracted and sequenced to give further insight as to what transcriptomic expression might be linked to this varying preference. With this information relating gene expression to preference and susceptibility toward ETOH, we will move one step closer to understanding how some people are more susceptible to the negative aspects of alcohol use.
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Asset Metadata
Creator
Polonus, Zachary Marshel
(author)
Core Title
Behavioral choice assays and alcohol preference in Drosophila melanogaster
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
06/27/2018
Defense Date
04/22/2016
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
alcohol use,Drosophila melanogaster,ethanol abuse,genetic differences,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Nuzhdin, Sergey (
committee chair
), Hacia, Joseph (
committee member
), Tokes, Zoltan (
committee member
)
Creator Email
zakpolonus@gmail.com,zpolonus@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-258473
Unique identifier
UC11280539
Identifier
etd-PolonusZac-4477.pdf (filename),usctheses-c40-258473 (legacy record id)
Legacy Identifier
etd-PolonusZac-4477.pdf
Dmrecord
258473
Document Type
Thesis
Format
application/pdf (imt)
Rights
Polonus, Zachary Marshel
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
alcohol use
Drosophila melanogaster
ethanol abuse
genetic differences