Urca Poster Turner
1
Introduction Abstract Results Methods Discussion Literature Cited Acknowledgements The rela(onship between Drosophila and Saccharomyces has been an area of evolu(onary study for decades. While the two’s 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 different 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 beneficial food proteins. To successfully survive passage through the flies’ diges(ve tract, yeast species must have previously undergone sporula(on. Considering that vegeta(ve cell survival through the fly gut is an anomaly, it gives the yeast strains a dis(nct evolu(onary advantage to aCract flies to their sporulated forms. The experiment conducted aimed to further prove Drosophilae’s 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. Differences 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. D. melanogaster Prepara(on Female drosophilae melanogaster are raised in vials filled with sugar rich solid media. Females were knocked out using FlyNap TM and isolated using microscopy into FlyCafe Vials. Eight flies were isolated per vial. Flies were starved for 6 hours 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 filtraGon 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. 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 The relaGonship between Drosophila and Saccharomyces has been an area of evoluGonary study for decades. While the two’s interacGons are well documented and discussed, the enGrety of their symbioGc relaGonship is yet to be fully explored and established. While the benefits 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 differenGate 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 areas 2 . 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, effecGve. While the assistances gained by the yeast through this relaGonship are quite obvious, the advantages befiWng the flies are less apparent. Studies have proven that adult drosophilae are able to use yeast as a sole source of food 2 , however this is rarely applicable in the natural world. Far more oXen flies feed upon fruit or tree sap, ingesGng yeast cells as a secondary event. To successfully survive passage through the flies’ digesGve tract, yeast species must have previously undergone sporulaGon. 3 Considering that vegetaGve cell survival through the fly gut is an anomaly, it give the yeast strains liVle evoluGonary advantage to aVract flies to their vegetaGve forms. It is reasonable to hypothesize that it would be evoluGonarily Howe HF (1986) Seed dispersal by ruitSeaGng birds and mammals. In: Murray DR, ed. Seed Dispersal. Orlando, FL: Academic Press. pp 123–190. Begon M (1986) Yeasts and Drosophila. In: Ashburner M, Carson H, Thompson JN, eds. The GeneGcs and Biology of Drosophila. London: Academic Press. pp 345–384. 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. 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 melanogaster’s 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 Student’s T-Test with an α value of 0.5. In order to test the effectiveness of our device, Drosophila’s 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 Spent- YPD 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 bi- products are released that may be involved in attracting drosophilae. Through resuspension in sterile spent-YPD, these specific biproducts were removed. Drosophila melanogaster’s 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. Proulx 2 * 1 University of California, Santa Barbara – Department of Molecular, Cellular, and Developmental Biology 2 University of California, Santa Barbara – Department of Ecology, Evolution, and Marine Biology beneficial to have spores appear more aVracGve to flies. 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 different 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 beneficial food proteins. 6 Figure 1: FlyCafe equipped with two media filled capillary tubes and two breathing tubes Figure 2: Example Consumption Measurement. Demonstrates difference in millimeters between two types of media exposed to starved female flies for 24hrs. Figure 4: Pure YPD vs. 1:100 YPD. D. melanogaster demonstrated strong preference to higher concentrations of sugar-rich media.. 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. 29.8 17.73 22.62 0 5 10 15 20 25 30 35 YPD vs. Diluted YPD VegetaGve M4330 vs. Spent YPD VegetaGve M4330 vs. Sporulated M4330 Average Total Consump(on (mm) 18.87857143 10.92142857 0 5 10 15 20 25 Pure YPD 1:100 YPD Average Consump(on (mm) 11.64705882 10.50980392 0 2 4 6 8 10 12 14 VegetaGve Sporulated Average Consump(on (mm) 11.47619048 6.261904762 0 2 4 6 8 10 12 14 YPD + Veg Pure YPD Average Consump(on (mm) Compra(ve Selec(vity Difference in Preference PQValue Degrees of Freedom YPD vs. Diluted YPD 7.957 0.000456968 138 VegetaGve M4330 vs. Spent YPD 5.2143 1.69038ES06 86 VegetaGve M4330 vs. Sporulated M4330 1.1372 0.032634678 202 Figure 5: YPD + Vegetative S. Cerevisea vs. Spent YPD. D. melanogaster had mild preference to spent YPD with vegetative S. cerevisea. Figure 6: Vegetative M4330 vs. Sporulated M4330. D. melanogaster demonstrated extremely light preference to vegetative S. cerevisea. Figure 3: Total Consumption. Shows relatively standard consumption over entirety of experiment. With 24 hour consumption times, 8 flies consistently ate ~20mm of media. S5 0 5 10 15 20 25 30 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 Consump(on (mm) Round 3 Trial Number
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Transcript of Urca Poster Turner
-
Introduction
Abstract Results
Methods
Discussion
Literature Cited
Acknowledgements
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$observed.$
$!
D.$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!!
!$$
Media$Prepara(on!Spent!YPD!was!created!by!allowing!
uninhibited!yeast!cell!growth!in!pure!YPD!for!one!week.!!Yeast!cells!were!then!removed!via!sterile!ltraGon!and!the!remaining!YPD!was!autoclaved.!!SporulaGon!of!M4330!was!induced!with!potassium!acetate.!!VegetaGve!and!sporulated!versions!of!M4330!were!equalized!via!spectroscopy!once!resuspended!in!spent!YPD$
Feeding$and$Analysis!Two!categories!of!media!were!introduced!
via!capillary!tubes.!!ConsumpGon!of!media!was!allowed!for!24!hours.!!Immediately!proceeding!consumpGon,!capillary!tubes!were!removed,!and!consumpGon!rates!were!measured!in!millimeters!consumed.!
!!$$$
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
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!
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,!1483314838!(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.!
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 Spent-YPD 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 bi-products are released that may be involved in attracting drosophilae. Through resuspension in sterile spent-YPD, these specific biproducts were removed.
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
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!!Figure$1:!FlyCafe!equipped!with!two!media!lled!capillary!tubes!and!two!breathing!tubes!
Figure 2: Example Consumption Measurement. Demonstrates difference in millimeters between two types of media exposed to starved female flies for 24hrs.
Figure 4: Pure YPD vs. 1:100 YPD. D. melanogaster demonstrated strong preference to higher concentrations of sugar-rich media..
Figure 2: Feeding Preferences and Significance Utilizing a student t test analysis, all results were found to be significant. YPD concentration was found to be the biggest factor in feeding behaviors, with addition of vegetative yeast boosting preference. Light preference was found between sporulated states, with drosophilae preferring sporulated cells.
29.8!
17.73!22.62!
0!
5!
10!
15!
20!
25!
30!
35!
YPD!vs.!Diluted!YPD! VegetaGve!M4330!vs.!Spent!YPD! VegetaGve!M4330!vs.!Sporulated!M4330!
Average$To
tal$C
onsump(
on$(m
m)$
18.87857143!
10.92142857!
0!
5!
10!
15!
20!
25!
Pure!YPD! 1:100!YPD!
Average$Co
nsum
p(on
$(mm)$
11.64705882!10.50980392!
0!
2!
4!
6!
8!
10!
12!
14!
VegetaGve! Sporulated!
Average$Co
nsum
p(on
$(mm)$
11.47619048!
6.261904762!
0!
2!
4!
6!
8!
10!
12!
14!
YPD!+!Veg! Pure!YPD!
Average$Co
nsum
p(on
$(mm)$
Compra(ve$Selec(vity$Dierence$in$Preference$ PQValue$
Degrees$of$Freedom$
YPD!vs.!Diluted!YPD! 7.957! 0.000456968! 138!
VegetaGve!M4330!vs.!Spent!YPD!5.2143! 1.69038ES06! 86!
VegetaGve!M4330!vs.!Sporulated!M4330! 1.1372! 0.032634678! 202!
Figure 5: YPD + Vegetative S. Cerevisea vs. Spent YPD. D. melanogaster had mild preference to spent YPD with vegetative S. cerevisea.
Figure 6: Vegetative M4330 vs. Sporulated M4330. D. melanogaster demonstrated extremely light preference to vegetative S. cerevisea.
Figure 3: Total Consumption. Shows relatively standard consumption over entirety of experiment. With 24 hour consumption times, 8 flies consistently ate ~20mm of media.
S5!
0!
5!
10!
15!
20!
25!
30!
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!
Consum
p(on
$(mm)$
Round$3$Trial$Number$
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