Dissertation final

DylanPenlington Biol387
Interplantsignallingandthe effectivenessof itsvolatilesproducedbyJasmonicAcidtreatment
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BIOL387 Project
Interplant signalling and the effectiveness
of volatiles produced by Jasmonic acid seed
treatment
Student: Dylan Penlington
Student ID: 33299633
Supervisor: Dr. Jane Taylor

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Glossaryof abbreviations
13-HPOT= 13-hydroperoxyoctadecatrieneoicacid
13-LPX = 13-lipoxy-genase
AC = AlisaCraig
ADJ = Adjacent
AOC= allene oxidecyclase
CON = control
GD = Gardner’sDelight
GPA = Greenpeach potatoaphid
JAZ= jasmonate zim-domainproteins
MeJA = Methyl jasmonate
NINJA = novel interactorof JAZproteins
OPDA=cis-(+)-12-oxophytodienoicacid
OPR3 = 12-Oxophytodienoate reductase3
JA = JasmonicAcid
TF= transcriptionfactors
TPL = toplessproteins
TSL = Two spottedladybirds

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Table of contents
1. Abstract
2. Literature review
i) What is the problem?
ii) VOCs (Volatileorganic carbons) Directand indirectdefence
a) Introduction
b) Direct defence
c) Indirectdefence
iii) Jasmonic acid (JA) structure, synthesis and cellularpathway.
a) JA structure
b) JA synthesis
c) Possible useful artificial modificationstostructure andartificial synthesisof JA
d) JA signallingcellular pathway and resultantresponses
iv) The costs and risks of jasmonic acid againstthe risks and costs of pesticides.
v) Model organisms, Tomato, green peach potato Aphids and Two spotted ladybirds
a) Tomato
b) Green Peach potato aphid (GPA)
c) Two spotted Ladybird (TSL)
vi) Aims and objectives
3. Materials and methods
4. Results
i) Primary data and leaves surfacearea and number
ii) GD (Gardeners Delight)
iii) AC (Alisa Craig)
iv) Summary
5. Discussion
i) Gardner’s Delight green peach potato aphid
ii) Gardner’s Delight two spotted ladybirds
iii) Alisa Craiggreen peach potato aphids
iv) Alisa Craigtwo spotted ladybirds
v) Decision vs No decision
vi) Leaf surfacearea and average leaf surfacearea
vii) Results compared to other literature
viii) Conclusion
6. Bibliography

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1. Abstract
Jasmonicacid(JA) isa signallingmolecule whichactsasa part the defence response inplants
duringinfestation. Itinducesthe indirectresponseof attractingnatural enemiesof
herbivorousinsects suchasthe two spottedLadybird(TSL) andpredatorymites.Whatisnot
yetknowniswhetherornot JA can induce pantstoproducesVOCscapable of interplant
signalling.Inthisstudy seedsof twotomatovarieties,Gardner’sDelight(GD) andAlisaCraig
(AC), where pre-treatedwithJA orethanol. Ethanol treatedplantswhere grown eitherclose
to the JA treatedplants asadjacentplants or where placedalongdistance away for controls.
Once grown theywere placedinaY-tube olfactoryassayto determine the response of
differentinsectspecies foreachplantvarietyandtreatmentpairs.Inseparate groupsthe
GreenPeachpotato aphids (GPA) andTSL were placedinthe apparatus for a choice test
betweentwoplantsof the same variety anddifferenttreatments. The resultsshowsa
minorityof insectsmade adecision.ForTSL forboth varietiesJA treatedplantsandthose
adjacentshowna significantly higherpreference comparedtothe control. ForGPA forGD JA
treatedplantsandthose adjacentshowna significantly higherpreference comparedtothe
control. For AC withGPA shownno significantdifference betweenthe control-JA and
adjacent-control pairings. There wasnosignificantdifference inchoice foranyof the JA
treatedandadjacentpairings. The conclusionof thisstudyisthat plantsare capable of
interplantsignallingandthere isadifference betweeneffectsof the plantssignalledto
betweenvarietiesandthose thathave beenJA treatedhasoninsectbehaviour.

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2. Literature review
i) What isthe problem?
Since 2009 there hasbeena directive fromthe EU to reduce overall use of pesticides, toidentify
pesticide alternatives.There isaparticularinteresttoprotect environmentallysensitive areas from
pesticides andthere is anever-growingconcernonthe effectsof pesticideson pollinatorssuchas
bees.(EU2009) There are risksinvolvedwiththe use of pesticides.For examplethere isan
evolutionaryrelationshipthatincreasedpesticide usage increasesthe chancesof pesticide
resistance ininsectpopulations,(Strantonovitchetal.2014) thiscausescrop managementissues.
(Leibee etal.1995) Alternatives topesticidesforpestcontrol hastobe considered,suchas
Jasmonicacid(JA) (Smartetal. 2013; Wakeil etal.2010.) JA isthe mainfocusof this review.The
aimsof thisreviewistodefine whatJA is,how itisproducedandto determine the potential usesit
will have inthe future incrop managementandhow interplantcommunication mightbe exploited
to reduce the economiccostsof usingJA.
ii) VOCs(Volatile organiccompounds) directandindirectdefence
a) Introduction
Volatile organiccompounds (VOCs) actsas airborne signalswhichinfluences the behaviourof
herbivoresnatural enemiesandneighbouringplants.JA actsas a switchthat up-regulates, the
synthesisof VOCsafterbioticstresses.The exogenousapplicationof JA caninduce production of
VOCsinplants. (Arimuraetal.2009.) The abilitytosynthesize VOCscanbe reducedorremoveddue
to mutation, forexample tomatomutantsthatlackthe JA biosyntheticpathway doesnotproduce
plantVOCsthat are induciblebyJA. (VanSchie etal.2007; Degenhardtetal. 2010.) VOCsproduced
duringinfestationcanconsistof terpenoids,fattyacidderivativesandaromaticcompounds.(Table
1) Thiscan produce a cocktail of VOCs that are emitted,whichare dependent onthe kindof damage
done and the locationof itwhichcan be causedby egg depositionordirectconsumption (Mithofer
and Boland2012.)

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b) Direct defence
Directdefence responsescausedbyJA are inducible.(Baldwin etal. 2002.) Crosstalk between
Jasmonicacidcross talksand ethylene,anothersignallinghormone,leadstothe build-upof direct
defence metaboliteswithinNicotianaattenuata.These metabolitesinclude trypsinproteinase
inhibitors,thisreducesthe nutritional value that herbivores gainleadingdue tointefreingwithits
digestive processleading tostarvation. Italsoleadsto Nicotianaattenuataproducingthe toxin
Nicotine whichlarge dosage killsattackingherbivores. The costof thisdefence onthe plantisthatit
leadstoa reducedgrowthrate (Figure 1) (Onkokesungetal.2010.)
Table 1 showsthe varioustypesof VOCsproducedbyplants.
(MithoferandBoland2012)
Figure 1: Schematicrepresentationof JA andethylenecross
talkand how theywork inconjunctionwhenwoundedand
havingdetectedherbivore elicitorcompoundstoproduce
directdefence metabolitesof trypsinproteinase inhibitors
and the toxinnicotine toactas defence againstherbivores
howeveralsoshowsthatthere isa cost inreducedgrowthto
defendagainstherbivores (Onkokesungetal. 2010.)

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c) Indirectdefence
InterplantsignallingviaVOCs, hasbeen shownin 30 species, (HeilandKarban2010.) Methyl
jasmonate(MeJA) helpsregulate plant’sdefence responses. MeJA hasbeenshowntobe takenin
plantsand metabolizedintoJA,jasmonoyl-isoleucine andjasmonoyl-leucine.These metabolic
conversions activatesVOCemissionswhich actas an indirectdefenceresponse.Inadditionto
signallingmolecule uptakeof MeJA some plantsalso take upothervolatile compoundswhich could
be re-emittedortransformedintootherby-products(Tamogamietal.2008.)
Interplantsignallinghasbeen demonstratedtobe inducedbybothinfectionandherbivory (Kostand
Heil 2006; Castelynetal.2015) VOCsproducedbyJA are capable of longdistance signallingbetween
differenttomatoplants (Fanetal.2015; Li etal. 2002.) By encouragingthe emissionof volatilesin
advance shouldbe plausibletoinitiatethe defence response in neighbouringplants. There are some
criticismsof doingexperiments aboutInterplantsignallingundertypical laborgreenhouse
conditions.Investigationsof volatilesusingthese conditionscanbe flawedif conditionsina
greenhouse are leftnotventilated.Thismay leadVOCsthe opportunitytobuildupwhichleadsto
the potential of increased interplantsignallingthanwhat wouldbe observedin anoutdoor
agriculture setting(Pascholdetal. 2006.) Such investigationsneedtouse greenhouses with
continuousairflow inmindinordertobettermodle volatilesignallingaswouldtake place outdoors
(Pettersonetal.1999; Pascholdetal. 2006.) Heil andton 2008 foundevidenceagainstinterplant
signallingbeingcausedbyvolatiles.The studyshownevidence thatsignallingforsome speciestakes
place only betweendistal partsof the plantwhere releasedvolatilesare the signals utilizedonlyby
the plantthat producesthem.Suggesting thatinterplantsignallingviavolatilesisnotalwaysclear
cut.
Whenattacked,plantsrelease volatilestoattractnatural enemiessuchas ladybirds (Thaler2002.)
Duringco-infestationthe effectivenessof plantsignalling isquestionable asinfestationcandisrupt
the volatile signalling bythe infesters,preventingeffective emissionof volatilesproducedthus
preventingthe herbivore’spredatorsbeing attractedtothe plant. (Miles,1999; Will etal. 2007; Will
et al.2009.) There are possible genderdifferencesthatcanoccur duringplantsignallingforexample
female waspsare more attractedthan malestothe volatile signalsof theirspecifictargetsfromdue
to theirneed tolayeggs withinthe insects. (Zhangetal.2013)

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iii) JA (Jasmonicacid) structure,synthesisandcellularpathway.
a) Jasmonicacidstructure
JA has the chemical structure asdepictedin (figure2a) (Donaryketal. 1995,) It’s3D intermolecular
structure can be manipulated,this influencesthe functionalityof JA whichcanbe influencedby
small changesindensitydue toitaffectingLogP whichaffectsitsabilitytoproperlybindtowhere
it’sneeded (Figure2b) (Li etal.2009.) JA’sfunctionalityisalsoaffectedwhenit becomesgaseous.It
signalstonatural predatorstowardsplantsherbivoreslike the plant ArabidopsisthalianausesJA
alongwithothervolatilestoattract the enemiesof attacking herbivores(Zhangetal.2013.)
#
Figure 2a) showsthe chemical structure of jasmonic acid(Donaryk etal.1995.) b)
showsthe 3D structure of Jasmonicacidmoleculesboundtogetherthe redareasare
the most susceptible tochangesinshape tothe overall Jasmonicacidstructure. (Liet al
2009)

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b) Jasmonicacidsynthesis
JA synthesisbeginsinthe chloroplastwhen leafsare wounded.Alphalinoleicacid isreleasedfrom
the chloroplastmembrane.Itundergoes aseriesof reactionswiththe supportof 13-lipoxy-genase
(13 –LOX) whichcatalysesoxygeninsertionatcarbon13 as the backbone thisleads tothe formation
of 13-hydroperoxyoctadecatrieneoicacid(13-HPOT.) The 13-LOXis convertedbyallene oxide
synthase (AOS) tocreate an unstable productwhichis quickly converted usingaseriesof allene
oxide cyclases(AOC) intocis-(+)-12-oxophytodienoicacid(OPDA) (figure 3) AOCisimportantsince it
formsthe final structure of JA. Since 2000 there are 58 knownAOCsequencesthatleadtodifferent
waysthat JA biosynthesisisregulated (Delkeretal. 2006.)
The second phase of JA biosynthesishappens usingperoxisomes. 12-Oxophytodienoatereductase3
(OPR3),thiscarriesa peroxisomal targetsequenceandactswithOPR3 to carry a peroxisomaltarget
sequence andacts only inthe cis-(+) form. Inthe final step the carboxylicacidside chainisshortened
inthree roundsof betaoxidation.Of whichonlyevennumberedOPC derivatives were convertedto
JA;which leadingtogene expression (Delkeretal.2006.)
Figure 3 Schematicof the JA biosynthesispathway.(Delkeret
al.2006)

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c) Possible usefulartificial modificationstostructure andartificial synthesisof JA
Syntheticanalogsof JA made artificiallyhave beenshowntoincrease the productionof nicotine in
tobacco. It may be possible tomodifythe overall structure of JA tomodifyitsfunctionality to
promote the productionof othersecondarymetabolites topromotesandimprove plantdefence.
Artificial methods toproduce JA hasbeendevelopedwhichinvolvesaseven stepprocesswhich
yields33%JA. (GullermoHet al 2015.) Thismethodinthe future hasthe potential tosignificantly
reduce the costs of JA leadingtopossiblyfuture agricultural industrywide adoptionof usingJA asan
alternative topesticides.
d) Jasmonicacidsignallingpathway andresultantresponses
JA is hormone that becomes convertedtoJA-lle inordertoactivate the pathwaythatproduces
Jasmonates.Inthe absence of JA, jasmonate zim-domainproteins(JAZ) are bound tonovel
interactorof JAZ proteins (NINJA), toplessproteins (TPL) andtranscriptionfactors(TF). These block
the JA respnse genesare switchedoff.Inthe presence of Jasmonicacid,causesthe activationof JAZ
proteinsandthe F-box protein SCFC011
thisfacilitatesthe recognitionof the E3ubiquitinligasewhich
causesthe degradationof the JAZproteins of the 26S proteosome.Thisreleases TFfromit causing
the Jasmonate response genestoturnon (figure 4.) Thiscausesthe productionof jasmonatesor
directdefence response dependingwhere the TFislocated,the responsesare highlightedinthe
defence section, Once JA isabsentJAZproteins are producedonce more andbindto the TF
therefore deactivatingthe JA response gene again (Wageretal.2012.)
Figure four:Inthe absence of JA and JA-lle jasmonate zim-domain(JAZ) proteinsbindstoTFand is
boundto co-repressorslikenovelinteractorof JAZproteins (NINJA) andtopless(TPL) proteinssupresses
gene transcription.WhenJA-lleispresentitcausesthe interactionbetweenJAZproteinsandthe F-box
proteinSCFCOI1 .Thiscausesit to be recognized.Causingthe 26Sproteasomestodegrade releasingTF
frominhibitionthuscausingthe activationof the JA responsivegene readyfortranscription.(Wageretal
2012)

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There are alsovarious downstreamevents thatcantake place fromthis initial signallingevent
dependingononwhichTFthe differentJAZproteinsbindto. (Figueroaetal 2012) For example
MYC2, a helix-loop-helixtranscriptionfactorthatacts as an acivator andrepressorof specificJA
responisve geneexpression events.ForexampleArabidopsis,where MYC2 possitivelyexpresses
oxidative strestolerance,flavenoidbiosynthesisandinsecthervivore resistancewhilstnegatively
regulatingtryptophanmetabolism (Dombrechtetal.2007; Lorenzoet al. 2004.)
iv) The costs and risksof jasmonicacid comparedto the risksand costs of pesticides.
SprayingJA ontoplantscosts a lot to treat because large quantitiesof JA hastobe used. (Wakeil et
al.2010.) Inorder to increase the commercial viability of JA treatment, pre-treatingthe seeds has
beenproposed.This significantlyreducesthe amountof JA beingused, thusdecreasingthe
economiccostof JA (Smart etal.2013.) The reason whyJA treatmentis a viable optionis thatJA
activatedplants hasdefence mechanismstohelpthe fightoff herbivoressuchasthe peach potato
aphidthroughinducingamixture of directandindirectdefence responses. (Onkokesungetal.2010;
Fan etal. 2015; Li etal. 2002.) Thisresultsinthe reduced use of pesticides(Smartetal.2013; Wakeil
et al. 2010.) It is knownthatplantsare capable of communicatingsignalsbynearbyplants (Paschold
et al.2006.) Thiscreatesanotheravenue of study to testif a JA pre-treatedseedwillbe capable of
causinginterplantsignallingwithitsproductionof Jasmonates,topotentiallyfurtherreduce the
overall costof JA treatmentof plants.
Throughthe use of bioassaysthere have beenargumentsthatpre-treatedseedsdoesnotalterthe
behaviourof predatoryinsectsbutinsteadreducethe effectivenessthatinsectscanconsume the
plantlike the Leaf Minorusingorganic tomatocultivars (Strapassonetal.2014.) Howeverasshown
inY tube experimentspredatorymitesare showntobe attractedto the volatile chemicalsproduced
by the JA treatedseeds.(Smartetal 2013) The reasonfor thisclashof evidence couldbe due tothe
differencesbetweenthe predatoryspeciesthatthese insectstargetsandtheirevolvedmechanisms
of searchingfortheirprey (Strapassonetal.2014; Smartet al.2013.) Meaningthatthismethodof
treatmentmayneedtohave a cost-benefitassessmentappliedtoeachspecieswhere the seedsand
local pestsare treatedbefore beingconsideredasamethodof treatmentdue tothe variousranges
of responsesobservedbydifferentpests.

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v) Model organisms, Tomatoplants,PeachpotatoAphidsandTwospottedladybirds
a) Tomato
Solanumlycopersicumoriginatedinthe Andes isseenthroughthe world andisaneconomically
importantcrop (Paduchuri etal.2010.) It giveshumans vital nutrients suchasvitaminCand has
beenknown foranti-oxidative effects(Raiolaetal.2014.) It isan importantmodel organismtotest
withbecause it’sgenome hasbeensequenced. Whichisadvantageousbecause itisnow possible to
lookat the gene interactionsthattake place withinthe plant foritsresponses. Furthermore it
makesfora goodmodel because ithasthe possibilityof growingunderdifferentcultivation
conditions. Ithasa short life cycle andlarge seedproduction.Meaningthatgenerationalstudiescan
be quickly assessed. Ithas a small genome,(950Mb) lack of gene duplication,highself-fertilityand
homozygosistsy.Meaningthatgene interactionscanquicklybe identifiedandthatall plantscan be
made to have similarDNA sequencescreatingmore reliabledatasets.Furtheradvantagesinclude
easyway of controllingpollinationandhybridization,asexual propagationbygraftingandpossibility
of regeneratingwholeplantsfromdifferentexplants. Meaningthatthe experimental batchworked
on will be easytomanagedandnot be taintedbyother tomatoesdue tothe additional meansof
controllingitspopulation (Bai andLindhout2007; The TomatoGenome Consortium2012.)
Tomato hassome unique featurescomparedtoothermodel organismssuchasArabidopsisorrice.
For example ithassympodial shootsandisthe onlymodel plantwithcompoundleafsalongwitha
large pool of tomato mutantswhichwere eitherspontaneousorinducedbychemicalsorirradiation.
(Lozanoetal. 2009) Literature onstudiesinvolvinghave alreadybeenproducedlikethe studyof JA-
deficientspr2mutantsof tomatoes. Itshowsa reducedresistance againstaphidscomparedto
control.Thisindicates thatJA does playa role in defence fortomatoes. (Avilaetal 2012)
b) GreenPeachAphids (GPA)
GPA otherwise knownasMyzuspersicae targets400 plantspeciesbelongingto50 plantfamilies
includingimportantcropslike potatoandtomatoplants(BlackmanandEastop. 2007.) Due to the
wide range of targetsand itsrapid abilitytoincrease inpopulationsize andresistance to large
numbersof pesticideshascausedthemtobecome a top agricultural pestinthe USA.( Georghiou
and Lagunes-Tejada,1991; Vasquez,1995; Silvaetal.,2012a,b; KleinandWaterhouse,2000)

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GPA are capable of reducingthe woundingresponse of plantsbysealingthe woundby usingit’s
saliva. Thisissecreted whenthe insectisfeeding,thusallowingthe aphidstofeedcontinuously
avoidingthe plant’simmuneresponse (Miles,1999; will etal.2007, Will etal. 2009.) The saliva
containseffectorsthatmanipulatehostphysiologytomake furthercolonizationeasierforother
aphids(RodriguezandBos 2013.) Furtherevidencesuggests whenthe genesof the protein
responsible forenhancingcolonizationMpC002of the GPA issilenced overall GPA colonizationwas
reduced (Pitinoetal.2011; PitenoandHogenhout2013.) Since these happenedpostinfestation the
ideaof pre-treatingthe seedswith JA inadvance beforeinfestation happenscanpotentiallygivethe
planta fasterimmune response againstGPA.
GPA iscapable of sexual andasexual (Pathenocarpic) reproduction.The GPAslife cycle utilizes
primaryand secondaryhosts.The sexual cycle iscompletedonstone fruitsuchaspeachesand
Canadianplums,the primaryhost.Secondaryhosts consistsof plantssuchas tomatoand potatoes.
(BlackmanandEastop 2007) GPA geneticsequencingisnear completion (Ramseyetal. 2007)
combinedwiththe developmentof plant-deliveredRNA-interference technologyforgene silencing
has ledtothe understandingof the geneticworkingsof GPA particularlyinplant-aphidinteractions.
(Pitinoetal.2011; PitinoandHogenhout 2013)
c) Two spottedladybird. (TSL)
Adaliabipunctata(TSL) isendemictoEurope,Asiaand Central America(Majerus1994.) TSL is a tree-
dwellingspecies. TSLfeedsdifferentinsectpreyincludingaphids (Hemptinne andDesprets 1986.)
TSL attaches to smoothservicesthrough by glandularswellingattheirposterior.The zygopodiumof
larvae prevents dislodgementfromplants. (Dixon2000) The larvae are attractedtowardsblue-green
light480-522nm wavelength thisimproves theirabilityof findinginfestedhostplants (Dimetryand
Mansour 1976.)
TSL are attracted to volatiles producedbyplants likethe sweetpepper.Thisattractioncanbe further
enhancedbysimultaneousinfestationof two species of the same genus infestingone plant (Oliveria
and Pareja2014.) It isnot yetknownthe impactof infestationof twodifferentgenus.The effectof
such infestationsmaydependuponthe ladybird’sdiet. Thislackof knowledge isfurthersupported
of the potential mixesof differentgenuscanbe demonstratedbycertaininsectssuchascaterpillars
whichare capable of inhibitingthe productionof VOCsbyplants. Whichmayalterthe cocktail of
VOCsproducedor inhibitthe productiontosuchanextentthatTSL are lesslikelytolocate infested
plants(Schwartzbergetal.2011.)

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There has beendebate amongstscientists whetherornotplantVOCs that act as an attractant to
herbivore predatorsbutinsectpheromones actas the attractant instead (Francisetal. 2004.) Some
evidence indicatesthatthisisnot the case is that the aphidpheromonessuchas C-beta-Farnsense is
whatattracts TSL towardsinfestedplants (Joachimetal. 2015.)
vi) Aimsandobjectives
The aim of thisprojectis to investigatethe effect thatJA pre-treatedseeds hasonattractingor
repellingGPA orTSL andwhetherornot JA treatedplantsare capable of interplantsignalling. The
firstobjective is tofigure outthe effectsof JA acidtreatedseedsondifferentvarietiesof tomato
plant,AlisiaCraigandGardendelightusingaY tube experiment tosee whetherornot GPA and TSL
are repelledorattractedtowards differentJA treatedplants;alsoobservehow GPA andTSL behaves
to each plantvariety.The secondobjective istoworkoutwhetherornot interplantsignalling
betweenJA treatedplantsandadjacentplantstake place andthenworkoutwhat thispotentially
meansina wideragricultural andevolutionary context.The thirdobjectiveistoworkout future
directionsof research couldgoand to planoutexperimentswhichcanbettermodel thisconcept.
The final objective istoassesswhichvarietyof seedwouldbe bettertogrow inan agricultural
environmentandassessthe reasonswhythiswouldbe the case. The hypothesis isthatboththe
Ladybirdsandgreenpeachaphidswill show greaterpreference overthe JA treatedandAdjacent
overthe control and no preference betweenthe adjacent-JA paringsof plants. The second
hypothesisisthatthere will be adifference inthe way bothinsectspeciesreacttothe different
tomatovarieties toJasmonicacidseedtreatment.

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3. Materialsandmethods
Seedpreparation
Two varieties of Solanumlycperisucm(Tomato) AlisiaCraig(AC) andGardenersDelight(GD) where
used.The seedswhere preparedbyimmersingtheninside 10ml of water30ul of ethanol or30ul of
1M JA wasadded.For eachvarietyandgroup (Adjacent(ADJ),control (CON) andJA treated(JA)) six
seedswhere preparedandfurthersampleswere preparedinsubsequentweeksforreservesamples
and to ensure all samplestestedwhereof the same age. Each sample where storedinafridge
overnightbefore plantation.Ondayof plantationthe seedswhere washedin500ml of water and
stirredfortenminutestoensure minimal JA orethanol influencesthe seedsdirectlyduringgrowth.
Growth conditions and plantation
Seedswere plantedinindividualpotsandeachpot was3cm apart from eachother,to prevent
interplantsignallingfromthe JA plantsthe controls where 6mawayfrom eachgroup (figure 5.) The
greenhouse grownin hadanaverage of 22 degreesCelsiusdaytime and18 degreesCelsiusnight
time witha photoperiodfor14 hours.Theywere leftfora weektogrow before beingusedinthe Y-
tube Setup (figure 6.)
Figure 5. Showsa diagramof the formationplantswhere setupinthe greenhouse.JA are
the JA treatedplants,ADJare the adjacent to JA treatedplantand CON standsfor the
controls.Whichwere placeddistancesfromeachotherasshownby the arrows.

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Y Tube set up and preparation steps.
For all plantsused,the numberof leafswhere countedandthe average surface areawasestimated
by measuringthe lengthandwidthof 10% of leafs.The average wasthentakenbetweenthose. The
Y tube experimentwasset upin accordance to figure 6. The Jars where sealed leftwiththe pump
on for1 hour before use.Thenalarge simple randomsample of TSLand GPA where collected.In
separate rounds6 insectsof eitherGPA or TSL are placedintothe Y tube of firstthe ladybirdsand
thenthe GPA. Every 6 minutes the decisionswhere recordedthiswasrepeatedforeachpairing until
60 data setswhere collected foreachcombinationof plants. The groupswhere setupas JA-ADJ,
ADJ- CON JA-CON thiswasrepeatedforeachgroup 3 timesusingthree differentsetsof plantsfor
each variety of plant. Leadingtoeachsetof data havingtested180 insectdecisions forboth GPA
and TSL for eachsample set.
Figure 6 showsthe Y tube setup.The tubingwasplastictubingwithoutholesandthe containerswhere made
out of glass. (Notmade to scale)

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4. Results
i) Primarydata and leaves data
60 decisionswere made fromeachof the three setsof plants,of which3 repeatswhere done usinga
separate setof three plantsfromtwo differentvarietiesof tomatoplant. Towork out the P values
the two tail T-testwasperformed.
On average lessdecisionswere made comparedtonodecisions forboth varieties of plantsandboth
speciesof insects. Forthe GPA for all pairsof GD the P value revealsthatthere isa significant
difference betweennodecisionsagainstdecision.(P<0.05) ForAC for GPA there was nosignificant
difference betweendecision (P>0.05) andnodecisionforthe CON-JA treatedand CON-ADJpairs
there wasa significantdifference betweenthe JA treated-ADJpairinfavourof nodecisions.
(P<0.05) For TSL there wasa significantdifference betweennodecisionsanddecisionsforboth
varietiesof plants(P<0.05.)
For leaf measurementsforACthere wasa significance difference forthe average surface areaforall
ADJ-JA,CON-JA andADJ-CON pairs.(P<0.05) For AC the average quantity of leavesthere wasa
significantdifferencebetweenthe ADJ-CON pair(P<0.05) andno significantdifference betweenthe
ADJ-JA andJA-control pairs.(P>0.05) For leaf measurements forGDplantsthere wasno significant
difference betweenADJ-JA,CON-JA andADJ-CON (P>0.05.) For GD for average quantity of leafs
there wasa significantdifference betweenADJ-control andJA control (P<0.05) For the ADJ-JA pair
there wasno significantdifferencebetweenleaf size.(P>0.05) (Table 2)
AC Average Leaf quantity Average surface area(cm^2)
ADJ/CON 18/22 8/5.5
ADJ/JA 14/15 7.9/7.6
CON/JA 18/16 5.7/6.9
GD
ADJ/CON 19/15 8.7/6.5
ADJ/JA 14/16 8/6.5
CON/JA 16/18 6.8/8.2
Table 2: The average leaf numberandaverage surface areaforboth
varietiesof plantsusedinthe Ytube experimentfromthe three pairsof
plantsusedforeach setup.

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ii) GD (Gardner’sdelight)
On average forthe GD plants there isa greaterpreference forthe JA treatedandADJ plantsoverthe
control for bothinsectspeciesandverylittle difference inpreference betweenthe JA treated-ADJ
pairs. For GD withGPA there wasno significantdifferencebetweenthe JA treated-CON andJA
treated-ADJ (P>0.05) forADJ-CON there wasa significance difference shown.(P<0.05) ForGD with
TSL there was nosignificantdifference between ADJ-JA pair, (P>0.05) there wasa significant
difference between JA treated-CON andADJ-CON (P<0.05.)

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Figure 7 mean number decisions oftwo spottedladybirds and greenpeachpotatoaphids betweenthree sets ofplants of GDJA andCON
(A) adjacent andCON (B), ADJ andJA (C) Made bythe average of 180 total decisions per plant turningto 60 total onaverage per repeat in
the Y tube set up(Figure 5) . The average leaf number andsurface area of each leaf is shown ontable 2.
0 10 20 30 40 50
Control
JA
Treated
No
decision
Aphid GD CON-JA
Average number of decisions
01020304050
Control
JA treated
No decision
Ladybird GD CON-JA
0 10 20 30 40
Control
JA
Treated
No
decision
Aphid GD CON-ADJ
0510152025303540
Control
Adjacent
No decision
Ladybird GD CON-ADJ
0 10 20 30 40 50
Control
JA
Treated
No
decision
Aphid GD ADJ-JA
01020304050
Adjacent
JA treated
No decision
Ladybird GD ADJ-JA
A
B
C

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iii) AC (AlisaCraig)
On average there isa preference frombothinsectsthe ACJA treatedandadjacentplantsoverthe
control whilstthere is nosignificantdifference betweenthe preferencebetweenthe JA-ADJsamples
for the AC plants. ForGPA there wasno significantdifference betweenCON-JA treated,CON-ADJ
and JA-ADJ.(P>0.05) For TSL there wasno significantdifferencebetweenJA-CONandADJ-JA
samples(P>0.05.) there wasa significantdifference betweenADJ-CON (P<0.05)

21
Figure 8 mean number decisions oftwo spottedladybird andgreen peach potatoaphids betweenthree sets of plants ofAC
adjacent andCON (A), ADJ andJA (B) andJA and CON (C) Made bythe average of180 total decisions per plant turning to 60 to tal
on average per repeat inthe Y tube set up (Fig.5 ). The average leafnumber andsurface area ofeachleaf is shownon Table 2.
0 10 20 30 40
Control
JA
Treated
No
decision
Aphid AC CON-JA
01020304050
Control
JA treated
No decision
Ladybird AC CON-JA
0 10 20 30 40
Control
JA
Treated
No
decision
Aphid AC CON-ADJ
010203040
Control
JA treated
No decision
Ladybird AC CON-ADJ
0 10 20 30 40 50 60
Control
JA
Treated
No
decision
Aphid AC ADJ-JA
0102030405060
Control
JA treated
No decision
Ladybird AC ADJ-JA
A
B
C

22
iv) Summary
For TSL for AC comparedwithGD forADJ-CON, ACmade more decisionscomparedwith GD,but
withGD TSL greaterpreferredADJ overthe control.Betweenthe ADJ-JA between TSLthere wasa
greaterpreference of choice forthe GD overthe JA.For the TSL there islittle difference inthe
numberof insectsthatchose the JA overthe CON butthere isa greaternumberof TSL chose the
control in the ACplantsthan the GD plants.
For GPA forAC and GD there islittle difference inpreferencebetweenthe twovarietiesof plants.
There isalso forGPA a strongerpreference forJA treatedplants of GD overthe AC butlittle
difference inthe preference inthe adjacentplantsbothvarieties. Forthe GPA a greaternumberof
insectschose the JA treatedof the AC overthe GD howevertheirwhere farlessthatchose the
control plantsof the GD overthe ACplants.

23
5. Discussion
i) Gardner’sDelight Peachpotatoaphid
For Gardner’sDelightwith greenpeachpotato aphids there is apreference betweenthe ADJ-CON
and no preferencebetweenJA treated-CON. (Figure 7A-C.) Thiscouldbe since the plantsAdjacent
to it have beenaffectedbyaunique volatilethatactedlike anaphid pheromone suchasC-beta-
farnsense makingADJplants more attractive to invasivespecies(Joachimetal.2015.) Insteadof
benefitingthe adjacentplant.The volatileemissions of ADJ effectively become ahoneytrap, for
attractinggreenpeachpotato aphids.Thisbenefitsthe JA treatedplantbecause itreducesthe aphid
populationinfestingit.Anothermechanismthatmayhave formedtopreventfurtherinvasion by
makingtheirscentsharderto distinguish betweenJA treatedplantsformed amechanismtomake
theirscentharderto smell like CON plants bynotproducingthese aphidattractingvolatiles.
HoweverwhentryingtodistinguishbetweenADJ-JA treated there wasnosignificantdifference. This
difficultytodistinguishbetweenthe twocouldarise since greenpeachpotato aphidsare generalists,
meaningtheirolfactorysystemsmaynothave evolvedanefficientmechanismtocompare similar
scentslike atomato specialistmayhave evolved. Itispossible thatspecialistaphidscandistinguish
betweenthe ADJ-JAtreatedpairthiswarrantsfurtherinvestigation. Other literatureseemsto
suggestthatgeneralistaphids dopreferthe scentof volatileemittingplantscomparedto none
volatile emittingonesbutonlytovolatilesemittedwheninfestedbyanotherspeciesof insect.
Furthermore noreferencesof thisproposed honeytrapmechanism seemtoexistincurrant
literature makingitanovel areaof research (Girlingetal.2006.) Most literature doesnotsuggest
aphidsare beingattractedto volatile emittingplants andsome suggeststhe opposite effectof them
beingrepelled.(Smartetal.2013; OliveriaandPareja2014; Schwartzbergetal.2011)
There wasno significantdifference betweenADJ-JA itcouldbe possible thatsince the scents
betweenthe twocouldbe due tothe experimentsetupthatthe scents are similarenoughtocreate
a maskingeffectdue tothe narrownessof the tubes. (Figure 7A-C.) Forfurtherinvestigation usinga
well ventilated greenhousetodetectpreference of insectinfestation betweenADJ,JA treatedand
CON plants overa longperiodof time andcountingthe numberof insectsinvesting the plants,could
see if there isa preference betweenthe twoVOCemittingplants.Thisisbecause comparedtothe Y
tube experiment wherethe volatilesare concentratedtoa place where the volatilesdispersed
meaningtheymaybe easiertodiscernandthe aphids will be inalessstressful environmentmaking
it more likely observe theirnormal behaviour andsee there populationdistribution. (Lopez-Sepulcre
and Kokko2005.)

24
Anotherpossibleexperiment todowould be to workout whatunique volatile organiccompoundsof
ADJ plantsproduce whichleadstothe masking and/orhoneytrapeffectswiththe use of gas
chromatographyandmass spectrometry.Thiswill make itpossibletocompare andcontrast the
volatilesproducedbyJA treatedplants.Whichwill grantthe abilitytofindanyunique volatilesnot
emittedbyJA andemittedbythe ADJplantsand vice versa. Thenuse Y tube experiment withthe
unique volatiles extractedfromgaschromatography andnoscentto findwhichscentcausesthe
honeytrap effect.Similarexperimentshave beenperformedtofigure outthe functionsof such
volatilesinthe past sothe methoddescribedshouldwork (Boldizsaretal.2011; Schaueret al.2006.)
ii) Gardner’sDelightTwospottedladybird
For Gardner’sdelight withthe twospottedladybird the paringsacceptedthe null hypothesisof ADJ-
JA whichwas expectedandthe null hypothesiswasrejectedforthe ADJ-CandC-JA pairingswhich
was expected.(Figure7A-C) The reasoningbehind astowhy greenpeachpotatoaphids shown
differentresultscouldbe because the scentreceptorsof greenpeachpotatoaphids are differentto
twospottedladybirdsolfactory receptors, thatare capable of detectingdifferentcompoundsto
greenpeachpotatoaphids.(Figure 7A-C) The reasonwhyJA treatedandADJ plants emitvolatiles
couldbe that theyevolvedthismechanismtocreate a largerfieldof effectforvolatileemission for
the two spottedladybirds andotherherbivore enemies todetect.A largersignal thatthisplantis
infectedhere inalarge openenvironmentispreferabletoa small signal whichatgreaterdistancesis
harderto detect. (Figure 9) Althoughthere appearstobe no suchliterature Thatshowsthiseffectit
may be possible toinvestigateitthroughthe use of Laser-basedinfraredspectroscopyandproton
transferreactionmassspectrometry due totheirhighsensitivitytolocate the difference between
concentrationsof VOCsinlarge groupsof plantscomparedto VOCemissionof anindividual interms
of range by testthemVOCemissionsatdifferentrangestoobserve whichVOCemissionrange isthe
widest(HarrenandCristescu2013.)

25
Figure 9: (Notto scale.) Showsthe possible areaof effectof the tomatoplantsif emittingvolatilesaloneorin
groups.A) Showsthe effectof an individual VOCemittingplantwhichshowsasmallerVOCareaof effect
where TSL wouldbe attractedto.B) Showsa group of VOCemittingplantsthathave communicatedwitheach
otherto produce a much widerare of effectcomparedwithA.Demonstratingthe advantage of interplant
communicationwhendealingwithapestissue.The widerarealeadstomore TSL potentiallybeingattractedto
the plants.

26
If this endsupbeingthe case the widerarea of signallinghasthe keyadvantage of attractingmore
twospottedladybirds todeal with infestation.Whichwouldshow that the keyadvantage of
interplantsignallinginthat the infested plantwouldbe capable of activatingthe nearbyplants
defence response. Similareventsof interplantsignallinghasbeendescribedbypastinvestigations
(Castelynetal.2015 ;Heil andKarban 2010; Kostand Heil 2006.) Thiscouldbe why ADJ plantsthat
produce VOCsthat are attractive to TSL because theyproduce the same kindof volatilesthatare
attract them.(Figure 7 A-C) Thishasthe addedbenefittothe adjacentplantstohelpthemprevent
infestationevenwiththe possible increasedrate of infestationcausedbythe honeytrapeffect. To
testwhetherornot thisisthe case a possible future experimenttodoisto compare and contrast
the effectivenessof asignal of a JA treatedwithadjacentplantsinattractingpredatorscomparedto
a JA treatedplantonits own ina greenhouseandobserve theirpopulationdistribution(Lopez-
Sepulcre andKokko2005.)
A group of volatile organiccompound emittingplants mayalsohave the advantage of havinghigher
concentrations of volatileorganiccompounds thanasingle plant. Whichmaycause the two spotted
ladybirds toperceive thatmore foodislocatedinlocationwiththe bigger volatileconcentration
than the locationwithalowervolatile concentration. Whichhassimilarlybeenseenin
bioengineeredArabidopsisthalianabutonan individual levelviabioengineeringwhere increased
concentrationsof volatileemissionof nerolidol,aplantvolatile organiccompounds ledtohigher
numbersof carnivoreslandingonthe plant(Houshyani etal.2013.) Thisindicates apossible
evolutionarymechanismastowhy signal amplificationtotwospottedladybirds wouldhave
originallybroughtaboutsuchbehaviour more foodina largerarea meanshigherchance tobread
leadingtoa higherpreference of highconcentrationvolatile areas,itpotentiallyindicatesahigher
qualityterritorytohabitatleadingtoa potentiallyhigherpopulationdistribution.

27
Since the plantsare releasingvolatilesnotbecause of infestationbutbecause of beingJA treatedor
adjacentto themthere islesschance of themhavinglongtermresidence due tothe absence of
food.But itincreasestheirchancesof goingtowardsthe tomatoplantand there isthe potential of
twospottedladybirds topotentiallypopulate the plantsthatare already infesteddue tothe food
presentthere.Howeverthe chance remains thatthisvolatileorganiccompound signallingisthatit
may cause the dispersal of the twospottedladybirdswhich will reduce the benefitsof the infested
plantdue to lesspatrollingTSL(Smartetal. 2013; Lopez-Sepulcre andKokko2005.) Therefore itmay
be possible thatthere isa pointof whichwhere the Adjacent-JA signallingstopstopreventdispersal.
A future experimenttodo wouldbe to testto see if populationdispersals of twospottedladybirds
on a largergroup of volatile organiccompound emittingplantsthatare notinfestedorcanbe done
througha Y tube experimentwhereagroupof plantsemittingvolatilescanbe testedagainstasingle
plantemittingvolatiles. (Lopez-Sepulcre andKokko2005)
iii) Greenpeachpotato aphids AlisaCraig.
For greenpeachpotatoaphids,the AlisaCraigplantsall three pairingsof plantsacceptedthe null
hypothesisthiswasunexpectedforthe C-JA andC-ADJpairingsandexpectedof the JA-ADJpairings.
(Figure 8 A-C) The resultsindicatesthatthe volatilesproducedbyACfor GPA donot produce a scent
that istheycan detect. Comparedwiththe Gardner’sDelight variety(Figure7A-C) indicatesthatthe
differentvarietiesof plantshave differenttraitswhichdealswithinfestationusingdifferent
strategies.
By producingnovolatilesattractive to greenpeachpotatoaphidsimplies thatgreenpeachpotato
aphidswill be lesslikelytolocate AlisaCraig comparedtoGardner’sDelight.Inan agricultural
settingthiswouldbe advantageousforusingAlisaCraigvarietycrops overGardner’sDelight because
there will be noincrease inthe numberof aphidsgoingtothe plantsbecause of the volatiles
produced.Whilstwith Gardner’sDelight there wouldbe the disadvantageof adjacentplantsbeing
attractedto the volatilesof ADJplants.Indicatingthat AlisaCraigwouldthe superiorcroptopre-
treat the seedsof jasmonicacid anduse commerciallyforJA treatment. These resultsindicatethat
AlisaCraigmay have evolvedamore mutualisticrelationshipbetweenitsneighbourswhen
comparedwiththe Gardner’sDelight where the honeytrapeffecttakesplace.

28
Howeverthe disadvantage of lackingof the honeytraptraitinthe AC varietycouldmeanthatonce a
plantisinfestedthe populationof Aphidsare lesslikelytodisperse due tochemical cues. Whichmay
come at a disadvantage because the plantsare more likelytodie due toinfestationbeing
concentrated.Whichcouldmeanthatonce the plantisdeadthe aphidswouldmove ontoother
plantsand will be able tokill inthe cropmore efficientlydue tothe lowerpopulationdistribution
than whatcouldbe observedwithGDplants. Therefore itwouldbe importanttocarry out a
possible experimenttotest tosee if the benefitoutweighsthe costhighlighted.Thiscanbe tested
betweenthe twopre-treatedseed varietiestocompare the effectivenessof aninfestationbetween
these twovarietiesinseparate greenhouseswithsimilarpopulationsof aphids tosee which
populationof aphidscausesthe mostdamage andcompare bothwitha control sample.
What thisexperimentfailedtodoisto testwhetherornot JA treatedplantsstill producesdirect
defence metabolitessuchastrypsinproteininhibitorsandsee if crosstalkcan be achievedbyalso
pre-treatingseedswithethylene tosee if youcaninduce multipledirectdefenceresponses.Thenby
testingthe physiological effectthishasoninsectsinfestingdifferentspeciesof plants.Thiscould
helpreduce the needforpesticidesbyallowingplantstodefendagainstattacksthemselves(EU
directive 2009; Onkokesungetal.2010.)
iv) AlisaCraigTwo spottedladybirds
For AlisaCraigwithtwospottedladybirds there wasnosignificantdifferencebetween ADJ-JA
treatedwhichwasexpectedandJA treated-controlwhichwasunexpected.The ADJ-control pair
showna significantdifferencewhichwasexpected. (Figure 8A-C) The reasoningbehindwhyJA
treated-control plantshadnosignificantdifference isthat JA treatedplantsmayhave undergone an
epigeneticchange frombeingpre-treatedwhichisonlypossibleforthisvarietydue toitsunique
geneticmakeup(Tomatogenome consortium2012; Heimesetal.2015.) Thischange may have
causedsome of its volatile emittingpropertiesandisanevolvedmechanismtosave energy.Butit
still emitscertain volatileswhichtransmitssignalstoother adjacentplantswhichproducesthe
volatilesitneedstoattract twospottedladybirds toitsgeneral vicinity.(Figure 9) Therefore even
thoughit isnot directlyemittingvolatilesitselfitstill gainsthe benefitsof the protectionvolatile
emissionsof otherplants byutilizingits’evolvedmechanism.

29
In an agricultural contextpre-treatingthe seedswithJA mayhave the riskof havingTSL beingless
likelytodetectthe infestedtomatoplant due the adjacentplantsproducingthe attractive scent.
Thismay create an lessevenpopulationdistributionacrossthe AlisaCraigcropsinorderto locate
foodfocusedonthe ADJ plants as whatcomparedto whatcould potentiallybe observedwith
Gardener’sDelight.Howeverthe reverse mayalsobe true itmay be by the JA treatedplantnot
producingvolatilesattractive itmaybe that itbecomeseasiertodiscernbetweenthe infestedplant
and non-infestedplantsinthisvariety.Thusmakingita more populousareawhich twospotted
ladybirdsfirstinvestigatesforaphidinfestation.Thisseems feasible because greenpeachpotato
aphidsduringinfestationproducesawoundsealingpropertiesintheirsalviamakingthe plantmore
noticeable becauseof its lackof attractive volatiles inareawhere astrongvolatile scentis the norm.
(Miles1999; will etal.2007, Will etal.2009.) Essentiallythismayleadtothe twospottedladybirds
to firsthone inthe volatile scentsandthentheymaythenhave evolvedthe behaviouratthe highest
concentrationsof volatilestobe able tolocate an area inthat area where lessscentisproducedin
an infestedorpre-treatedseedwhichmimicsthiseffectinordertofindtheirprey. Howeverinthis
situationcouldhave anagricultural disadvantageinthe contextof pre-treatingthe seeds because it
couldmeanan unevenpopulationdistributionof twospottedladybirds beingfocusedonthe JA
treatedplantscouldleadtothe othernearbyplantsbeing more prone toinitial infestationmeaning
the ADJ plantswouldneedtobe firstinfested inordertoattract two spottedladybirds tothe plants
emittingstrongerscents.Inordertoinvestigate whichone isthe case itwouldmeantaking two
spottedladybirds intoagreenhousesettingorganise the plantslike figure 5insee measure
populationdistributionof TSLfor AC(Lopez-Sepulcre andKokko2005.)

30
v) Decisionvsnodecision
Withinbothvarietiesof plantsandbothspecies of insects there wasasignificantdifference between
makinga decisionagainstnodecisionof whichmostinsectsmade nodecision. The possiblereason
for thispreference tomake nodecisionis thatthey were placedoutside of theirnormal habitat
leadingtoabnormal behaviour. Furthermore someof the twospottedladybirds aswell hadtobe
shippedinanduseddue to time constraintsleadingtofurtherconfuse theirnormal behaviour. It
alsocan be due to the lack of otherstimuli suchas seeingthe tomatoplantswhichaffectedtheir
decisionprocessesbecause itmaybe thatmore stimuli increasesthe chancesof makingthe decision
to pursue the scent.A secondcause forindecision fromvolatile organiccompounds producedbythe
plantsmixedtogetherleadingtosome possibleconfusioninthe insectsolfactorysystemwhich
wouldparticularprevalentinthe ADJ-JA pairsforGD plants.(Figure 7C) To testto see if itis due to
the lack of otherstimuli one cantry to add stimuli inanew tube setup to incorporate the visual
stimulusof the plantwiththe volatilesandanotherwithoutvolatilesandjustthe visual stimulusof
the plantand a third groupwithjustvolatiles andacontrol withno stimuli tosuggestnearbyplants
to observe whetherornot addingextrastimuli doeseffectthe decisiveness of the insects.
Indecisioncouldhave also occurred due tothe diversityinboth insectspecies genepools. Thismay
have ledto varyinglevelsof sensitivityintheirolfactorysystemsthatare usedtodetectthe
difference of the twogroupsof volatile organiccompounds.Itmaybe possible thatsome may have
recently fedtherefore where nothungryenough toleadthemtospendenergylookingfora new
foodsource. It mayalso be possible thatforsome insectgenotypes are notbornwiththe capability
of detectingvolatileorganiccompounds andfavourothermethodsof detection. Itwouldbe
possible totesttosee suchvariabilitydoesexistinsome of the methodsdescribedbyGetahunetal.
2013, whichusedextracellularsingle sensillumrecordingtomeasure thissensitivity.Thenfrom
those methodsfilteroutthe differentgroupsof insectsinaccordance totheirsensitivityof each
insectspeciesandtestthe numberof decisionstheymake againstthe numberof nodecisionsusing
the same methodsinthe materialsandmethodssectionof thisinvestigation.

31
There isa greaterlevel of significance displayedbyJA treated-ADJpairof insectsmakingnodecision
as seenin Figures7C and8C. Thiscouldbe due to theirscentsbeingsimilarthatitcausedgreater
confusionamongstthose insectsunlesstheyare variantsbornwiththe correctgenescapable of
producingan olfactorysystem detectingthe difference betweenJA treated-ADJ. thereforecausing
higherratesof indecision.Howeverinthe case of AlisaCraigthe ADJ-JA treatedpairisless
pronounced thisindicatesthatforthe twospottedladybirdsare more decisive inchoosing Alisa
Craig comparedwithGardner’sDelight since the difference betweenthe decisionsvsthe no
decisionsismore pronounced.Whichindicatesthatfor AlisaCraigplantsandladybirdsthatthey
have a higherchance of attractingladybirdsincomparisontothe Gardner’sdelight whichinstead
appearsto utilize the ADJplants withthe honeytrapeffectdescribedbefore toprotectthemagainst
infestation.
For AlisaCraigwithpeachpotatoaphids the JA treated-CON andADJ-CONare exceptionstothis
trendsince there wasno significantdifference betweenthe decisionsandnodecisions.Thiscouldbe
due to the JA treatedandADJ plantsproducedamuch more differentiated andstrongerscentin
comparison tothe control plants. Thisshowsthatfor aphidsandAlisaCraigplantsthat JA treated
and the ADJ plants volatiles appeartobe more randomcomparedto Gardner’sdelight.Thisis
supported bythere beingnosignificantdifference betweenthe choicesmade betweenthe JA
treated-CON andADJ-CON. Howeveritisstill arguable that since the null isacceptedthatthey
appearto move more comparedto than when toGardner’sDelightpairs whichindicatesthatthe
volatilesproducedmay have aninfluence intheirbehaviour. A potentialexplanationtothiscould
be that AC may have a unique mutualisticrelationshipwithone ormore bacterial speciesunique to
the plant,similartohowrhizobacteriainterfereswiththe attractionof parasitoids thankstoplant-
bacteriainteractionsthankstoJA signalling(Pinedaetal.2013)

32
To investigate if thispossibleplant-bacteriainteractionhasanimpacton aphidbehaviour,wouldbe
to testa JA treatedplantthat hasbeentreatedwithantibacterialagentsonthe surface of the plant.
Thenusingsimilarmethodsof the Ytube set upin thisinvestigation(Figure 5) see whetherornot
thishas an effectoninsectbehaviourcomparedwithacontrol and a plantnot treatedwith
antimicrobial agents, tosee if thishasanimpacton aphidbehaviour.If itisthe case that there isan
effectthe nextstepwouldbe toisolate the microbiome andfindthe bacterial speciesthatproduces
thiseffect.Fromthere furtherresearchcouldbe done tosee if thisbacteriacan have potential
future agricultural applicationstopossiblytryandreduce the ratesof infestationonplants orisolate
the specificcompoundsproducedbythe bacteriaandproduce itforfarmers.Thiscouldhelpreduce
the rates of infestation of aphidsandmayhelpinthe EU directivesaimtoreduce pesticide byuse of
sustainable alternatives (EU2009.)
vi) Leafsquantity andAverage surface area
The average quantity of leafsforAlisaCraigandGardner’sDelightmostof the resultsrejectedthe
null hypothesis. Thismayinfluence the behaviourof the insectsdue to volatile organiccompounds
productionbeingaffectedsince leavesproduce jasmonatevolatileorganiccompounds. Thisis
shownbyEffmertetal 2008 whichdemonstratesthatareductioninleaf numberreducedoverall
volatile emission. Thiscouldreduce the accuracyof the resultssince the large difference inleaf
numbercouldaffectthe resultsbecause one plantinanyexperimental paircouldhave produced
significantlymore volatilesthananother.Therefore if thisexperimentwhereperformedagain
statistical testsshouldbe done inadvance betweenselectedplantstoensure thatthe error due to
biasdoesnot appear.Eveninthe case of the control planetsthathave notbeentreatedstill emits
othervolatile organiccompounds.
For leaf surface areafor the AlisaCraigand Gardner’sDelight there are resultsthatrejectthe null
hypothesiswhichnegativelyeffectsthe accuracyof the results.Thisisbecause surface areais
anothertraitwhichinfluences the releaseof volatiles,since the largerthe surfacesareathe greater
the release of volatiles(Constable etal.1999.) Whencombinedwiththe measurementsseenwith
the average quantity of leavesthiscanpotentiallyleadinto agreaterinaccuracyinthe resultsdue to
the impact ithas on the difference of volatilesemittedbetweentwoplants.

33
vii) Discussionof resultscomparedtootherstudies
In Zhanget al 2013 showsthatJA signallingforthe attraction of enemiesof herbivores hasbeen
demonstratedinotherspeciesof plantsuchas Arabidopsisthaliana.Where the female, Diadegma
semiclasumconfirmshowstheyhave agreaterpreference overJA treatedplantsover acontrol.
Similartohowthe two spottedladybirdsshow a greaterpreference overthe JA treatedovera
control for bothvarietiesof plants.(Figure 7and 8) The groups inZhang etal 2013 where separated
ingreenhouses anddidnotuse a Y tube set up(Figure 5) makingthe resultsmore reliable.Butthis
studyhas no reference towhetherornotplantscommunicate toeach otherso cannotbe usedto
predictthe behaviourof the behaviourof Diadegmasemiclasumtoadjacentplantsorhow
Arabidopsisthalianainfluencesotherinsectsbehaviour.
Zhang etal. 2013 putsintoquestionthe efficacyof whetherornotthiswouldbe feasible by
observingthatthose plantsthatwhere infestedwiththe white-fliesappearedtosupressthe ability
of the crop abilitytoattract these wasps. Similartohow GPA suppressesthe plantsabilityto
produce VOCs.(Miles,1999; will etal. 2007; Will etal.2009.) Howeverthe potential advantageof
pre-treatingtheseplantscouldoutweighthe costthisisbecause byhavingJA treatedplantsout
there inadvance before infestation willmeanapopulationof predatorsof interestwill be more
likelytobe aroundthese plantstodeal withearlyinfestation. Aninvestigationonthe effectsof pre-
treatingseedsof otherspeciescanbe performedtosee if similareffectscanbe inducedinother
crops. Whichwill be goodforthe agricultural industrybecause itwill reduce theiroverall needtouse
pesticidesforothercropsinaccordance to the EU directive (EU2009.)
In Paschold etal 2006 showsthatplantsare capable of communicating witheachotherbyplacing
themwithinanair conditionedchamber whenwounded.These experiments show it’spossible for
wounded plantstoemitvolatilestothe unwoundedplants,andinduce aresponse inthem.Thisis
similartothe effectsobservedinthisinvestigation of the JA treatedplantssignallingtothe adjacent
plants. Since woundingresponsecausesthe transmissionof Jasmonatesaswell gaschromatography
resultsof the receivershownthatthe receiverplantsdidreleasevolatiles.Whichfurthersupports
the resultswhichsuggeststhatADJplants can influenceinsectdecision byproducingvolatiles
emittedbyJA treatedplants.

34
Smart etal 2013, shows an experimentsimilartothisexperimentthroughthe use of aY tube setup
and pre-treatingseedswithJA.(Figure 5) Theyusedadifferentvariety of tomato,the moneymaker,
it wasshownin these resultsthatusingpredatorymites there isaclearpreference inthe predators
for targetingthe JA treatedplants overthe control samples due toVOCsproduced.Whencomparing
these resultswithTSL,withGD andAC withresultsinCON-JA (Figure7and 8) alsoshowna
significantdifferencebetweenthe pairwithgreaterpreference towards JA treatedplants.This
showsthat thisattractionfor tomatoplants VOCsisnot relatedtojust to TSL but to otherspeciesof
predatoryinsects.
Vii) Conclusion
The research showsthe firsthypothesiswasincorrectforshownchoice preference thataphidsand
ladybirdschose between plantpairs. The secondhypothesishasshowntobe correct since JA treated
plantsare capable of interplantsignalling thisisbecause ADJplants wherecapable of signallingto
ladybirdsandaphids. Thisinvestigationhasadded towhatisknownto the subject,itshowsthat JA
treatedseedsare capable of producingvolatiles capable of signallingtoplants. Italsoshowsthat
differentvarietiesof tomatoplant signal toeachotherindifferentways,like withGardner’sDelight
havingthe honey trapeffectandAlisaCraighavingitsownabilitytodirectlyattractladybirds
silencedbutisstill emitsvolatilesthatcommunicate withadjacentplantsto attract ladybirds.The
shortcomingsof the investigationwasthatthere was a significantdifference foraverage leaf surface
area and average leaf quantity forthe majorityof plants whichmayhave ledtoan unbalanced
emissionof volatiles.The researchhasleftseveral unansweredquestions,suchashow doesJA seed
treatmentaffectdirectdefence responsesof plants,whatspecificvolatile orgroupof volatilesin
Gardner’sDelightvarietyproducesthe honeytrapeffectandhow wouldthe insectsreacttoJA
treatedplantsina more realisticsettinglikeagreenhouseintermsof populationdistribution.A few
more questionswhichhave beenleftunansweredare whichvarietyof plantbestworkswithJA
treatmentandwhetherornot bacteria-plantinteractionshave beenaffectedbyJA treatment.The
resultsshowthatJA treatmentpotentiallyhasause in agriculture asa possible alternativefor
pesticideshoweverfurthertrialsonhow thistreatmentwouldinfluence insectbehaviourinthe real
worldneedstobe done.

35
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