ChemotacticImprintinginNematode

Worms
Sweeney,Z.Wells,G.Gerrish,J.Hunt,S.
Introduction
Thenematodeworm,C.elegans,isapopularexperimentalorganismforneuroscience
researchduetoitsrelativelysimpleandwellunderstoodnervoussystem.C.elegans possess
32presumedchemosensoryneuronswhicharededicatedtodetectingthepresenceof
chemicalsintheenvironment.Theseincludetheprecursorstomorecomplexvertebrate
olfactoryandgustatorysensorysystems.AccordingtoBargmannetal,morethan5%ofits
genesaredevotedtotherecognitionofenvironmentalchemicals.Onceachemicalstimulus
reachestheneuron,itactivatesaGproteindependentenzymaticcascade(Bargmann,1).

Therefore,anematodewormcandetectcertainstimuliandalteritsbehaviorinthe
followingways.Itiscapableofmovingtowardorawayfromfoodsourcesandchemicalcues,
alteringitsgeneraldegreeofmovement,andevenchangingdevelopmentalcoursebyentering
anextendedlarvalphasecalledtheDauerdevelopmentalstage(Bargmann,1).
Thisexperimentdemonstratesthatcertainstimulielicitaspecificeffect.Glucoseelicits
taxistowardthefoodsource,E.colibacteria.NaClaloneelicitskinesis,akanondirectional
movement.
ThisexperimentconditionedC.elegans toassociateNaClasaprecipitatingstimulusfor
theintroductionofglucose.Initially,NaClaloneproducedgeneralizedkinesishowever,
followingrepeatedexposuretoNaClfolloweddirectlybytaxiselicitingglucose,theworms
respondedwithtaxistotheNaClalone.Theirresponseincreasedindegreescommensurate
withtheamountofNaClintroduced.ThisbreakthroughdemonstratesthatC.elegansare
capableofclassicalconditioning:associationofaneutralstimulus,NaCl,withanincentive
stimulus,glucose.

Hypothesis
Alinkagebetweentwochemicalstimulicanbeimprintedonlarvalnematodeworms,
whowillrespondasadultstothesamestimuluslinkage.

ResearchQuestions
WhatresponsedonematodewormsexhibitwhenexposedtoNaCl?Toglucose?
Cannematodesbeconditionedtoassociateonestimuluswiththeother?
Ifexposedinthelarvalphase,willnematodesrecalltheirconditionedassociationsand
exhibitthesameresponsesasadults?
Howdodifferentconcentrationsandcombinationsofchemicalstimuliaffectthebehavior
ofnematodeworms?
HowdolongtermandshorttermexposurealterthebehaviorofC.elegans?

Expectedoutcomes
Potentialdeathduetoglucoseoverdose.
AssociationofNaClwithglucosesothatthestimulipresentedtogetherwouldelicita
differentresponsethanNaClpresentedalone.

Safety
Nitrilegloveswereused.Passagingimplementwassterilizedwithethanolandflame.

Procedure
I.
Makesolutions
A. Astocksolutionof0.1Mdextrosewaspreparedbycombining3.96gramsof
FlinnScientific#S0061labgradedextrosewith200mLofdistilledwater.
B. Asolutionof0.005Mglucosewaspreparedbydiluting25mLof0.1Mdextrose
stocksolutionwith475mLofdistilledwater.
C. Astocksolutionof0.1MNaClwaspreparedbycombining1.169gramsofFlinn
Scientific#D0005NaClwith200mLofdistilledwater.
D. Astocksolutionof0.075MNaClwaspreparedbycombining0.438gramsof
FlinnScientific#D0005NaClwith100mLofdistilledwater.
E. Stocksolutionswereseriallydilutedinto100mLbeakersandtransferredonto
individualpetridishesusingglassdropperpipets.
1. Serialdilutions,50mLeach
a) 0.06MNaCl
b) 0.05MNaCl
c) 0.01MGlucose

II.Prepareplatesofdifferentconcentrationsandcombinations
A. SixOP50(astrainofecoliusedtofeedC.elegans)seededngmliteplateswere
purchasedfromCarolinaLabs.
B. Eachplatewasidenticalinitiallyhowever,eachplatewasdesignatedforspecific
concentrationsofNaClandGlucosetobeaddedtothem.
a. Plate1:Fivedropsof.01Mglucoseandfivedropsof.05MNaClwere
addedoneachofthetendaysduringwhichtheexperimentwas
conducted.
b. Plate2:Fivedropsof.005Mglucoseandfivedropsof.075MNaClwere
addedoneachofthetendaysduringwhichtheexperimentwas
conducted.
c. Plate3:Fivedropsof.005Mglucoseandfivedropsof.06MNaClwere
addedoneachofthetendaysduringwhichtheexperimentwas
conducted.
d. Plate4:Fivedropsof.005Mglucoseandfivedropsof.05MNaClwere
addedoneachofthetendaysduringwhichtheexperimentwas
conducted.

e. Plate5(Controlofglucose):Fivedropsof.005Mglucosewereadded
eachofthetendaysduringwhichtheexperimentwasconducted.
f. Plate6(ControlNaCl):Fivedropsof.05MNaClwereaddedoneachof
thetendaysduringwhichtheexperimentwasconducted.

III.Growandpassagenematodes
A. AllowthewildtypeCEleganstogrowforaweek
B. Aregionofwormsinaplatewasobservedandidentified
C. Thepassagingimplementwasdippedinethanolandignitedinordertosterilizeit
D. Thepreviouslyidentifiedregionofwormswascutfromtheplate
E. Thechunkwastransferredwiththeimplementtothenewplateandplacedwormsdown
F. Thiswasrepeatedfor5otherplateswithanequalsectionofworms
G. Wormswereobservedtoverifysuccessfultransfer
H. Allplateswereincubatedfor48hours

IV.Observeplatesundermicroscope
A. AnAmericanOptical40StereoMicroscopewasused
B. Theplateswereobservedevery2days
C. Every2days,respectivemolaritiesofNaClandglucosewereaddedanddatawastaken
D. Plateswereobservedbefore,during,andafterdropsofNaClandglucosewere
administered
E. ThedropsofNaClwasaddedfirst,thentheplateswereobservedfor5minutes,thenthe
glucosewasaddedandtheplateswereobserved

V.Recordobservations
A. Allmovementwasclassifiedaseithertaxisorkinesis
B. Scalesfrom110,with1beingclosetonegligible,and10beingextreme,wereusedfor
rateoftaxisandkinesis
C. Percentageswereusedforamountofpopulationexhibitingtaxisorkinesis
D. Datawereinitiallyrecordedonpaper,thentransferredtoatable

Data
Thedatawereinterpretednumericallybyfirstdescribingtheintensityoftaxisand
kinesis,thenobservingtheamountofwormsexhibitingeithertaxisorkinesis.Fromtheverbal
explanation,anumericalvaluewasassignedforeachdatapoint.Tablesarecompiledbelow.

Plate #1 Table
Kinesis Kinesis Taxis
Sp.
Pop
Sp.

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose
5/20 Initial
NaCl
Glucose
5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

2
6
6
2
6
6
4
4
8
4
9
6
4
8
7
5
8
5
5
8
6
5
9
10
5
9
10
5
8
8

75%
60%
60%
75%
60%
60%
75%
65%
75%
50%
75%
65%
50%
75%
60%
60%
70%
60%
55%
65%
45%
60%
65%
75%
60%
55%
50%
70%
60%
55%

0
2
6
0
6
6
4
4
8
4
6
9
2
4
7
1
8
8
1
8
8
1
9
8
1
9
8
1
8
7

Taxis
Pop.

0%
10%
25%
0%
10%
30%
10%
15%
25%
30%
25%
45%
10%
25%
40%
10%
20%
35%
10%
20%
50%
10%
20%
15%
10%
35%
40%
10%
30%
35%

N/A
Pop.

25%
30%
15%
25%
30%
10%
15%
20%
0%
20%
0%
0%
40%
0%
0%
30%
10%
5%
35%
15%
5%
30%
15%
10%
30%
10%
10%
20%
10%
10%

Pop.
Alive

100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
95%
95%
95%
95%
95%
95%
90%
90%
90%
90%
90%
90%
90%
90%
90%
85%
85%
85%

Additional

Plate #2 Table
Kinesis Kinesis Taxis
Sp.
Pop
Sp.

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose
5/20 Initial
NaCl
Glucose
5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

2
5
5
3
5
5
4
6
5
5
7
7
5
7
7
6
7
7
6
9
8
6
8
8
6
8
8
6
8
8

30%
50%
50%
35%
50%
50%
40%
50%
45%
50%
35%
25%
55%
65%
60%
55%
70%
65%
60%
60%
65%
65%
65%
60%
70%
65%
60%
65%
60%
60%

1
5
5
1
5
5
1
5
7
2
6
8
1
6
7
2
6
8
1
8
9
2
7
8
2
8
8
1
9
8

Taxis
Pop.

0%
10%
15%
0%
10%
15%
0%
20%
25%
10%
50%
60%
0%
35%
40%
10%
25%
30%
0%
30%
35%
10%
35%
40%
10%
30%
35%
0%
40%
40%

N/A
Pop.

Pop.
Alive

70%
40%
35%
65%
40%
35%
60%
30%
30%
40%
15%
15%
45%
0%
0%
45%
5%
5%
40%
0%
0%
25%
0%
0%
20%
5%
5%
35%
0%
0%

100%
100%
100%
100%
100%
100%
95%
95%
95%
95%
95%
95%
95%
95%
95%
90%
90%
90%
90%
90%
90%
90%
90%
90%
90%
90%
90%
85%
85%
85%

Additional

Plate #3 Table
Kinesis Kinesis
Sp.
Pop

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose
5/20 Initial
NaCl
Glucose
5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

1
5
6
2
5
5
2.5
6
6
3
7
5.5
4
6.5
6.5
4
6.5
7
5
7
6.5
5
7
6
5
6
7
5
6
7

20%
90%
10%
20%
80%
10%
20%
70%
10%
20%
75%
10%
20%
70%
10%
20%
70%
10%
25%
70%
10%
25%
70%
15%
25%
70%
20%
30%
65%
15%

Taxis
Sp.

0
1
5
0
3
6
0
6
7
0
3
6.5
0
3.5
7.5
0
5
8
0
5
7
0
5.5
6.5
0
6
8
0
6
8

Taxis
Pop.

0%
5%
90%
0%
15%
90%
0%
25%
90%
0%
20%
90%
0%
25%
90%
0%
30%
90%
0%
30%
90%
0%
30%
85%
0%
30%
80%
0%
35%
85%

N/A
Pop.

80%
5%
0%
80%
5%
0%
80%
5%
0%
80%
5%
0%
80%
5%
0%
80%
0%
0%
75%
0%
0%
75%
0%
0%
75%
0%
0%
70%
0%
0%

Pop.
Alive

100%
100%
100%
95%
95%
95%
90%
90%
90%
80%
80%
80%
75%
75%
75%
60%
60%
60%
55%
55%
55%
45%
45%
45%
35%
35%
35%
25%
25%
25%

Additional

Note: the amount of

e-coli on the plate is
markedly diminished
amount of e-coli on the
plate has markedly
diminished
amount of e-coli on the
plate has markedly
diminished

Plate #4 Table
Kinesis Kinesis
Sp.
Pop

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose
5/20 Initial
NaCl
Glucose
5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

1
4
4
1
5
5
2
5.5
5
2.5
5.5
5.5
3.5
5.5
5.5
4
5.5
5.5
4.5
5.5
5.5
5
6
6
5.5
6
6
5.5
5.5
5

10%
80%
10%
15%
75%
10%
25%
70%
10%
30%
65%
10%
30%
65%
10%
30%
60%
10%
35%
30%
10%
30%
40%
10%
35%
40%
10%
40%
40%
10%

Taxis
Sp.

0
0
5
0
4
5.5
0
5
6.5
0
5
6.5
0
5.5
7
0
5.5
7
0
6
7.5
0
6
7.5
0
6.5
7.5
0
7
8

Taxis
Pop.

0%
0%
90%
0%
15%
90%
0%
20%
90%
0%
30%
90%
0%
30%
90%
0%
35%
90%
0%
60%
90%
0%
55%
90%
0%
55%
90%
0%
55%
90%

N/A
Pop.

90%
20%
0%
85%
10%
0%
75%
10%
0%
70%
5%
0%
70%
5%
0%
70%
5%
0%
65%
10%
0%
70%
5%
0%
65%
5%
0%
60%
5%
0%

Pop.
Alive

100%
100%
100%
95%
95%
95%
90%
90%
90%
85%
85%
85%
75%
75%
75%
65%
65%
65%
60%
60%
60%
55%
55%
55%
45%
45%
45%
40%
40%
40%

Additional

Plate #5 Table
Kinesis Kinesis Taxis
Sp.
Pop
Sp.

Taxis
Pop.

N/A
Pop.

Pop.
Alive

Additional
Taxis was observed for
about a minute after

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose

0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0

0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%

0
N/A
3
0
N/A
3
0
N/A
5
0
N/A
5
0
N/A
6
0
N/A
7

0%
N/A
75%
0%
N/A
75%
0%
N/A
80%
0%
N/A
80%
0%
N/A
85%
0%
N/A
85%

100%
N/A
25%
100%
N/A
25%
100%
N/A
20%
100%
N/A
20%
100%
N/A
15%
90%
N/A
5%

100%
N/A
100%
100%
N/A
100%
100%
N/A
100%
100%
N/A
100%
100%
N/A
100%
90%
N/A
90%

5/20 Initial
NaCl
Glucose

0
N/A
2

0%
N/A
5%

0
N/A
7

0%
N/A
80%

90%
N/A
5%

90%
N/A
90%

5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

0
N/A
2
0
N/A
2
0
N/A
2

0%
N/A
5%
0%
N/A
5%
0%
N/A
2.50%

0
N/A
7
0
N/A
7
0
N/A
7

0%
N/A
75%
0%
N/A
70%
0%
N/A
75.00
%

85%
N/A
5%
80%
N/A
5%
80%
N/A
2.50
%

85%
N/A
85%
80% more worms had died
N/A
80%
80% fewer worms exhibiting
kinesis
N/A
80%

Same result as previous

day

N/A

Same result as previous

observation

Slightly higher taxis than

previous observation

Some worms had died,

taxis slightly higher than
previous observation,
large majority of living
population exhibiting
taxis
slight kinesis in small
worms (likely newly born
worms with no exposure
to glucose)
More worms had died,
small worms still
exhibiting kinesis

Plate #6 Table
Kinesis Kinesis Taxis Taxis N/A
Sp.
Pop
Sp.
Pop. Pop.

5/6 Initial
NaCl
Glucose
5/7 Initial
NaCl
Glucose
5/11 Initial
NaCl
Glucose
5/13 Initial
NaCl
Glucose
5/15 Initial
NaCl
Glucose
5/18 Initial
NaCl
Glucose
5/20 Initial
NaCl
Glucose
5/22 Initial
NaCl
Glucose
5/26 Initial
NaCl
Glucose
5/29 Initial
NaCl
Glucose

0
3
N/A
0
3
N/A
0
3
N/A
0
3
N/A
0
4
N/A
0
4
N/A
0
4
N/A
0
4
N/A
0
4
N/A
0
4
N/A

0%
30%
N/A
0%
30%
N/A
0%
30%
N/A
0%
50%
N/A
0%
50%
N/A
0%
50%
N/A
0%
50%
N/A
0%
50%
N/A
0%
50%
N/A
0%
50%
N/A

0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A
0
0
N/A

0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A
0%
0%
N/A

100%
70%
N/A
100%
70%
N/A
100%
70%
N/A
100%
50%
N/A
100%
50%
N/A
100%
50%
N/A
100%
50%
N/A
100%
50%
N/A
100%
50%
N/A
100%
50%
N/A

Pop. Alive

100%
100%
N/A
100%
100%
N/A
100%
100%
N/A
90%
90%
N/A
90%
90%
N/A
90%
90%
N/A
80%
80%
N/A
80%
80%
N/A
70%
70%
N/A
70%
70%
N/A

Additional
Exhibited taxis, to a smaller
degree than glucose

Same result but for longer

Minimal kinesis

Small worms=taxis, big

worms=kinesis, slow

Minimal kinesis (only big

worms present)

Minimal kinesis

Minimal kinesis

Minimal/moderate kinesis

Minimal/moderate kinesis

Minimal/moderate kinesis;
4th/3rd most surviving
worms (Note: tied with
Plate 5-5)

AnalysisandConclusion
Upon analysis the experiment showed a correlation between adding NaCl followed by
glucose, and taxis in the worms, whereas only kinesis was observed when NaCl alone was
added, and taxis alone was observed when only glucose was added.The experiment showed
that NaCl would cause kinesis on its own and the concentration of NaCl was directly
proportional to the intensity of kinesis observed. Additionally, with every time that the
experiment wasperformedthespeedoftaxisas wellasthepopulationundergoingtaxisbrought
about by the addition of the NaCl increasedfollowingpriordayswhenglucosewas addedafter.
Furthermore the exposure to glucose increased the speed of taxis of the wormsoneveryplate
with glucose. Initially, all plates experienced a minor degree of kinesis before the experiment
was conducted, this can be assumed to be a natural amount of kinesis experienced by a
population of C. elegans. Also, upon successive exposure to NaCl, the amount of worms
experiencing kinesis and the speed of kinesis increased every day the experiment was
conducted. The glucose only plates experienced taxis which increased with higher
concentrations of glucose, this increase eventually plateaued until the worms died from an
overdose of glucose. This confirmed that an over addition of glucose could lead worm
populations to die offduetoexhaustionbroughtaboutbyrapidmotionforanextendedperiod of
time,whichdamagedtheirchemosensoryneurons.
As for the effects of NaCl alone on C. elegans, the worms on plate #6 showed that the
addition of NaCl alone will not lead to any taxis nor will it result in anypermanentalterationsin
worm behavior in the absence of NaCl. Furthermore, upon successive exposuretoNaClmore
worms began to undergo kinesisaswellasatamoderatelyhigher ratealthough,the changein
population experiencing kinesis and speed of kinesis was not as significant as upon other
plates. Due to the inability to trace each individual worm, it is uncertainastowhethersomeC.
elegans resist kinesis from NaCl naturally, or if, instead, during different days worms reacted
differentlyastherewasalwaysanonactivepopulation.
In plate #5, which saw the addition of glucose alone, not only did worms begin to
experience taxis upon the addition of glucose, but with eachsuccessiveadditionofglucosethe
rate of taxis as well as population experiencing taxis increased. Furthermore,towardsthefinal
daysofthe experiment,glucosealso beganto causekinesistoasmalldegreewhichwouldhelp
explain the increased speedof kinesiswiththeadditionofNaClandglucosetoplates14which
sawincreasedrateofkinesiswhileoverallpopulationundergoingkinesisdecreased.
Interestingly, in both control plates (#s 5 and 6) the addition of NaCl or glucose didnot
lead to any initial kinesis or taxis when the worms were observed each day before the
respective amounts of glucose or NaCl were added. However, plates with both glucose and
NaCl (#s 14) did experience an increasing rate of taxis and kinesis and increasing population
experiencing taxis and kinesis initially upon each day during which the experiment was
conducted. This revealed that the effects of NaCl or Glucose alone will not permanentlyaffect
the behavior of worms with regards to kinesis and taxis however, NaCl and glucoseinconcert
willpermanentlyaffecttheinitialbehaviorofC.elegans.
Additionally, the results showed that onplateswithhigherconcentrationsofglucoseand
NaCl, the population as well as supplies of ecoli began to decrease. The depletion of ecoli
was likely caused by an overabundance of worms undergoing taxis, whereintheworms moved

towards the ecoli to eat it and, over the duration of the experiment, diminished food supplies.
Decline in populationcouldhaveresultedfromthisdepletionofecoliaswellasduetotoogreat
a concentration of glucose and NaCl, which have been demonstrated as harmful toC.elegans
in large amounts. Furthermore, a decline in taxisoccurredwhentheecolipopulationdeclined,
likely because the worms had no food to move towards, and therefore, no taxis would be
possible. It could also be possible that surviving worms were more resistant to glucose and
NaClthantheotherworms.
Due to the lack of proper equipment to isolate individual C. elegans and to test larval
phase imprinting on these worms, the results of were inconclusive as to whether worms would
respond to NaCl with taxis as adults given exposure during their developmental phase.
However, given the successful shortterm imprinting on both adult and larval worms, the
possibility that adolescent worms would carry this memory into adulthood appears promising.
Further research is necessarytoconfirmthisassertion,andthis research couldbeconducted to
yield more conclusive results with accesstotracers,strongmicroscopes,andequipmentforthe
passage of individual worms to isolated plates. Another potential research inquiry is to see if
the membrane soluble molecules of NaCl and Glucose could be used to imprint on embryonic
C.elegansworms.

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