Drosophila melanogasters feeding preferences to unique cell-surface

molecules of Saccharomyces cerevisiae vegetative and sporulated states

Turner S. Baker*1, Cameron A. Gable, Victor H. Quach, Sarah Seagrave, Kelly M. Thomasson, and Stephen R. Proulx2
*1University of California, Santa Barbara Department of Molecular, Cellular, and Developmental Biology
2University of California, Santa Barbara Department of Ecology, Evolution, and Marine Biology

Abstract

Results

The$rela(onship$between$Drosophila$and$Saccharomyces$
has$been$an$area$of$evolu(onary$study$for$decades.$$While$the$
twos$interac(ons$are$well$documented$and$discussed,$the$
en(rety$of$their$symbio(c$rela(onship$is$yet$to$be$fully$explored$
and$established.$$Previous$studies$have$found$Drosophila*
Melanogaster$to$be$able$to$dis(nguish$between$dierent$
concentra(ons$of$sugar$sources$and$show$a$preference$to$sugar$
types.$Drosophila$has$also$been$shown$to$be$able$to$recognize$
areas$of$faCy$acids,$and$other$benecial$food$proteins.$$To$
successfully$survive$passage$through$the$ies$diges(ve$tract,$
yeast$species$must$have$previously$undergone$sporula(on.$
Considering$that$vegeta(ve$cell$survival$through$the$y$gut$is$an$
anomaly,$it$gives$the$yeast$strains$a$dis(nct$evolu(onary$
advantage$to$aCract$ies$to$their$sporulated$forms.$
The$experiment$conducted$aimed$to$further$prove$
Drosophilaes$ability$to$recognize$varying$levels$of$sugar$
concentra(on$and,$primarily,$to$establish$a$preference$for$
sporulated$or$vegeta(ve$Saccharomyces$Cerevisiae.$$
Instruments$referred$to$as$FlyCafes$were$u(lized$to$
quan(ta(vely$compare$feeding$consump(ons$of$varying$media.$$
Dierences$in$consump(on$were$considered$a$direct$correla(on$
to$preference.$$While$Drosophilae$Melanogaster$demonstrated$
a$preference$for$higher$concentrated$sugars,$no$preference$to$
either$state$of$Saccharomyces$Cerevisiae$was$observed.$
$

Dierence$in$
Preference$

PQValue$

Degrees$of$
Freedom$

7.957!

0.000456968!

138!

5.2143!

1.69038ES06!

86!

1.1372!

0.032634678!

202!

Compra(ve$Selec(vity$
YPD!vs.!Diluted!YPD!
VegetaGve!M4330!vs.!Spent!YPD!
VegetaGve!M4330!vs.!Sporulated!
M4330!

Figure 2: Feeding Preferences and Significance Utilizing a student t

test analysis, all results were found to be significant. YPD concentration
was found to be the biggest factor in feeding behaviors, with addition of
vegetative yeast boosting preference.
Light preference was found
between sporulated states, with drosophilae preferring sporulated cells.

Average$Total$Consump(on$(mm)$

35!

Figure$1:!FlyCafe!equipped!with!two!media!
lled!capillary!tubes!and!two!breathing!tubes!

D.$melanogaster$Prepara(on!
Female!drosophilae!melanogaster!are!
raised!in!vials!lled!with!sugar!rich!solid!media.!!
Females!were!knocked!out!using!FlyNapTM!and!
isolated!using!microscopy!into!FlyCafe!Vials.!
Eight!ies!were!isolated!per!vial.!!Flies!were!
starved!for!6!hours!!
!
$
$

30!
25!
20!
15!

29.8!
22.62!

10!

17.73!

5!
0!
YPD!vs.!Diluted!YPD!

VegetaGve!M4330!vs.!Spent!YPD!

VegetaGve!M4330!vs.!Sporulated!M4330!

Figure 3: Total Consumption. Shows relatively standard consumption

over entirety of experiment. With 24 hour consumption times, 8 flies
consistently ate ~20mm of media.

Introduction
Average$Consump(on$(mm)$

25!
20!
15!
10!

18.87857143!
10.92142857!

5!
0!
Pure!YPD!

1:100!YPD!

Figure 4: Pure YPD vs. 1:100 YPD. D. melanogaster demonstrated

strong preference to higher concentrations of sugar-rich media..

Average$Consump(on$(mm)$

14!
12!
10!
8!
6!

11.47619048!

4!

6.261904762!

2!
0!
YPD!+!Veg!

Pure!YPD!

Figure 5: YPD + Vegetative S. Cerevisea vs. Spent YPD. D.

melanogaster had mild preference to spent YPD with vegetative S.
cerevisea.

Average$Consump(on$(mm)$

14!
12!
10!
8!
6!

11.64705882!

10.50980392!

4!
2!
0!
VegetaGve!

Sporulated!

Literature Cited
Howe!HF!(1986)!Seed!dispersal!by!ruitSeaGng!birds!and!mammals.!In:!Murray!DR,!ed.!Seed!Dispersal.!
Orlando,!FL:!Academic!Press.!pp!123190.!
Begon!M!(1986)!Yeasts!and!Drosophila.!In:!Ashburner!M,!Carson!H,!Thompson!JN,!eds.!The!GeneGcs!
and!Biology!of!Drosophila.!London:!Academic!!
Press.!pp!345384.!!
Coluccio!AE,!Rodriguez!RK,!Kernan!MJ,!Neiman!AM!(2008)!The!Yeast!Spore!Wall!Enables!Spores!to!
Survive!Passage!through!the!DigesGve!Tract!of!Drosophila.!PLoS!ONE!3(8):!e2873.!doi:10.1371/
journal.pone.0002873!
Masek,!P.!&!ScoV,!K.!Limited!taste!discriminaGon!in!Drosophila.!Proc.*Natl*Acad.*Sci.*USA107,!14833
14838!(2010).!
Sanchez,!Maria!Gabriela!De!Brito,!Esther!Lorenzo,!Songkun!Su,!Fanglin!Liu,!Yi!Zhan,!and!MarGn!Giurfa.!
The!Tarsal!Taste!of!Honey!Bees:!Behavioral!and!Electrophysiological!Analyses.!Fron=ers*in*Behavioral*
Neuroscience*(2014):!n.!pag.!Web.!
Masek,!Pavel.!"Drosophila!FaVy!Acid!Taste!Signals!through!the!PLC!Pathway!in!SugarSSensing!
Neurons."!PLOS*Gene=cs:.!10.1371/journal.pgen.1003710,!12!Sept.!2013.!Web.!04!Oct.!2014.!

30!
25!

Consump(on$(mm)$

The!relaGonship!between!Drosophila!and!Saccharomyces!has!
been!an!area!of!evoluGonary!study!for!decades.!!While!the!twos!
interacGons!are!well!documented!and!discussed,!the!enGrety!of!
their!symbioGc!relaGonship!is!yet!to!be!fully!explored!and!
established.!!While!the!benets!received!by!saccharomyces!are!
apparent,!there!may!be!solid!evidence!that!supports!the!idea!of!a!
nonScommensal!relaGonship!between!them!and!drosophila!
species.!!This!project!aims!to!compile!substanGal!data!that!would!
help!to!prove!that!Drosophila*Melanogaster!has!the!ability!to!
dierenGate!sporulated!Saccharomyces*Cerevisiae!from!its!
vegetaGve!cell!form.!
!Prior!research!has!clearly!indicated!that!yeast!
species!are!able!to!uGlize!drosophila!movements!to!transport!to!
new!nutrient!rich!areas2.!!This!uGlizaGon!nearly!guarantees!
exposure!to!new!food!sources!with!liVle!energy!expenditure.!!
While!some!transportaGon!is!acquired!through!the!limbs!of!
drosophila,!it!has!been!well!documented!that!survival!through!the!
gut!may!be!equally,!if!not!more,!eecGve.!While!the!assistances!
gained!by!the!yeast!through!this!relaGonship!are!quite!obvious,!
the!advantages!beWng!the!ies!are!less!apparent.!Studies!have!
proven!that!adult!drosophilae!are!able!to!use!yeast!as!a!sole!
source!of!food2,!however!this!is!rarely!applicable!in!the!natural!
world.!!Far!more!oXen!ies!feed!upon!fruit!or!tree!sap,!ingesGng!
yeast!cells!as!a!secondary!event.!!!
!To!successfully!survive!passage!through!the!ies!
digesGve!tract,!yeast!species!must!have!previously!undergone!
sporulaGon.3!Considering!that!vegetaGve!cell!survival!through!the!
y!gut!is!an!anomaly,!it!give!the!yeast!strains!liVle!evoluGonary!
advantage!to!aVract!ies!to!their!vegetaGve!forms.!!It!is!
reasonable!to!hypothesize!that!it!would!be!evoluGonarily!
benecial!to!have!spores!appear!more!
aVracGve!to!ies.!!By!enGcing!drosophilae!
to!consume!areas!of!sporulated!cells,!the!
yeast!has!a!greater!chance!of!survival.!!
This!is!not!a!unique!idea!within!the!natural!
world.!!Fruits!and!berries!were!similarly!
designed!to!aVract!birds!to!increase!
dispersal.1!!And!the!honey!bee,!an!insect!
similar!to!drosophila*melanogaster,!is!able!
to!disGnguish!between!wide!varieGes!of!
substrates!in!order!to!beVer!acquire!
pollen!and!sugar.5!
!Studies!have!found!
Drosophila*Melanogaster!to!be!able!to!
disGnguish!between!dierent!
concentraGons!of!sugar!sources!and!show!
a!preference!to!one!sugar!over!another.4!!
Drosophila!has!also!been!shown!to!be!
able!to!recognize!areas!of!faVy!acids,!and!
other!benecial!food!proteins.6!
!

Discussion
Further understanding the inter-specie interactions amongst
the most common model organisms is an important area of
study within evolutionary biology. The eventual goal of
identifying molecular reasons inducing evolved traits begins
with discerning minute behavioral traits amongst organisms.
Within this experiment, no statistically relevant selectivity
was present within Drosophilae melanogasters traits in
response to saccharomyces cerevisae sporulation. The lack
of ability to discriminate between saccharomyces forms
suggests Drosophilae is unable to identify extracellular
proteins of microorganisms.
Consumption was measured in the unit of millimeters
and was considered to be a direct representation of feeding
preference. All data sets were statistically analyzed with a
Students T-Test with an value of 0.5. In order to test the
effectiveness of our device, Drosophilas ability to detect and
prefer higher concentrations of sugar-rich media was tested.
Pure yeast extract peptone dextrose (YPD) was compared
to 100-fold diluted YPD. The raw data demonstrated that
flies preferred higher concentrated YPD by an additional
+7.957mm on average, with daily averages ranging from
+14.75mm to 2.22mm. The student T-Test demonstrated
high significance (p=.00046) These results were
successfully replicated using spent-YPD as a media.
Vegetative M4330 was grown and resuspended SpentYPD media. The flies had a very mild attraction to the
addition of the yeast cells to spent-YPD. The overall
average preference was found to be 5.21mm, with individual
daily ratings ranging from +3.33mm to +8.286mm. This
markedly reduced average preference demonstrates that
Drosophila would prefer medias with higher sugar
concentration than those with vegetation. However, with
some preference it indicates there must be some ability to
detect the presence of yeast in media.
When comparing feeding preference between the
sporulated and vegetative state of M4330, drosophilae light
preference for vegetative cells. The average preference
towards vegatative was +1.13mm. This data, while
significant, does not indicate a strong preference between
sporulated and vegetative external protein structures.
Although this data suggests S. cerevisea does not
actively attract D. melanogaster to its sporulated states, this
research did not encompass the entirety of sporulation
conditions. During sporulation a variety of unique biproducts are released that may be involved in attracting
drosophilae. Through resuspension in sterile spent-YPD,
these specific biproducts were removed.

20!

Acknowledgements

15!
10!
5!
0!
S5!

1! 4! 7! 10! 13! 16! 19! 22! 25! 28! 31! 34! 37! 40! 43! 46! 49! 52! 55! 58! 61! 64! 67! 70! 73! 76! 79! 82! 85! 88! 91! 94! 97!100!
Round$3$Trial$Number$

Figure 6: Vegetative M4330 vs. Sporulated M4330. D. melanogaster

demonstrated extremely light preference to vegetative S. cerevisea.

Our greatest appreciations go to Dr. Stephen Proulx and

Kelly Thomasson, who mentored us through the entire course
of the project. We would like to thank. We are also extremely
grateful for the contribution of the UC Santa Barbara
Undergraduate Research and Creative Activities (URCA) unit,
as well as the Faculty Research Assistance Program (FRAP),
as this project would have been impossible without their
generous funding

Methods

Media$Prepara(on!
Spent!YPD!was!created!by!allowing!
uninhibited!yeast!cell!growth!in!pure!YPD!for!
one!week.!!Yeast!cells!were!then!removed!via!
sterile!ltraGon!and!the!remaining!YPD!was!
autoclaved.!!SporulaGon!of!M4330!was!
induced!with!potassium!acetate.!!VegetaGve!
and!sporulated!versions!of!M4330!were!
equalized!via!spectroscopy!once!resuspended!
in!spent!YPD$

Feeding$and$Analysis!
Two!categories!of!media!were!introduced!
via!capillary!tubes.!!ConsumpGon!of!media!was!
allowed!for!24!hours.!!Immediately!proceeding!
consumpGon,!capillary!tubes!were!removed,!
and!consumpGon!rates!were!measured!in!
millimeters!consumed.!
!!
$
$
$

Figure 2: Example Consumption

Measurement. Demonstrates
difference in millimeters between
two types of media exposed to
starved female flies for 24hrs.