The resistance of lettuce
totheaphid
Nasonovia ribisnigri
Maarten van Helden
CENTRALE LANDBOU
W CATALOGUS
000006113043
^f oqS
j
Promotor:
Dr. L.M. Schoonhoven, Hoogleraar inde Entomologie
Co-promotoren:
Dr.W.F.Tjallingii, Universitair Hoofddocent in de Entomologie
Dr.T.A. van Beek, Universtitair Hoofddocent indeOrganische Chemie
iJJIJO^'O l \ & %
MaartenvanHelden
The resistance of lettuce
totheaphid
Nasonovia ribisnigri
Proefschrift
ter verkrijging van degraadvan doctor
inde landbouw- en milieuwetenschappen
opgezagvan de rector magnificus,
Dr.C.M.Karssen,
in het openbaar te verdedigen
opwoensdag 22februari 1995
des namiddagste vier uurindeAula
van de Landbouwuniversiteit teWageningen.
^n
BIBLIOTHEEK
LANDBOUWUNIVERSITEIT
WAGENINGEN
CIP-DATA KONINKLIJKE BIBLIOTHEEK, DENHAAG
Helden, Maarten van
The resistance of lettucetothe aphid Nasonovia
ribisnigriI Maartenvan Helden -[S.I. :s.n.].- III
Thesis Wageningen.-With ref. -With summary inDutch.
ISBN 90-5485-349-2
Subject headings:Aphids / resistance / lettuce
uuo82oi,>8^
STELLINGEN
1.
EenAIO heeft geen luizenbaantje.
Dit proefschrift
2.
The resistance of lettucetothe aphid Nasonovia ribisnigriis
based onachemical factor inthe phloemsap.
Dit proefschrift
3.
The assumption that phloem sap is poor insecondary plant
compounds iscaused by ageneral lack of knowledge on
phloem sapcomposition.
4.
The ingestion rate of aphids during phloem feeding is much
lower thanthesap exudation rate from amputated stylets.
Therefore,the relation between aphid resistance and
exudation from amputated stylets in Medicago sativa as
suggested by Girousse &Bournoville ispremature.
Girousse,C.&R. Bournoville, 1994. Roleofphloem sapquality and
exudation characteristics on performance of pea aphidgrownon
lucerne genotypes. Entomol. Exp.Appl.70:227-235
5.
There are noscientific arguments to prefer the AC feeding
monitor to a DC-EPG amplifier for the recordingof
homopteranfeeding behaviour.
Reese,J.C.,Tjallingii, W.F., Helden,M.van& E. Prado,1994.
Waveform comparison amongAC and DCsystems for electronic
monitoring of aphidfeeding behaviour. In: E.A. Backus &G.Walker,
Homopteran feeding behaviour: Recent research advances andexperimentaltechniques. Special issues Ent.Soc.Am.:inpress)
6.
The results of experiments with aphids on artificial diet should
not be extrapolated directly to the situation onthe plant,
especially inview ofthe compartmentalisation of chemicals in
planttissues.
7.
Naturally occurring host-plant resistance is the most
widespread plant protection method and therefore, deserves
more research and plant breeding effort.
8.
Deeltijdwerken vergroot de efficiëntie per gewerkte
tijdseenheid.
9.
De beslissing om op een project een AIO dan wel een Postdoc aan te stellen wordt te vaak genomen op financiële
gronden.
10.
Een opgeruimd humeur is het halve werk.
11.
Veel wetenschappers zijn te eigenwijs om dat zelf toe te
geven.
12.
Het houden van exotische huisdieren dient rigoureus te
worden beperkt door de invoering van zogenaamde
positieflijsten die zowel voor handelaren als houders gelden.
13.
De toepassing van fonteinen en filters in tuinvijvers is uit
ecologisch oogpunt ongewenst.
14.
Het belang van een flinke lichamelijke inspanning voor de
geestelijke ontspanning wordt sterk onderschat.
Stellingen behorende bij het proefschrift "The resistance of lettuce
to the aphid Nasonovia ribisnigri " door Maarten van Helden.
Wageningen, woensdag 22 februari, 1995
CONTENTS
1. Introduction
1
2. Bionomics of N.ribisnigri
11
3. Tissue localisation of the resistance
21
4. Feeding behaviour of free N.ribisnigri
39
5. Phloem sapcollection
55
6. Phloem sap chemistry
77
7. The development of abioassay
97
8. Summary andConclusions
115
Samenvatting enconclusies
117
Publications
119
Nawoord
121
Curriculum Vitae
127
1. INTRODUCTION
APHID-PLANT INTERACTIONS
Aphidsarehighly specialisedphloemsapfeeders.Thisspecialisation on
the phloemsap has madeit very difficult tostudy thefeeding behaviour in
detail.What happens duringthe penetration ofthestylets throughthe
different plant tissues ontheir waytothesieve elements was unknown.The
only waytoobserve ingestion was bythe recording of honeydew excretion
(see Pollard,1973for a review). Knowledge ofthe events which occur during
the period betweenthe insertion ofthestylets inthe plant and ingestionfrom
a sieve element isessential to understand aphid-plant interactions.
The introduction ofthe "feeding monitor" by McLeanand Kinsey (1964,
1965) was a major breakthrough.Byconnectingthe aphidandthe plant in
anelectrical circuit it was possible to monitor electrical resistance fluctuations
during probing.Freddy Tjallingii greatly improvedthis methodwiththe
introduction ofthe Direct Current (DC) Electrical Penetration Graph (EPG)
amplifier (Tjallingii, 1978, 1985).The DC-EPG amplifier made it possible to
identify many more different waveforms, including so-called Electro Motive
Force (EMF) components.Through combination withvarious other
techniques (TEM,radioactive tracer studies,virustransmission,stylectomy
etc.) EPGwaveforms could becorrelated withaphidactivities,stylet location
and plant histology (Kimmins&Tjallingii, 1985;Spiller etal.,1990;Tjallingii,
1985, 1988, 1990, 1994;Tjallingii &Hogen Esch,1993;Tjallingii &Mayoral,
INTRODUCTION
1
1992; Prado &Tjallingii, 1994).Thiswork hasenriched our knowledge of
aphidfeeding behaviour enormously.
We now knowthat aphidstylets penetrate extracellular, viathe cellwalls.
Ittakes aconsiderable amount oftime,and often several different penetrations before asieve element is reachedforthe first time.The stylets
frequently puncture cells duringthe extracellular probingtothe phloem,but
the aphid retracts itsstylets after afewsecondstocontinuetheir extracellular
route until asieve element is reached.Salivation intothe sieve element
precedesthe actual ingestion of phloemsap.Often several sieve elements
are punctured before sustained ingestionoccurs.
Increased knowledge of theevents during feeding behaviour multipliedthe
number of questions. Understanding ofthe aphid-plant interactions during
stylet penetration andtheir role inhost plant acceptance or rejection is still
very poor. Doesthe aphidtaste extracellular fluidduring probing or doesit
ingest fluidduringthe cell punctures? Howdoaphids locate and recognise
the phloem sieve elements,isthisstrictly bytrialanderror or arethere plant
cuesto guidethe aphid?Why isthere astylet sheathexcreted during stylet
penetration andwhat isthe role of salivary enzymes? Why does probing
sometimes induce (hypersensitive) plant reactions and how couldthese
influencethe aphid? Howdoaphids avoid defence reactions like P-protein
gelation and callose formation duringtapping of the phloem sap?
Because ofthis shortage of fundamental knowledge, it isdifficult to
interpret the behaviour during probing.The behavioral sequencethat leadsto
plant acceptance appears complex and highly variable.The early theory of
aphids makingonly ashort test probeto decide onhost-plant suitability was
certainly anoversimplification. Especially on resistant varieties of normally
susceptible plant species aphids usually make numerous penetrations and
often reachthe phloem before differences inbehaviour appear (Caillaudet
al.,1992;Cole, 1993). Rejection is not alwaysstraightforward and,evenon
absolutely resistant plants,aphids cancontinue probing whiletheir
motivational state gradually shifts andfinally reaches apoint wherethe aphid
decides to leave the plant.On partially resistant plants changes in behaviour
2
CHAPTER 1
can besmall and difficult totrace inthe large natural variationthat occurs.
Aphid-plant interactions during host-plant selectionform adynamic process,
the result of plantsuitability, aphid motivation andstochastic variability,which
may vary intime.
RESISTANCE MECHANISMS
Several possible resistance mechanisms have been proposedforvarious
aphid-plant combinations, rangingfromvolatiles which deter alightingtotoxic
compounds inthe phloem.In most cases aphids seemto landat randomon
green/yellow surfaces (Moericke, 1962;Müller, 1964) and penetration is
necessary to evaluate plant resistance (Klingauf, 1972, 1987;Müller, 1962).
The mechanisms that play arole duringstylet penetration tothe phloem
might beeither chemical or mechanical.Achemical resistance mechanism
acting inthis stadium would implythat aphids do ingest fluid during
penetrationto betested bythe contact chemoreceptors inthe epipharyngeal
cavity (Wensler &Filshie, 1969;Tjallingii, 1985).Though ingestion during cell
punctures has never beenshown unequivocally, it probably occurs,aphids
can acquire viruses duringthese punctures (Powell, 1991). Mechanical
resistance mechanisms could lay inthetoughness of thetissue,especially of
the secondary cellwallthroughwhichthestylets penetrate. Dreyer and
Campbell (1984) suggestedthat a higher degree of polymerisation of the
pectin inwheat caused resistance tothe greenbug Schizaphisgraminum by
making penetration more difficult.
Once anaphid reaches aphloem sieve element and attempts to establish
ingestion, blocking ofthesieve element orthe aphids stylets by plant
defence reactions like P-protein gelation or callose formation can occur.
Aphidsalivation intothesieve element (Prado &Tjallingii, 1994) might
normally avoidthis.
Chemical composition ofthe phloem sap isvery important.This isthe
only foodof the aphidandtherefore itshould contain allthe dietary
INTRODUCTION
requirements ofthe aphid.Phloemsap is rich insugars andcontains many
different amino acids,organic acids andvitamins. Sophloem sapseemsto
contain everything neededfor theaphid's metabolism andsome additional
essential compounds aresynthesized bysymbionts (Douglas, 1990;Houk&
Griffiths, 1980).
Apart fromthe nutritional aspectsthechemicals inthe phloemsap
influencethe acceptance ofthe plant bytheir gustatory properties. Sucrose
andsome amino acids arefeedingstimulants whilethestrongfeeding
stimulancy of other aminoacids canbeshownonly incombination with
sugars (Mittler & Dadd,1964).
Many secondary plant metabolites canalso play animportant role inhost
plant acceptability. Phenolic acids,hydroxamic acids,alkaloids and many
other compounds show feeding deterrent action inartificial diet experiments
(Nault &Styer, 1972;Schoonhoven & Derksen-Koppers, 1976;Argondonaet
al.,1980;Herrbach,1985;Mittler, 1988;Harrewijn,1990;Niemeyer, 1990
and many others).The artificial diet assays usedforthese experiments are
very different from the natural situation,especially inthe physical aspects
(pressure,compartmentation).Therefore,the effect of acompound inan
artificial diet cannot beextrapolated directly tothe natural situation.Some
plant specific secondary compounds stimulate feeding of monophagous
species while deterring others (Klingauf etal.,1972;Nault &Styer, 1972),
but the preference of anaphidfor acertain plant species is usually
unexplained. Host plant recognition of mono-andoligophagous species is
expectedto bebased onsecondary compounds rather than ondifferences in
primary nutrients.
Phloem sap isthought to be poor insecondary plant chemicals.
Knowledge about the role of secondary compounds inaphid-plant relations is
very limited (Herrbach,1985;Wink &Witte, 1991; Cole, 1993).Whether the
compounds whichshow allomonal effects in vitro are present inthe phloem
sap or inother plant compartments sampled bytheaphid during penetration,
and inconcentrations highenoughto affect the aphid,is usually unknown.
Often correlations betweenthe concentration of acertain compound inwhole
4
CHAPTER 1
plant extracts andplant resistance werethought to represent causal
relationships (Todd etal.,1971;Argandona, 1980;Dreyer &Jones, 1981;
Herrbach, 1985;Leszczynski etal.,1985;Niemeyer 1990;Kanehisha etal.,
1990; Luczak &Gaweda, 1993),evenwhile it isinsome cases unlikely that
these compounds are present inthe phloemsap.
Research inthis field ishampered by problems of phloem sapcollection,
which is necessary to allow conclusions aboutthe biological activity of its
compounds. Differences inphloem sapcomposition will not show inwhole
plant analysis since phloem sap isonly avery smallfraction ofthetotal
plant.
THE MODEL SYSTEM
The aphid-plant combination chosenforthis work consisted ofthe aphid
Nasonovia ribisnigri(Mosley) and lettuce (Lactuca sativaL.) onwhichthis
aphid isa major pest inlarge parts of theworld (Forbes &Mackenzie,1982;
MacKenzie &Vernon, 1988; Reinink &Dieleman, 1992). Chemical controlis
difficult because aphids settle insidethe headofthe lettuce wherethey
cannot be reached by insecticides. Frequent spraying (oftentwice weekly) is
necessary for effective control.Aphidfeeding deforms plants,aphidscan
transmit viruses,and- most important -the presence of aphids in lettuce
makes it unmarketable. Plant resistance could beagood alternative for
chemical control providedthat the level of resistance isvery highor even
absolute,the plant contains resistance against all important aphidspecies,
andthe resistance isdurable.
Duringthe last 25 yearsabreeding program has been performed atthe
CPRO-DLOto increase aphid resistance inlettuce (Eenink etal.,1982a,b;
Eenink &Dieleman, 1982;Reinink & Dieleman 1990, 1992).Complete
resistanceto N.ribisnigriwasfound inLactucavirosa L, awild lettuce
species.This resistance wastransferredto L.sativavia interspecific crosses
using Lactuca serriolaL. asabridge.The resistance is basedonasingle
INTRODUCTION
5
dominant gene (Nr gene, Eenink etal.,1982a,b).The resistance was not
effective against other aphid species like Myzuspersicae(Sulzer) or
Macrosiphum euphorbiae (Thomas). Breeding iscontinuingto improve
resistanceto other species (Reinink &Dieleman, 1990).Sofar, fieldtrials
have not shown any signof breaking ofthe resistance by N. ribisnigri
(Reinink & Dieleman,1992).Lettuce lineswiththe Nr-gene are usedby
seed-breeding companies tryingtodevelop commercial lines withaphid
resistance. Segregation,occurring after several generations of inbreeding,
producedsetsof near isogenic resistant (genotype NrNr) and susceptible
lines (nrnr) asaby-product.These sets are usedfor comparison of the aphid
feeding behaviour andchemistry.
This modelsystem hasseveral important characteristics.The absolute
resistance isaguarantee forclear differences inbehaviour ofthe aphids on
these lines,andtherefore probably also between lettuce lines.The
availability of (near) isogenic lineswithandwithout monogenic resistance is
animportant advantage,sincethe riskof differences between resistant and
susceptible plants which are not relatedto resistance is reducedtoa
minimum.Simultaneously the genetic differences areassmall aspossible,
reducingthe riskthat differences relatedto resistance areso largethat
identification of asingle factor isimpossible. Still, it isimportant to realise
that statements about the isogenicity areonly based on breeding
experiments and notona moreelaborate genetic analysis,so genetic
difference will besomewhere betweenone set of chromosomes (out of9)
and one gene,depending onthe amount of crossing-over.
OBJECTIVES
The main goals of the research project described inthis thesis wasthe
identification ofthe resistance mechanism to Nasonovia ribisnigriinlettuce.
This includedthe localisation of the resistance inthe plant,the effect of the
resistance onthe aphid's feeding behaviour, and-if possible-the
CHAPTER 1
identification ofthe responsible chemical compounds (allomones).When
successful,the results couldbe usedtoexplain whythis resistance is not
effective against other species like Myzuspersicae or Macrosiphum
euphorbiae.
Identification of the mechanism couldalso enable predictions about the
durability ofthe resistance, important for possible commercial useofthe
resistance gene.
EXPERIMENTS
The work described inthisthesis started in 1988.The project proposal on
which it was originally basedwas entitled "The role of secondary plant
substances inthe resistance of lettuceto aphids".The plant lineswere newly
developed inthe breeding program of aseed-breeding company.Therefore,
the study started withabasic characterisation of the resistance using aphid
developmental parameters (Chapter 2).After this Electrical Penetration
Graphs were usedto localise the resistance inthe plant (Chapter 3).Since
aphids are connectedto awire during EPG recording,they arevery
restricted intheir movements. Inchapter 4wecompared EPG recordings
withvisual observations ofthe behaviour of freely moving (not wired) aphids.
Chapter 5describes our efforts to collect phloemsapfrom lettuce plants
usingdifferent methods,with some unexpected results.The phloem sap
samples were chemically compared forsugars,amino acids,proteins and
secondary compounds inchapter 6. Chapter 7finally describesthe
development of a bioassay based onphloemsap.
REFERENCES
Argondofia, V.H., J.G. Luza, H.M. Niemeyer
& L.J. Corcuera, 1980.Role of Hydroxamic
acids in the resistance of cereals to aphids.
INTRODUCTION
Phytochemistry 19: 1665-1668
Caillaud, CM., J.P. Dipietro, B. Chaubet &
CA. Dedryver, 1992. Feeding behaviour of
the aphid Sitobion avenue on resistant and
susceptible wheat. Proc. 8th International
Symposium on Insect-Plant Relationships,
Kluwer academic publishers, Wageningen,
The Netherlands: 295-296
Cole, R.A. 1993.Identification of a resistance
factor in the brassica species Brassica
/maculosa and B. spinescens to the cabbage
aphid Brevicorynebrassicae.Entomol. exp.
appl. 71:23-32
Douglas, A.E. 1990.Nutritional interactions
between Myzuspersicae and its symbionts.
In: R.K. Campbell & R.D. Eikenbary (eds.),
AphidPlant Genotype Interactions, Elsevier
Science Publishers B.V. Amsterdam
Dreyer, D.L. & B.C. Campbell, 1984.
Association of the degree of methylation of
intercellular pectin with plant resistance to
aphids and with induction of aphid biotypes.
Experientia 40: 224-226
Dreyer, D.L. & K.C. Jones, 1981.Feeding
deterrency of flavonoids and related
phenolics towards Schizaphisgraminumand
Myzuspersicae: aphid feeding deterrents in
wheat. Phytochemistry 20: 2489-2493
Eenink, A.H., R. Groenwold &F.L.
Dieleman, 1982a. Resistance of lettuce
{Lactuca)to the leaf aphid Nasonovia
ribis-nigri 1:transfer of resistance from L.
virosato L. sativa by interspecific crosses
and selection of resistant breeding lines.
Euphytica 31:291-300
Eenink, A.H., R. Groenwold, &F.L.
Dieleman, 1982b.Resistance of lettuce
{Lactuca)to the leaf aphid Nasonovia
ribis-nigri2: Inheritance of the resistance.
Euphytica 31: 301-304
Eenink, A.H. & F.L. Dieleman, 1982.
Resistance of Lactuca accessions to leaf
aphids: components of resistance and
exploitation of wild Lactuca species as
sources of resistance. Proc. 5th. Symp.
Insect-Plant Relationships Pudoc
Wageningen
Forbes, A.R. &J.R. Mackenzie, 1982.The
lettuce aphid, Nasonoviaribisnigri
(Homoptera: Aphididae) damaging lettuce
crops in British Columbia. J. Entomol. Soc.
Brit. Columbia 79: 28-31
Harrewijn, P., 1990.Resistance mechanisms
of plant genotypes to various aphid species.
In: R.K. Campbell & R.D. Eikenbary (eds.),
Aphid-Plant Genotype interactions, Elsevier
Science Publishers B.V., Amsterdam
Herrbach, E. 1985.Rôle des sémiochimiques
dans les relations pucerons-plantes. II.Les
substances allélochimiques. Agronomie 5:
375-384
Houk, E.J.& G.W. Griffiths, 1980.
Intracellular symbiotes of the Homoptera.
Annu. Rev. Entomol. 25: 161-187
Kanehisa, K., H. Tsumuki, K. Kawada &
M.A. Rustamani, 1990.Relations of
gramine contents and aphid populations on
barley lines. Appl. Ent. Zool. 25:251-259.
Kimmins, F.M. & W.F. Tjallingii, 1985.
Ultrastructure of sieve element penetration
by aphid stylets during electrical recording.
Entomol. exp. appl. 39: 135-141.
Klingauf, F., 1972. Die Bedeutung von
peripher vorliegenden Pflanzensubstanzen
für die Wirtswahl von phloemsaugende
Blattläusen (Aphididae). Zeitschrift für
Pflanzenkrankheiten 79:471-477.
Klingauf, F., 1987. Host plant finding and
acceptance. In: Minks, A.K. & P. Harrewijn
(eds.): Aphids & their biology, natural
enemies and control. World Crop Pests 2a:
209-223. Elsevier Science Publishers B.V.,
Amsterdam
Klingauf, F., C. Sengonca, & H. Bennewitz,
1972. Einfluss von Sinigrin auf die
Nahrungsaufnahme polyphager und
oligophager Blattlausarten (Aphididae).
Oecologia (Bert.) 9: 53-57
Leszczynski, B.,J. Warchol & S. Niraz, 1985.
The influence of phenolic compounds on the
preference of winter wheat cultivars by
cereal aphids. Insect Sei. Applic. 6: 157-158
Luczak, I. & M. Gaweda, 1992. The
relationship between the chemical
composition of sugar beet leaves and the
development of the black bean aphid Aphis
fabae. IOBC/WPRS Bulletin (Working
group "Breeding for resistance to insects
and mites") 16: 178-183
CHAPTER 1
Mackenzie, J.R. &R.S. Vernon, 1988.
Sampling for distribution of the lettuce
aphid, Nasonoviaribisnigri
(Homoptera:Aphididae), in fields and within
heads. J. Entomol. Soc. Brit. Columbia 85:
10-15
McLean, D.L. &M.G. Kinsey, 1964. A
technique for electronically recording aphid
feeding and salivation. Nature 202:
1358-1359
McLean, D.L. & M.G. Kinsey, 1965.
Identification of electrically recorded curve
patterns associated with aphid salivation and
ingestion. Nature 205: 1130-1131
Mittler, T.E. 1988.Applications of artificial
feeding techniques for aphids. In: A.K.
Minks and P. Harrewijn (Eds.). Aphids,
their biology, natural enemies and control.
Elseviers Science Publishers B.V.,
Amsterdam
Mittler, T.E. & R.H. Dadd, 1964. Gustatory
discrimination between liquids by the aphid
Myzuspersicae (Sulzer). Entomol. exp.
appl. 7: 315-328
Moericke, V. 1962. Über die optische
Orientierung von Blattläusen. Z. angew. Ent.
50: 70-74
Müller, HJ. 1962. Über die Ursachen der
unterschiedlichen resistenz von Viciafaba 1.
gegenüber die bonnen blattlaus,Aphis
(Doralis)fabae Scopp. VIII. Das Verhalten
geflügelter Bohnenläuse nach der landung
auf wirten und nichtwirten. Entomol. exp.
appl. 5: 189-210.
Müller, HJ. 1964. Über die Anflugdichte von
Aphiden auf farbige Salatpflanzen. Entomol.
exp. appl. 7: 85-104
Nault, L.R. & W.E. Styer, 1972. Effects of
sinigrin on host selection by aphids.
Entomol. exp. appl. 15:423-437
Niemeyer, H.M., 1990.The role of secondary
plant Compounds in aphid-host interactions.
In: R.K. Campbell & R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam, pp.
187-205.
Pollard, D.G., 1973.Plant penetration by
feeding aphids (Hemiptera, Aphidoidae): a
INTRODUCTION
review. Bull, entomol. Res. 62: 631-714
Powell, G., 1991.Cell membrane punctures
during epidermal penetrations by aphids:
consequences for the transmission of two
potyviruses. Ann. appl. Biol., 119, 313-321
Prado, E.B. & W.F.Tjallingii, 1994. Aphid
activities during sieve element punctures.
Entomol. exp. appl.72: 157-165
Reinink, K & F.L. Dieleman, 1990.
Inheritance of resistance in lettuce to leaf
aphids. IOBC/WPRS Bulletin (Working
group "Breeding for resistance to insects
and mites") 13:65-71
Reinink, K & F.L. Dieleman, 1992. Survey of
aphid species on lettuce. IOBC/WPRS
Bulletin (Working group "Breeding for
resistance to insects and mites") 16:56-68
Schoonhoven, L.M. & I. Derksen-Koppers,
1976. Effects of some allelochemicals on
food uptake and survival of a polyphagous
aphid,Myzuspersicae. Entomol. exp. appl.
19: 52-56
Spiller, N.J., Koenders, L. & W.F. Tjallingii,
1990. Xylem ingestion by aphids -a
strategy for maintaining water balance.
Entomol. exp. appl. 55: 101-104.
Tjallingii, W.F., 1978.Electronic recording of
penetration behaviour by aphids. Entomol.
exp. appl. 24: 521-530.
Tjallingii, W.F., 1985. Stylet penetration
activities by aphids. Thesis Wageningen
Agricultural University, Wageningen, The
Netherlands.
Tjallingii, W.F., 1988.Electrical recording of
stylet penetration activities. In: A.K. Minks
& P. Harrewijn (eds.), Aphids, Their
Biology, Natural Enemies and Control, vol.
2B, Elsevier Science Publishers B.V.
Amsterdam, pp.95-108.
Tjallingii, W.F., 1990. Continuous recording
of stylet penetration activities by aphids. In:
R.K. Campbell &R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam.: 8999.
Tjallingii, W.F., 1994. Sieve element
acceptance by aphids. Eur. J. Entomol.91:
47-52.
Tjallingii, W.F. &Th. Hogen Esch, 1993.
Fine structure of the stylet route in plant
tissue by some aphids.Physiol. Entomol.
18: 317-328
Tjallingii, W.F. &A. Mayoral, 1992. Criteria
for host plant acceptance by aphids. Proc.
8th Int. Symp. Insect-Plant Relationships.
Kluwer acad. publ. Dordrecht, The
Netherlands, pp 280-282
Todd, G.W., A. Getahun, A. & G.C. Cress,
1971. Resistance in barley to the greenbug
Schizaphis graminum. 1.Toxicity of
phenolic and flavonoid compounds and
related substances. Ann. Ent. Soc. Amer. 64:
718-722
Wensler, R.J. & B.K. Filshie, 1969. Gustatory
sense organs in the food canal of aphids. J.
Morph. 129:473-492.
Wink, M. & L. Witte, 1991.Storage of
quinolizidine alkaloids inMacrosiphum
albifrons and Aphis ganistae (Homoptera:
Aphididae). Ent. Gener. 15:237-254
10
CHAPTER 1
2. BIONOMICS OF
N. RIBISNIGRI
ABSTRACT
The intrinsic rateof increase (revalues), mean relative growth ratesand
mortality of Nasonovia ribisnigri(Mosley) (Homoptera,Aphididae) on different
lines of lettuce (Lactuca sativaL.) were determined.Near isogenic resistant
and susceptible lines,plustheir ancestors,were used.Bionomics of N.
ribisnigrionthe resistant lines (NrNr) withthe dominant Nasonovia resistance
gene (Nr-gene) differed clearly fromthesusceptible lines (nmr). Mortality was
high, nolarvae reachedadulthood,and no reproduction nor honeydew
production was seen onthe resistant lines.Transfer of aphidsto susceptible
plants after aperiod of 2days onthe resistant linesshowed nosignsof
intoxication of aphids.Apparently there is nofeedingonthe resistant lines.It
is not clear whether the aphids can not reachthe phloem or do not accept it
onthe resistant line.
INTRODUCTION
Aphids area major problem inlettuce growing.Aphids transmit viruses
and aphid-infested headsare unmarketable.The aphid N.ribisnigrioccurs
frequently andcauses severe problems inWestern Europe and Canada
(Eenink era/, 1982a, 1982b, Forbes &Mackenzie, 1982).
BIONOMICS OF N. RIBISNIGRI
ÏT
During abreeding program atthe Institute for Horticultural Plant Breeding
(IVT) absolute resistanceto N.ribisnigriwastransferred fromthewildL.
virosaXo the cultivated L.sativa(Eenink etal.,1982a,Eenink &Dieleman,
1982a).This resistance isbasedonasingle dominant gene (Nrgene,
Eenink &Dieleman,1989;Eenink etal.,1982b) and isextremely effective
against N.ribisnigribut it gives little or noprotection against other important
aphidspecies suchas Myzuspersicae andMacrosiphum euphorbiae
(Reinink & Dieleman,1989).Breeding companies arecurrently developing
commercial lineswiththis resistance gene. Inthis studythe effect ofthe
resistance gene ondevelopmental parameters of N.ribisnigriwas
determined. Future experiments ontheaphid resistance will focus on its
localization inthe plant andidentification of the mechanism (chemical or
mechanical).
MATERIALS AND METHODS
All experiments and rearing of aphids were conducted in agreenhouse at
20± 2°C, r.h. 70%and L16:D8photoperiod (extra light provided by SONT
high pressure sodium lamps).Seedswere sown inastandardized soil
mixture intrays andtransferred to 15cmplastic pots (1.35 I) inthe 2-3 leaf
stage. Plantsfor experiments were about fiveweeks oldand hadabout 6
full-grown leaves. Forthetransfer experiment 4week oldplants (3-4fullgrown leaves) were usedfrom asoilless culture system asdescribedby
Harrewijn & Dieleman (1984) inagreenhouse at 18± 1°C,r.h. 70%,L16:D8
photoperiod.
Plantgenotypes: Three susceptible lines (A,B,C,genotypes nrnr) andone
resistant line 411 (genotype NrNr) were used inabackcross breeding
program to produce acommercial line.Pedigree:
[F6((((F3((C*F1(B*411)))*A))))]. Segregation inthe F6generation resultedin
two near isogenic lines:RES(NrNr) and SUS (nrnr).The susceptible cultivar
"Taiwan" (nrnr) was usedasacontrol.These seven lines were used inour
experiments.
12
CHAPTER 2
Aphids:Mass rearing of anorange biotype of N.ribisnigri(WNr1)was performed onlettuce plants of cv.Taiwan.Neonate (<24 h) larvae were selected
by placing adultvirginoparae (alates) inclip-on cages on lettuce plants orin
plastic containers onexcisedleavesfor 24 hoursafter whichadults were
removed andlarvaewere usedfor experiments.
MeanRelative GrowthRate(MRGR): Fiveyoung larvae (<24 h,total weight
250±19u.g)were placedina2.5 cmdiameter clip-on cage.After 8daysthe
aphids were countedandweighed.MRGR wascalculated as {Ln(mean
weight per aphid at day 8) - Ln(meanweight peraphidat day 1)}/8(Kogan,
1986). Four replicates of arandomized block designwiththe seven lettuce
lines andfive plants (=blocks) per line were performed.Oneclip-oncage
was usedper plant resulting in25aphids per line per replicate and4x25=
100aphids per line intotal.
Intrinsic rateofincrease andlarvalmortality:
Experimentaldesign: Six replicates were performed withthe seven lines in
a randomized block designwithone plant as ablock.There werefive (repl.
1-4) orseven (repl.5and6) plants per line ineach replicate.
Larvaldevelopment time(D)andmortality (LJ: Fiveyoung larvae (age<
24 h) were placed ina2.5 cmdiameter clip-on cage.Two clip-on cageswere
used per plant. Eachday the number of surviving larvae and adultswas
determined and adults were removed.Mortafity per plant wascalculatedas
the fraction of initial number of larvae (10)that moultedto adults:(10-totalnr.
of adults)/10. Results were basedonatotal of 34 plants and 340aphids per
line.
Reproduction (RJ:Two adults from the larval development time and
mortality experiment were used.They were placed individually in clip-on
cages on newplants of thesamegenotype. Fortests onthe resistant lines,
where all larvae died,adultvirginoparae from cv.Taiwanwere usedto
provide aphids in replicates 1to 4. Every secondday the offspring was
counted and removedto avoidcrowding effects. Rd wasthetotal numberof
larvae produced inaperiodequaltothe larval development time D(Dis
depending of lettuce line asdetermined in larval development time and
mortality experiment). Estimates ofthe number produced duringthetime
BIONOMICS OF N. RIBISNIGRI
13
betweentwo observations were made by linear interpolation. Reproduction
was basedon68virginoparae per lineonatotal of 34plants.
Calculation ofintrinsic rateofincrease (rj: Theformula developedby
Wyatt &White (1977) was usedtocalculatethe intrinsic rate of increase
(Birch, 1948) onthe susceptible lines:rm =0.738 * Ln(Rd)/D.
Honeydewproduction: 30 Larvae (age96±12 h)were placed individually in
honeydew production cages asdescribed by Eenink etal.(1984) on plantsof
the lines RESand SUS.The number of honeydew droplets was counted
every 24 hoursduring 3days.
Transferexperiment. Onday 0, larvae reared onTaiwan (age 96±12 h,mean
weight 163±32u.g)wereweighed and placedindividually in 1cmclip-on
cages onsmall plants (4weeks old,3-4 full grown leaves) of the lines SUS
and RES (50larvae per line).After 2daysthey wereweighed andtransferredto plants of the other genotype.At day 4they were weighedagain.
The larvae from RESweretransferredto SUS andat day 6they were all
weighed forthe last time (resultingtreatments RES->SUS->SUS and
SUS->RES->SUS). Asacontrol 10aphids per line were nottransferred to
the other genotype andweighed only at day 4of the experiment (continuous
treatments RES^,,,and SUSconl) orweighed at day 2and 4and placed back
onthe same plant but onanew leaf (nottransferred treatments RESntand
SUSJ. Weights onday 6were either not detectable because all adults died
(onthe resistant line) or not determined because they had moultedto adults
(onthe susceptible line).
Statistics: Inall experiments one plant represented one block.All results
werefirst calculated per plant. Means andstandard deviations werethen
calculated over all plants.Kruskal-Wallis tests with multiple comparison (a<
0.05) (Conover, 1980) were usedto comparethe linesfor each parameter.
Forthe MRGR experiment Tukey tests with multiple range analysis were
usedto show differences betweenlines.
14
CHAPTER 2
RESULTS
The results of all developmental parameters are listed inTable 1.
MRGR: Theweight growth (MRGR) ofthe aphids onthe resistant lineswas
only half oftheweight growthonthe susceptible lines.There were no
significant differences amongthesusceptible lines.The results forthe resistant lineswere basedononly very few surviving aphids (3on411and8on
RES).
Larvaldevelopment time(D)andmortalityLm:The larval development time
onthe susceptible (nrnr) lines rangedfrom 8.20 (A)to 8.44 (SUS) days
(Table 1).These minor differences were not statistically significant. Onthe
resistant lines (NrNr) only 7(line 411) or 3(Line RES) out of 340 larvae
reached adulthood,therefore Dcould not bedetermined.The mortality
rangedfrom 15.15%(LineA)to 30.47%(Taiwan) onthe susceptible lines,
andwas always >99%onthe resistant lines.
Reproduction: Rd values onthesusceptible lines did not differ significantly.
Onthe resistant lines (RESand411) very few larvae were produced during
replicates 1to 4whenvirginoparae of Taiwanwere usedfor the reproduction
experiment to make uptheshortage of aphids developed onthese lines.In
replicates 5and6 no larvae were produced. Usually the adults diedwithin4
days onthe resistant lines.
Intrinsic rateofincrease: Therm showedsmall but significant differences
amongthe susceptible lines. Forthe resistant lines it was not possible to
calculatethe revalues.
Honeydewproduction: Essentially no honeydew was produced onthe
resistant line RES;onthesusceptible line SUS 7-8 droplets per aphidper
day were produced (Table2).
Transferexperiment. The results of this experiment showed a complete
absence of weight gainandeven asmall decrease inweight, onthe resistant
BIONOMICS OF N. RIBISNIGRI
15
Table 1. Bionomics of Nasonoviaribisnigri on
Line
A
B
C
411
RES
SUS
Taiwan
Genotype
MRGR
D
nrnr
nrnr
nrnr
NrNr
NrNr
nrnr
nrnr
.365*
.358*
.380*
.181"
.164"
.364*
.389*
8.20*
8.42*
8.28*
N.D.
N.D.
8.44'
8.39'
seven different lettuce lines
Rd
36.06"
34.41*"
37.14 *
3.85 d
0.97 d
32.79*
39.35 c
rm
L*
0.310"
15.15*
0.297*"
17.35*
0.307'"
17.50*
N.D.
99.36 c
N.D.
99.85 c
0.296'
18.52*
0.310"
30.47"
MRGR = Mean Relative Growth Rate, D = Larval Development Time (days), Rd = Mean
reproduction in time eq. D (nr),rm = Intrinsic rate of increase (formula by Wyatt and
White, 1977)(/day), L,,,= Larval mortality in percentage. N.D. = Not determinable. Values
that have no letter in common are significantly different (Kruskal-Wallis test with multiple
comparison a < 0.05, Conover, 1980).
lines (Fig. 1).After 2days onthe resistant line andtransfer tothe susceptible
linetheweight growthwas resumedatthesame rate (parallel lines) asfound
onthe susceptible line (Fig.1),resulting inequal meanweights of 285u.g on
day four forthetreatments RES->SUS->SUS and SUS->RES->SUS.This
weight wascomparabletothe weight ofthetreatments SUSnt and
SUS->RES->SUS ondaytwo.
DISCUSSION AND CONCLUSIONS
Differences between resistantandsusceptible lines:Forall parameters the
performance onthe resistant lineswas muchlessthan onthe susceptible
lines. Inadditionthe MRGRvalues onthe resistant lines (Table 1)were
overestimated becausethey were basedonly onthesurviving aphids
whereas 94%ofthe aphids died .Over ashorter periodwhen no mortality
was observedthe weight gain waszero (seetransfer experiment, Fig1.).
However someaphids didgrow andapparently reached adulthood onthe
resistant lines.
16
CHAPTER 2
Table 2. Honeydew production ofN.ribisnigrion RES and SUS during 24 hours (Mean
standard error, N=30)
Line
SUS
RES
# droplets on day:
1
7.41 ± 1.08
0.04 ± 0.04
2
8.28 ± 1.27
0.05 ± 0.05
3
7.11 ± 1.11
not detected
Onthesusceptible linesthere were significant differences forthe Rd, rm
and Ld,but these differences were notvery large.Onlythe mortality was
clearly higher onTaiwan compared tothe other susceptible lines.Nevertheless,the smalldifferences amongthesusceptible lines appear negligible in
comparisontothe differences betweensusceptible and resistant lines.
The resistant linesshowed nosignificant differences.The resistance
appearedto be basedexclusively onthe Nrgene.Noloss ofthe resistance
occurred duringitstransfer from the411parent linetothe resistant line RES.
Noevidence of any change inthe resistance duringthistransfer wasfound.
The absence of honeydew onthe resistant line RESindicatedthat there
was acomplete absence of uptake of phloem sap or any other plant fluidon
this line.
Inthetransfer experiment atotal absence of weight gain onthe resistant
lines wasfound,indicatingthat there was either nofood uptake onthese
lines and/or very short termtoxic effects.The immediate resumptionof
growth onthe susceptible linesshowedthat therewas no lasting intoxication
onthe resistant line.
The results showthat the resistance isprobably based on atotal absence
of feeding onthe resistant line.The complete absence of weight increase
and honeydew production indicatesthat there is nofood uptake onthe
resistant lines ratherthanapoorfoodquality or atoxic effect of the ingested
food inwhichcasea reducedweight gainand honeydew would have been
present.The course ofthe mortality ofthe aphids onthe resistant lines is
comparable tothe mortality inapetri dishwith some moist cottonwool inthe
total absence of food (unpublished results) andsimilar tothe resultsof
BIONOMICS OF N. RIBISNIGRI
17
800
SUS->RES->SUS
•
RES->SUS->SUS
•--•--
700 -
SUScont
600
SUSnt
- —o—.
REScont
—•&•—
500
RESnt
-A
E
ca
O)
o
k_
p
o
"E
400
g>
300 -
200
U
100 "••
1
dead
r
3
4
Day number
r
6
Figure 1.Weight of N. ribisnigrion resistant and susceptible lettuce lines with transfers
from one genotype to the other at two-day intervals. For explanation of treatments see
Materials and Methods. Aphids of RESnl and REScomdied after 3 to 4 days. Points
represent mean ± standard errors.
18
CHAPTER 2
Eenink &Dieleman (1982b) whoworkedwith adifferent lettuce carryingthe
same resistance gene.Atoxic effect would also have influencedthe growth
after transfer for acertain period.Although nearly all aphids diedonthe
resistant lines,thosethat survivedfor alonger periodshowedsome growth
(MRGR experiment) suggestingthat feeding is possible but stronglysuppressed. N.ribisnigriisaspecialist withonly Lactuca species andsome
related compositeaeassecondary hosts.Thereforethe presence of a
feeding deterrent or absence of afeeding stimulant are possible resistance
mechanisms,interferingwith hostplant discrimination inthismonophagous
species and not giving resistance against other more polyphagous species
like M. persicae or M.euphorbiaewhich are not influenced bythe resistance
(Reinink & Dieleman,1989).Other mechanisms likefoodquality ormechanical resistance of the leaf during penetration probably would effect all species
to a certain degree. However, it is notyet clear whether the absenceof
feeding isdueto problems locating or reachingthe phloem-vessels onthe
resistant lineordueto problemsfeedingonoracceptingthe phloem.Thisis
investigated inastudy ofthe Electrical Penetration Graphs (EPG's) on both
the resistant andsusceptible line (van Helden, 1990;van Helden &Tjallingii,
1990).Behavioral studies and EPG recording will give more informationof
the possible nature and location of this resistance mechanism.
REFERENCES
Birch, L.C., 1948.The intrinsic rate of natural
increase of an insect population. Journal of
Animal Ecol. 17: 15-26.
Conover, W.J., 1980.Practical nonparametric
statistics. Wiley, New York. 493pp.
Eenink, A.H. & F.L. Dieleman, 1982a. Resistance of Lactuca accessions to leaf aphids:
components of resistance and exploitation of
wild Lactuca species as sources of resistance. Proc. 5th. Symp. Insect-Plant Relationships, Pudoc Wageningen, The Netherlands.
Eenink, A.H. & F.L. Dieleman, 1982b.Resistance of Lettuce to leaf aphids:Research on
components of resistance, on differential
BIONOMICS OF N. RIBISNIGRI
interactions between plant genotypes and the
environment, aphid genotypes and the environment and on the resistance level in the
field after natural infestation. Med. Fac.
Landbouww. Rijksuniv. Gent 47/2: 607-615
Eenink, A.H. &F.L. Dieleman, 1989. Comparison of sources of resistance to leaf
aphids in lettuce (LactucasalivaL.).
Euphytica 40: 21-29.
Eenink, A.H., F.L. Dieleman, R. Groenwold,
P. Aarts & B. Clerkx, 1984. An instant
bioassay for resistance of Lettuce to the leaf
aphid Myzuspersicae. Euphytica 33:825831.
Eenink, A.H., R. Groenwold & F.L.
19
Dieleman, 1982a. Resistance of lettuce
(Lactuca)to the leaf aphid Nasonovia
ribis-nigri 1:transfer of resistance from
L.virosa to L.sativa by interspecific crosses
and selection of resistant breeding lines.
Euphytica 31: 291-300.
Eenink, A.H., R. Groenwold & F.L.
Dieleman, 1982b.Resistance of lettuce
(Lactuca)to the leaf aphidNasonovia
ribis-nigri2: Inheritance of the resistance.
Euphytica 31: 301-304.
Forbes, A.R. &J.R. Mackenzie, 1982.The
lettuce aphid Nasonovia ribisnigri
(Homoptera Aphididae) damaging Lettuce
crops in the Goverdale area of British Columbia. J. Entomol. Soc. Brit Columbia 79:
28-31
Harrewijn, P. & F.L. Dieleman, 1984. The
importance of mineral nutrition of the host
plant in resistance breeding to aphids. Proc.
Vllh int. Congr. on soilless culture: 235-244
Helden, M. van, 1990. Resistance of lettuce to
the aphid Nasonovia ribisnigri:Are electrical penetration graphs (EPGs) helpful to
find the origin of resistance. Proceedings
Vil"1Int Symp. Insect-Plant Interactions.
Budapest Hungary 1988. Symp. Biol. Hung.
39: 473-474.
Helden, M. van & W.F.Tjallingii, 1990.
Electrical penetration graphs of the aphid
Nasonovia ribisnigrion resistant and susceptible lettuce (Lactucasaliva). Proceedings Int. Symp. Aphid - Plant Interactions:
Population to Molecules. Stillwater
Oklahoma U.S.A. 1990: 308
Kogan, M., 1986. Bioassays for measuring
quality of insect food. In: J.R. Miller &
T.A. Miller (Eds.) Insect plant Interactions,
Springer Verlag, New York.: 155-189
Reinink, K. & F.L. Dieleman, 1989. Comparison of sources of resistance to leaf aphids in
lettuce (L. sativa L.). Euphytica 40: 21-29
Wyatt, I.J. & P.F. White, 1977. Simple estimation of intrinsic increase rates for aphids
and tetranychid mites. J. Appl. Ecol. 14:
757-766.
20
CHAPTER 2
3. TISSUELOCALISATION
OF THERESISTANCE
ABSTRACT
Electrical penetration graphs (EPG's) of Nasonovia ribisnigri(Mosley)
(Homoptera,Aphididae) onresistant andsusceptible lettuce (Lactuca sativa,
Compositae) showed alarge reduction inthe duration of thefood uptake
pattern (E2) onthe resistant line. Nodifferences in EPG's were observed
beforethe phloemwas reached.Therefore, resistance is believedtobe
located inthe phloemvessel.Both mechanical blocking of the sieve element
after puncturing andadifference incomposition of the phloem sap are
possible resistance factors. However, achemical factor seems more likely
because ofthespecificity of the resistance against N. ribisnigri.
INTRODUCTION
Absolute resistance to N.ribisnigriwastransferred from Lactucavirosato
L.sativa(Eenink etal.,1982b).The resistance isbased onasingle
dominant gene (Nr gene) (Eenink & Dieleman, 1982;Eenink etal., 1982a;
Reinink &Dieleman, 1989). Herewestudiedthe resistance usingthe direct
current electrical penetrationgraph (DC-EPG) technique asdevelopedby
Tjallingii (1978, 1985, 1988).Thistechnique is especially suitable for studying
possible resistance mechanisms because it provides information about both
TISSUE LOCALISATION OF THE RESISTANCE
2?
the behaviour ofthe aphidandthestylet tip positions inthe plant during
penetration (Montllor &Tjallingii, 1989;Niemeyer, 1990;Spiller era/., 1990;
Tjallingii, 1978, 1985, 1988,1990).Table 1provides asummary of the
different waveform patterns whichcanbedistinguished inthe EPG (modified
after Tjallingii, 1990). Electrical Penetration graphs of Nasonoviaribisnigrion
a resistant andasusceptible lettuce (L.sativa)linewere compared.These
lineswere isogenic except forthe resistance gene (Van Helden&Tjallingii,
1990).Thefrequency of occurrence, meanandtotal duration ofthe
waveform patterns,their sequence andother derivedparameters were
comparedto get information aboutthe possibletissue location ofthe
resistance.
MATERIALS AND METHODS
Aphidsandplants.Mass-rearing of N.ribisnigrion L.sativacv "Taiwan"and
culture of plants were performed asdescribed by Van Helden etal.(1992a).
The plants used intheexperiments weretwo nearly isogenic lines (RES and
SUS) whichdiffered only inthe Nasonovia resistance gene (Nr gene) (Van
Helden etal,1992a). Plantswere approximately 6weeks old (5-6 leaves)
whenused.
Production ofalates. Alates were produced on L.sativa cv "Taiwan"by
placing 5-7 females ina2.5 cm clipcage.After two daysthe clip cages and
adults were removed.Morethan 90%of the larvae developed asalates due
to crowding inthe first larvalstage. Recently moulted (< 2hours) young
alates were used inthetests.
Pretreatment oftheaphids. Sincethe extensive handling of the aphids before
the EPG recording canstrongly influence their behaviour, special care was
takentostandardize it as muchas possible (seediscussion).Teneral alate
virginoparae were collected with abrushfromtheir food plant (lettucecv
"Taiwan") intoapetri dish between 8.30 and9.00 a.m.They were tethered
onthethorax (not onthe abdomen) to a20\imgoldwire using avacuum
device bysucking them ontoa pipettetip and attaching them tothe wire
22
CHAPTER 3
Table 1.Summary ofEPG patterns andtheir correlations toaphid
after Tjallingii,1990)
EPG
PATTERN
CORRELATIONS
PLANT TISSUE
np
stylets notinserted
non penetration
(walking etc.)
A
epidermis
B
epidermis /mesophyll
electrical on/off
stylet contacts
sheath salivation
C
all tissues
pd
all living cells
Ele
unknown
activities during
stylet pathway
stylet tippuncture
of cell membrane
unknown
El
sieve elements
unknown
sieve elements
(watery?) salivation
(passive) ingestion
E2
P1
w
F
G
p'
w
tf
APHID ACTIVITY
activities (modified
REMARKS
first waveform only.
1 A,B, and C
1 overlap:
1 in analysis
1 often pooled
all tissues
mechanical stylet
work
"penetration
difficulties"
xylem
unknown
active ingestion
"drinking"
il
*P= peaks, w= waves.
usingwater-based conductive silver paint (Tjallingii, 1988).Aftertethering,
the aphids were placed back on aTaiwan lettuce plant for four hours.The
EPG recordings started between 13.00and 14.00 p.m.Totransfer the aphids
tothetest plantthey werecarefully lifted off theTaiwan plant by pullingthe
goldwire.Theelectrode wasconnectedtothe amplifier placed ina
micromanipulator andthe aphidwas loweredonthe abaxial side of a
reversed,fully expanded leaf ofthetest plant (RESor SUS) immediately.
TISSUE LOCALISATION OFTHE RESISTANCE
23
EPGrecording. The recordingswereperformed inthe laboratory at 22± 1°C
under continuous artificial illumination (HFfluorescent tubes ca4000Lux).
The amplifier usedwasa 109Ohminput impedance DCamplifier (Tjallingii,
1988). Eachday three EPG'swere recordedfor 16hours onsusceptible and
resistant plantssimultaneously. Fifteen recordings were made onplantsof
the susceptible line (SUS) and 20onthe resistant line (RES).
EPGanalyses. EPG'swereanalyzedwiththe computer program STYLET
(Tjallingii & Hogen Esch,1993). Inthe EPG'sthe patterns np(non
penetration),A, B, C(with numerous potential drops), E1e(E1at
extracellular voltage level), E1 (intracellular or pdlevel), E2, Fand Gcould
bedistinguished (seeTable 1andTjallingii, 1990for anoverview). For
analysis A, Band Cwere regarded asone pattern (ABC) becausethey
always occurredtogether atthe start of apenetration and were not always
clearly separated.Theterm Epattern refersto any Epattern atthe
intracellular level,either E1or E2.Thesequence of Epatterns onasuitable
host plant isashort period of E1, followed by ashort transient periodanda
long E2(Tjallingii, 1990).Sometimes shifts from E2backto E1did occur,
mostly onthe resistant line.Thetransition from E1to E2andviceversawas
not always clear, particularly onthe resistant line andthetwo patterns
sometimes occurred simultaneously. Ambiguous mixtures were classifiedas
E1and only clear E2patterns as E2.The actual aphid activities during E1
are notyet known (Tjallingii,1990).
The parameters extractedfromthe EPG's can bedivided intotwogroups.
The "Non Sequential" parameters (Table 2),like totaltimeand frequencyof
each pattern,do nottake into account the order ofthe events intime but use
the pooleddata of each EPG.The "Sequential"parameters (Table 3),like
registration timeto 1 stE(time from start of experiment to 1st E), refertothe
sequence of the patterns asthey occur inthe EPG.Several ofthese
sequential parameters areexplained infigure 1.
Calculations andstatisticalanalysis. The different parameters were
determined orcalculated per aphid,andafter that the meanandstandard
errors over all aphids ofthetreatment were calculated.Mann-Whitney rank
sumtest was usedtotest forsignificance.The patterns whichwere
24
CHAPTER 3
1
2
np { H HABC F i E
Pattern:
Penetration:
5
3
789
6
III
IM
IV
IV
T= 0
10
IV
IV
Time
A Startofexperiment
Endofexperiment ^
Figure 1.Simplified overview of an EPG and some parameters.
After placing the aphid on the leaf the recording starts (T=0). It takes some time before
penetration starts (I s ' np period).Usually several penetrations without E pattern
(pen. 1and 2)precede the first "successful" penetration (3) showing E. Registration time
to 1stE is the time between T=0 and the Is' E pattern (arrow I) and includes several
penetrations. Penetration time to 1stE is the time between the start of the successful
penetration (3) and the start of E (arrow II).During an EPG several penetrations showing
E patterns can occur with a different penetration time to E (arrows III). To calculate the
Potential E index (see text) the sum of arrows IV is calculated as a percentage of arrow V.
terminated by the end of the experimental period (after 16 hours) and not by
the aphid itself, were not excluded from the calculations (see discussion).
RESULTS
Table 2 presents the total time, frequency and mean duration (= total
time / frequency) of each pattern during 16 hour of EPG recording.
Significant differences between the resistant and susceptible lines occurred
TISSUE LOCALISATION OF THE RESISTANCE
25
Table 2.Total time,frequencyand meandurationofeach EPG pattern inthe 16h EPG's
of N. ribisnigrionresistant andsusceptible lettuce
PARAMETER
RESISTANT (N=20)
TOTALTIME(inseconds x103)
np
ABC
Ele
El
E2
F
G
17.3
33.6
0.05
2.5
2.1
1.0
1.8
±
±
±
±
FREQUENCY
np
ABC
Ele
El
E2
F
G
64.9
70.2
0.1
4.6
2.2
1.1
1.3
±
±
±
MEAN' DURATION (in seconds)
np
287
ABC
510
Ele
45
El
623
621
E2
F
716
G
1350
+
±
±
+
±
±
±
±
±
±
±
±
±
±
SUSCEPTIBLE (N=15)
2.4
2.3
0.04
1.3
0.8
0.4
0.4
9.9
23.3
±
±
1.7
3.6
1.4
22.1
0.2
1.8
±
±
±
±
0.4
5.0 **
0.2
1.1
5.1
5.3
0.1
0.7
0.4
0.6
0.3
52.8
58.4
±
9.0
9.7
5.7
3.9
0.3
1.1
±
±
±
±
1.0
0.8
0.2
0.5
50
41
37
448
213
407
399
*
*
ND
184
467
207
9548
113
574
+
ND
+
20
+
77
ND
± 45
+ 3743 ***
+
77
264
±
Time in seconds, Mean± standard error. Mann-Whitney tests forcomparison of2
samples, *=p<0.05; **=p<0.005; ***=p<0.001. ND=notdetected.
in totaltimeof non penetration (np) (p <0.05),ABC (p <0.05) and E2
(p<0.005), and inthe meanduration of E2(p<0.0001). InTable 3the
parameters associated withthe startoftheEPGinclude: Duration of 1 stnp,
duration of thefirst penetration period andofthesecond non penetration
26
CHAPTER3
period (Table 3 :Start of EPG).Nosignificant differences wereobserved.
The parameters associated withthef' E(Table 3) were:The penetration
time,fromthestart ofthe "successful"penetration,tothe 1 stEpattern;the
time fromthestart of the registrationto 1 st E;the number of penetrations
precedingthe 1 st E;the duration ofthe 1 st Eandthe duration ofthe npperiod
after the 1 st E.These parameters showed nosignificant differences between
RESand SUS,except forthe penetrationtimetofirst E,whichwas longer on
the susceptible line (2.47x 103versus 1.34 x 103s, p<0.05). Similar
parameters are providedforthe 1st E2(Table 3).The duration ofthe 1 stE2
patternwas much longer onthe susceptible line (7.7versus 1.1 x103,
p <0.005).The last part ofTable 3 {AllE)shows some other parameters
relatedto all intracellular Epatterns (both E1intracellular and E2).No
significant differences wereobserved inthetime fromthe start of the
penetrationto E,the number of penetrations showing E,the numberof
penetrations showing E2andthe percentage of penetrations showing E.
Onthe resistant linethe Eperiods were difficult to separate in E1andE2
patterns.Onthe susceptible linewe usually foundthe characteristic
sequence of ashort (< 1min) E1pattern andashort "transient period"(ca.
15s) preceding a long E2pattern (many minutesto hours) asalso described
byTjallingii (1990).Onthe resistant linethis sequence showed many
irregularities.Atthe beginning of asieveelement puncture E1wasalways
found but quiteoftentheshift to E2did not occur or occurredvery late.
Irregular shifts from E1to E2and back occurred andsometimes the EPG
signal showedcharacteristics of both patterns. Patterns E1e, F,andG,
occurred inonly afew cases and no relation withsusceptibility was
observed.
DISCUSSION
Pretreatment. The pretreatment (from rearingto finaltransfer tothetest
plant) isknownto strongly influence the aphid's behaviour, in particular the
duration of thefirst nonpenetration period as reported by Montllor and
Tjallingii (1989).A number of different methods have beenreported,
especially with regards tothe wiring and handling of the aphids. Inorderto
TISSUE LOCALISATION OF THE RESISTANCE
27
Table 3.Other parameters and events during 16hEPG recording ofN. ribisnigri on
susceptible and resistant lettuce
EVENT
STARTOF EPG
Duration of 1stnpperiod
Duration of 1"penetration period
Duration of 2ndnpperiod
IST E
time to Is'E
a. from start registration (x 103 s)
b. from start penetration (x 103 s)
Nr ofpenetrations preceding 1stE
Duration of Is'E(x 103 s)
Duration ofnp after 1st E
IST E2
Time to Is' E2
a. from start registration (x 103s)
b. from start penetration (x 103s)
Nr. ofpenetrations preceding Is' E2
Duration of 1st E2 (x 103s)
Duration ofnp after Is' E2
All E
Time toEfrom start penetr.(x 103 s)
Nr. ofpenetrations showingE
Nr. ofpenetrations showingE2
%ofpenetrations showingE
RESISTANT (N=20)
SUSCEPTIBLE (N=15)
209.1
59.6
237.6
± 49.7
± 15.0
± 39.0
178.2
115.6
210.7
± 33.9
± 56.7
± 68.7
17.8
1.34
19.9
1.0
322.9
± 3.0
± 0.18
± 3.3
± 0.5
± 90.2
9.7
2.47
17.7
5.2
313.5
± 2.9
± 0.52
± 5.5
± 3.8
± 134.1
20.0
1.49
20.1
1.1
191.8
± 3.8
± 0.21
± 3.8
± 0.6
± 70.3
11.8
1.80
19.3
7.7
122.4
± 3.2
± 0.18
± 5.1
± 4.0
± 19.2
1.56
3.2
1.8
5.42
±
+
±
±
0.20
0.5
0.3
0.85
1.57
3.7
2.6
8.84
±
±
±
±
0.15
0.7
0.6
1.70
Time in seconds unless otherwise stated, Mean ± standard error. Mann-Whitney testsfor
comparison of2samples, *=p<0.05; **=p<0.005; ***=p<0.001. Noticethe
difference between lstE which refers toonly one event peraphid (thefirsttime itreaches
E)andALLEwhich may betheresult ofseveral penetrations showing Epatterns forone
aphid.
attachthe goldwiretothe aphidthree methods have been used: inactivation
ofthe aphid by cooling or anaesthetization (Harrewijn,1990, pers.comm.),
immobilizing theaphidbyapplying avacuum (inthis case) or attaching the
wire onthe walking aphid (C.B. Montllor &W.F.Tjallingii pers.comm).After
wiring the aphidwas either used directly (Tjallingii, 1988),after a periodof
28
CHAPTER3
recovery onaplant (Montllor &Tjallingii, 1989) orstarvedfor acertain period
(McLean &Kinsey, 1967). Durations ofthe first nonpenetration periodofup
to 15 minutes have beenfoundwhenaphidswere cooled beforetethering(P.
Harrewijn, 1990).Wedevelopedapretreatment withafour hourperiodon
the Taiwan plant betweenthe wiringandthe EPGrecordingto recover from
the stress experienced duringtethering.This reducedthe first non
penetration periodto aboutthree minutes.
Morphs. Mateswere chosenbecausethey arethe migrating morphand
therefore most relevant, andthey might be moresensitiveto host-plant
differences.Alates of N.ribisnigri have more receptors ontheir antennae
thanapterae (Bromley etal.,1979).Thewire hadto beconnectedtothe
thorax because thewings overthe abdomen of the alates interfered withthe
"normal"wire connection ontheabdomen.Attaching thewire onthesmall
thorax isslightly more difficult than connection onthe abdomen,butthis
seemedto reducethe burden ofthewire onthe aphids movements.
Totaltime,frequencyand meanduration (Table 2).These "nonsequential"
parameters showedthat the only clear difference observed inthe EPG'swas
a strong reduction of the duration ofthe E2pattern onthe resistant line
(p<0.001). This resulted inastrongly reducedtotaltime in E2onthe
resistant line of 0.5 hourversus 6hoursonthe susceptible line (p<0.005).
Pattern E2 reflects passivefood uptakefrom asieve element (Tjallingii,
1985, 1988),sofeeding was almost completely absent onthe resistant line.
These results are comparable tothe results of Montllor andTjallingii (1989)
who useddifferent lettuce lines butthe same resistance gene.They didnot
distinguish between E1and E2pattern but usedthe old classification E(pd)
which consists of both E1and E2(Tjallingii, 1990).
The calculation of the meandurations was somewhat biased bythe
termination of the EPG recording after 16hours. However, wedecided notto
excludethe patternthat was inprogress at the end of the EPGfromthe
calculations ofthe meanduration.Thiswould haveinfluencedthe results
even more,inparticular forthe meanduration of the E2pattern.Six long(>
2 h) E2patterns were endedonthe susceptible line bythe endof the
experimental time,against one onthe resistant line.Therefore,the actual
TISSUE LOCALISATION OF THE RESISTANCE
29
Table 4. Potential indices of np, ABC, E and E2
A. Potential E indices as percentage of time used as E (both El and E2) or only E2
after subtraction of registration time to 1"E or Is'E2
B. Percentage of total time in np and ABC andfrequency of non penetrations corrected
for pattern E. Total time in E is subtracted from total EPG time
PARAMETER
RESISTANT
SUSCEPTIBLE
A.
Pot.E index(%)
Pot. E2index(%)
11.5
7.4
+
4.0
2.3
48.5
55.0
± 9.3 ***
± 10.0 ***
B.
%Time in np
%Timein ABC
Freq/h of np
31.7
62.9
4.3
±
±
±
4.1
3.8
0.3
30.0
66.9
5.7
±
±
+
±
3.4
3.5
0.6
Mean ± standard error. Mann-Whitney tests for comparison of 2 samples, *** = p <
0.001.
duration of the E2patternonthe susceptible linewas longer thanwe have
calculated. Forother patterns the errors are less important because their
frequency is usually higher andtheir meanduration much shorter.
Another methodto overcome this problem isto calculate a"potentialE
index"and a"potential E2index",this isthe percentage of time spent in Eor
E2after subtraction of thetime neededto reach Eor E2forthefirst time,in
other wordsthetotaltimespent in E(or E2) divided bythetotal length of the
EPG minusthetimetofirst Eor E2(table4a,Formula:Potential Eindex =
100 *Totaltime in E/(Total EPGtime -timeto 1st Efrom start registration)).
Infig. 1the potential Eindex can becalculated asthe sum of all arrows IV
divided by arrow V.These indicestake into account that ittakes considerable
time to reach an Epattern (Tjallingii &Mayoral, 1993)forthe first time.SoE
can never occur at thestart of the registration andthetime available for Eis
shorter thanthe totaltime ofthe experiment.Theoretically it ispossible to
find ashorter totaltime in Eonasusceptible linethanona resistant linejust
because ittakes longerto reach Epattern onthe susceptible line. Inthat
30
CHAPTER 3
case most ofthe Epatternwouldoccur atthe endandafter the experimental
period, leadingtothefalse conclusionthat there is lessfeeding.The
potential Eand E2indices showedvery clear significant differences.The
indices were muchlarger forthe susceptible line (48.5 and55.0%
respectively) thanforthe resistant line (11.5and 7.4%). Noticeable isthefact
that if only E2was concernedthe difference was clearerthan withpooledE1
and E2,whichagainsuggest that the E2pattern is more important.
Duringthe analysis ofthe results one hasto bear in mindthat nearly all
parameters influence eachother. Ifthe E2pattern is40%of the experimental
time onthesusceptible lineandonly 5%onthe resistant line,thenthe
number of penetrations will be higher onthe resistant line.Careful
interpretation of the dataof these dependent variables istherefore necessary
to revealcausal relationships. Inthis casethe longer totaltime spent innp
andABC pattern onthe resistant linewassimply duetothe "extra"time
available for patterns otherthan E.Whenthe percentage oftime in npand
ABCwas calculated withthetotaltime in Esubtractedfrom thetotal EPG
time there was nodifference betweenthe lines (seeTable 4,Formula:
X= 100xTotaltime in np (orABC)/(Total EPGtime -Totaltime in E)).This
also holds forthe number of nonpenetrations per hour after thesame
correction (Table 4b,Formula:X=Total number of np/(16 hours -Totaltime
in E)).Therefore,these parameters indicated astrongly reduced feeding
period but did not showtheorigin ofthis reduction during the EPG.To
determinethe originofthis reductionthesequence ofthe EPGpatterns had
to bestudied indetail.
StartoftheEPG(Table 3).The duration ofthe 1 stnon penetration periodof
the EPGcangive information onthe influence ofthe plant phyllosphere on
the aphid (volatiles, leaf structure,colour etc).Nodifferences were observed
inthis experiment. However, weconsiderthe use ofthis parameter in EPG
recording rather limited becausethe 1 st npperiod isstrongly influencedby
the aphids pretreatment and aphidstendto probe onany leaf surface to
determine the possibility offeeding (Moericke, 1955). However, large
differences inthe 1 stnonpenetration period may indicate differences
observed bythe aphid before penetration.This can not be investigated
further by EPG's but requires separate behavioral studies under more natural
TISSUE LOCALISATION OF THE RESISTANCE
3?
conditions.Theothertwo parameters (durationof 1 stpenetration periodand
duration of 2ndnpperiod) showed nodifference, indicatingthatthe
unsuitability ofthe resistant plantswas notyet discovered bytheaphid.
1s'E. Duringan Epattern,either E1or E1followed by E2,the stylets are
located inasieve element (Kimmins&Tjallingii, 1985;Tjallingii & Hogen
Esch, 1993).The occurrence of an Epatternisthefirst clear proof of asieve
element puncture. Nearly allaphids (32out of 35) reachedasieve element
duringthe EPGasshownbythe presence of an Epattern.However, during
the precedingABC patternbrief sieveelement punctures canoccur as
potential dropswhich are indistinguishable from potential dropsof
intracellular punctures inany other type of cell (Tjallingii &HogenEsch,
1993).Though likely, it is not clear whether cells aresampled bythe aphids
during potential drops. Itseems asif duringthese cell punctures sieve
elements are not immediately recognized assuch.
Thetime neededforthe aphidto reachan Epatternforthe first timewas
2.7to5 hoursand notsignificantly different betweenthe two lines.This
periodcontained a number of penetrations (not different between lines)
showing mostly ABCpatternwith numerous potential drops (cell punctures)
(see fig.1).Therefore weconcludethat the sieve elements were not harder
to locate onone ofthe lines and nochemical or mechanical factor inhibiting
penetration orfeedingwas present outside thephloem.
Thetimetothe 1 st Epatternwithinthefirst successful penetration (seefig.
1, arrow II; Table 3) wassignificantly different (p<0.05), beingtwice aslong
onthe susceptible line (22 minversus 41 min). However,whenthis
parameter was extendedtoall penetrations reaching pattern E(see fig. 1,
arrows III) nodifference wasfound (Table 3:Timeto Efrom startof
penetration).Therefore,the difference foundinthis parameter forthefirst
successful penetration was considered an artefact and it was concludedthat
there was no mechanical barrier present which made it harder to reachthe
phloem.A resistance mechanism outsidethe phloem,issuggested in other
aphid-plant combinations wherethe resistance was relatedtothetime
needed bythe aphidto reachthe phloem (Dreyer and Campbell,1984;
Niemeyer, 1990),butthis seems unlikely here.Niemeyer (1990) suggested a
chemical factor outside the phloem inthe case of Rhopalosiphum padion
32
CHAPTER 3
barley (possible roleof hydroxamic acids). Inthe resistance of sorghum
against greenbugs (Schizaphisgraminum) a mechanical barrier (e.g.pectin
composition) outsidethesieve elementswassuggested (Dreyer &Campbell,
1984). Inbothcasesthe authors did not distinguish between registration
time,fromstart ofthe experiment, andpenetrationtime,from thestart ofthe
successful penetration. It ispossible that anincreased registrationtime
before reaching asieve element occurswith anequal penetrationtime in
whichcasethere aresimply moreorlonger penetrations precedingthe
successful one. Ifthis occurs andthe penetrationtime fromthestart of the
successful penetration is notdifferent then mechanical differences canbe
excluded asa resistance mechanism.
The duration ofthe 1 stnpperiodafter an Epattern could beinfluencedby
the uptake of feeding stimulants or deterrents withthe food, signallingtothe
aphidthat the plant isahost or anon-host. Our results did notshow any
difference betweenthe lines inthis respect.
7s'E2(Table 3).The E2pattern is knownto be relatedto passive phloem
sap ingestion (Tjallingii, 1985).Theduration ofthe 1 st E2pattern onthe
susceptible line (Table 3) was 2 hours,showing clear food uptake.The mean
duration of 1st E2onthe resistant lineshowedthat there wassomefood
uptake but muchshorter (20min,Table 3).Sothe longer duration ofthe E2
patterns ingeneral (Table2) was also reflected inthe 1 st E2.Thiswasthe
first occasion duringthe EPGwhere adifference occurred betweenthe
lettuce lines.Apparently duringthe 1 st E2(thefirst feeding) the host plant
suitability wasperceived bythe aphid.Thissuggested a resistancefactor in
the phloemvessel,presumably relatedtocharacteristics or compositionof
the phloemsap.
E1versus E2patterns. The irregularity inthe Epattern onthe resistant line
also indicatedthat duringthat stage of behaviour the unsuitability of theplant
was interfering withfeeding.Nothing is known about the activities of the
aphidduring E1. It ispossiblethat during E1the aphid issamplingthe
phloem sapor preparingthesieve element for feeding.Nodataonfood
uptake,saliva excretion or muscle activities during E1are available so far.
TISSUE LOCALISATION OF THE RESISTANCE
33
Moredetailed knowledge of E1is necessary sincethis phase of the EPG
seemsto beof crucial importance inthis case of resistance.
Duration ofEPG's. Theduration ofthe EPG's inthis experiment (16 h)was
considerably longerthanthe lengthinearlier reports (4h) (Van Helden,
1990).Wedecidedto make longer EPG's because inthe short EPG's notall
patternscouldbeseen.For most N.ribisnigriaphids ittook morethanfour
hoursto reachan Epatternfor thefirst time. Ina naturalsituation anaphid
may leavethe plant whenit has notedthat it is not asuitable host plant. In
that casethe aphidwouldleave after asieve element puncture. However it is
still possiblethat afree aphid respondsto moresubtlestimuli whichoccur
before it reaches the phloem which are masked inthe EPGduetothe
tethering.Agood comparative experiment withfreeaphids istherefore
necessary. A minimum EPGlengthof 8 hours seems necessary inthiscase.
Fromthis experiment it could not be madeclear whether the second partof
the EPGyielded muchextrainformation. Normally onewould expect a
equilibrium inthe distribution of thetime overthe different patternsto be
established bythen. However, between hour 9and 12of the EPG (clock
time ± 22.00 hour) aclear change inthe distribution of the different patterns
occurred.This could be dueto adiurnal rhythmicity inthe plant ortheaphid,
inspite of the continuous light provided duringthe experiment. The lengthof
the EPG's inthis kindof experiments should include a reasonable amountof
phloemfeeding onthe susceptible line andampletimeto reachthe phloem
onbothlines.Agoodway to decide onthe duration ofthe EPG's wouldhave
beento determinethetime anaphid needstostart the productionof
honeydew onthesusceptible line.The EPGshouldthen be recordedfor a
period of about twice as long.Thiswas not done inthiscase.
Correlation with bionomics. Van Helden etal(1992a) describedthe
bionomics of N.ribisnigrionthese same lines.Their experiments suggested
atotal absence of feeding onthe resistant line but notoxic effect of the
resistance onthe aphid.The EPGresults didagreewiththeir conclusions.
The possibility of achemical resistance factor assuggested inVan Heldener
al(1992a) isconsistent withthe EPG's. Inthis case a relation betweenthe
amount of feeding andthe growth rate (Van Helden eral,1992a) could not
34
CHAPTER 3
beestablished becausethe EPG reflected only the initial phase of aphid
plant interaction whilethe mean relative growth rate was determined overa
periodof severaldays.
Correlation with behaviour. The behaviour ofthe aphid duringthe EPGcould
beinfluenced bythetreatment. Especially thetethering caninfluencethe
aphid (Tjallingii, 1985). Itislikely that the aphidwould leave a resistant plant
after it hasclassified it assuch,inthecase of N.ribisnigrion lettuce afterthe
1 st E2pattern.Tjallingii (1985) showedadecreased sensitivity ofthe aphidto
plant suitability ina host versus non-host situation. Inorderto evaluate the
possible disturbance oftheaphids behaviour anadditional behavioral study
is needed (Van Helden etal,1992b).
Resistance mechanism. Aresistance factor inaphloem vessel can beeither
a mechanical or achemical barrier.A mechanical barrier inthe form of sieve
element or aphid stylet blocking after puncturing still is apossible resistance
mechanism. However,the resistance gene isspecific against N.ribisnigriand
gives noclear resistance against other lettuce attacking aphids likeMyzus
persicae,Macrosiphum euphorbiae or Uroleucon sonchi(VanHelden,
unpubl.; Reinink &Dieleman, 1989).Thissuggests avery species specific
mechanism. Itispossible thatthe stylet penetration activities of the aphid
induce a resistance mechanism inthesieve elements by elicitors inthe
saliva of the aphidor alternatively elicitors may be released from by plant in
response tothe aphids salivary enzymes.A purely mechanical mechanism
seems notvery likely.Apossible mechanical barrier duringthe penetration
and beforethesieve element is reachedcanbeexcluded basedonour
results.The possible uptake of smallvolumes of plant sap during penetration
which canthen betasted bythe chemoreceptorsinthe pharyngeal cavity
(Tjallingii, 1985;Wensler &Filshie, 1969) may provide information for the
aphid and influence its behaviour (Niemeyer, 1990).Though it seems likely,
noclear proof exists for sap uptake during penetration and beforethe
phloem has been reached. Inthis case nodifference was observed during
the penetration tothe phloem.Thissuggests that the aphid did not gainany
information about the resistance inthis stage.The phloem sap may contain
many clues about the host plant suitability. Duringthe E2pattern uptakeof
TISSUE LOCALISATION OF THE RESISTANCE
35
phloemsaptakes place andcontact withthe pharyngeal chemoreceptors
occurs andwetherefore hypothesizethat thechemical composition of the
phloem was responsible forthe resistance.Asshownearlier (Van Heldenet
al,1992a)there was noproof for intoxication oftheaphid onthe resistant
line,whichsuggests afeeding deterrent (orabsence of afeeding stimulant)
asa possible mechanism.The absence of long E2patterns supports this
theory andtherefore ourstudieswill nowconcentrate onthe phloem sap
composition ofthe lettucelines.
REFERENCES
Bromley, A.K., J.A. Dunn &M. Anderson,
1979. Ultrastructure of the antennal sensilla
of aphids. I. Coeloconic and placoid
sensilla. Cell Tissue Res. 203:427-442.
Dreyer, D.L. & B.C. Campbell, 1984.
Association of the degree of methylation of
intercellular pectin with plant resistance to
aphids and with induction of aphid biotypes.
Experientia 40: 224-226.
Eenink, A.H. & F.L. Dieleman, 1982.
Resistance of lettuce to leaf aphids:
Research on components of resistance, on
differential interactions between plant
genotypes, aphid genotypes and the
environment and on the resistance level in
the field after natural infestation. Med. Fac.
Landbouww. Rijksuniv. Gent 47/2:607-615.
Eenink, A.H., R. Groenwold & F.L.
Dieleman, 1982a. Resistance of lettuce
{Lactuca)to the leaf aphidNasonovia
ribis-nigri 1:transfer of resistance from
L.virosa to L.sativa by interspecific crosses
and selection of resistant breeding lines.
Euphytica 31:291-300.
Eenink, E.H., R. Groenwold &F.L. Dieleman,
1982b. Resistance of lettuce {Lactucd) to the
leaf aphid Nasonovia ribis-nigri 2:
Inheritance of the resistance. Euphytica 31:
301-304.
Harrewijn, P. 1990.Resistance mechanisms of
plant genotypes to various aphid species. In:
36
R.K. Campbell &R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam.: 117130.
Helden, M. van. 1990. Resistance of lettuce to
the aphid Nasonovia ribisnigri:Are
electrical penetration graphs (EPGs) helpful
to find the origin of resistance. Proc. VIIIh
Int. Symp. Insect - Plant Interactions.
Budapest, Hungary 1988. Symp. Biol.
Hung. 39:473-474.
Helden, M. van & W.F. Tjallingii, 1990.
Electrical penetration graphs of the aphid
Nasonovia ribisnigrion resistant and
susceptible lettuce (Lactucasativd). In:
Peters, D.C., J.A. Webster & CS. Chlouber
(eds). Proc. Int. Symp. Aphid - Plant
Interactions: Populations to Molecules.
Stillwater, Oklahoma, U.S.A.: 308
Helden, M. van, W.F. Tjallingii & F.L.
Dieleman, 1992a. The resistance of lettuce
{Lactucasativa 1.) to Nasonoviaribisnigri:
Bionomics of N. ribisnigrion near isogenic
lettuce lines. Ent. exp. appl. 65: 53-58
Helden, M. van, M.H. Thijssen & W.F.
Tjallingii, 1992b.The behaviour of
Nasonovia ribisnigri on resistant and
susceptible lettuce lines. In: Menken, S.B.J.,
J.H. Visser & P. Harrewijn (eds.) Proc. 8th
Int. Symp. Insect-Plant relationships. Kluwer
CHAPTER 3
acad. publ. Dordrecht, The Netherlands.
286-288
Kimmins, F.M. & W.F. Tjallingii, 1985.
Ultrastructure of sieve element penetration
by aphid stylets during electrical recording.
Ent. exp. appl. 39: 135-141.
McLean, D.L. & M.G. Kinsey, 1967. Probing
behaviour of the pea aphid Acyrtosiphon
pisum. I: Definitive correlation of
electronically recorded waveforms with
aphid probing activities. Ann. Entom. Soc.
Am. 60:400-406.
Moericke, V., 1955.Über die
Lebensgewohnheiten der geflügelten
Blattläuse (Aphidina) unter besonderer
Berücksichtigung des Verhaltens beim
Landen. Zeitschrift für angewandte
Entomologie 37: 29-91.
Montllor, C.B. & W.F. Tjallingii, 1989. Stylet
penetration by two aphid species on
susceptible and resistance lettuce.Ent. exp.
appl. 52: 103-111.
Niemeyer, H.M., 1990. Secondary plant
chemicals in aphid host interactions. In:
Peters, D.C., J.A. Webster & CS. Chlouber
(eds). Proc. Int. Symp. Aphid - Plant
interactions, populations to molecules.
Stillwater Oklahoma U.S.A.: 101-111.
Reinink, A.H. & F.L. Dieleman, 1989.
Comparison of sources of resistance to leaf
aphids in lettuce (LactucasalivaL.).
Euphytica 40:21-29.
Spiller, N.J., L. Koenders & W.F. Tjallingii,
1990. Xylem ingestion by aphids -a
strategy for maintaining water balance. Ent.
exp. appl. 55: 101-104.
Tjallingii, W.F., 1978.Electronic recording of
penetration behaviour by aphids. Ent. exp.
appl. 24: 521-530.
Tjallingii, W.F., 1985.Stylet penetration
activities by aphids.Thesis Wageningen
Agricultural University, Wageningen, The
Netherlands.
Tjallingii, W.F., 1988.Electrical recording of
stylet penetration activities. In: A.K. Minks
& P. Harrewijn (eds.), Aphids, Their
Biology, Natural Enemies and Control, vol.
TISSUE LOCALISATION OF THE RESISTANCE
2B, Elsevier Science Publishers B.V.
Amsterdam, pp.95-108.
Tjallingii, W.F., 1990. Continuous recording
of stylet penetration activities by aphids. In:
R.K. Campbell &R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam.: 8999.
Tjallingii, W.F. &Th. Hogen Esch, 1993.
Fine structure of the stylet route in plant
tissue by some aphids.Physiol. Entomol.
18: 317-328
Tjallingii, W.F. & A. Mayoral, 1992. Criteria
for host plant acceptance by aphids. In:
Menken, S.B.J., J.H. Visser &P. Harrewijn
(eds.) Proc. 8th Int. Symp. Insect-Plant
relationships. Kluwer acad. publ. Dordrecht,
The Netherlands.280-282
Wensler, R.J. & B.K. Filshie, 1969. Gustatory
sense organs in the food canal of aphids. J.
Morph. 129:473-492.
37
4. FEEDINGBEHAVIOUR
OFFREEH. RIBISNIGRI
ABSTRACT
Thefeeding behaviour of Nasonoviaribisnigri(Mosley) alates was studied
to findthe behavioural phase where rejection of anabsolutely resistant
lettuce (Lactuca sativaL.) line occurred.Nodifferences were observed
between landing percentages or behaviour duringthe first 30 minutes on
resistant andsusceptible plants.Longer observations revealedthat aphids
were more mobile onthe resistant lineandstartedto leave after 4 hours.
Most aphids stayedfor morethan 48 honthe susceptible plants.
Ina previous study, using electrical penetration graphs,weshowedthat
differences inprobing behaviour occurred duringthefirst attempted phloem
feeding.Thefirst observable sieve element puncture usually occurred after
5h. Through comparison of free andtethered aphids weshowedthat
tethering didaffect probing behaviour. Rejection of the plant byfree aphids
occurred after 4to 24 hr,which corresponded wellwiththetime after which
free moving aphids startedto leavethe plant.Wetherefore conclude,that
rejection of the resistant plant occurs after ingestion from aphloem sieve
element.
FEEDING BEHAVIOUR OF N. RIBISNIGRI
39
INTRODUCTION
Absolute resistance inlettuce totheaphid N.ribisnigri(Van Helden etal.,
1993) isbasedonasingle dominant gene (Nrgene, Eenink &Dieleman,
1982; Eenink etal.,1982;Reinink & Dieleman,1989).The resistance
mechanism isstill unclear. Electrical Penetration Graphs (EPG's) (Tjallingii,
1988)from N.ribisnigrionresistant andsusceptible lettuce showedthat
probing behaviour differed only aftertheaphid hadreachedthe phloem (Van
Helden&Tjallingii, 1993).Duringthe EPGthe aphid isattachedto agold
wirewhich hampers its movements andwill probably influence its behaviour
(Tjallingii, 1985, 1986).Theexperiments presented here aimtostudy atwhat
point free moving aphids reject the resistant plant andwhether free aphids
andtethered aphids behave similarly.
MATERIALS AND METHODS
Aphidsandplants.Mass-rearing of N.ribisnigrionlettuce (cv."Taiwan") and
culture of plants were performed asdescribed by Van Helden eral.(1993).
Alateswere produced asdescribed by Van Helden&Tjallingii (1993).
Recently-moulted alateadultswereused.The plantsweretwo nearly
isogenic lines (RESand SUS)whichdiffered only inthe Nasonovia
resistance gene (Van Heldenetal.,1993). Plantswere about 4weeks old(68fully expanded leaves). Intheflight chamber another susceptible lettuce
line ("Taiwan") and a non-host plant Chinese cabbage (Brassica cernua
(Thbg) cv. "Granaat") were alsoused.
Landing behaviour
Pretreatment. Teneral alatevirginoparae were collected witha brushfrom
their food plant andtransferredto asmall piece of leaf ( 3 x 3 cm) ina Petri
dish at 22°Cfor 24 hours,after whichthe leaf fragment was removed.The
aphids were used after another 24 hours of starvation whenthey showed
muchflight activity inthe Petri dish.
Flightchamber. A 1x 1x 1mflight chamber was constructed comparable
tothe one described by Legge (1962) and Halgren & Rettenmeyer (1967).
40
CHAPTER 4
The walls,floor and ceilingwere coveredwith white cloth.Windspeed
rangedfrom 0.50 m/s (directly underthe air inlet) to0.05 m/s onthebottom.
Light intensity from HFFluorescent tubes was 36n.E.m"2.s'1(ceiling under
funnel) to 6n.E.m"2.s"1(floor).Temperature was25± 3°C.
Protocol. Aphids were released individually from asmall platform (5 mmin
diameter) onthetip of awhitecone at20cmfromthe floor of the chamber
and 20cmfromthe left wallofthe chamber.A plant was placedat 20cm
fromthe right wall withawhite paper cover (11cm high) aroundthe pot.
Whenthe aphidstayedonthe platform for morethantwo minutesthe
observation was discarded.Most aphidsflew off soonand landedeitheron
the plant (endof observation), onthewalls,or onthe pot cover. Ifthey
stayedthere for morethantwo minutesthe observation was ended.Some
aphids walkedtothe plant after landing nearby.About 100aphids were
released intheflight chamber for eachplanttype.
Reflection-spectra. Reflection spectra (350-1100 nm) of boththe adaxial and
the abaxial leaf surfaces were measured at two different dateswitha
spectrophotometer.
Behavioural observations
Experiments were performed inthe laboratory at 22± 1°Cunder HF
fluorescent tubes at 48nE.m"2.s1Lux.
Pretreatment oftheaphids. Teneral alatevirginoparae were collected witha
brushfromtheir food plant (lettuce cv "Taiwan") into a Petri dish between
8.30 and9.00 am.They were markedwithwater soluble paint in different
colours usingavacuum device (Van Helden &Tjallingii, 1993;Van Helden&
Tjallingii, 1994) and placed onafreshTaiwan plant for at least 3hto recover
(Pretreatment identical toVan Helden&Tjallingii, 1993).
Continuous observations (0.5h).One aphidwas carefully picked upwitha
fine brush andtransferredto apaper platform (8 mmindiameter) onthe
adaxial side of the fourth leaf of the plant (fromthe growing point).
Observation started whenthe aphidwalkedoff the platform.The behavioural
sequences observed (Table 1a) were comparablethose described forAphis
fabaeI.(Scop.) (Ibbotson &Kennedy, 1951,1959). Behaviour was recorded
FEEDING BEHAVIOUR OF N. RIBISNIGRI
4?
Table la.Events recorded during continuous behaviourial observations (0.5h).
EVENT CLASS
ACTIVITY
EVENT
Walking
Penetration
No activity
Aphid walking,proboscis notextended
Stylet penetration: Proboscis incontinuous contact with leaf surface without
moving, antennae backwards
Tapping leaf with proboscis, antennae
upright
None of the previous activities
Adaxial
Abaxial
Stem
Growingtip
Adaxial (upper-)side ofleaf
Abaxial (under-)side ofleaf
Stem ormain vein near tothestem
Smallest still folded leaves (1or 2)
Tapping
LEAF POSITION
PENETRATION SITE
DESCRIPTION
Nopenetration
Main vein
Side vein
Lamina
No proboscis contact (occurs only when
ACTIVITY isother than penetration)
First order (central) vein ofleaf
Second andthird order veins which were
clearly raised from theabaxial leaf-surface
Lamina orvery small vein (notraised
from theabaxial surface of the leaf).
for 30 minutes usingaportable computer and "THE OBSERVER"™(Noldus,
1991) software.
Intervalobservations (48h).The start ofthe experiment was identical tothe
previous observations.8aphids,eachonadifferent plant,were observedat
0.5, 1,2,4,8, 16,24,32,40,and48 hours.The events are listed inTable
1b.To prevent aphids walking off the plant andthen returningto it,a ringof
plastic tapewith Fluon™was placedaroundthe base of the plant.Aphids
walking onthe Fluon™droppeddown into adishfilled withwater andwere
recorded as having left the plant.
42
CHAPTER4
Table lb. Events recorded during interval behaviourial observations (48 h)
EVENT CLASS
RELOCATION
EVENT
No relocation
Relocation
PLANT POSITION
LEAF POSITION
PENETRATION SITE
Growingtip
Younger leaf
Start leaf
DESCRIPTION
Aphid in same position aslast
observation
Aphid shifted tonewposition
Older leaf
Smallest still folded leaves (1or2)
Leaf younger than starting leaf
First fully extended leaf (usuallynr.4
fromtip)
Leaf older than starting leaf
Adaxial
Abaxial
Stem
Growingtip
Adaxial (upper-)side ofleaf
Abaxial (under-)side ofleaf
Stem ormain vein near tostem
Smallest still folded leaves (1 or 2)
Nopenetration
Main vein
Side vein
Lamina
No proboscis contact
First order (central) vein ofleaf
Second andthird order veins which were
clearly raised from the leaf-surface
Lamina orvery small vein which did not
raise from thesurface.
Statistics. All parameters were first calculated peraphid andthen averaged
over all aphids. Landing andbehavioural dataweretested for significance
with Mann-Whitney rank-sumtests or Fisher's exact test.
RESULTS
Landingbehaviour. Aphidstook off readily andflew inthe chamber for
periods of uptoten minutes before landing.Direct landing frequencies (Table
2) on Chinese cabbage andTaiwan lettucewerecomparable (25.7and
27.6%) and lowerthanon RESand SUS (42and35%).Many aphidswalked
FEEDING BEHAVIOUR OFN. RIBISNIGRI
43
Table 2. Results of the flight experiment.
Plant
N
Taiwan
RES
SUS
Chin. Cabbage
105
100
100
105
% ARRIVING AT THE PLANTS
Landing
Walking
Total
27.6
42.0
35.0
25.7
47.6
38.0
48.0
32.4
75.2
80.0
83.0
58.1
tothe plants after landing nearby.Alltogether the number of aphids arriving
atthe plant wascomparable for all lettuce lines andsomewhat higherthan
for Chinese cabbage (Table 2).Thecolour spectra of the plants were almost
identical with apeak inthevisible spectrum at 550-560 nm.The abaxial side
allways showeda higher reflection.REShadaslightly higher total reflection
than SUS (e.g.34versus 31% at 555 nm)and bothwere somewhat higher
than Chinese Cabbage andTaiwanwhich were almost identical (20-23%at
555 nm).
Continuous observations (0.5h).Onlytheduration of the first stylet
penetration showedasignificant difference, being almost twice as longon
the resistant line (43.1versus 23.9s) (Table 3a;"free").The aphids were
probing almost 75%ofthetime.
These visual observations of "free"aphids could becompared with results
oftethered aphids in EPG recordings (Table 3a) derived from earlier
experiments (Van Helden&Tjallingii, 1993).Significant differences were
apparent especially inthe duration of thefirst non-penetration period (the
time beforethefirst penetration) whichwas over threetimes aslongforthe
tethered aphids.Thetotaltime spent instylet penetration (Table 3a:total
time sp) was alittle longer (andconsequently thetotaltime innonpenetration shorter) forthefree aphids.Although significant only for the
resistant line,the meanduration of the individual non-penetration periods
(interval between penetrations,table 3a:meanduration np) was somewhat
44
CHAPTER 4
Table 3.Results ofcontinuous observations offree aphids (0.5h)andcomparison with
tethered aphids (extracted from earlier EPG data)
A. CONTINUOUS OBSERVATIONS (0.5h)AND EPG DATA
SUSCEPTIBLE
FREE
tethered
Nr. ofobservations
Total timenp
Number ofnp
Mean durationnp
Total timesp
Number ofsp
Mean Durationsp
Dur. firstnp
Duration first sp
33
458 ± 34
4.2 ± 0.2
118 ± 10
1342 ± 34
4.1 ± 0.2
365 ± 23
55.5 ± 6.8
23.9 ± 1.8#
15
* 777 ±105
5.1 ± 0.4
149 ± 17
* 1023± 105
4.8 ± 0.3
* 248± 40
* 179.9 ±32.2
115.6 ±54.8
RESISTANT
FREE
tethered
34
504 ± 41
4.9 ± 0.3
116 ± 11
1296 ± 41
4.8 ± 0.3
307 ± 22
68.5 ±12.7
43.1 ± 7.2#
20
* 746 ± 83
4.9 ± 0.4
* 164 ± 24
* 1054 ± 83
4.8 ± 0.4
252 ± 34
* 205.2 ± 49
47.4 ± 10
B. INTERVAL OBSERVATIONS (48h)AND 16h EPG DATA
Nr. of observations
% time insp
% ofobs. in sp
% aphids leaving
36
92.7
33.3
15
77.0
0
33
81.1
97.4
20
74.2
0
Time inseconds. Mean± standard error, np=non-penetration, sp=stylet penetration.
Total time is sumofallindividual periods peraphid (e.g. £(time probing)), mean duration
is average ofduration individual periods (e.g.£(time probing)/nr ofprobes)). * =
Significant difference between free andwired aphids onthesame line (p<0.05).
# =Significant difference between aphids onresistant andsusceptible plants (p<0.05).
longer forthetethered aphids.The meanduration ofthe individual stylet
penetrations (Table 3a:meandurationsp) was alittle longer forthefree
aphids but only significant onthesusceptible line.Nearly allfree aphids
changedfrom adaxial to abaxial ofthe leaf inthefirst minutes ofobservation,
often without penetrating first ("Seitenwechsel", Klingauf, 1972) (Table4).
FEEDING BEHAVIOUR OFN. RIBISNIGRI
45
Table 4. Aphids switching to abaxial side of leaf in continuous observations.
PARAMETER
SUSCEPTIBLE
% showing side switch
100
mean time to switch (s± SE)
67 ± 10
% performing a switch without penetrating
first ("Seitenwechsel")
55
RESISTANT
94
73 ± 9
53
Intervalobservations (48h).Onthesusceptible plants 66.7%of the aphids
stayed onthe plant for 48 h.(Table 3b, Fig.1a).Onthe resistant plant
97.4%left the plant (Table 3b, Fig.1b),i.e. only one aphidstayed.The
aphids left betweenthe 4thandthe 24th hour of the experiment (Fig.2).
Overthe whole observation period aphids onthe resistant line consistently
showedsignificantly higher mobility (80-90%oftheaphids relocated between
two observations) thanonthe susceptible line (50-60%relocation).The
aphids remained longer onthe 4th (start) leaf onthe susceptible line whereas
they moved earliertothe growingtip onthe resistant line (Fig. 1a,b).Stylet
penetrations were mostly onthe mainveinorthe larger sideveins
(Fig. 1c,d).Compared withthe percentage of time in non-penetration from
EPG registrations (Table 3b) freeaphids seemedto probe aslightly larger
percentage of time although comparison of (continuous, 16h) EPGand
visual (interval,48 h) observations isdifficult. Aphids were almost exclusively
foundonthe abaxial side oftheleaves.
DISCUSSION.
Landingbehaviour.
Aphids areattractedto green-yellow surfaces, often irrespective of the
plant species (Moericke 1955, 1962;Müller, 1962). Inour experiment the
small differences inlanding percentages onthe different plants probably
reflect thesmall differences inbrightness (Müller 1964).Althoughthe
experimental arrangement is not really comparableto a natural situation no
indication wasfoundthat the aphidswere ableto distinguish between
46
CHAPTER 4
RESISTANT
SUSCEPTIBLE
a
100
80
-a
Q- 60
CD
CD
<S •
c
CD
Ü
i_
CD
a.
20
16 24 32 40 48
RESISTANT
SUSCEPTIBLE
CD
O. 20
• I" " I " " I " " l " " I " " I
0
Time (h)
0.5
1
2
4
8
i
i
i
i
i
16 24 32 40 48
Time (h)
Figure 1.Interval observations (48h). Aphid position on susceptible (a) orresistant (b)
plants and penetration sites on the leaves of susceptible (c) and resistant (d) plants at
different observation times.Decreasing column lenghts show aphids leaving theplant.
FEEDING BEHAVIOUR OF N. RIBISNIGRI
47
susceptible and resistant lettuce before alighting.The same result has been
shownfor several other aphid-plant combinations (Müller, 1962).
Continuous observations (0.5h)
Resistant versus Susceptible. The behaviour ofthefree aphids on RES
and SUSlettuce showedvery little difference duringthefirst half hour.The
duration ofthefirst non-penetration period (time before probing) was not
different, soeffects of host-plant differences inthis stage were notshown.
Severalauthors have reportedsignificant differences inthis parameter ina
host/non-host situation,always withalonger time before probingon non-host
plants (Klingauf, 1972,Goffreda era/., 1988).Similarly, alonger firstnonpenetration periodwas reportedon resistant lettuce lines withthe same Nrgeneaswe used here (Harrewijn,1990),but hislineswere not near-isogenic
sodifferences might beattributedto differences not relatedtothe resistance
gene.
The longer durationofthefirstpenetration onthe resistant plants
suggeststhat the aphids didencounter some difference inplant stimuli
duringstylet penetration,before phloem contact. Klingauf (1972) even
suggestedthat alonger first probe reflects better plant acceptance. However,
apart fromthissingle parameter weobserved noimportant effects onthe
subsequent behaviour ofthe aphids (see also interval observations).
After landing onthe adaxial side of the leaf aphidswalk tothe edge and
changetotheabaxial side,often without afirst probe ("Seitenwechsel",
Klingauf, 1972).Suppression or delay ofthis behaviour was considered a
sign of plant deterrence (Klingauf, 1970;1972).Nodifferences were
observed inthis behaviour on SUSversus RESlettuce.
Thus,although some behavioural differences showed between aphids on
resistant andsusceptible plants before reachingthe phloemthis did not lead
to host-plant rejection (as measured by leavingtheplant).
Freeversus EPG.Many differences were observedwhen freeand
tethered aphids werecompared.The pretreatment (handling,paint marking,
starvation etc.) may influence the results,especially the parametersduration
ofthefirstnon-penetration (time before probing) and durationofthe first
penetration (Van Helden&Tjallingii, 1993).The pretreatment ofthe free and
tethered (EPG) aphids wasalmost identical,since eventhe free aphids were
48
CHAPTER 4
givenaspot of paint ontheirthorax, similarto thewiredaphids in EPG
recordings. However, several causes may explainthe observed differences in
these parameters:
a.Thetransfer of theaphidstothetest plant wasslightly different. The
"free" aphidwas picked upwithabrush andplacedonapaperplatform.
Observation started only aftertheaphidwalkedoff the platform. Inthe EPG
experiment the aphidwas lifted fromtheTaiwan lettuce plant bythe gold
wire andlowereddirectly onthe leaf ofthetest plant.The pretreatment can
haveanimportant effect onsubsequent behaviour (Van Helden&Tjallingii,
1993).
b. Behavioural observations startedonthe adaxial side ofthe leaf, free
aphids often walkedseveral cm and morethan 50%even changedleaf
surface before probing (Table 4).Tethered aphids were dearly hamperedin
their movements,often pullingthe goldwire for awhile before probingforthe
first time.Surface changes were impossible forthese aphids andthe surface
exposedtothem was limited bythe lengthof the goldwire.
The EPG results showed nosignificant difference inthe duration ofthe
first penetration periodon resistant versus susceptible plants. However,
variation inthe results is large andthe number of replicates issmall (Van
Helden&Tjallingii, 1993). it ispossiblethat thetethering reducesthe impact
of asubtle stimulus inthis phase ontheaphid,thus reducingthe difference
inbehaviour on RESversus SUS,aswas also concluded byTjallingii (1985).
The larger percentage oftime spent in non-penetration (andconsequently a
shortertime instylet penetration) bytethered aphids indicates ageneral
"restlessness".Thetotaltime in non-penetration (or stylet penetration) isa
combination of frequencyand mean duration, which did not always show a
significant difference individually (Table 3a).Thedifference inthese
parameters can beattributed tothe direct physical effect ofthetether onthe
insect which acts not only during non-penetration periods (whenthe aphid
cannot movefurther because ofthe length of thewire) but alsoduring
probing, constantly pullingtheaphid.
We concludethat the difference inthe lengthofthefirst non-penetration
period isattributable to bothdifferences inthe pretreatments andtether
effects whereas variation inother parameters was mainly caused bytether
effects.
FEEDING BEHAVIOUR OF N. RIBISNIGRI
49
Intervalobservations (48 h)
Resistant versus Susceptible. Already atthe first observation (after 0.5 h)
the aphids showed a higher mobility having moved moretothe growingtip
onthe resistant plants.Thisconfirms our observationthat adifference in
plant quality affected aphid behaviour asfoundinthe previous experiment.
However,thisdid not result ina rejectionofthe resistant plants inanyform.
Though generally a higher mobility isconsidered asignof lower plant
acceptance (Müller, 1962)the reactionalmost resembled afaster acceptance
andsubsequent relocationtothe preferredfeeding site (growingtip) onthe
resistant plant. However,thestimulus which causedthis small difference
might play arole inthe many (poorly understood) sequential steps leadingto
final plant acceptance or rejection (Klingauf, 1987). Itisalso possible that the
stimuli involved are related (genetically or chemically) to larger differences
encountered during phloem contact.This requires more knowledge onthe
nature ofthe resistance and host-plant evaluation bythe aphids during
different phases of penetration.
Ina host versus non-host situation onewould expect the aphids to leave
the plant quitesoon after the first plant contact (Müller, 1962; Klingauf 1970,
1987).While comparingtwo lines of Vicia fabaI. Müller (1958)foundthat
nearly all aphids left the more resistant line after 1-10 minutes (clearly before
contactingthe phloem bundle). Inour experiment the aphid initially seemed
to accept the resistant plant.
Althoughthe level isquite different,thetime course (shape of the curve)
ofthose aphids leaving wassimilar for both lines suggestingthat the decision
moment (timeafter plant access) wasthesame (Fig.2).
Freeversus EPG.
Using EPG recordings Van Helden&Tjallingii (1993) found no difference
inpenetration behaviour on RESversus SUS beforethe phloem was
reached.Asshown in Fig.2most aphids reachedthe phloem within 1-8
hours inthe EPGstudy.This iswell withinthe range of time that thefree
aphids left the plant, corroborating the conclusionthat phloem contact is
necessary before rejectionof the plant. Noultimate proof canbeobtained
that the aphids do reachaphloem sieve element without an EPG recording
and eventhen not every sieve element contact canbe recorded since brief
50
CHAPTER 4
0.8
o F
o> S
c o
*f,S*
Susceptible
0.6
ctio
chi
e H' 0 4
03 TO
ir o)
u. >-
Resistant
12
16
20
24 28
Time(h)
32
36
40
44
48
Figure 2. Time course of the departure of free aphids on RES and SUS compared to the
aphids showing E pattern (stylets located in a sieve element) in EPG experiments. (EPG
data on RES and SUS were pooled since no differences were found)
punctures of phloem sieve elements do occur intermixed and indistinguishable from other cell punctures (Tjallingii 1990; Tjallingii & Hogen Esch, 1993).
Interval recording of free aphids (Montllor & Tjallingii, 1989) was impossible
because the aphids moved to the growing tip where they could not be
reached by an electrode.
Since tethered aphids cannot leave the plant there were large differences
between free and tethered aphids due to free aphids leaving after several
hours.
In EPG experiments the aphids had access to a limited surface area on
the abaxial side of fully expanded leaves (Van Helden & Tjallingii, 1993)
which is not the preferred feeding site (Mackenzie & Vernon, 1988). What
determines this preference is unknown but both plant structure, phloem
composition, microclimate and shelter will be different in the growing tips or
in the heads of lettuce. Tjallingii & Mayoral (1992) showed differences in the
time needed to reach the phloem on different plant parts.
FEEDINGBEHAVIOUR OFN. RIBISNIGRI
51
CONCLUSIONS
1. Aphids encounter differences between resistant andsusceptible plants
beforethe phloem is reached butthis does not result inplant rejection.
2. Rejection ofthe resistant plants occursonly after phloem contact.
Therefore,further researchshouldfocusondifferences inthe phloem
(either chemical or mechanical) between resistant andsusceptible lines.
3.Tethering effects occur during EPG's, mainly dueto locomotion restraints.
REFERENCES
Eenink, A.H. & F.L. Dieleman, 1982.
Resistance of lettuce to leaf aphids:
Research on components of resistance, on
differential interactions between plant
genotypes, aphid genotypes and the
environment on the resistance level in the
field after natural infestation. Med. Fac.
Landbouww. Rijksuniv. Gent 47/2:607-615.
Eenink, E.H., Groenwold, R. & F.L.
Dieleman, 1982.Resistance of lettuce
(Lactuca)to the leaf aphid Nasonovia
ribis-nigri2:Inheritance of theresistance..
Euphytica 31: 301-304.
Goffreda, J.C., Mutschler, M.A., W.M.
Tingey, 1988.Feeding behaviour of potato
aphid affected by glandular trichomes of
wild tomato. Ent. exp. appl. 48: 101-107.
Halgren, L.A. & C.W. Rettenmeyer, 1967. An
insect flight chamber. J. Econ. Entomol. 60:
1165-1167.
Hardie, J., Holyoak, M.,Taylor, N.J. & D.C.
Griffiths, 1992.The combination of
electronic monitoring and video-assisted
observations of plant penetration by aphids
and behavioural effects of polygodial. Ent.
exp. appl. 62:233-239.
Harrewijn, P., 1990. Resistance mechanisms
of plant genotypes to various aphid species.
In: R.K. Campbell & R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
52
Science Publishers B.V., Amsterdam.
Helden,M. van & W.F.Tjallingii, 1993.
Tissue localisation of lettuce resistance to
the aphid N. ribisnigri using electrical
penetration graphs. Ent. exp. appl. 68:269278
Helden, M. van, and W.F. Tjallingii, 1994.
The use of electrical penetration graphs in
plant resistance research, in: E.A. Backus &
G. Walker, Homopteran feeding behaviour:
Recent research advances and experimental
techniques. Special issues Ent. Soc. Am.: In
press.
Helden, M. van, Tjallingii, W.F. & F.L.
Dieleman, 1993.The resistance of lettuce
{Lactucasativa 1.) to Nasonoviaribisnigri
(Mosley, Homoptera, Aphididae):
Bionomics of N. ribisnigrion near isogenic
lettuce lines. Ent. exp. appl. 66:53-58.
Ibbotson, A. & J.S. Kennedy, (1951). Aggregation in Aphis fabae (Scop.): Aggregation
on plants. Ann. Appl. Biol. 38:65-78.
Ibbotson, A. & J.S. Kennedy, (1959). Interaction between walking and probing in Aphis
fabae (Scop.) Exp. Biol. 36 (2): 377-390.
Klingauf, F., 1970. Zur Wirtswähl der Grünen
Erbsenlaus, Acyrthosiphonpisum (Harris)
(Homoptera: Aphididae). Zeitschrift für
Angew. Ent. 65:419-427.
Klingauf, F., 1972. Die Bedeutung von perip-
CHAPTER 4
her vorliegenden Pflanzensubstanzen für die
Wirtswahl von phloemsaugende Blattläusen
(Aphididae). Zeitschrift für Pflanzenkrankheiten 79:471-477.
Klingauf, F.A., 1987. Host plant finding and
acceptance. In: Minks, A.K. & P. Harrewijn
(eds.): Aphids &their biology, natural enemies and control. World Crop Pests 2a.
Elsevier Science Publishers B.V., Amsterdam, pp209-223.
Legge, J.B., 1962. An aphid flight chamber.
Nature 194:706.
Mackenzie, J.R. &R.S. Vernon, 1988.Sampling for distribution of the lettuce aphid,
Nasonovia ribisnigri (Homoptera:
Aphididae), in fields and within heads. J.
Entomol. Soc. Brit. Columbia 85:10-15.
Moericke, V. 1955. Über die Lebensgewohnheiten der geflügelten Blattlause (Aphidina) unter besonderer berücksichtigung
des Verhaltens beim Landen. Zeitschrift für
angew. Ent. 37: 29-91.
Moericke, V. 1962. Über die optische Orientierung von Blattläusen. Zeitschrift für angew. Ent. 50:70-74.
Montllor, C.B. & W.F. Tjallingii, 1989. Stylet
penetration by two aphid species on susceptible and resistance lettuce. Ent. exp. &
appl. 52: 103-111.
Müller, H.J. 1958.The behaviour of Aphis
fabae in selecting its host plants, especially
different varieties of Viciafaba.Ent. exp. &
appl. 1:66-72.
Müller, H.J. 1962. Über die Ursachen der
unterschiedlichen resistenz von Viciafaba 1.
gegenüber die bonnen blattlaus,Aphis
(Doralis)fabae Scop. VIII. Das Verhalten
geflügelter Bohnenläuse nach der landung
auf wirten und nichtwirten. Ent. exp. appl.
5: 189-210.
Müller, HJ. 1964. Über die anflugdichte von
aphiden auf farbige salatpflanzen. Ent. exp.
appl. 7: 85-104
Noldus, L.P.J.J., 1991.The Observer: a
software system for collection and analysis
of observational data. Beh. Res. Meth.,
Instr. & Comp., 23:415-429
Reinink, K. & F.L. Dieleman, 1989.
FEEDING BEHAVIOUR OF N. RIBISNIGRI
Comparison of sources of resistance to leaf
aphids in lettuce (Lactucasativa L.).
Euphytica40: 21-29
Tjallingii, W.F., 1985.Stylet penetration
activities by aphids. Ph.D.-Thesis
Landbouwhogeschool Wageningen.
Tjallingii, W.F., 1986.Wire effects on aphids
during electrical recording of stylet
penetratioa Ent. exp. appl. 40: 89-98.
Tjallingii, W.F., 1988.Electrical recording of
stylet penetration activities. In: A.K. Minks
& P. Harrewijn (eds.), Aphids, Their
Biology, Natural Enemies and Control, vol.
2A, Elsevier Science Publishers B.V.
Amsterdam, pp.95-108.
Tjallingii, W.F., 1990. Continuous recording
of stylet penetration activities by aphids. In:
R.K. Campbell &R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam.
Tjallingii, W.F. &Th. Hogen Esch, 1993.
Fine structure of the stylet route in plant
tissue by some aphids. Physiol. Entomol.
18: 317-328
Tjallingii, W.F. & A. Mayoral, 1992. Criteria
for host plant acceptance by aphids. In:
S.BJ. Menken, J.H. Visser, P. Harrewijn
(eds.). Proceedings 8th Int. Symp.
Insect-plant relationships. Kluwer acad.
publ. Dordrecht, The Netherlands, pp
280-282.
53
5. PHLOEMSAP
COLLECTION
ABSTRACT
Three methods to collect phloem sapondifferent lettuce lineswere
optimized anddescribed indetail.Success ratiofor stylectomy(1) of aphids
was over 80%throughthecombination of aspecially designed setup and
Electrical Penetration Graphsto monitor phloemsap ingestion.Onsome
lettuce linesstylets nevershowedsustained exudations for unknown
reasons.Clear differences instylet exudation showedbetweentwo aphid
species onthesame lettuce line. Honeydew(2) collection in hexadecane
made accurate quantitative analysis possible,samples were largeandclean
but biotransformed.The EDTAchelation method (3) produced large samples
but dilution,oxidation andimpurities fromthe woundsurface reducedthe
reliability.
INTRODUCTION
Lettuceresistance: Theabsolute,monogenic resistance of lettuce(Lactuca
sativaL.) tothe aphid Nasonovia ribisnigri(Mosley) (Nr-gene, Eenink etal.,
1982; Reinink and Dieleman, 1989;Van Helden etal.,1993) isbased onan
early interruption of phloem sap uptake afterthe aphid-stylets have reached
the sievetubes of the plant (Van HeldenandTjallingii, 1993;Van Heldenet
PHLOEM SAP COLLECTION
55
ai 1992). Possible resistance mechanisms are (1) amechanical blockingof
the aphidstylets orthesievetube after puncturing bythe aphid,or (2) a
difference inchemical compositionofthe phloemsap between susceptible
and resistant lettuce (Van HeldenandTjallingii,1993).
Phloem sapcollection: Several collection methods of phloem sap have been
described inliterature. Direct collection of phloemsap after incisions inthe
plant asdescribedfortrees (Zimmerman, 1957),cucurbits (Richardson et ai,
1982) or legumes (Pate etai, 1974) is not possible for lettuce becauseof
the presence of largequantities of lactiferous ducts aroundeventhe smallest
veins,whichimmediately exude latex atwounding.The remaining methods
therefore are amputation of aphidstylets (stylectomy, Downing andUnwin,
1977; Unwin,1978; Fisher and Frame, 1984),collection of honeydew (Banks
and Macaulay, 1964) and"facilitated exudation"through EDTA chelation
(King andZeevaart, 1974;Girousse etai, 1991;Groussol etai, 1986).
The objectives of this study were:(1)the optimisation of thesethree
methods for lettuce, (2) aqualitative andquantitative comparison ofthe
samples and (3) comparison ofthe results for different lettuce lines and
aphidspecies in relationto aphid resistance.
The detailedtechnical description of allthree different phloem sap
collection methods andtheir results may be useful in research onplant
physiology andaphid-plant interactions in relationto host-plant resistance.
MATERIALS AND METHODS
Plants. The plants used inthe experiments were lettuce plants of the lines
"Taiwan" (susceptible,genotype nmr), "411" (resistant, genotype NrNr) and
two set of isogenic lettuce lines RES(genotype NrNr) and (SUS,nrnr) (Van
Helden etai, 1993) and RES2 (NrNr) and SUS2 (nrnr).The second set is
different fromthe first set (apart fromthe source ofthe resistance gene) and
was selected for partial resistanceto Myzuspersicae(Sulz.) (Reinink and
Dieleman, 1989;Reinink etai, 1988).The culture of plants was as described
by Van Helden etal.(1993). Plantswere used ina4-5 leaves stage
(stylectomy) or ina6-8 leaves stage (honeydew and EDTA chelation).
56
CHAPTER 5
Aphids.Mass-culture andsynchronized culture of adults ofthe aphids N.
ribisnigri(biotype WN1),M.persicae(biotype WM1) and Macrosiphum
euphorbiae (Thomas.) (WMe1) (Reinink and Dieleman,1989) were
performed asdescribed byVan Heldenetal.(1993).Aphid rearing plants
were L sativa cv. "Taiwan"for N.ribisnigri, Brassica napusL cv. "Olymp"for
M.persicaeand L.sativa cv."Snijsla"for M.euphorbiae. Forstylectomywe
alsotriedAulacorthum solani(Kltb.) and Uroleucon sonchiL.from amassculture on L sativacv."Taiwan"andoriginating from alocal fieldpopulation.
Stylectomy
Setup.Experiments were performed inthe labat 22± 1°C, RH± 60%and
4000 Luxfrom HFfluorescent tubes. Insome casesthe humidity was raised
to over 95%RHusing aultrasonic humidifier. Plants were potted insmall(5
cm) square pots and mounted inaspecially designed setup.The abaxial
side ofthe fourth (fully expanded) leaf of the plant was fixedover a
cylindrical perspex support inthe centre of the setup (fig. 1)which madeit
possibletoturnthe aphid"target" inevery direction (arrows) while keepingit
infocus of a horizontally viewing stereomicroscope (notshown).
Electrical penetration graphs (EPG,Tjallingii, 1988) were usedto monitor
the feeding behaviour of the aphids.This method enables recordingof
different waveform patterns during penetration which can be relatedto
different aphidactivities andstylet locations inthe plant tissue (Van Helden
andTjallingii, 1993). Phloem sap ingestion occurs during waveform pattern
E2(Tjallingii, 1990).Six aphids werewired andviaachoice switch each
aphidcouldbeconnectedto an EPGamplifier (DCsystem, 1GQinput,
Tjallingii, 1988) coupled with astorage oscilloscope to monitor itsfeedingbehaviour. Stylets were cutthe following day whenaphids showed phloem
sap ingestion patternE2.
Stylectomy was performed with a highfrequency microcautery unit (48
MHz,circa 25Watt, Syntech).Output of the microcautery unit was reduced
to around 50%of the maximum output (output circa 10-15Watt) ,using a0.2
s pulse. Needles were prepared from 0.2 mmtungsten wire and
electrolytically sharpenedto produce aneedle withalong (1.5cm) tapered
thintip (Brady, 1965).Afeed/eposition. The aphidwas approached frontally,
carefully movingthetipa little beyondthe labium.Just beforethe amputation
PHLOEM SAP COLLECTION
57
Figure 1.Design of the stylectomy setup and microcautery unit. The plant can be turned in
every direction (arrows). Six aphids can be connected over a choice switch to an EPG
amplifier to record the EPG signal. Detail shows needle position and microcapillary just
before amputation.
58
CHAPTER 5
TOP COMPARTMENT
LEAF
MESH
CLIP
HONEYDEW
BOTTOM
Figure 2. Design of the honeydew collection cage.
the needlewasslowly movedlaterally against the narrowest part (atthe
second labial segment) ofthe proboscis (fig.1,detail),firmly touchingthe
proboscis, andcausing aslight deflection of the needle tip andthe proboscis.
Thenafoot-switch activatingthe microcautery pulse was pressedimmediately. Insome cases iswas necessary to bendthe needleto reachthelabium.
Other needle locations onthe proboscis weretried.
Phloem sapcollection. Acapillary (20u.lMicrocaps"11,52 mmlong, outer
diameter 0.97 mm,inner diameter 0.69 mm,with atapered tip,brokenat
outer diameter 0.15 mm,filledwithsilicon oil) was placed over the exuding
stylets,suchthat these were positioned inthe oilfilled part.The positionof
the upper oil meniscus was determined every 5-15 minutes.Volumes were
estimated bythe extension of the fluid-column (precision 0.04 mmor 15nl
using a measuring Eyepiece).Anoverestimationof volume andrate
appeared whenthe oil did not completely fillthetapered end.When
exudation stoppedthe capillary was emptied in 100|il of 50%methanolfor
sugar analysis. Samples were frozen at -80 °C until analysis.
Statistics. Comparison between aphidspecies were made by Mann-Whitney
Utests (a <0.05). Comparison amongveins for eachspecies were made
usingthe Kruskal-Wallis test for multiple comparison (a <0.05). Fortables
and statistical tests concerning duration,sample size andexudation rateonly
PHLOEM SAP COLLECTION
59
Table 1.Overview of the stylectomy results
Result of attempt
Total number of attempts
E2 continued until microcautery
Succesfull amputation
Exudation from stump
Long exudation (> 1min)
Samples > 1jü
N
337
322
284
245
124
48
100.0
95.5
84.3
72.7
37.7
14.2
values largerthan zerowere usedandsome discordant values were
excludedwithduration >200min(5casesexcluded),samplesize > 15uJ (1
case) or rate > 1nl/srespectively (5cases).
Honeydewcollection
Honeydew was collected inthe greenhouse at 22± 2°Cand RH70%
under continuous illumination. Honeydew collection cages were prepared
from 5cm Petri dishes modified after Eenink etal.(1984) (fig.2).5-10 adults
were placed inthe bottom compartment (anempty base of a Petri dish),and
left there for 24 hoursto allow settling and reproduction of theaphids onthe
leaf piece.The next daythe bottom part was replaced by afreshonefilled
withtwo mlof n-hexadecane (C16H34,sg 0.77 g/ml, mp 18°C).At 24h
intervals the honeydew was collected inacapillary andstored at -80°C
under n-hexadecane. Different aphidspecies were used,depending onthe
lettuce line (see results).Thetop ring ofthe clip-on cagewas made into a
second cageby closing itwith nylon mesh (fig.2,topcompartment). Before
andduring afew collections onthe lines RES, RES2,SUSand SUS2thetop
compartment wasfilledwith 5-10 N.ribisnigriadults.
EDTA chelation
Leaves werecut closetothe baseandthe cut surface was rinsedin
water for afew seconds. Leaveswereweighed and placed in 1.5mlvials
containing 0.8 ml of an EDTA solution buffered with5 mM phosphate buffer
(pH6).All vials were placed inalargetranslucent container (RH >95%,22
60
CHAPTER 5
Table 2.Overview of the results per aphid-plant combination
Aphid Species
Lettuce line
TAIWAN
M.persicae
N S. size
147 1.3 ±0.4(54)
N. ribisnigri
N S. size
88 2.8 ±0.4(47)
RES
19
##
**
SUS
13
##
12
RES2
0
**
SUS2
0
11
411
TOTAL
10 2.0 ± 1.5(2)
189
##
Other
N S. size
1
Total
N
236
27
##
46
6
##
31
0
0
2
##
13
**
1
##
11
111
37
##
337
N = number of attempts, S. size = sample size of long exudations (> 1 min) in
ul ± SE(number of samples), ** = incompatible aphid-plant combination, ## = No long
exudation observed (Sample size < 1nl).
± 2°C,and artificial illumination) for 18-24 h .Different EDTA concentrations
and pH's weretried.Thetime course ofthesugar yieldandtheyieldper
gram of leaf and its relationto leaf agedetermined.This methodwas
comparedto collection indarkness or collection with only ashort (2h) EDTA
exposure after which leavesweretransferredto avial containing wateronly.
Final collectionwas in8mM EDTA and5 mMphosphate buffer at pH6.
Sodium metabisulfite Na2S205 (5 mM) was used asan antioxidant.
Sugarconcentration. Total sugar concentration ofthe EDTA sampleswas
determined using acolorimetric reactionwith anthrone.After heating for 10
minutes at 90°Cthe reaction mixture shows equal extinction per gramsugar
for different sugars (Van Handel, 1967)andtherefore is agood measureof
total sugar content.
Results of a more detailed chemical analysis of thesamples are given
elsewhere (Van Helden etal., 1994)
PHLOEM SAP COLLECTION
61
RESULTS
Stylectomy
Asummary ofthe stylectomy isgiven intables 1and2.The resultsof
337 attemptswere recorded,mainly on"Taiwan"with N.ribisnigriand M.
persicae.
Needlepositioning. Theoptimal approach ofthe needlewasfromthefront
side ofthe aphid,amputating atthesecondlabialsegment.Whenthe needle
accidentally touchedthe aphidit sometimes endedthe penetration,but more
often disturbance was only shown inthe EPGsignal which temporarily
changedfrom waveform E2(phloem ingestion) to E1(stylets insieve
element,salivation but noingestion, Prado andTjallingii, 1994) (table 1:E2
continued until microcautery). Usually E2was resumed after afew minutes
andthe attempt could becontinued.Sometimes the aphid raised andswayed
itsabdomen and moved its legswithout achange inthe EPGwaveform.
Amputation. The overallsuccess ratiofor amputation was 84.3%(table 1).
Usually the proboscis was partially, andthe stylets completely cut allowing
the aphidto retract thesevered proboscis andwalk off leaving only the stylet
stump. Inonethird oftheamputations the proboscis was also cut.The
remnants could usually be removedwith afine brush,if not removablethe
capillary wassimply placedoverthem. Inaround 10%of all casesthe
mandibular/ stylets separatedfromthe maxillary ones,bending away.When
a pulsewasfired but amputationfailedthis was usually attributable to
inadequate contact between needleandproboscis.
Other places ofthe proboscis thanthethird segment were either harderto
reach (proximal segments) or gave alower success ratio of amputations
(distalsegments). Bendingthe needlewith smooth curves did not affect the
efficiency of the microcautery but madethe positioning difficult. Stronger
and/or longer pulses caused damagetothe plant surface.
Exudation success. Nearly all successful amputations were followed bysome
exudationfromthe stylet stump (245 of 284).Whenamputation was
accidentally performed during an EPGwaveform other than E2exudation
was neverobserved.
62
CHAPTER 5
Duration ofexudation. 50%of all exudations (124of 245) stopped after
around 10seconds,whenaonlytiny droplet (diameter 0.05-0.15 mm,i.e.
volume± 1nl) hadbeenformed.Whenexudation continued for morethana
minute,so beyondthis onedroplet (further referredtoas "long exudation")
total samplevolume was mostly smaller than 1uJ,with durations of 5to 120
minutes (mean53 minutes,median30 minutes). Exudations producing
volumes of 1-5 u.loccurred irregularly (48) withdurations of 30-200 minutes.
One exceptionally longexudation of three daysoccurred.
Exudation rate.Theexudation rate (table 3) of longexudations wasvery
variable (range0.05 to0.8 nl/s). Exudation rate usually decreased withtime
but sometimes increasedtemporarily e.g.whenthecapillary waschanged.
Miscellaneous observations. Thephloem sapflowing out ofthe stylet stump
adheredtothewall ofthe capillary,quickly forming acomplete disk with
menisci atthe oil interfaces and pushingthe upper meniscus upwards when
exudation continued.Whenthe phloemsap droplets did nottouchthe wallof
the capillary they could beobserved sinking tothe lower oil/air interface.
Exudation from levels belowthetop ofthestump occurred inafewcases.
Sometimes afew seconds delay precededthe appearance of the phloem
sap atthetip ofthe stump. Increasingthe humidity did not increase
exudation rate or duration.Whenthe mandibular stylets separated fromthe
maxillary ones exudation seemedtostop earlier thanwhenthey remained
joined.
Differences amonglettuce lines.Success ratio for amputation or exudation
did not differ among lines (table 2)."Taiwan"lettuce showed long exudations
quite often whilethe lines RES,SUS,RES2 and SUS2never exuded phloem
sap for morethan afewseconds. Line411 (whichcontains the resistance
gene) showed long exudations ontwo (out often) occasions.
Differences between aphidspecies. Tables 3and4summarize a numberof
parameters to compare N.ribisnigriand M.persicae on"Taiwan"lettuce.
Stylet stumps of N.ribisnigriwere longer, showed higher exudation ratesand
yielded larger samples.Thesuccess ratiofor long exudations seemed
somewhat higher for N.ribisnigri (47out of 88versus 54out of 147forM.
persicae).N.ribisnigripreferred the larger veins while M.persicaeoften
probed inthe lamina (table4).
PHLOEM SAP COLLECTION
63
Table 3.Differences between Nasonoviaribisnigri and Myzuspersicae during stylectomy
on Taiwan lettuce.
Parameter
Nr of attempts
Nr disturbed
Nr of successful amputations
Nr of stylets exuding
Nr exuding > 1min
Stump Length (m)
Dur. of exudation (min)
Sample size (1)
Estimated speed (nl/s)
M. persicae
147
5
135
128
58
154 ± 4
44.6 ± 6.2
0.84 ±0.12
0.33 ± 0.22
84
57
53
52
*
*
*
*
N.ribisnigri
88
7
79
68
48
304 ± 10
73.5 ± 8.2
2.75 ± 0.38
0.46 ± 0.04
65
44
47
43
Values are mean ± standard error. Some discordant values excluded (see text). *
significant difference between species ( a = 0.05, Mann-Whitney test).
Differences amongveins. The size,exudation rate andduration of samples
collected ondifferent veins of "Taiwan"lettuce leaves showed no statistically
significant differences (table 4) butthe number of replicates waslow.
Honeydew
The honeydew collection methodyielded 10to 50u.lper cage per day.
Adult aphids produced large quantities of offspring which contributed tothe
honeydew. Onasuccessful aphid-plant combination many droplets ofvery
different sizeswere collected. Honeydew droplets dropping inthe oil sankto
the bottom andfusion of individual dropletsfalling ontop of eachother
occurred frequently. A minimum temperature of 21 °Cwas necessary to
avoidsolidification ofthe hexadecane (mp 18°C). Contamination bythe
aphids and exuviae which haddropped intothe hexadecane was avoidedby
the manual collection. Duetothe affinity betweenthe glass capillary andthe
aqueous honeydew the droplets were automatically sucked intothe capillary
together withsome oil.Other methods like filtration or centrifugation of the
oil/honeydewsuspension were nottried, both for the risk of contamination
andthe needto washthe droplets fromthe Petri dish (dilution). Dry matter
content ofthe collected honeydew wasaround 11.5%.
64
CHAPTER 5
Differences amongaphid-plantcombinations. Combination of aphidspecies
and lettuce genotype gave different yields,dueto differences innumberof
larvae produced inthecageand number of aphids dropping intothe oil.On
the lines RESand RES2 N.ribisnigricould not beused.However also M.
persicaeshowedalot of aphids dropping inthe oilonthese lineas it didon
the line SUS2,resulting insmallyields.Onthese lines M.euphorbiaegave
better yields.Nodifference inaphidbehaviour or honeydew productionwas
observedwhen N.ribisnigriwas introduced intothe adaxial top compartment
(see fig.2) onthe resistant lines.
EDTA chelation.
Optimisation. The highest sugar yieldwas reachedat 8 mM EDTA and
decreased at higher concentrations (fig.3A).Alinear relationship appeared
between leaf-fresh weight andthetotalamount ofsugar inthe extract (sugar
yield (mg) = 1.33 *leaf weight (g) -0.06, r=0.57). Theyoung,notfully
expanded leaves yielded30to 60%lowerquantities of sugar per gramof
freshweight comparedtofully grown leaves.Thetime course of sugar yield
showedthat the rateof sugar exudation rose untilthe sixth hourof EDTA
exposure and remained constant untilthe endofthe collection period(fig.
3B). Norelation wasshownbetween pHand yield.When exudationwas
performed inthe dark yields were reduced by 50%.Inexperiments witha
short exposure (2h) to EDTA andsubsequent collection inbuffered water no
sugar could befound inthe extract after 20 h.Adding C0 2 (1%) tothe
container did not increase sugar yield. Sodium metabisulfite(Na2S205)
suppressedthevisible browning of the extracts at 5 mMwithout affecting
sugar yield.
Yield. EDTA chelation yieldedsamples of 0.2-0.6 mlper leaf. The sugar
content of thesamples was0.3 to0.9%(w/v,sucrose asstandard) or around
1.3(0.3-2.4) mgper gram of leaf. Large leaves yieldedsmall samples with
highsugar concentrations.
Differences among lettucelines. Twodifferences were observed between
resistant andsusceptible plants. 1. Thesugar yield (per gram of leaf) from
the resistant plants wasonaverage around30percent lowerthanon
susceptible plants.2. Leaves of resistant plants remainedturgescent during
the EDTA chelation while leaves of all susceptible plants lost turgor.
PHLOEM SAP COLLECTION
65
5
6
7
8
9
10
EDTA concentration (mM)
4
25
6
50
100
24
Time(h)
Figure 3.Results of the EDTA chelation experiments on Taiwan lettuce.
A. Sugar yield per gram of leaf material at different EDTA concentrations.
B. Time course of sugar exudation.
66
CHAPTER 5
DISCUSSION
Stylectomy
Setup. The highsuccess ratioofthestylet amputations canbe attributed
tothe special setupwiththeaphidandthe plant located inthe centre ofthe
setup wherethey can beturnedinevery directionwithout movingthem outof
the focus point ofthe microscope. Disturbance of aphids duringthe
positioning ofthe needle,which isconsidered adrawback of radio-frequency
microcautery (Fisher and Frame, 1984) rarely occurred.The use of laser
microcautery (Fisher and Frame, 1984) was not considered becausethe
anatomy of lettuce makes it impossibleto avoiddamageto theplant.The
observed behaviour of someaphids movingtheir body and antennae(e.g.
whenthe needle accidentally touchedthe aphid.) wasvery similartothe
effect of alarvawalking by andtouchingthe adult.
Thethinwallofthe Microcaps*71capillary reduced refraction problems
duringthe positioning overthe stylet stump.Thethintip makes it possibleto
movethestylet stump slightly to anoptimal position with oil layers onboth
sides of the exuded phloem sap.The oil avoided evaporation and enabled
estimation ofvolumes.Weighing could not be usedbecause oftheoil.
Silicon oil does not interfere withthe chemical analysis and its density (0.98
g/ml) was expectedto beclosetothat ofthe phloemsap. However it
appearedthat the phloem sap is heavierthanthe oil.
Amputation. The success ratio for amputation is much higher than any result
published sofar using microcautery (Girousse etal.,1991, Rahbé etal.,
1990,Chino etal.,1991) andcomparable tothe results of Kawabe etal.
(1980) who used laser equipment, eventhoughthe aphids we usedare
smallerthanthe insect species they used (Acyrtosiphon pisumHarris,
Macrosiphum albifrons Essigand Nilaparvata lugens Stal.).
The narrowest part of the proboscis atsegment 2appearedthe best place
for amputation because itscuticle islesschitinisedthanthe distal (darker)
proboscis segments asusedby Mentink etal.(1984).Amputation at these
distal proboscis segment required higher aoutput power fromthe
microcautery and increasedthe risk of plant damage.The electrical contact
betweenthe needle andthe proboscis should not be broken duringthe
PHLOEM SAP COLLECTION
67
microcautery pulse.Thiswas achieved by using needles withalongflexible
tapered point andthefirm lateral contact against the proboscisjust before
amputation.Whenonlythevery tip ofthe needlewas positioned against the
proboscis (Downing and Unwin,1977)the electrical contact wasoften broken
duringthe pulse resulting ininsufficient damage tothe stylets. Sometimes
littlevisible damagetothe proboscis (lowelectrical resistance) still gave
successful stylet (high resistance) amputation.The bleeding ofthe stylet
stump belowthetop indicatesthat the maxillary stylets weresometimes cut
or damaged at a lower levelthanthe mandibular/ stylets.
Differences amongaphid-plantcombinations. Thesuccess ratiofor
amputation depended onthe aphidspecies and host-plant suitability. Some
species were more restless (U.sonchii) or hadavery short proboscis when
feeding (A.solani).Some aphid-plant combinations could not beused
because of complete resistance (A/,ribisnigrion RESand RES2) or hada
low amount of aphidsshowing E2(e.g.M.persicae onSUS2and RES2),
apparently because of partial resistance (Reinink etai, 1988).
Exudation success. The high success ratio for exudation from asuccessful
amputation (over 90%on"Taiwan") canonly beachieved bythe useofthe
EPG methodto ascertain phloemfeeding before amputation (Mentink eta/.,
1984).Without EPG recording exudation success ratio's of 0.05to 55%have
been reported (Winter etal.,1992;Girousse etal.,1991,Rahbe etal.,1990,
Hayaschi and Chino, 1986,Fisher and Frame, 1984).The extrawork
involved withthe useof EPGs pays off bythe certainty that the "target" is
indeed ingesting phloemsap andthat feeding iscontinued untilthe
amputation.We never observed exudation whenwaveform E1(which always
precedesthe actual ingestion waveform E2,Tjallingii, 1990) was present,
confirmingthe hypothesisthat noingestion occurs duringthis waveform
(Prado andTjallingii, 1994). Evenwhenthe waveform E2continued untilthe
moment of amputationsomestylets did not produce phloem sap. Itis
possiblethat the stylets weretranslocated orwere mechanically blocked
duringthe amputation.The latter possibility seemsto beconfirmed bythe
short delay inthe exudationwhichsometimes occurred after amputation asif
anobstacle hadto beexpelled withthe first outflow.
Duration ofexudation. Many exudations stopped after about 5-20 secondsas
was also reported by Girousse era/. (1991).Apparently the amputation itself
Ü
CHAPTER 5
orthe outflow ofthe phloemsap causedaplant reaction,blockingthe
outflowvery soon after amputation.This reaction could betriggered bythe
sudden decrease ofturgor pressure inthe sieveelement cell (Peel,1975)or
byenergy generated duringthe pulsewhich istransported downthe stylets
intothe plant,either aselectrical discharge or as heat. Calloseformationor
P-protein gelation (Girousse etal.,1991) have been mentioned but callose
formationseems unlikely because ofthespeedofthe reaction (<10
seconds).Tjallingii and Hogen Esch (1993) observed fibrousstructures inthe
very tip ofthefood-canal of cauterisedstylet (which hadshown only ashort
exudation) similarto P-protein bodies inthe sieveelements.
Long exudations apparently occur only whenthis short term blocking
mechanism fails completely or is incomplete. It is not clear wetherthese
same mechanisms stopthe outflow from long exudations (see hereafter).
Exudation rate.The meanexudation rate of longexudations was 0.33 and
0.46 nl/sfor M.persicae and N.ribisnigrirespectively (range 0.2-0.8 nl/s).
Earlier reportsshow very variable results (Rahbe, 1990:0.1-0.3 nl/sfromM.
albifronsEssigon lupine; Peel,1975:> 1nl/sfrom Tuberolachnussalignus
Gmelinonwillow;Tjallingii, 1994:0.09 nl/sfor N.ribisnigrionlettuce;
Girousse etal.,1991;0.07 nl/s of A.pisumon Medicago sativaL; Fisher
and Frame, 1984:0.3 nl/sfor T.salignus and0.03 nl/sfor Quadrapidiotus
astraeformisCurtis bothonwillow).Thesedifferences arethoughtto be
caused byvariation inthe lengthanddiameter ofthefoodcanal inthe stylet
stumps (Fisher and Frame,1984). However, thediameter ofthefood canal
of a large aphid like T.salignus (1-2p.mi-d., Peel,1975) is notvery different
from M.persicae and diametersvariedfrom 0.6 - 1.4 urnamong 16
measured species (Forbes, 1977) whichcannot explain allthevariation in
exudation rate.Large differences inexudation rateforthe same species on
different plants of thesame genus (T.salignus onwillow, Peel,1975, Fisher
and Frame, 1984;N.ribisnigrionlettuce,Tjallingii 1994,our data) showthat
plant quality plays animportant role.
The exudation rateof long exudations was comparable tothe estimated
exudation rate directly after amputation (estimate basedonexudation ofthe
first droplet).This suggests anabsence of blocking reactions.Theslow
decrease of the exudation rate overtime can becaused by plant blocking
reactions but also by evaporation fromthe air-exposed part ofthestylet
PHLOEM SAP COLLECTION
69
stump betweenthe leaf surface andthe capillary.Thisseemsto be
confirmed by Downing and Unwin (1977),whofounda higher osmotic
strength ofthe sapwhencollected inanoilfilledcapillary (identical to our
method) comparedtosapcollected inanoil droplet coveringthewhole
stylet.The increase inexudation whichsometimes occurred after changing
the capillary andthefactthat separation of the mandibular stylets from the
maxillary onescaused exudationto stopsooner support this hypothesis and
showthat blocking canoccur higher upthestylet stump,abovetheplant
surface.Closingthe air gap by placingthe capillary onthe plant surface
failed becausethe oilflowedout ofthe capillary ontothe leaf surface.
The meanexudation rate is much larger thanthe estimated feeding rate
of around 25pl/sfor M.persicae(Tjallingii, 1994). Itshows that aphids
actively reducethe flow rate duringfeeding,possibly to avoid plant defence
(blocking) reactions.
Differences amonglines.Unfortunately, wewere unableto obtain usable
stylectomy samples fromthetwo sets of isogenic lines dueto immediate
blockage of the outflow. Since resistant andsusceptible plants of bothsetsof
near isogenic lines possesthis characteristic it ispossibly linkedwiththe
resistance gene,e.g.located onthesame chromosome.Apart fromthe
source of resistance gene (L virosa), thetwo sets aregenetically different.
From line 411, whichcontainsthe resistance gene,some samples couldbe
collected. Mentink era/. (1984) also reportedamputated stylets "producing
sap" (duration not specified) ona resistant lettuceline.
Differences between aphidspecies. N.ribisnigriis bigger than M. persicae
and produced longer stylet stumps.Thefoodcanal of N.ribisnigriseems
somewhat larger than reportedfor M.persicae(0.7u.mhalfway thestylets,
Forbes, 1977).The diameter of the food canal (TEM pictures of N. ribisnigri,
Tjallingii and Hogen Esch,pers.comm) variedfrom 0.5 x0.8 p.m(oval) near
thetipto 1.1|xm(round) just abovethe leaf surface,and salivary canalfrom
0.3 to 0.4 |xmatthe same locations. Dataare incomplete andexact
comparison isdifficult. This might explainthe differences inexudation rate,
duration andsample size but aphid-plant interactions can play arole.
Salivation into asieve element during E1 (Prado andTjallingii, 1994) could
preparethe sieve element for sapflow (e.g.injection of anti-coagulants) by
suppressing plant defense (blocking) reactions. Efficacy coulddiffer among
70
CHAPTER 5
Table4.Relationbetweenpenetration siteontheleaf andexudation parameters for
Nasonovia ribisnigri andMyzuspersicae onTaiwanLettuce.
Veinclass
Total Duration(min)
N
N
Myzuspersicae
MainVein
Second order
Third order
Smallest visible vein
Lamina
13
38
21
12
47
Nasonovia ribisnigri
Main Vein
Second order
Third order
Smallest visible vein
Lamina
21
36
18
5
7
17 ± 4
33± 6
69 ±20
51 ±35
49± 14
5
15
8
5
16
Sample(nl)
N
0.6 ± 0.2
0.7 ± 0.1
1.4 ± 0.4
0.8 ± 0.3
0.8 ± 0.3
115+ 22
9
4.0± 1.0
2.9 ± 0.5
75± 10 20
51 ± 14 11
1.5±0.5
50± nd
1
3.9 ± 3.2
25± 5 2
0.7 ± 0.5 2
4
14
6
5
16
Rate(nVs)
N
0.5 ± 0.1
0.3 ± 0.0
0.3 ± 0.1
0.3 ± 0.1
0.3 ± 0.0
4
14
6
4
16
11
0.4 ± 0.1
21
0.5 ± 0.1
10
0.5 ± 0.1
2
0.3 ± 0.1
0.5 ± 0.2 2
10
18
10
2
Values are mean ± standard error. Some discordant values excluded (see text).No
significant differences among veins (Kruskall-Wallis multiple comparison, a = 0.05)
species or aphid-plant combinations. N.ribisnigri might be better adaptedto
"Taiwan" lettucethan M.persicae because it was reared onthis plant.This is
supported bythe fact that the higher outflow ratefound with N. ribisnigri
apparently did not cause exudationto stop sooner whichwould be expected
if plant reactions play a roleinthetermination of longexudations.
N.ribisnigripreferredthe mainveins and larger sideveins whereasM.
persicaepenetrated moreoftenonthe lamina (table4), presumably into
minorveins.This might indicate adifference insuitability or chemical
composition (Rahbé etal., 1990).
Differences amongveins. The sieve elements of different veins might react
differently to penetration bystylets. However, for eachaphidspecies,
exudation rate,samples size and duration of exudation did not differ
PHLOEM SAP COLLECTION
71
significantly amongveins (table 4).Chemical composition canbe different
amongdifferent plant parts (Rahbé etal., 1990).
Summarizing,it seemsthat the differences observed betweenthetwo
species onthe sameplant inexudation rate,duration andsample size are
caused byadifference inaphid-plant interactions ratherthan plant
characteristics or stylet dimensions.
Honeydew
The collection methoddescribed hereiscomparabletothat of Banks and
Macauley (1963) and Fisher etal. (1984).Although manual collection makes
the method rathertime consuming,ityields largequantities of honeydew
(ml), makingquantitative analysis of chemical compounds possible.
Hygroscopic uptakeor evaporation of water was prevented bythe oil layer.
Fisher etal. (1984) suggested adecrease of volume dueto evaporationof
10%in2hoursduring collection in hexadecane, butthis wasconcludedfrom
osmolality measurements, ignoring possible osmolality changes dueto
continued enzymatic activity inthe droplets (Eschrich and Heyser, 1975,Van
Helden etal., 1994).Though unlikely,it is possible that some apolar
components did dissolve inthevery apolar hexadecane. Banksand
Macaulay (1964) reportedaspecific gravity of 1.040g/mlforthe honeydew.
Itistherefore not clear whythe droplets floatedjust under thesurface of their
Castor motor oil (0.89g/ml),but inthe lighter hexadecane (0.77g/ml) sank
immediately.The dry matter content of the honeydew was muchlowerthan
measured for phloem sap (Van Helden etal., 1994) indicating important
changes made bytheaphids.
Differences amongaphid-plantcombinations. The number of aphids which
dropped intothe oil differed amongspecies and lettuce lines. N. ribisnigri
does not produce honeydew onthe resistant lines (Van Helden etal.,1993)
but eventhe partial resistance to M.persicae which is present inthe lines
RES2and SUS2 (Reinink etai, 1988) causedanincreased restlessness
andtherefore more aphids dropped inthe oil and honeydew yieldwasvery
low.
Whenstudying aphid resistance,finding asuitable aphid-plant
combination can bedifficult, anddifferent aphid species will surely bioprocessthe phloemsap differently orfeed onother sieve elements.
72
CHAPTER 5
EDTA chelation
The methodof EDTA chelation,asdescribed by KingandZeevaart
(1974),isapplicablefor lettuce.Howeverthe optimal conditions for lettuce
areobviously different fromthosefor PerillacrispaThunb. Lightexposureof
the leaves during collectionalmost doubledthesugar yield.Atemporary
exposureto EDTAwasapparently not enoughtoestablish exudationfor long
periods.Adding carbondioxide as usedbyTully and Hanson(1979) to
reduce evaporation fromthe leaves didnot increasethe sugar yield.
Ittook afew hoursto reachthe highest rate of exudation whichthen
continuedfor alongtimewith no noticeable decrease (Fig.3b,Groussolet
al.,1986).The influence ofthe uptakeandévapotranspirationofthe
collectionfluidthroughthe leaf onsugar yield isunclear.This method
produces largequantities of sugar withvery littlework involved.Oneplant
withfiveorsix leaves (total mass 6-8 g)yielded around9 mgof sugar
thought to beequivalent toat least 45|il of phloem sap (assuming20%
sucrose). Disadvantages arethe strong dilution ofthe samples andtheir
unknown contamination with substances fromthe cut surface ofthe petioleor
the submerged part ofthe petiole whichshowedsometissuedegradation.
Nolarge scalecontamination of thesamples by lactiferous ductswas
observed. Latex was released immediately after excision but stoppedsoon
andwaswashed off bythe rinsing inwater. Itis unknownwether major
physiological changes of phloem sap occur duetothe excision of the leaves.
Difference amonglines. The lower sugar yield onthe resistant lines may be
caused bythefactthatthe resistant plants wereslightly smaller. Inaddition
totheir lower weight,these smaller leaves may have been physiologically
younger andtherefore give a lower yield per gram of leaf material.Wether
the absence of turgor lossonthe leaves of the plants 411, RESand RES2
comparedto "Taiwan",SUSand SUS2 hasany direct relationwiththe
mechanism of aphid resistance is not clear. However, it may have adirect
relation withthe lower sugar yield.
PHLOEM SAP COLLECTION
73
CONCLUSIONS
Thethree methods producevery different samples,both inquantity andin
quality. Stylectomyisvery laborious.Althoughthesuccess ratioof
amputation and exudationwas exceptionally high ityielded onlyvery small
(but presumably pure) samples,oftentoosmall for analysis.The numberof
cut stylets yielding ausablesample isvery unpredictable andcertain plants
show nolongexudations,inspite ofthefactthat theaphids show sustained
phloemsap ingestion.Honeydewcollection produces relatively large
samples.Agood aphid-plant combination is necessary. Collection in
hexadecane makes quantification of compounds possible. EDTA chelation
produces large but very diluted samples with unknown contaminations.
Excision might influence the phloem sapcomposition.
Which method ispreferred depends largely onthe intended use of the
samples. For major compounds of the phloem sapfor whichsensitive microanalysis methods are available (sugars,amino acids,Van Helden etal.,
1994) thevery purestylectomy samples are optimal. For identification of
(unknown) minor compounds stylectomy samples aresimply toosmall.The
other methods yield larger samples (interms of phloem sap equivalents) but
their reliability is unclear. Noneofthese methods isideal when studying
aphid resistance caused by differences inphloem sap composition as inour
model.When resistance isinduced byaphid attack, collectionwithout using
aphids (EDTA chelation) may not show any significant differences. Using
other aphidspecies (honeydew orstylectomy) might not induce the defence
reaction.Togain moreinsight inthe differences amongsamples fromthe
three collection methods achemical comparison was performed which will be
reported inVan Helden etal.(1994).
REFERENCES
Banks, C.J. and Macaulay, E.D.M., 1964.The
feeding, growth and reproduction of Aphis
fabae Scop, on Viciafaba under
experimental conditions. Ann. Appl. Biol.
53: 229-242.
74
Brady, J., 1965.A simple technique for
making very fine, durable dissecting needles
by sharpening tungsten wire electrolytically.
Bull. W.H.O. 32: 143-144.
Chino, M„ Hayashi, H., Nakumura, S.,
CHAPTER 5
Oshima, T., Turner, H., Sabnis, D„
Borkovec, V., Baker, D„ Girousse, G.,
Bonnemain, J.L. and Delrot, S., 1991.
Phloem sap composition. In: J.L.
Bonnemain, S. Delrot, W.J. Lucas and J.
Dainty (eds). Recent advanced in phloem
transport and assimilate compartmentation,
Quest Editions.
Downing, N. and Unwin, D.M., 1977. A new
method for cutting the mouthparts of
feeding aphids.Physiol. Entomol. (2):
275-277.
Eenink, A.H., Groenwold, R. and Dieleman,
F.L., 1982. Resistance of lettuce (Lactuca)
to the leaf aphid Nasonovia ribis-nigri1:
transfer of resistanca from L.virosato
L.sativa by interspecific crosses and
selection of resistant breeding lines.
Euphytica 31: 291-300.
Eenink, A.H., Dieleman, F.L., Groenwold, R.,
Aarts, P. and Clerkx, B., 1984. An instant
bioassay for resistance of lettuce to the leaf
aphid Myzuspersicae. Euphytica 33:
825-831.
Eschrich, W. and Heyser, W., 1975:
Biochemistry of phloem constituents. In:
Zimmerman, M.H. and Milburn, J.A. (Eds.).
"Encyclopedia of plant physiology" 1.
Transport in plants:Phloem transport.
Springer verlag, Berlin
Fisher, D.B. and Frame, J.M., 1984. A guide
to the use of the exuding-stylet technique in
phloem physiology. Planta 161: 385-393.
Fisher, D.B., Wright, J.P. and Mittler, T.E.,
1984. Osmoregulation by the aphid Myzus
persicae: Aphysiological role for honeydew
oligosaccharides. J. Insect Physiol. 30:387393.
Forbes, A.R., 1977.The mouthparts and
feeding mechanism of aphids. In: Harris,
K.F. and Maramorosch, K. (Eds.). Aphids as
virus vectors. Academic press, New York.
Girousse, C, Bonnemain, J.-L., Delrot, S. and
Bournoville, R., 1991.Sugar and amino acid
composition of phloem sap ofMedicago
sativa L.: a comparative study of two
collecting methods. Plant Physiol. Biochem.
29: 41-49.
PHLOEM SAP COLLECTION
Groussol, J., Delrot, S., Caruhel, P. and
Bonnemain, J.-L., 1986.Design of an
improved exudation method for phloem sap
collection and its use for the study of
phloem mobility of pesticides. Physiol. Vég.
24: 123-133.
Handel van, E., 1967. Determination of
fructose and fructose-yielding carbohydrates
with cold anthrone. Anal Biochem. 19:
193-194.
Hayashi, H. and Chino, M., 1986. Collection
of pure phloem sap from wheat and its
chemical composition. Plant Cell Physiol.
27: 1387-1393.
Helden, M. van, Thijssen, M.H., and
Tjallingii, W.F., 1992.The behaviour of
Nasonovia ribisnigrion resistant and
susceptible lettuce lines. In: Menken, S.B.J.,
Visser, J.H. and Harrewijn, P. (Eds).Proc.
of the 8th Int. Symp. on Insect-Plant
relationships. Kluwer academic press,
Dordrecht, the Netherlands.
Helden, M. van and Tjallingii, W.F., 1993.
Tissue localisation of lettuce resistance to
the aphid Nasonovia ribisnigriusing
electrical penetration graphs. Entomol. Exp.
Appl. 68:269-278.
Helden, M. van, Tjallingii, W.F. and
Dieleman, F.L., 1993.The resistance of
lettuce (Lactucasativa L.) to Nasonovia
ribisnigri (Mosley, Homoptera, Aphididae):
Bionomics of N. ribisnigrion near isogenic
lettuce lines. Entomol. Exp. Appl. 66:
53-58.
Helden, M. van, Tjallingii, W.F. and Beek,
T.A. van, 1994. Phloem sap collection from
lettuce (Lactucasativa): Chemical
comparison among collection methods. J.
Chem. Ecol. 20: 3191-3206
Kawabe, S., Fukumorita, T. and Chino, M„
1980. Collection of phloem sap from stylets
of homopterous insects severed by YAG
laser. Plant cell Physiol. 21: 1319-1327
King, R.W. and Zeevaart, J.A.D., 1974.
Enhancement of phloem exudation from cut
petioles by chelating agents. Plant Physiol.
53:96-103.
Mentink, P.J.M., Kimmins, F.M., Harrewijn,
75
P., Dieleman, F.L., Tjallingii, W.F., van
Rheenen, B. and Eenink, E.H., 1984.
Electrical penetration graphs combined with
stylet cutting in the study of host plant
resistance to aphids. Entomol. Exp. Appl.
35:210-213.
Pate, J.S., Sharkey, P.J., and Lewis, O.A.M.,
1974. Phloem bleeding from legume fruits:
A technique for study of fruit nutrition.
Planta 120:229-243.
Peel, A.J., 1975.Investigations with aphid
stylets into the physiology of the sieve tube.
In: Zimmerman, M.H. and Milbum, J.A.
(Eds.). "Encyclopedia of plant physiology"
1.Transport in plants: Phloem transport.
Springer verlag, Berlin
Prado, E. and Tjallingii W.F., 1994. Aphid
activities during sieve element punctures.
Entomol. Exp. Appl. 72: 157-165
Rahbé, Y., Delobel, B., Calatayud, P.-A. and
Febvay, G.. 1990.Phloem sap composition
of lupine by aphid stylectomy:
Methodology, variation in major constituants
and detection of minor solutes. In: Peters,
D.C., Webster, J.A. and Chlouber, CS.
(Eds.) Proc. Aphid-plant interactions:
populations to molecules. Stillwater,
Oklahoma.
Reinink, K., Dieleman, F.L. and Groenwold,
R., 1988.Selection of lines of lettuce
(Lactuvasativa L.) with a high level of
partial resistance toMyzuspersicae (Sulzer).
Euphytica 37:241-245.
Reinink, K. and Dieleman, F.L., 1989.
Comparison of sources of resistance to leaf
aphids in lettuce (LactucasativaL.).
Euphytica 40:21-29.
Richardson, P.T., Baker, D.A. and Ho, L.C.,
1982. The chemical composition of cucurbit
vascular exudates. J. Exp. Bot.33:
1239-1247.
Tjallingii, W.F., 1988.Electrical recording of
stylet penetration activities. In: A.K. Minks
and P. Harrewijn (eds.), Aphids, Their
Biology, Natural Enemies and Control, vol.
2A, Elsevier Science Publishers B.V.
Amsterdam, pp.95-108.
Tjallingii, W.F., 1990. Continuous recording
76
of stylet penetration activities by aphids.. In:
R.K. Campbell and R.D. Eikenbary (eds.),
Aphid-Plant Genotype Interactions, Elsevier
Science Publishers B.V., Amsterdam.: 8999.
Tjallingii, W.F., 1994.Regulation of phloem
sap feeding by aphids. In: R.F. Chapman
and G. de Boer. Insect Feeding. Behavioral
and physiological mechanisms. Chapman
and Hall,New York, in press.
Tjallingii, W.F. and Hogen Esch, Th., 1993.
Fine structure op aphid stylet routes in plant
tissues in correlation with EPG signals.
Physiol. Entomol 18: 317-328
Tully, R.E. and Hanson, A.D., 1979. Amino
acids translocated from turgid and
water-stressed Barley leaves. Plant Physiol.
64: 460-466.
Unwin, D.M., 1978. Aversatile high
frequency radio microcautery. Physiol,
entomol 3:71-73.
Winter, H., Lohaus, G. and Heldt, H.W. 1992.
Phloem transport of amino acids in relation
to their cytosolic levels in barley leaves.
Plant Physiol. 99: 996-1004
Zimmermann, M.H., 1957.Translocation of
organic substances in trees 1:The nature of
sugars in the sieve tube exudate of trees.
Plant Physiol. 32: 288-291.
CHAPTER 5
6. PHLOEMSAP
CHEMISTRY
ABSTRACT
The chemical composition of phloem sapfrom lettuce,collected bythree
different methods,wascompared.Phloemsapfromstylectomy samples
contained sucrose and 14amino-acids. Honeydew and EDTA chelation
samples showed considerable breakdown of sucrose intofructose and
glucose,several additional amino-acids andlarge differences in relative
concentrations of amino acids,whencomparedtostylectomy samples.
Honeydew contained considerable amounts of other oligosaccharides, and
few proteins inlow amounts,while EDTAsamples showed many proteins.
HPLC chromatogramsshowed numerous unidentifiedsecondary plant
compounds inhoneydew and EDTA samples.Comparison of phloem sap
samples from near-isogenic susceptible and resistant lines showed no
relation of phloem sap composition with monogenic resistancetothe aphid
Nasonovia ribisnigri.
INTRODUCTION
The absolute, monogenic anddominant resistance (Nr-gene) of lettuceto
the aphid Nasonovia ribisnigriis basedonthe interruption of phloem feeding
(Van HeldenandTjallingii, 1993,Van Helden etal.,1992, 1993) probably
PHLOEM SAP CHEMISTRY
77
caused byadifference inchemical composition ofthe phloem sieve element
sap.
The composition ofthephloemsapof lettuce (Lactuca sativaL) canbe
derivedfromsamples collected bystylectomy (Downing and Unwin,1977),
from aphid honeydew (Banks and Macaulay, 1965) andfrom samples
collected by "facilitated exudation"through EDTAchelation (Kingand
Zeevaart, 1974). Inaprevious paper we reported onthe optimisation of
these methodsfor maximum yield (Van Helden eral.,1994),without
comparingtheir chemical composition.The collection of phloem sapby
stylectomy failed ontwosetsof (near isogenic) resistant and susceptible
lettuce lines (lines inoneset differ only inthe Nasonovia resistance gene,
Van Helden etal.,1994).Onasusceptible lettuce line "Taiwan"stylectomy
was successful andsamples of threecollection methods are compared in
this paper.
Chemical comparisons betweenstylectomy and EDTA chelationsap
samples are available forseveral plant species (Weibull etal.,1990,
Girousse etal.,1992).Sofar only Rahbé etal.(1990) comparedthese three
methods simultaneously, concentrating onsugars and amino acids.Though
stylectomy samples areconsidered asthe best representation of real phloem
sap,thistechnique does notwork onall plants ((Van Helden etal., 1994)
andsamples size isvery limited (usually < 1u.l)(Fisher and Frame, 1984,
Van Helden etal.,1994).The reliability of sampiesfrom other methods is
unclear. EDTA chelation samples from excised leaves are contaminated with
compounds releasedfromthe woundsurface,and honeydew isaphloem
sap which is bio-transformed bythe aphid usedforcollection.
The objectives of this study were:(1) Qualitative comparison of three
collection methods (stylectomy, EDTA chelation and honeydew) for sugars,
amino acids,proteins and UVabsorbing secondary metabolites, (2)comparison of phloemsap samples of (near isogenic) resistant andsusceptible lines.
MATERIALS AND METHODS
Samples. Inanearlier paper (Van Helden etal.,1994) we reportedonthe
methodology andyieldof phloem sap collection methods.Samples were col78
CHAPTER 6
lectedfrom lettuce line "Taiwan"for comparison of collection methods,and
two sets of near-isogenic lettuce lines:RES (genotype NrNr) with SUS (nrnr)
and RES2(NrNr) with SUS2(nrnr) respectively,forcomparison of resistant
andsusceptible plants (Van Helden etal.,1993).Thesetwo sets have
distinctly different genetic backgrounds apart fromthesource of the
resistance gene whichoriginatesfrom L.virosaL.
Stylectomysamples (phloem sapvolume 0.1 - 1 uJ,stored in50%MeOHor
2%sulfosalicylic acid) wereavailablefromlettucecv. "Taiwan"usingtwo
aphidspecies, Nasonovia ribisnigriand Myzuspersicae (Sulzer). Honeydew
sampleswerecollected inhexadecane. Samples from lettuce line"Taiwan"
were collected fromthe aphids M.persicae, N.ribisnigriand Macrosiphum
euphorbiae (Thomas).Onthe other lines honeydew was collected from M.
persicae.EDTA sampleswere collected in8 mM EDTA, pH6,yielding 0.20.6 ml/leaf, total sugar content 0.3 -0.9%or around 1.3mg/g of leaf.
Samples werestored at -80 °C (honeydew andstylectomy) or -20 °C(EDTA)
before analysis.Alltests with 2-4 independent replicates pertreatment.
Sugaranalysis was performed witha highperformance anion-exchange
chromatography system (Dionex CarboPac PA-1Column,PED/PADDetector) asdescribed byGruppen etal.(1992),concentrating onsucrose,
glucose andfructose. Samples from stylectomy (±0.5 u.lin 100u.l 50%
MeOH) were lyophilised and redissolved in 1ml H20shortly beforeanalysis.
EDTA samples were diluted 50fold, honeydew samples 1000fold before
analysis. Injectionvolume 20 uJ.
Aminoacidanalysiswas performed with anion-exchange amino acid
analyzer (Beekman). Proteins were removed by adding of (forthe EDTA
samples),or dilution in (forstylectomy and honeydew),2%sulfosalicylic acid
andcentrifugation. Injectedsamples (100u.l)contained =2.5 |xlstylectomy
samples (several collections pooled),5 ]LLIof honeydew, or undiluted EDTA
samples. Some other products (NH3)were alsodetected.
Protein analysis. Proteins in EDTA samples (5 ml) and honeydew (0.5 ml)
fromthe lines RESand SUSwere precipitated by addition of acetone upto
80% (Joosten,1991)andcentrifugation.SDS-PAGEwas performed ona
12% mini-slab gelwitha4%stacking gel.Proteins were silver-stained
accordingto Morrissey (1981).
PHLOEM SAP CHEMISTRY
79
Secondaryplantchemicals. EDTA and honeydew samples were separated
using a250 x4 mm C18RP-HPLC(nucleosil 120-5C18)column (MachereyNagel)witha30 mmpre-columnusingwater/acetonitrileor water/methanol
gradients,or isocratic mixtures withtrifluor acetic acid (TFA, 0.1%, pH2)or
tetra butyl ammonium ions (addedaschloride or hydrogen sulphate,0.5%,
pH5). Flowrate 1ml/min, runtime 35-40 min.UVdetection at 200-400nm
was performedwith adiodearray detector (Waters 911). Injectionvolumes
were 20 uJ of undiluted EDTA samples or 10folddiluted honeydew.
Stylectomy samples were notavailable.
RESULTS
Sugars.
The mainsugar in most samples was sucrose (fig.1Aand B),withthe
exception of some honeydew samples. Fructose andglucose were identified
in nearly allsample types from alllines.
Differences amongcollection methods. Thetotal sugar concentration inthe
stylectomy and EDTAsamples was 13.9to 18.0%(mean 16.2%,n=4) and
0.19to 0.71% (mean 0.41%, n=28) respectively. Inhoneydewthe combined
sucrose,fructose,glucosequantity was 3.4 to 6.2%(mean 5.1%, n=24).
Inthe stylectomy samples nearly all sugar was sucrose and onlytracesof
glucose,fructose occurred (fig.1A,left columns),whilethe EDTA collected
samples showed fructose and glucose in roughly equal shares of 0to 15%of
thetotalsugar content each (fig.1A,B).Variation inprofiles between
replicates was largerthanamong lines. Honeydew samples showed little
variation between replicates but variation amongaphid-plant combinations
was considerable (fig.1A,right bars, Fig 1B).Fructose concentrations were
3-8 times higher thanglucose (fig.1A,B). Large amounts of other
(unidentified) oligosaccharides (possibly representing polymerisation seriesof
glucose containing oligosaccharides, Fisher etai, 1984,Ammeraal etal.,
1991),werefound (not quantified but upto 50%oftotal peak areaof
chromatogram).Asmall unidentified peak (probably a monosaccharide witha
retentiontime betweenthose of glucose andfructose) was sometimes
observed.
80
CHAPTER 6
a 100
H
glucose
I | fructose
f ü sucrose
MP
NR
STYLECTOMY
EDTA
RES
SUS
HDEWEDTA
HDEWEDTA
MP NR ME
HONEYDEW
RES2
SUS2
1
b oo
HDEWEDTA
HDEWEDTA
Figure 1.Relative sucrose/fructose/glucose concentrations of samples from different
collection methods, a. Comparison on "Taiwan" lettuce using stylectomy ofMyzus
persicae (MP) andNasonoviaribisnigri(NR),EDTA chelation, and honeydew ofM.
persicae (MP),N. ribisnigri(NR) and Macrosiphum euphorbiae(ME), b. Comparison of
two sets of isogenic lines, using M.persicae honeydew (HDEW) and EDTA chelation.
PHLOEM SAP CHEMISTRY
81
30
STYLECTOMY
?5
2
F
c
o
TS
rCB
o
c
o
o
• Nasonovia
• Myzus
20
15
10
b
T—i—r
i
11,,11,.iwfti
i i i i i i r
i—iH'1' i n ' r i—i—r
2
EDTA
b
i"
-
trat
c
o
CD
o
i—i
C 0.5
O
n
hr ' T ' - T M' T T ?
0
1 ni—r nr
1
i T1
i
i
TÛT
,nn,ffî,
n
i
i
30
HONEYDEW
25
E.
20
<
c>
o
15
>
10
Ü
(11
CO
CD
tr
• Nasonovia
D Myzus
M Macrosiphum
S
m L j i - fih
T
cr^io-trtrz^
Q
>•
UJ<hCOIUlCfl-l-J<
(r>\-iu<>-<n<Oü<
a.
Q-
C3
< tr 3 -J ço
- j I - £0 < >< n < > Ü
T*i i - r
tr < tr
U 1 3 C t l U < 3 < n 2tr» 0 3 > - Œ O
O
H Ü <
aminoacid
Figure 2. Amino acid composition of phloem sap samples from "Taiwan" lettuce using
different collection methods, a. Absolute concentrations of stylectomy samples ofMyzus
persicae andNasonoviaribisnigri. b. absolute concentrations of EDTA chelation samples.
c. Relative concentrations of honeydew of M.persicae,N. ribisnigriandM.euphorbiae.
82
CHAPTER 6
Differences amongaphidspecies. Stylectomy samples of M. persicaeand
N.ribisnigrishowed near equal concentrations andprofiles ofsugars.
Differences insugar profiles among honeydew samples of aphidspecies
wereconsiderable (fig.1A,right bars).N.ribisnigrihoneydew from"Taiwanshowed nosucrose at all.The combinedsucrose,fructose,glucose quantity
in honeydew from N.ribisnigrion"Taiwan"(5.3%) was higherthanforM.
persicae(3.5%) and M.euphorbiae (3.9%).
Differences amonglettucelines.Fig.1Bshowsthe results of a comparison
ofthetwo sets of isogenic lettuce lines,using EDTAsamples and honeydew
of Myzuspersicae.Stylectomy samples were impossibleto obtainfromthese
lines (Van Helden eral.,1994).Thesugar profiles of the EDTA samplesof
all lines were identical.The lines RESand RES2showedtotal concentrations
of 0.19%and 0.22%respectively against 0.34,0.52 and 0.49%onthe lines
SUS, SUS2and "Taiwan".The honeydew samples showed a high proportion
offructose online SUS2(and"Taiwan",fig. 1A).The combinedsucrose,
fructose,glucose quantity in honeydew from isogenic lines was comparable
(mean5,2%, range 4.2-6.2%) alittle higher thanon "Taiwan"(4,2%)
Amino acids
Differences amongcollection methods. Thetotal concentrations of amino
acids inthe original samples were 54 mMfor honeydew (range 32-72 mM,
n=24),5.4 mMfor EDTA samples (range 2.2-8.8 mM, n=30) andan
estimated 130mM(range 125-168 mM, n=4) for stylectomy samples.Amino
acid profiles of stylectomy samples (fig.2A) showed 14amino acids.Main
amino acids were glutamine,glutamic acid,serine andy-isobutyric acid
(GABA).Ammonia was also present infairly high concentrations (6mM).
Proline was notdetected.Nounexpected peaksweredetected.
EDTA samples on "Taiwan" (fig.2B) consistently showed substantial
quantities of one extraamino acid (arginine,rel.cone.2%) andtrace
quantities of ot-aminobutyric-acid,methionine andtryptophan.Comparedto
stylectomy samples they showed anincrease inthe glutamine/glutamic acid
ratio, lower relative quantities of serine,tyrosine,andespecially of GABA and
ammonia.
Honeydew samples of different aphidspecies on"Taiwan"(fig.2C)
showedthesame amino acids asthe stylectomy samples plustracesof
citrulline andarginine. Relative amounts of alanine,tyrosine, GABA and
PHLOEM SAP CHEMISTRY
83
CO
3
CO
CO
LU
DC
•D
CM
CO
3
CO
•
CM
CO
ULI
<r
H
- uvo
-
AH1
SIH
SA1
NHO
EHN
c
o
vavo
navi-8
viv-a
<
3Hd
Q
W
dAi
(%|OLU) '0UO0 9A!}E|9y
s
ê
"3
o
ammoniawerestrongly reduced,and phenylalanine wassomewhat reduced,
for allaphidspecies.
Differences amongaphidspecies. Amino-acidlevelsand profiles in
stylectomy samples of N.ribisnigriand M.persicae on"Taiwan"were almost
identical (fig.2A). Honeydewamino-acid profilesforthree aphidspecies on
"Taiwan"weresimilar (fig.2C)thoughthetotal concentration variedfrom
32.4 (N.ribisnigri) through 46.9 (M.persicae) to 72.8 mM(M. euphorbiae).
Honeydew of M.persicaeshowedasparagine (2out of 3 replicates) and
lower relative quantities of glutamic acidand higher concentrationsof
leucine.
Differences amonglettuce lines.Comparison of EDTA samples of isogenic
lines showedsimilar amino acid profiles for all lines (fig.3) apart fromthe
presence oftrace amounts of ornithine in RES2and SUS2.Sometimes minor
compounds were not detected (glycine, methionine,tryptophan) but no
relationwith resistance appeared.Thelines RES,RES2,SUSandSUS2
differed from "Taiwan"bythe presence oftraces ofphospho-ethanolamine.
Thetotal concentrations of amino acids inthe EDTAsamples were50%
lower onthe resistant lines (RES 2.3 mM,RES23.6 mM) comparedtothe
susceptible lines (SUS6.3 mM,SUS27.1 mM, "Taiwan"7.4 mM).
Proteins
EDTA and M.persicaehoneydew samples of the lines RESandSUS
showedvery low amounts of protein (EDTA:20|ig/ml, Honeydew: 80 |ig/ml).
SDS-PAGE showedalarge number (>30) of proteins inthe range of 50to
200 kD inthe EDTAsamples. Honeydew contained only afew proteins (510), most of which were alsofound inthe EDTA samples. Nodifference was
observed between resistant andsusceptible plants.
Secondary Plantcompounds
The results fromour analysisfrom honeydew and EDTAsamples showed
many peaks whichwere not primary compounds (sugars,proteins or amino
acids) asjudged bytheir UVspectrum and retention times.A numberof
them probably represented secondary plant compounds. Noattempt was
madeto identify individual compounds but weconcentrated on differences
between resistant andsusceptible lines. Differences between EDTA samples
and honeydew samples from "Taiwan",SUSand RESwere mainly inthe
PHLOEM SAP CHEMISTRY
85
ratio of different peaks. Noconsistent differences wereobserved between
resistant andsusceptible plants.
DISCUSSION
Sugars
Stylectomy. Thestylectomy samples showthat sucrose isthe only sugar
present inthe phloemsapof lettuce,asexpectedfor most plants (Ziegler,
1975).Thesmall quantities of fructose and glucose occurring inthe
stylectomy samples of M.persicae areprobably the result of a contamination
ofthe sample by micro-organisms (invertase) duringcollection. Invertase is
not present inthe phloem sap (Ziegler, 1975).The size of the stylectomy
samples was difficult to determine exactly (Van Helden etal.,1994).
Therefore, our measurements are only anestimate of the exact quantity of
sugars.Ourfindings (16.2%) correspondwell withanexpectedvalue of 1520%.
EDTA. The near equal amounts of fructose and glucose inthe EDTA
samples showthe result of sucrose breakdown. Invertase activity inthe
sample can occur dueto microbial contamination or release of plant
enzymes fromthe woundsurface ofthe leaf. Thoughthe sugar concentration
inthe EDTA samples is muchlower than in honeydew, EDTA samples area
better representation of the phloem sugars (see below), andthey are also
easier to collect.Assuming 16%sugar (assucrose) inthe real phloemsap
EDTAsamples are approximately diluted 40fold.
Honeydew. Honeydew sugar composition isvery different from phloem sugar
dueto biotransformation bythe aphid,the combined activity of invertase from
the aphids alimentary tract (Srivastava andAuclair, 1962b),and
biotransformation.The resultingtotal sucrose/fructose/glucose content ofthe
honeydew (5%) is low compared tothestylectomy samples.A higher
concentration was expected (Lamb and Kinsey, 1959; Peel,1975).
Sometimes atotal breakdown of sucrose inthe honeydew occurred with
nearly all glucose "missing" (e.g.honeydew of N.ribisnigrifrom "Taiwan").
Reports on honeydew sugars,from "dry" collections nearly allshow sucrose,
but atotalabsence of sucrose hasbeen reported aswell (Auclair, 1963;
86
CHAPTER 6
Lamb, 1959). Enzymatic activity (invertase) inthe honeydew hasbeen
reported (Srivastava andAuclair, 1962b;Rahbé etal.,1994). Fisher etal.
(1984) suggestedthat after invertase actionalarge proportion ofthe glucose
istransformed bythe aphidtoglucosecontainingoligomersfor
osmoregulation.This is inlinewiththe observed predominance of fructose
over glucose,andthe presence of largequantities of higher order
oligosaccharides in honeydew samples (polymerization series of 5to 7
peaks, retention timessuggest one glucose unit extrafor eachpeak).
Identification andquantification ofthese oligomers isdifficult butthe peak
area ofthese compounds suggeststotalconcentrations of upto 5% (w/v)
(Ammeraal eral.,1991)or 50%of allsugars present. It is notclear whether
the sucrose breakdown andglucose polymerization occurred exclusively in
the aphids alimentary tract or continue after excretion.The increase in
osmolality of honeydew droplets asobserved by Fisher etal.(1984) (see
also Van Helden etal.,1994) suggests that invertase activity doescontinue.
Enzymatic activity inthe honeydew could also becaused by microbial
contamination which isprevented bythe hexadecane cover, but might occur
inthe aphidalimentary tract (Grenier etal.,1994,Srivastava and Auclair,
1962a).
Differences amonglettucelines. The observed difference insugar concentration between EDTAsamples of resistant andsusceptible plantswasnot
reflected inthe honeydew samples.This suggeststhat the difference is
caused byvariation inthe efficiency ofthe EDTA collection method rather
than adifferent sugar concentration inthe phloemsap (also confirmed bythe
equally lower amino-acidconcentration (see hereafter). However, agenetic
coupling seemsto exist since resistant lines of bothsets possessthistrait
(Van Helden etal.,1994), possibly asaleftover fromthe introduced partof
the L. virosa genome (Eenink era/. 1982a,b).
Differences amongaphidspecies. The differences in honeydew sugars
among aphids on"Taiwan"are remarkably large,asarethe differences
between lettuce lines.Thissuggests anextensive difference in
"biotransformation" ofthe phloemsugar, by differences ininvertase activity
(Srivastava andAuclair, 1962b) andglucose utilization (for assimilation and
bypolymerization) amongaphidspecies.Another possibility isthat the
amount of phloemsap ingested,andtherefore,the degree of utilisation is
PHLOEM SAP CHEMISTRY
17
very different whichcould bechecked by acomparison of honeydew
production.
Summary. Forsugars onlystylectomy gives agood representation.EDTA
samples areanacceptable substitute,though collection efficiency can differ
among lines. Honeydew sugars cannot becomparedto real phloemsap.
Sugar composition or concentration insamples from different the lettuce lines
cannot explainthe resistance.
Amino acids
Stylectomy. The estimated 125mMof amino acids inthe phloem sap is in
the same range asfoundfor most other plants (Ziegler, 1975; Rahbé eral.,
1990, 1994;Riens etal.,1991).The resemblance inamino acid profiles from
stylectomy samples ofthetwo different aphidspecies seems self-evident.
Still,differences between species could have occurred dueto different
"preparation" of asieve element prior to sap ingestion andthus stylectomy
(Prado andTjallingii, 1994),or different aphid species selecting different
sieve elements for feeding (Van Helden etal.,1994). More sensitive methods
for amino acid analysis (Riens etal.,1991)would haveallowed analysisof
individual stylectomy samples andbetter quantification oftraceamounts.
Differences between samples may have been reduced bythe poolingof
samples.
The presence of relatively largequantities of GABA inthe stylectomy
samples issomewhat surprising.Chino era/. (1991), Rahbé etal.(1990) and
Girousse etal.(1991) claim GABA to beanartifact of the EDTA extraction
method.GABA has beenfound in honeydew on many occasions (seeAuclair
1963).Sofar,the relative amount of GABA reported instylectomy samples
hasalways been lowerthan in EDTA samples.Ourfindings suggest that
GABA isone ofthe major amino acids present inlettuce phloem sap,though
our identification isonly based onretention times.The concentration of
ammonia inthe stylectomy samples is higher thanexpected,other reports
showed lower amounts (Kuo-Sell, 1989;Weiner etal.,1991).This may be
explained by our collection method,which prevents evaporation.
Contamination by atmospheric ammonia was prevented bythe oil layers,so
the ammonia iseither present inthe phloem sap or a result of breakdownof
nitrogenous compounds duringcollection andstorage.
88
CHAPTER 6
EDTA.It is notevident fromour resultsthat the amino acids foundin EDTA
samples areof phloemoriginand notfromother plant parts.The relative
concentrations of amino acids inphloemsapis usually not morethana
factor 2different fromthecytosol (Riens etal.,1991;Winter etal.,1992;
Weiner etal.,1991). However, it seems unlikely that (except for
macromolecules likeproteins) somecompounds present inthe phloem sap
wouldexude (sugars) andothers not.This isconfirmed bythefactthat
concentrations of bothsugars andamino-acidsare 50%lower inthe RES
and RES2samples.The presence of arginine andtraces of 3otheraminoacids,whichwere notfound instylectomy or honeydew samplessuggest that
contamination occurs,though amounts might have been underthe detection
limit inthe stylectomy samples.
Increase intheglutamine/glutamicacid ratio in EDTA (and honeydew)
samples has been reported byseveralauthors (Weibull etai, 1990;
Girousse etal.,1991).The EDTAsamples might differ from the other
collection methods becausethe phloemsap iscollected at other locations on
the plant,which might haveadifferent amino acid profile.This wasshownfor
oat (Kuo-Sell, 1989),but notfor lupin (Rahbé etal., 1990).
Honeydew. Honeydewamino acids arethe result of utilisationand
biosynthesis of new aminoacids (Sasaki etal.,1990).Total concentrations in
the honeydew are 50to 75%lowerthan inthe phloem sap.Thevolumeof
the excreted honeydew issupposedly lessthanthe actual ingestedvolume.
Tjallingii (1994) suggests around50%,butthe difference, mainly dueto
evaporation,will bevariable.This showsthat avery large proportion is
utilised bythe aphid.Surprisingly,the ammonia concentration inthe
honeydew is lowerthan inthestylectomy samples suggesting that the
ammonia isof plant origin rather thanaresult of amino acid breakdown by
the aphid (Sasaki era/., 1990).
Differences amongaphidspecies. Theamino acidprofiles of honeydew of
different aphidspecies showeddifferences inbiotransformation of amino
acids among species (e.g.low glutamineandglutamic acid and high leucine
and isoleucine concentration for M.persicae).However, the differences in
total concentration of amino acids were much larger.
Differences amonglines.Comparison ofthe EDTAsamples of resistant and
susceptible lettuce showedvery little differences amongtwo lines ofthe
PHLOEM SAP CHEMISTRY
89
sameset andlittle variation between sets,apart fromtraces of ornithine in
the lines RES2and SUS2 (fig.2). Differences with "Taiwan"lettuce (fig1C)
aresomewhat larger. Resistance isobviously not basedonaminoacids.
Summary. Both honeydew and EDTAsamples are not avery goodrepresentation of phloem sap amino-acids. Itisdifficult tojudge which sampling
technique isabetter representation,anddifferences inamino acids in
honeydew from different aphids arevery large.
Proteins
EDTA.Based uponthesugar content,Van Helden etal.,1994estimated
that EDTA samples are a40folddilution ofthe realphloem sap.The
observed protein concentration of around 20|xg/mlwould meanthat the
lettuce phloem sap contains around0.8 mg/mlof protein,which is inthe
same range as reportedforthe phloem sap of several plant species collected
by incision (Ziegler, 1975) orstylectomy (Fisher etal.,1992).The numberof
proteins isvery high but comparableto Fisher etal.(1992) who reported
several hundreds of different proteins in phloem sap from wheat, collectedby
stylectomy orfrom brokenpedicels.
It is not evident that allthese proteins are normally present and mobile in
the phloem sap.The loss of turgor pressure upon incision of aphloem strand
(andpresumably also excision of aleaf for EDTAsamples or stylectomy)
might causeconsiderable leakage of enzymes fromthe surrounding phloem
bundle cells intothe sieve elements (Eschrich and Heyser, 1975),and
proteins whichare normally immobile inthesieve elements, incorporatedor
boundtostructural elements (e.g.P-proteins fibrils) can be released bythe
wounding or EDTA action.Tjallingii and Hogen Esch (1993) observed
filamentous structures, probably P-proteins, blocking the food canalof
amputated aphidstylets. Finally,contamination of EDTA samples canoccur
with proteins releasedfromthe woundsurface.
Honeydew. The honeydew showed low amounts andfew proteins compared
tothe EDTA samples.Aphid alimentary tracts do not contain proteinases
(Srivastava andAuclair, 1963)so proteins ingested bythe aphidare
expected to passthe aphid unchanged or accumulate inthe aphid (Rahbé
and Febvay, 1993). Itseems unlikely that nearly all proteins which were
present inthe EDTA samples were accumulated. Ifthe aphidwould ingest
phloem sapwith800u.g/ml(EDTA sample estimate) of protein and excreteit
90
CHAPTER 6
without metabolising,thenthe protein content of the honeydew wouldbe
higherthanthevalue found inour experiments (80 u.g/ml).Normal aphid
feeding ratesare afactor 10lowerthanthe outflow from amputated stylets
(Van Helden etal.,1994;Tjallingii, 1994) inwhichcase no leakingof
proteins fromthe companion cellstothe phloemsieve element is expected
(Eschrich and Heyser, 1975) and nowound reaction (P-proteingelation,
responsible forthefirst fast wound reaction) will occur.This confirmsthe
hypothesisthat the EDTAsamples contain proteins which are normally not
present or not mobile inthe phloemsap and aretherefore not ingestedby
the aphid.Additional proteins,present inthe honeydew but not inthe EDTA
samples,are produced bythe aphidor itssymbionts.
Summary. Moreexperiments are necessary tojudge whichsampling method
providesthe best representation of the proteins of phloem sap (oraphid
food). Honeydew might bethe best sample,even better thanstylectomy,
though some proteins might accumulate intheaphid.
Secondary Plantcompounds
The main reasonfor aphidsto usephloemsap asfoodsource isthought
to bethe absence or low concentrations of secondary compounds inthe
phloem sap ascomparedtoother plant parts. Many different groupsof
secondary substances have been reportedfrom lettuce (phenolics;
flavonoids;sesquiterpene lactones;alkaloids;sterols,Gonzalez, 1977),but
nothing is known about their presence inthe phloemsap.
The analysis method using HPLCwithadiode array detector islimited,
only substances having UVabsorption,present insufficiently highamounts
andseparatedwith our column/solvent system doshow up inthe
chromatogram.
The resemblance between EDTA and honeydew samples indicatesthat
aphids doexcrete most ofthe ingestedsecondary substances unchanged
throughthe honeydew. Atthe sametime it confirmsthat EDTAsamples are
mainly of phloem origin,thesecondary compounds originating fromthe
phloem sap were not overrated by contamination fromthe woundsurfaceby
highconcentrations from vacuoles of damagedcells.The effect of aphid
digestion onsecondary compounds is unclear although several secondary
plant compounds are reportedto pass unchanged (Molyneux et al. 1990).
Polyphagous aphids might possess detoxification mechanisms breaking
PHLOEM SAP CHEMISTRY
9?
downsecondary compounds andleaving notraces inthe honeydew while
monophagous species do not accept these plants. Hussain etal.(1974)
reported 18different phenolic acids present inaphid honeydew feeding on
radish,five of whichwerefound in radishseedlings together withfour other
phenolic acids.Therefore,they suggestedthat most ofthe phenolics present
inhoneydew were breakdown products. However,they usedwhole plants
and not phloem sapsamples. Compounds andconcentrations inphloemsap
areexpectedto differ widelyfromwhole plantsamples.
Difference amonglettucelines. Wewere unabletoshow consistent
differences between lettuce lines.Therefore a relation with resistance isnot
clear. Because of the limitations ofthe analysis methodthis is noproof that
secondary plant compounds are not involved in resistance.
Overallcomparison ofcollection methods.
The outcome ofthe chemical analysis of different compounds revealsthat
the question of which collection methods is most reliable cannot beanswered
easily and differs amongchemicalgroups.
Stylectomy. Sofarstylectomy hasbeenthought to represent the real phloem
sap. However, it is possiblethat the aphid changes the composition of the
phloem sap prior to or duringfeeding.Auclair (1963) reports ona 10-50%
reduction ofthe amino acidcontent ofthe honeydew dueto crowded aphid
attack.The aphid's penetration behaviour, together with salivary sheath
formation andsalivation intothe sieve element (PradoandTjallingii, 1994)
might influencethe plant. Phloemsapcollected bystylectomy depicts the
phloem sap of anaphidattacked sieve element. Eventhen,some
compounds normally immobile (proteins) might betranslocated dueto the
high outflow rate (orthesuddenturgor changes),which is much higher than
under normalaphidfeedingcircumstances.Thetime-consuming and often
rather uncertain yields (Van Helden etal.,1994;Fisher and Frame, 1984)
are anobvious disadvantage.Thesmall sample size and shortageof
replicates pose aproblem forthe chemical analysis of many compounds,
especially secondary plant substances.
EDTA.EDTA collected samples appear to beagood representationof
most compounds of the phloem sap apart from proteins.What exactly
happens withthe proteins remains obscure.Attention should bepaidto
92
CHAPTER 6
possible reactions inthecollectionvials (invertase,oxidation) andleakageof
compounds from other cells.Thequality might be improved by adding more
inhibitory compounds (antioxidants,enzyme inhibitors) tothesolution,
thoughthese compounds might influencethe metabolism of the leaf. Leaking
of compounds fromthewoundsurface seems limited,evenfor secondary
compounds.Washing ofthesurface after cutting isadvisable.Cuttingthe
petiole againafter ashort EDTAexposureto enhance exudation (Girousseet
al.,1991) isunnecessary andwillcauseextracontaminationfromthewound
surface. EDTAcollection is byfarthe easiest method,yieldingthe largest
quantities of phloemsapequivalents.
Honeydew. Honeydew is"bio-processed" phloem sap.The profile and
concentration of sugars andamino acids differs profoundly fromthe real
phloem sap. Biotransformation occurs inthe aphidand might evenbe
continued inthe honeydew. Contamination ofthe honeydew could alsobe
caused by ingestionfromothertissues duringthe cell-punctures which occur
duringthe penetrationtowards the phloem.The number of these punctures is
high (Tjallingii and Hogen Esch,1993) andthetime spent penetrating
towards the phloem isalarge part of feeding behaviour (Van Heldenand
Tjallingii, 1993). If ingestionoccursduringthese punctures it will befrom
vacuoles wherethe concentration of secondary compounds is high.Toavoid
possible contamination,honeydew should becollected preferably from
individuals which have beenfeeding continuously for a longperiod.
Honeydew collection is mucheasierthanstylectomy and usually a
homopteraninsect feeding onthe phloem can befound.The honeydew
collection methodcoversthe possibility of short term or local inductionof
plant reactions whichthen might cause differences in honeydew composition.
Comparison oflettucelines: Variation inphloem sap compounds seemsvery
limited between lettuce lines,though minor differences do occur. No relation
with resistance has beenobserved sofar. Comparison of phloem sapfor
unknownsecondary compounds proved notto beauseful methodto find
resistance factors duetotheinfinite number of possibilities.The most
promising optionto show chemical differences relatedto resistance,would
beto attempt to develop abioassay using honeydew (Dorschner and Kenny,
1993) or EDTA samples addedto anartificial diet,guiding chemical isolation
and identificationtechniques.
PHLOEM SAP CHEMISTRY
93
REFERENCES
Ammeraal, R.N., Delgado, G.A., Tenbarge,
F.L. and Friedman, R.B., 1991.Highperformance anion-exchange
chromatography with pulsed amperometric
detection of linear and branched
oligosaccharides. Carbohydr. res. 215: 179192.
Auclair, J.L., 1963.Aphid feeding and
nutrition. Ann. Rev. Entomol. 8:439-490.
Banks, C.J. and Macaulay, E.D.M. 1965.The
ingestion of nitrogen and solid matter from
Vicia fabae by Aphisfabae Scop.. Ann.
appl. Biol. 55:207-218.
Chino, M , Hayashi, H., Nakumura, S.,
Oshima, T., Turner, H., Sabnis,D.,
Borkovec, V., Baker, D., Girousse, G.,
Bonnemain, J.L. and Delrot, S., 1991.
Phloem sap composition. In: J.L.
Bonnemain, S. Delrot and W.J. Lucas & J.
Dainty (eds).Recent advanced in phloem
transport and assimilate compartmentation,
Quest Editions
Dorschner, K.W. and Kenny, S.T. 1992.
Occurrence of hop aphid phago-stimulants
in the honeydew of aphids. Entomol. exp.
appl. 65:49-55.
Downing, N. and Unwin, D.M., 1977. A new
method for cutting the mouthparts of
feeding aphids. Physiol. Entomol. (2):
275-277.
Eenink, A.H., Groenwold, R. and Dieleman,
F.L., 1982a. Resistance of lettuce (Lactuca)
to the leaf aphid Nasonovia ribisnigri 1:
transfer of resistanca from L. virosato L.
sativa by interspecific crosses and selection
of resistant breeding lines. Euphytica 31:
291-300.
Eenink, E.H., Groenwold, R. and Dieleman,
F.L.„ 1982b. Resistance of lettuce (Lactuca)
to the leaf aphid Nasonovia ribisnigri2:
Inheritance of the resistance. Euphytica 31:
301-304.
Eschrich, W. and Heyser, W., 1975. Sealing
systems in phloem. In: M.H. Zimmermann
and J.A. Milburn (eds.), Transport in plants
94
1.Phloem transport. Springer, Berlin
Fisher, D.B.and Frame, J.M., 1984. A guide
to the use of the exuding-stylet technique in
phloem physiology. Planta 161:385-393.
Fisher, D.B., Wright, J.P. and Mittler, T.E.,
1984. Osmoregulation by the aphid Myzus
persicae: Aphysiological role for honeydew
oligosaccharides. J. Insect Physiol. 30:387393.
Fisher, D.B., Wu, Y., and Ku, M.S.B., 1992.
Turnover of soluble proteins in the wheat
sieve tube.Plant. Physiol. 100: 1433 - 1441.
Girousse, G, Bonnemain, J.-L., Delrot, S. and
Bournoville, R., 1991.Sugar and amino acid
composition of phloem sap of Medicago
sativa L.: a comparative study of two
collecting methods.Plant Physiol. Biochem.
29: 41-49.
Gonzalez, A.G., 1977. Lactuceae, a chemical
review. In: Harborne, J.B. and Turner, B.L.
(Eds.). The biology and chemistry of the
compositae (2). Academic Press, New York:
1081-1095.
Grenier, A.-M, Nardon, C. and Rahbé, Y.,
1994. Observations on the micro-organisms
occurring in the gut of the pea aphid
Acyrtosiphonpisum. Entomol. Exp. & Appl.
67: 91-96.
Gruppen, H., Hoffmann, R.A., Kormelink,
J.M., Voragen, A.G.J., Kamerling, J.P. and
Vliegenthart, J.F.G., 1992. Characterization
by H-NMR-spectroscopy of
enzymatically-derived oligosaccharides from
alkaline-extractable wheat-flour
arabinoxylan. Carbohydrate research 233:
45-64.
Helden, M. van, Thijssen, M.H., and
Tjallingii, W.F., 1992.The behaviour of
Nasonovia ribisnigrion resistant and
susceptible lettuce lines. In: Menken, S.B.J.,
Visser, J.H. and Harrewijn, P. (Eds). Proc.
of the 8th Int. Symp. on Insect-Plant
relationships. Kluwer academic press,
Dordrecht, the Netherlands.
Helden, M. van, Tjallingii, W.F. and
CHAPTER 6
Dieleman, F.L., 1993.The resistance of
lettuce (Lactucasativa 1.) toNasonovia
ribisnigri (Mosley, Homoptera, Aphididae):
Bionomics of N. ribisnigrion near isogenic
lettuce lines. Entomol. Exp. & Appl (66):
53-58.
Helden, M. van and Tjallingii, W.F., 1993.
Tissue localisation of lettuce resistance to
the aphid N. ribisnigriusing electrical
penetration graphs. Ent Exp. & Appl: 68:
269-278.
Helden, M. van, W.F.Tjallingii, Beek, T.A.
van, 1994. Phloem sap collection from
lettuce (Lactucasativa): methodology and
yield. J. Chem Ecol. 20: 3173-3190
Hussain, A., Forrest, J.M.S. and Dixon,
A.F.G., 1974. Sugar, organic acid, phenolic
acid and plant growth regulator content of
extracts of honeydew of the aphid Myzus
persicae and of its host plant,Raphanus
sativus. Ann. Appl. Biol. 78: 65-73.
Joosten, M.H.A.J., 1991.TheCladosporium
fulvum - tomato interaction: Physiological
and molecular aspects of pathogenesis.
Thesis Wageningen Agricultural University
King, R.W. and Zeevaart, J.A.D. 1974.
Enhancement of phloem exudation from cut
petioles by chelating agents. Plant Physiol.
53: 96-103.
Kuo-Sell, H.-L., 1989. Aminosäuren und
Zucker im Phloemsaft verschiedener
Pflanzenteile van Hafer (Avenasativa) in
Beziehung zur Saugortpreferenz von
Getreideblattlausern (Hom., Aphididae). J.
Appl. Entomol. 108: 54-63.
Lamb, K.P. and Kinsey, M.G., 1959.
Composition of the honeydew of the aphid
Brevicoryne brassicae (L.) feeding on
swedes (Brassica napobrassicaDC). J. Ins.
Physiol. 3: 1-13.
Molyneux, R.J., Campbell, B.C. and Dreyer,
D.L., 1990.Honeydew analysis for detecting
phloem transport of plant natural products.
Implications for host-plant resistance to
sap-sucking insects. J. Chem. Ecol. 16:
1899-1909.
Morrissey, J.H., 1981. Silver stain for proteins
in Polyacrylamide gels: a modified
PHLOEM SAP CHEMISTRY
procedure with enhanced uniform
sensitivity. Anal. Biochem. 117:307-310.
Peel, A.J., 1975.Investigations with aphid
stylets into the physiology of the sieve tube.
In: Zimmerman, M.H. and Milbum, J.A.
(Eds.). "Encyclopedia of plant physiology"
1.Transport in plants: Phloem transport.
Springer verlag, Berlin
Prado, E. and Tjallingii W.F., 1994. Aphid
activities during sieve element punctures.
Entomol. Exp. Appl. 72: 157-165
Rahbé, Y., Delobel, B., Calatayud, P.A. and
Febvay, G. 1990. Phloem sap composition
of lupine analyzed by aphid stylectomy:
methodology, variations in major
constituants and detection of minor solutes.
In: D.C. Peters, J.A. Webster & CS.
Chlouber (eds.), Proc. Symposium
Aphid-Plant Interactions: Populations to
Molecules, Stillwater, Oklahoma, U.S.A.
Rahbé, Y. and Febvay, G. 1993.Protein
toxicity to aphids: an in vitrotest on
Acyrtosiphonpisum. Entomol. Exp. & Appl.
67: 149-160.
Rahbé, Y., Febvay, G., Delobel, B. and
Bonnot, G., 1994. Amino acids and proteins
as cues in aphid-plant interactions.
Proceedings of the XIX international
congress of Entomology 1992. Homopteran
feeding behaviour: Recent research advances
and experimental techniques, in press
Riens, B.,Lohaus, G„ Heineke, D. and Heldt,
H.W., 1991.Amino acid and sucrose
content determined in the cytosolic,
chloroplastic, and vacuolar compartments
and in the phloem sap of spinach leaves.
Plant Physiol. 97:227-233.
Sasaki, T., Aoki, T., Hayashi, H. and
Ishikawa, H. 1990. Amino acid composition
of the honeydew of symbiotic and
aposymbiotic pea aphids Acyrtosiphon
pisum. J. Insect Physiol. 36: 35-40.
Srivastava,, P.N. and Auclair, J.L., 1962a.
Amylase activity in the alimentary canal and
honeydew of the pea aphid, Acyrtosiphon
pisum. J. Ins. Physiol. 8: 349-355.
Srivastava, , P.N. and Auclair, J.L., 1962b.
Characteristics of invertase from the
95
alimentary canal of the pea aphid,
Acyrtosiphonpisum. J. Ins. Physiol. 8:527535.
Srivastava,, P.N. and Auclair, J.L., 1963.
Characteristics and nature of proteases from
the alimentary tract of the pea aphid,
Acyrthosiphonpisum. J. Ins. Physiol. 9:
469-474.
Tjallingii, W.F., 1994.Regulation of phloem
sap feeding by aphids. In: R.F. Chapman
and G. de Boer. Insect feeding: Behavioral
and physical mechanisms. Chapman and
Hall, New York, in press
Tjallingii, W.F. and Hogen Esch, Th., 1993.
Fine structure of aphid stylet routes in plant
tissues in correlation with EPG signals.
Physiol. Entomol 18:317-328.
Weiner, H., Blechschmidt-Schneider, S.,
Mohme, H., Eschrich, W. and Heldt, H.W.,
1991. Phloem transport of amino acids.
Comparison of amino acid contents of
maize leaves and of sieve tube exudate.
Plant Physiol. Biochem. 29(1): 19-23.
Weibull, J., Ronquist, F. and Brishammar, S.,
1990. Free amino acid composition of leaf
exudates and phloem sap. Plant Physiol. 92:
222-226.
Winter, H., Lohaus, G. and Heldt, H.W.,
1992. Phloem transport of amino acids in
relation to their cytosolic levels in barley
leaves.Plant Physiol. 99:996-1004.
Ziegler, H., 1975.Nature of transported
substances. In:M.H. Zimmerman & J.A.
Milburn (eds.),Transport in plants. I.
Phloem transport, p. 59-100. Springer,
Berlin
96
CHAPTER 6
7. THEDEVELOPMENT
OFA BIOASSAY
ABSTRACT
The resistance of lettucetothe aphid Nasonovia ribisnigri(Mosley) is
located inthe phloem.Since chemical analyses ofthe phloem sap had
shown nodifferences between resistant andsusceptible lines,abioassay
was developed inorder totest samplesfrom resistant andsusceptible plants
onaphidfeeding.Whole plant extracts, honeydew, and EDTA collected
phloem extracts weretriedand asensitive bioassay was developed using
EDTA samples.The EDTAwas removed,samples were addedto asimple
sucrose solution orto acomplex artificial diet, and presented inachoice
situation comparing extracts from resistant andsusceptible plants.Samples
from susceptible plants were preferredtothose from resistant plants.The
resistance is probably based onafeeding deterrent action ofthe phloemsap
inthe resistant plant.
INTRODUCTION
Resistance of plantsto aphids isdifficult to investigate because ofthe
highly specializedfeeding behavior ofthese insects. In many casesthe
resistance is basedonaninterruption of thefeeding behavior after the
phloem sieve element hasbeen reached bythe insects mouthparts
THE DEVELOPMENT OF A BIOASSAY
97
(Kimmins, 1989;Padgham etal.,1990;Caillaud etal.,1992;Cole, 1993;Van
HeldenandTjallingii, 1993). Elucidation of the (chemical) background ofthe
resistance mechanism isvery difficult (Dreyer and Campbell 1987,Harrewijn,
1990,Wink andWitte, 1991).Sofar investigations into resistanceto
homopteraninsects have often usedcorrelations oftotal plant levels of
certain chemical compounds with performance traits (Todd etal.,1971;
Argandona, 1980;Dreyer andJones, 1981; Herrbach, 1985;Leszczynski et
al.,1985;Niemeyer, 1990;Kanehisa étal., 1990,Luczak andGaweda,
1993). Phloem sap,the only plant component ingested asfoodbytheaphid,
will beonly asmall part oftotal plant extracts.When resistance is basedon
achemical component ofthe phloem sap,it will very likely be lost inwhole
plant preparations.
The absolute,monogenic resistance of lettuce {Lactuca sativaL.) tothe
aphid N.ribisnigri(Nr-gene,Eenink era/., 1982;Reinink and Dieleman, 1989;
Van Heldenetal.,1993) isbasedonanearly interruption of sap uptake after
the aphidstylets have reachedaphloemvessel (Van HeldenandTjallingii,
1993).Although a mechanical resistance mechanism,like blocking of the
stylets or sieveelements during attempted feeding,cannot beexcluded asa
possible resistance mechanism,our current working hypothesis isthat a
chemical difference inthe phloem sap may interfere with host plant
recognition or acceptance inthe resistant plants (Van HeldenandTjallingii,
1993;Van Helden etal.1994a,b;Wink andWitte 1991).
Phloem sapsamples were obtained bystylectomy, EDTA chelation and
honeydew collection,andanalysed for sugars,amino acids,proteins andUV
absorbing secondary plant compounds,whichshowed noconsistent
differences between resistant andsusceptible lettuce lines (Van Heldenet
al.,1994a,b).Lettuce contains many secondary compounds (Gonzalez,
1977) but little is knownabout their presence inthe phloem andtheir
biological activity. Exhaustive analysis of secondary compounds inthe
phloem sap of lettuce,without knowledge ofthe nature of the active
substances (allomones),would bevery timeconsuming.
The development of abioassay using EDTA collected phloem sap
extracts ofthe plants asdescribed here,might makea"classical"bioassayguided-fractionation, isolationandidentification ofthe allomones involved in
the resistancefeasible.
98
CHAPTER 7
MATERIALS AND METHODS
PlantsandAphids
Plants weregrownandaphids were rearedas described earlier (Van
Helden etal.,1993,1994a). Synchronized larvae were usedwhenfive days
old (L3stage) unless otherwise stated.Two setsof near isogenic lettuce
lines were used:SUS(ceptible,genotype nmr) and RES(istant,NrNr) (SET1),
SUS2 (nrnr) and RES2 (NrNr) (SET2),lettuce line "Taiwan" (susceptible,
nmr) and oilseed rape cv. "Olymp" (Brassica napusL.) (Van Helden etal.,
1994a,b).Thetwo isogenic setsaregenetically distinctly different (except for
the source ofthe Nr-gene,Van Helden etal.,1994a).
Plantandphloem extracts
Whole plantextracts. Whole plants were heated ina microwave ovenfor
20seconds. Sapwas pressed fromthe leaves,filtered and useddirectly.
Methanol extracts were preparedfrom 0.2 glyophilized leaves and 10 mlof
MeOH,followed by ultrasonic mixingforseveral minutes,centrifugation,
filtration andvacuumdrying.
Honeydew. Honeydew from oilseed rapeand "Taiwan"lettuce was
collected using M.persicae Sulzer (biotype WM1) andfrom the lines RES
and SUS using M.euphorbiae Thomas (biotype WMe1). Honeydew was
collected in hexadecane (Van Helden etal.,1994a),stored at -80 °Cand
lyophilized priorto use.Occasionally honeydew was collected directly on
Petri dishes which were placed underneath leaves with large quantities of
aphid larvae.This "dry collected" honeydew was dissolved ina water/MeOH
(1:1) mixture, MeOHwas evaporated in vacuo andthe aqueous extract was
lyophilized.
Samplepreparation fromcrudeEDTA extracts. Phloem sap exudatewas
collected by placing excised leaves inanaqueous solution with8 mMEDTA
and 5 mMphosphate buffer at pH6for 20 h(King andZeevaart, 1974;
Groussol etal.,1986;Van Helden etal.,1994a). Initially 5 mMsodium
metabisulfite (Na2S205) was usedasananti-oxidant but later thiswas
omitted because of toxicity tothe aphids.The "crude EDTA extract" was
stored at -20 °C.To removethe EDTA fromthe crude EDTA extract the
following procedure was used:Aweak cation exchange column (Carboxylic
acid) was activated with 5mlammonium buffer (pH 10),washed withH20
THE DEVELOPMENT OF A BIOASSAY
99
and loadedwith 10mlof a0.01 Mollead nitrate solution.After washing with
H 2 0,10 mlof crude EDTA extract wasaddedand2 mlfractions were
collected. Fractionsweretestedforthe presenceof EDTA (seebelow),
EDTAfreefractions (usually thefirst 4) werecombined,lyophilized and
stored at -20°C until use.These EDTAfreesamples will be referredtoas
"Ed-samples" (EDTA derived samples) throughout this paper.After cleaning
with 10%formic acidinwater,the columns werereused.
Controltreatments: Together withthe Ed-samplestwo different control
treatments were used.Thefirst was ablank control ("BLANK") (nothing
added).Thesecondcontroltreatment ("CONTROL") was usedto
compensate for the amino-acidsandsugars inthe Ed-samples,andto
counterbalance the possible artifacts introduced bythe EDTA extraction
procedure.Acomplex artificial diet (consisting of 15%sucrose,aminoacids,
vitamins andsalts, Harrewijn and Noordink, 1971),was diluted around 30
times inthe original EDTA chelation solution (8 mM EDTA and 5mM
phosphatebuffer).Thissolution wasthentreated identically tothe crude
EDTAextracts (Pb-EDTA extraction,lyophilization,storage).The resulting
samples are referredto as Ed-Diet.To makethe 'CONTROL"treatments,
this Ed-Diet wasdissolved inthe sugar (sugartests) or inacomplex diet
(diettests) inthe sameamounts asthe Ed-samples (seebelow).
EDTADetection: EDTAwas detected using acolor reaction with
Eriochrome BlackT asanindicator. Inatest tubetwo droplets of indicator
(0.1% w/v), 1ml H20andtwo droplets of ammonium buffer weremixed.
Lead nitrate solution wasadded untilthe color changedto purple.Totest for
the presence of EDTA five (or more) droplets of afraction were addedto a
test tube.Acolor changeto blue indicatedthe presence of EDTA (detection
limit ± 2\iM of EDTA).
Preparation oftests
Alltests were prepared inalaminar flow cabinet. Materials were cleaned with
70%ethanol priorto use.Alltest solutions werefilteredthrough a0,22 urn
micropore filter.
Nochoicetests. Test solutions consisted of 15%sucrose inwater withone
of the following additions:whole plant sap (5or 10% v/v), methanol extracts
(50 mg/ml),crude EDTA extracts (10%v/v), EDTA at 0, 5, 10,25, 100or
200 mM,or Na2S205 at 0, 5, 10,25or 100 mM.Test solutions were
TÖÖ
CHAPTER 7
presented betweentwo layers of Parafilm,stretched over a26 mmdiameter
plastic ringtoform atest cage.8freshly molted adult aphids were introduced
andsurvival and reproduction werescored after 48h.
MultipleChoice testswereprepared accordingto Dorschner andKenny
(1993).Six parafilm sachets with different sucrose concentrations (0,5,10,
20%w/v in H20)were presentedthrough holes inthe lid of a9cm Petri dish
anda largequantity (>100) of aphids of mixedage were introduced intothe
dish. Distribution ofthe aphids overthe6sachets was determined after 24
and 44 h. Plant extracts were nottested.
DualChoice tests.Forthesetests weconstructed sachets withtwo adjacent
test solutions separated by only afew millimeters werethe two layers were
firmly pressedtogether using a rubber wheel (fig.1)
Twenty larvae of N.ribisnigriwere introduced andthetests were placed in
a 24 °C incubator withweak illumination fromthetop.Transparent yellow
plastic foil was placed belowthe light sourceto stimulate settling onthe
parafilm surface.Aphiddistribution was determined several times (after 8to
72 hours). Usually at least six replicates were performed (see results).
Honeydewtests. Test solutions were made by adding freeze dried honeydew
(10 mg/ml)to asucrose solution (15%in H20).Pairwisetests also included
15%sucrose solution ("BLANK").
Testing ofEd-samples.
Acceptabilitytest.Ed-diet was dissolved at 20 mg/ml inartificial diet, and
tested against artificial diettowhich 20 mg/ml of lyophylised artificial diet (not
treated onthe column) wasadded.
Sugartests:Tests were performed using Ed-samples from RESandSUS
(treatment RESand SUS) dissolved at 5mg/ml in 15%sucrose inwater.
These were compared withtwo controls:ablank control ("BLANK", 15%
sucrose with nothing added) andasecond control ("CONTROL") consisting
of a 15%sucrose solutiontowhich 5mg/ml of Ed-diet was added.This
makessix possible pairs.Thesame combinations weretested at 20mg/ml.
The extracts of RES2and SUS2weretested inthe same combinations at
5 mg/ml and 20mg/ml.
Artificialdiettests:Sametreatments andcombinations (RES,SUS,and
RES2,SUS2). Inthis case however, allsamples were dissolved inacomplex
artificial diet.There was no BLANK controltreatment andthe control
THE DEVELOPMENT OF A BIOASSAY
101
Figure 1.Cross section of the test cage with a dual choice sachet. 20 larvae of N.
ribisnigrican freely select a feeding site on a test solution.
treatment ("CONTROL") consisted ofthe same complex artificial diet to
which 5(or 20) mg/mlof Ed-diet was added,sotherewere only three
possible pairs pertest.
Statistics
Choicetest results werecompared by Wilcoxon's signed ranktests at 5%
significance level.
RESULTS
Nochoicetests. Tests withwhole plant sap or methanol extracts showed 10
to 50%mortality of the adults andvery low reproduction as comparedto
complex artificial diet. Nodifferences showed between mortality or
reproduction ontest solutions containingthe resistant or susceptible
whole-plant extracts.
EDTA affected survival at concentrations over 10 mM,at 100 mMor
higher most aphids diedwithin 24 hours. Little mortality occurred at lower
concentrations but reproduction was low (< 1larvae per adult per day).The
anti-oxidant sodium metabisulfitecaused high mortality, evenat
concentrations below 10mM.
102
CHAPTER 7
Tests withcrude EDTA extracts (containing EDTA and anti-oxidant)
showed lowacceptance ofthetest solutions, low reproduction and high
mortality. Whenthe anti-oxidant was omittedduringcollection andthe EDTA
extracted, nomortality wasobservedandmorethan80%oftheaphids
settled onthetestsolutions.
MultipleChoice testsgave nodiscrimination amongsucrose solutions of
different strengths.Aphids settled poorly andappeared restless.
Dualchoice tests.
Observations after 8,20or 28 hshowed no or less pronounced
differences,whereas during later observations (>48 h) microbial
contamination of thetest solutions wassometimesvisible.
Acceptabilitytest. The acceptability tests suggestedthat the lyophilized
diet was preferredtothe Ed-diet (distribution after 40hr
Diet :Ed-Diet = 10.1 :6.6, n=11)) butthis difference was not significant.
Honeydewtests.Honeydew of host ("Taiwan" lettuce) and nonhost plants
(oilseed rape) wasstrongly favored overthe sucrosetreatment "BLANK"(fig.
2a),and lettuce honeydew was preferredto oilseed rape honeydew. When
testing honeydew of isogenic resistant (RES) andsusceptible (SUS) lines
these were bothselected over sucrose only and honeydew of resistant plants
was preferredtosusceptible plants (fig.2b).
Ed-samples insugartests.Inthetests basedon 15%sucrose,comparing
Ed-samples ofthe SET1, aphids showed aslight preference for SUSto RES
at low concentration (5 mg/ml) while all other combinations showedno
significant difference (fig.3a).At the higher concentration Ed-samples of
SET1were preferredto BLANK but nottothe Ed-diet ("CONTROL") and
SUSwasselectedover RES(fig.3b). Ed-diet wasselected over BLANK at
the high concentration (fig.3b andd).
The tests of SET2showedthat RES2was preferred bythe aphidsat
5 mg/ml (fig.3c) while none of the other combinations showed discrimination
bythe aphids atthis low concentration,not even Ed-diet against BLANK.At
higher concentrations (fig.3d) RES2was not favored over SUS2 but allof
the other combinations showedasignificant deterrence of Ed-samples
comparedto BLANK or evenCONTROL.
Ed-samples inartificialdiettests. Thetests based onthe addition of extracts
to acomplex artificial diet (fig.4) showedvery distinct discriminationof
THE DEVELOPMENT OF A BIOASSAY
103
20
a
8
7
*
8
*
*
15 <o
•g
-
le
a.
i
«10
w
CD
,
E
C
i
5 -
.
BLANK - RAPE
20
b
'
•
o
-
en
number of aphids
6
6
*
6
*
15 -
BLANK - TAIWAN
RAPE-TAIWAN
- 1
•
H^
H
|
Cl
RES -BLANK
RES-SUS
SUS -BLANK
Figure 2.HONEYDEW TESTS. Results of thechoice tests using lyophilized honeydew at
10mg/mlin 15%sugar solution, a. Comparison of different plant species using honeydew
produced byM.persicae;b.comparison ofresistant andsusceptible lettuce using M.
euphorbiae. Bars represent mean number of aphids oneach ofthe test solutions after 44h.
Numbers above bars arenumber ofreplicates. *= significant difference at5%level.
104
CHAPTER 7
different test solutions bythe aphids,except forthe low concentration ofthe
SET1 Ed-samples (fig.4a).At 5 mg/mlsamples of SET2showeda
preference for SUS2to RES2 but SUS2was not selected over Ed-diet (fig.
4c).Atthe highconcentration Ed-diet wasselected over Ed-samples andthe
Ed-samples from susceptible plants over resistant plants (fig.4b andd).
DISCUSSION
Whole plant extracts weretoxic, probably duetosecondary compounds
present incellvacuoles and released oractivated uponthe crushing of the
cells during extraction (Matile, 1984). Plant sap consists mainly of vacuolar
fluid, richinsecondary metabolites,which normally is not ingested bythe
aphidduringfeeding.Wedid not succeed indesigning atest which resulted
inadiscrimination of whole plantsamples.
A bioassay requires relatively large amounts of phloem sap.Ofthe
methods availableto collect phloem sapfrom lettuce only honeydew and
EDTA chelation yieldsufficient quantities (van Helden etal.1994a,b).
Honeydew isphloem sapthat iscollected and bio-processed bytheaphids.
EDTA chelation exudates are consideredto be reliable representations of
phloem sap.Contamination withsubstances from other plant partsandfrom
the cut (wounded) surface ofthe petiole might occur but this has not been
shownto beof major importance (Fisher and Frame, 1984;Weibull era/.,
1990;Girousse era/., 1991;Van Helden etal.,1994a,b).
The crude EDTA extracts were not accepted bythe aphids,because of the
presence of EDTA andsodium metabisulfite.Omission of the anti-oxidant
during EDTA exudation increasedthe browning of the extracts but apparently
did not affect the biological activity of Ed-samples.
Duringthe processing of thesamples the EDTAwas retained bythe lead
ions onthe columnasan Pb-EDTA complex. It is possible that other
components ofthe crude EDTA extracts may have been retained onthe
column,some column components may have been released (lead ionsor
Pb-EDTA complex),and EDTA removalduringthe EDTA extraction
procedure may have been incomplete (below detection limit). Ininitial
experiments traces of lead (precipitation test with Kl) could beshown inthe
THE DEVELOPMENT OF A BIOASSAY
105
20
12
12
16
*
•g 15 le
Q.
CO
O
10
"•
H
IfcMEl
RES-BLANK
N.T.
RES-SUS
SUS-CONTROL
SUS-BLANK
RES-CONTROL
BLANK-CONTROL
RES-BLANK
RES-SUS
SUS-BLANK
SUS-CONTROL
RES-CONTROL
BLANK-CONTROL
Figure 3aandb. SUGAR TESTS OFSET 1.Results of the choice tests using Ed-samples
from SETI orEd-diet (CONTROL), alldissolved in 15%sugar solution. BLANK = 15 %
sucrose with nothing added. Seetext fordetails onprocessing ofextracts, a.RES, SUSor
ED-diet at5mg/ml andBLANK; b.at20mg/ml; Bars represent mean number of aphids
on each of the test solutions. Numbers above bars arenumber ofreplicates. *= significant
difference at5% level, N.T. =nottested.
106
CHAPTER 7
RES2- BLANK
RES2 - SUS2
SUS2 - CONTROL
SUS2 -BLANK
RES2 -CONTROL
BLANK - CONTROL
20
d
3
3
6
6
*
6
*
6
*
CL
co
»10
E
c
• 1
1.
. 1
RES2 - BLANK
RES2- SUS2
SUS2 - CONTROL
SUS2 - BLANK
RES2- CONTROL
BLANK - CONTROL
Figure 3c and d. SUGAR TESTS OF SET 2. Results of the choice tests using Ed-samples
from SET2 or Ed-diet (CONTROL), all dissolved in 15%sugar solution. BLANK = 15%
sucrose with nothing added. See text for details on processing of extracts, c. RES2, SUS2
or Ed-diet at 5 mg/ml and BLANK; d. at 20 mg/ml (d).Bars represent mean number of
aphids on each of the test solutions. Numbers above bars are number of replicates.
* = significant difference at 5%level.
THE DEVELOPMENT OF A BIOASSAY
107
en
aphids
zu
a
6
6
6
-
'S
CD
|
^ ^ _
^^^^^^H
10
• •
3
C
^^H
5
n
RES-SUS
in
aphids
zu
b
6
*
RES-CONTROL
6
*
^^H
SUS-CONTROL
6
*
-
o
number
o
5
n
RES-SUS
RES-CONTROL
SUS-CONTROL
Figure 4a and b. DIET TESTS OF SET 1.Results of the dual choice tests using
Ed-samples from SETI and Ed-diet (CONTROL), all dissolved in complex artificial diet.
See text for details on processing of extracts, a. RES, SUS or Ed-diet at 5 mg/ml; b.at
20 mg/ml; Bars represent mean number of aphids on each of the test solutions. Numbers
above bars are number of replicates. * = significant difference at 5% level..
108
CHAPTER 7
RES2-CONTROL
rt
l£
*
o
*
SUS2-CONTROL
6
*
«15
CL
CO
_g10
•
E
c
RES2-SUS2
RES2 - CONTROL
SUS2 - CONTROL
Figure 4c and d. DIET TESTS OF SET 2. Results of the dual choice tests using
Ed-samples from SET2 and Ed-diet (CONTROL), all dissolved in complex artificial diet.
See text for details on processing of extracts, c. RES2, SUS2 or Ed-Diet at 5 mg/ml; d. at
20 mg/ml. Bars represent mean number of aphids on each of the test solutions. Numbers
above bars are number of replicates. * = significant difference at 5% level.
THE DEVELOPMENT OF A BIOASSAY
109
"EDTAfree samples"when apure EDTA solution was processed.IntheEdsampleswesometimes observed aprecipitation,presumably of leadphopsphate.Thoughwe did not observe asignificant preference for Ed-Diet
over non-treated diet (acceptability test),this maystill have affected the
aphids. However,sincethetreatment of all samples includedthestep of
EDTA removal,artifacts will occur inall Ed-samples and Ed-diet in roughly
equal amounts,sothis does not affect our conclusions
Designofthe test
The bioassay of Dorschner and Kenny (1992),which demonstrated
preference for hop (Humulus lupulusI.)honeydew bythe hop-aphid
Phorodon humiliSchrank,turned out unsatisfactory for N.ribisnigriand
lettuce.Aphids often showed aggregation behavior andaccumulated onone
ortwo of thesix available sachets (irrespective of thesugar concentration).
Therefore we changedto adual choicetest (Mittler and Dadd,1964;
Harrewijn and Noordink, 1971).Our setup,usingtwo parafilm membranes
pressedtogether inthe middle,usesvery little solutionto fillthetwo
compartments and has noborder betweenthe adjacent compartments which
can act as abarrier (fig.1).Therefore,the aggregation behavior will not
influencethe results strongly. Stillthe individual aphid cannot be considered
as independent fromthe others inthe sametest.Thesimplicity of thedual
choice test makes it possibleto make many replicates, necessary to show
statistically significant differences.
Unequal distribution of the aphids overthe twosides became apparent
only after twenty hoursor more.This is muchlater than observed by Mittler
and Dadd (1964) onartificial diet,andalso much laterthan expected from
the rejection ofthe phloem sieve element oncethis is reached bythe aphid
(Van HeldenandTjallingii, 1993).The latter may beexplained bythe levelof
compounds inthe choicetests which iswill be around 10times lower thanin
the real phloem sap,only 20 mg/mlof Ed-sample isadded while normal
lettuce phloem sap hasadry weight of around 20%(200 mg/ml). It cannot
beexcludedthat thetest solutions didchange overtime,e.g.becauseof
continued enzymatic activity (Van Heldenetal.1994b), but this would also
meanthat there isachemical difference betweenfractions.
110
CHAPTER 7
Test results
The preferencefor lettuce honeydew (Taiwan) overthe nonhost plant
(oilseed rape) showsthat the host-plant honeydew has afeeding stimulant
effect (Dorschner and Kenny, 1992). Preference forthe honeydewto sucrose
only ("BLANK") (fig.2) isprobably largely causedbythe feedingstimulancy
of the amino acids present inthe honeydew (Dorschner and Kenny, 1992;
Mittler and Dadd,1964;Van Helden etal.,1994b).The choice for RES
honeydew over SUS honeydew (fig.2b) showsthat there is achemical
difference. Thechoicefor RES honeydew over SUS honeydew issurprising
sincethe inverse wouldbeexpected (discussed below).
The sugartests at 20 mg/ml (fig.3b,d),just likethe honeydewtests,
clearly show the attractiveness of acombination of sucrose withaminoacids.
Inthetest combinations which include aBLANK treatment,thesmall quantity
of amino acids present inthe Ed-samples orthe Ed-diet makesthat these
treatments arestrongly preferred to "BLANK", without any amino acids.At
the low concentration (5 mg/ml) most choices show nosignificant preference.
The absence of response in BLANK versus Ed-Diet at 5mg/ml (fig.3c)
suggest that this might becaused bythe lowquantity ofthe compounds
added.
The comparison of Ed-samples with Ed-diet ("CONTROL") gives more
valuable results since little interference dueto amino-acid concentration
differences (or EDTA extraction artifacts) will occur. Ed-samples (fig.3b,d)
are not preferredto Ed-diet,andinsomecases not evento BLANK (fig3d),
suggesting the presence of feeding deterrents inthe Ed-samples.
We decidedto use acomplex artificial diet as abasefor thetests inorder
to decreasethe sensitivity tovariations inamino acids andsugars inthe
Ed-samples,which is not relatedtothe resistance (Van Heldenetal.,
1994b).This could also increasethesensitivity to other compounds which
might only show biological activity ina"phloem like"environment. The results
ofthese artificial diettests (fig.4) show aprofound increase inthe response
of the aphids.The artificial diet test demonstrated significant preferences for
Ed-diet to plant extracts.Thissuggeststhat all Ed-samples (of both resistant
andsusceptible plants) contain acertainfeeding deterrence.The preference
for SUSand SUS2to RESand RES2 respectively proves that there is a
chemical difference betweenthese extracts,which (at20 mgof
THE DEVELOPMENT OF A BIOASSAY
TÏT
Ed-sample/ml) causes astronger feeding deterrence inthe Ed-samples from
resistant plants.Theabsence of response inthetests of SET1at 5mg/ml
(fig. 4a) andthe SUS2-CONTROLchoice (fig.4c) suggeststhat the
concentration of the feeding deterrents is underthe behavioralthresholdfor
the aphid,just like ininthe lowconcentration sugartests (fig.3a,c).
Inafew cases extracts from resistant plants were preferredto susceptible
extracts (honeydew tests, Ed-samples insugar tests of SET2 at 5mg/ml).
Feeding deterrents have been reportedto act as afeeding stimulants at low
concentrations (Bodnaryk, 1991).Sodeterrence ofthe resistant extractsat
20 mg/ml couldchangeto feeding stimulanceat 5 mg/ml,andthese
allomones could also bepresent inthesusceptible plant but at lower
concentration.Totest this hypothesis moretest series should beperformed,
comparing different concentrations of Ed-samples from the sameor different
plants,and incombination withappropriate controls. However, possible
interactionwithother biologically activesubstances ,will not makethis easy.
Also,afeeding deterrent could be metabolized and excreted inthe
honeydew, itsdegradation products acting asfeeding stimulants,orthe
reverse incase of afeedingstimulant. Since little is known about the fateof
secondary plant chemicals inaphids (Wink and Witte, 1991, Givovich et ai,
1992,Van Helden etal.,1994b) this remainsspeculative.
The sensitivity of thetest might beimproved by adjustment of the amino
acid composition ofthe complex artificial diet (andthe Ed-diet) tothat of
lettuce phloem sap (Van Helden era/., 1994b),andtotal removal of possible
interfering compounds fromthe Ed-samples. Using higher concentrations of
the samples would require largequantities of plants.
Althoughthe results of the honeydew andsugar tests cannot be explained
easily,the diettest results showthat there is aclear chemical difference
between extracts from resistant andsusceptible plants.This allows some
optimism that this bioassay can beusedto isolatethe active substances
through bioassay guidedfractionation. Still,the physical- and chemical
resemblance (pressure,concentration etc.) of thetest withthe in vivo system
remainsvery incomplete andthe EDTA sample processing needsto be
improved. Direct extrapolations of the in vitro test resultsto theinvivo
situation are hazardous.The phloem sap origin of any isolated compound
needsto beconfirmed andthe modeof action should be studied inthe plant.
TÏ2
CHAPTER 7
REFERENCES
Argandofta, V.H., Luza, J.G., Niemeyer, H.M.
and Corcuera, L.J., 1980.Role of
Hydroxamic acids in the resistance of
cereals to aphids. Phytochemistry 19:
1665-1668.
Bodnaryk, R.P., 1991.Developmental profile
of sinalbin (p-hydroxybenzyl glucosinolate)
in mustard, Sinapis alba L. and its
relationship to insect resistance. J. Chem.
Ecol. 17: 1543-1556.
Caillaud, CM., Dipietro, J.P., Chaubet, B. and
Dedryver, CA., 1992. Feeding behavior of
the aphid Sitobion avenae on resistant and
susceptible wheat. In: Menken, S.B.J.,
Visser, J.H. and Harrewijn, P. (eds.). Proc.
8th International Symposium on Insect-Plant
Relationships, Kluwer academic publishers,
Wageningen, The Netherlands pp. 295-296.
Cole, R.A. 1993.Identification of a resistance
factor in the brassica speciesBrassica
fruticulosa and B. spinescensto the cabbage
aphid Brevicoryne brassicae.Entomol. Exp.
appl 71:23-32.
Dorschner, K.W. and Kenny, S.T., 1992.
Occurrence of hop aphid phagostimulants in
the honeydew of aphids. Entomol. Exp.
Appl. 65:49-55.
Dreyer, D.L. and Campbell, B.C., 1987.
Chemical basis of host plant resistance to
aphids. Plant Cell Environment 10: 553-561.
Dreyer, D.L. and Jones, K.C., 1981.Feeding
deterrency of flavonoids and related
phenolics towards Schizaphisgraminumand
Myzuspersicae: aphid feeding deterrents in
wheat. Phytochemistry 20:2489-2493.
Eenink, A.H., Groenwold, R. and Dieleman,
F.L., 1982. Resistance of lettuce(Lactuca)
to the leaf aphid Nasonovia ribis-nigri2:
Inheritance of the resistance. Euphytica 31:
301-304.
Fisher, D.B. and Frame, J.M., 1984. A guide
to the use of the exuding-stylet technique in
phloem physiology. Planta 161: 385-393.
Girousse, C , Bonnemain, J.-L., Delrot, S. and
Bournoville, R., 1991.Sugar and amino acid
THE DEVELOPMENT OF ABIOASSAY
composition of phloem sap of Medicago
saliva L.: a comparative study of two
collecting methods. Plant. Physiol. Biochem.
29: 4149.
Givovich, A., Morse, S., Cerda, H., Niemeyer,
H.M., Wratten, S.D. and Edwards, P.J.,
1992. Hydroxamic acid glucosides in
honeydew of aphids feeding on wheat J.
Chem. Ecol. 18:841-846.
Gonzalez, A.G., 1977.Lactuceae, a chemical
review. In: J.B. Harborne and B.L. Turner
(eds.). The biology and chemistry of the
compositae (2).Academic Press, New York
pp. 1081-1095.
Groussol, J., Delrot, S., Caruhel, P. and
Bonnemain, J.-L., 1986. Design of an
improved exudation method for phloem sap
collection and its use for the study of
phloem mobility of pesticides. Physiol. Vég.
24: 123-133.
Harrewijn, P., 1990. Resistance mechanisms
of plant genotypes to various aphid species.
In: R.K. Campbell and R.D. Eikenbary
(eds.), Aphid-Plant Genotype interactions,
Elsevier Science Publishers B.V.,
Amsterdam pp 117-130.
Harrewijn, P. and Noordink, J.Ph.W., 1971.
Taste perception of Myzus persicae in
relation to food uptake and developmental
processes. Entomol. Exp. Appl. 14:
413-419.
Helden, M. van, Thijssen, M.H. and Tjallingii,
W.F., 1992.The behavior of Nasonovia
ribisnigri on resistant and susceptible lettuce
lines. Proceedings 8th international
symposium on Insect-Plant Relationships,
March 9-13, 1992. Wageningen, The
Netherlands. Kluwer academic publishers:
286-288.
Helden, M. van, Tjallingii, W.F. and
Dieleman, F.L., 1993.The resistance of
lettuce (Lactucasativa L.) to Nasonovia
ribisnigri (Mosley, Homoptera, Aphididae):
Bionomics of N. ribisnigrion near isogenic
lettuce lines. Entomol. Exp. appl 66:53-58.
113
Helden, M. van, and Tjallingii, W.F., 1993.
Tissue localisation of lettuce resistance to
the aphid N. ribisnigriusing electrical
penetration graphs. Entomol. Exp. Appl.68:
269-278.
Helden, M. van, Tjallingii, W.F. and Beek,
T.A. van, 1994a. Phloem sap collection
from lettuce (Lactucasativa): Methodology
and yield. J. Chem. Ecol. in press.
Helden, M. van, Tjallingii, W.F. and Beek,
T.A. van, 1994b.Phloem sap collection
from lettuce (Lactucasativa): Chemical
comparison among collection methods. J.
Chem. Ecol. in press.
Herrbach, E. 1985.Rôle des sémiochimiques
dans les relations pucerons-plantes. II. Les
substances allélochimiques. Agronomie 5:
375-384.
Kanehisa, K., Tsumuki, H., Kawada, K. and
Rustamani, M.A., 1990. Relations of
gramine contents and aphid populations on
barley lines. Appl. Entomol. Zool.25:
251-259.
Kimmins, F.M., 1989. Electrical penetration
graphs from Nilaparvata lugensonresistant
and susceptible rice varieties. Entomol. Exp.
Appl. 50: 69-79.
King, R.W. and Zeevaart, J.A.D., 1974.
Enhancement of phloem exudation from cut
petioles by chelating agents.Plant Physiol.
53: 96-103.
Leszczynski, B.,Warchol, J. and Niraz, S.,
1985. The influence of phenolic compounds
on the preference of winter wheat cultivars
by cereal aphids. Insect Sei. Applic. 6:
157-158.
Luczak, I. and Gaweda, M., 1992.The
relationship between the chemical
composition of sugar beet leaves and the
development of the black bean aphid Aphis
fabae. IOBC/WPRS Bulletin (Working
group "Breeding for resistance to insects
and mites") 16(5): 178-183.
Matile, P., 1984. Das Toxische kompartiment
der pflanzencelle. Naturwissenschaften 71:
18-24.
Mittler, T.E., 1988. Applications of artificial
feeding techniques for aphids. In: Minks,
114
A.K. and Harrewijn, P. (eds.). Aphids, their
biology, natural enemies and control.
Elseviers Science Publishers B.V.,
Amsterdam, pp. 145-162.
Mittler, T.E. and Dadd, R.H., 1964. Gustatory
discrimination between liquids by the aphid
Myzuspersicae (Sulzer). Entomol. Exp.
Appl. 7: 315-328.
Niemeyer, H.M., 1990.The role of secondary
plant compounds in aphid-host interactions.
In: R.K. Campbell and R.D. Eikenbary
(eds.), Aphid-Plant Genotype Interactions,
Elsevier Science Publishers B.V.,
Amsterdam, p.p. 187-205.
Padgham, D.E., Kimmins, F.M., and Barnett,
E.A., 1990. Resistance in groundnut and its
wild relatives toAphis craccivora, and its
relevance to groundnut rosette disease
management. Proceedings of the Brighton
crop protection conference, pests and
diseases vol 1: 191-196.
Reinink, K. and Dieleman, F.L., 1989.
Comparison of sources of resistance to leaf
aphids in lettuce (L. sativa L.). Euphytica
40: 21-29.
Todd, G.W., Getahun, A. and Cress, G.C.,
1971. Resistance in Barley to the greenbug
Schizaphisgraminum. 1.Toxicity of
phenolic and flavonoid compounds and
related substances. Ann. Entomol. Soc.
Amer. 64:718-722.
Weibull, J., Ronquist, F. and Brishammar, S.,
1990. Free amino acid composition of leaf
exudates and phloem sap. Plant Physiol. 92:
222-226.
Wink, M. and Witte, L., 1991.Storage of
quinolizidine alkaloids in Macrosiphum
atbifrons and Aphis ganistae (Homoptera:
Aphididae). Entomol. Gener. 15:237-254.
CHAPTER 7
8. SUMMARYAND
CONCLUSIONS
The resistance of lettucetothe aphid Nasonovia ribisnigriis basedona
single, dominant gene,the Nr-gene.Onthe resistant plant aphids diedwithin
a few days,without any honeydew production.Transfer-experiments witha
short stay ona resistant plant followed by a relocationto asusceptible plant
showedthat noweight increase occurred onthe resistant plant, but weight
gain immediately resumedonasusceptible plant (Chapter 2).Sono
intoxication seemedto occur onthe resistant plant. Behavioral observations
of (tethered) aphids using Electrical Penetration Graphs (EPG) (Chapter 3)
and comparison withfree living aphids (Chapter 4) showedthat onthe
resistant plantthe aphid's stylets did penetrate tothe phloem sieve elements.
Onthe resistant plants hardly any E2pattern (phloem sap ingestion)
occurred andthe aphidfinally died of malnutrition,or left the plant (Chapter
4). Itwas concludedthat the resistance isbased onafactor inthephloem.
This can be either achemical factor like afeeding deterrent or a mechanical
factor which obstructs ingestion. Experiments concentrated onchemical
differences inthe phloemsap of resistant andsusceptible (isogenic apart
from the resistance gene) lettuce lines.Amputation of aphidstylets
(stylectomy) during feeding canyieldsmall but pure phloem sapsamples.
However, onthe resistant andsusceptible isogenic lines no phloem sap
samples could be collected because outflow fromthestylet stump stopped
after afew seconds. Larger phloem sap samples were obtained by EDTA
chelation and honeydew collection (Chapter5).
SUMMARY AND CONCLUSIONS
115
Chemical analysis ofthecomposition ofthe phloem sap samples showed
nodifferences insugars,amino acids,proteins and UVabsorbing
compounds (Chapter 6). Exhaustive analysis of phloem sap compounds was
not feasible,not only because ofthe number of possible compounds,but
also becausethe sample size andquantity waslimited.
Ina bioassay aphids wereoffered achoice between EDTA collected
phloem sapsamples of resistant andsusceptible plants,addedto a complete
artificial diet (chapter 7).Aphids showedaclear aversiontothe extract of the
resistant plant.This suggeststhatthe resistance is basedonthe presenceof
feeding deterrents inthe phloemsapofthe resistant plant. Hopefully,these
substances can be isolatedand identified withthe help of the bioassay.
116
SUMMARY AND CONCLUSIONS
SAMENVATTING EN
CONCLUSIES
De resistentievan slategende bladluis Nasonovia ribisnigriberust opéén
dominant gen,het Nr-gen.Op de resistente plant sterft de bladluis binnen4
dagen,erwordt geen honingdauw geproduceerd.Tijdens eenverblijf van
twee dagen opde resistente plant vindt er geen gewichtsgroei plaats,na
terugplaatsing op eenvatbare plant iserdirect weer "normale" groei
(hoofdstuk 2). Er lijkt dusgeensprakete zijnvaneengiftige stof inde
resistente sla.Observaties van (aangelijnde) bladluizen met behulp van
Elektrische Penetratie Grammen (EPG) (hoofdstuk 3),envergelijking met
vrijlevende bladluizen (hoofdstuk 4)tonen aandat de bladluisstiletten ookop
de resistente plant wel degelijk penetreren tot indezeefvaten van het
floëem.Opde resistente plant isechter nauwelijks voedselopname (E2
patroon) waarneembaar, ende bladluis sterft uiteindelijk aanondervoeding,
ofverlaat de plant (hoofdstuk 4). Deresistentie berust dus op eenfactor in
het floëem van de plant. Dit kaneenchemische factor zijn, bijvoorbeeld een
smaakvergallendestof, of een mechanische factor die opname verhindert.
Het onderzoek heeft zichgericht op hetvindenvanchemische verschillen in
het floëem vangenetisch identieke (op het resistentie-gen na) resistenteen
vatbare planten.Amputatievan bladluisstiletten (stylectomy) tijdens de
voedselopname levert normaal zeer kleine -maar ergzuiverefloëemsapmonsters.Opde resistente envatbare planten kondenopdeze
manier geen monsters wordenverzameld doordat de uitstroom uit de
SUMMARY AND CONCLUSIONS
117
stiletstomp al naenigesecondenstopte.Via EDTAchelatie enhet
verzamelen van honingdauw kondenwelsapmonstersvaneen redelijke
volume wordenverzameld (hoofdstuk 5).
Analyses vandeze monsters liet geen reproduceerbare verschillen zienin
suikers,aminozuren,eiwitten en UVabsorberende stoffen (hoofdstuk 6).Het
isechter ondoenlijk om uitputtende analyses uittevoeren,niet alleen omdat
het aantal mogelijke inhoudsstoffen enorm groot is maarook doordat de
hoeveelheid engroottevande monsters beperkt is.
Ineenbiotoets werdaanbladluizen eenkeuzegebodentussen EDTA
verzamelde floëemsapmonstersvanvatbare en resistente planten diewaren
toegevoegd aan kunstmatig dieet (hoofdstuk 7). Hierbijvertoonden de
bladluizen eenduidelijke afkeer voor het extract vande resistente plant.
Hieruit kan geconcludeerdworden datde resistentie waarschijnlijk berust op
deaanwezigheid vaneensmaakvergallende stof(fen) in hetfloëemsapvan
de resistente plant. Mogelijk kunnendeze stoffen met behulp van chemische
fractionering incombinatie metde biotoets geïsoleerd en geïdentificeerd
worden.
118
SUMMARY AND CONCLUSIONS
PUBLICATIONS
The following chapters of this dissertation have been publishedor
submitted asjournal articles.
Chapter 2: Helden, M.van,Tjallingii,W.F.&Dieleman, F.L. 1993.The
resistance of lettuce (Lactuca sativaL.) to Nasonoviaribisnigri(Mosley,
Homoptera,Aphididae):Bionomics of N.ribisnigrion near isogenic lettuce
lines. Ent.exp.appl.66:53-58
Chapter 3: Helden,M.van &Tjallingii,W.F. 1993.Tissue localisationof
lettuce resistancetothe aphid N.ribisnigriusingelectrical penetration
graphs. Ent.exp.appl.68:269-278
Chapter 5: Helden,M.van,Tjallingii,W.F.&T.A. van Beek, 1994.Phloem
sap collection from lettuce (Lactuca sativa):Methodology andyield.
J. Chem. Ecol. 20:3173-3190
Chapter 6: Helden,M.van,Tjallingii,W.F.&T.A. van Beek, 1994. Phloem
sap collection from lettuce (Lactuca sativa):Chemical comparison among
collection methods.J.Chem.Ecol.20:3190-3206
PUBLICATIONS
119
Chapter 7: Helden, M. van, Heest, H.P.N.F van, Beek, T.A. van & W.F.
Tjallingii, 1995. The development of a bioassay to test phloem sap
samples from lettuce for resistance to Nasonoviaribisnigri (Homoptera,
Aphididae).J.Chem. Ecol. submitted
OTHER PUBLICATIONS
Other publications on relatedsubjectsto whichthe author hascontributed:
Helden, M.van,1990. Resistance of lettucetotheaphid Nasonovia ribisnigri.
Are Electrical Penetration Graphs (EPGs) helpful to find the origin of
resistance? Symp. Biol. Hung 39: 473-474 (short version) and
IOBC/WPRSBulletin 1990 XIII: 101-104
Helden, M. van & W.F. Tjallingii, 1990. Electrical Penetration Graphs of the
aphid Nasonoviaribisnigrion resistant and susceptible lettuce {Lactuca
sativa). Proceedings Symposium Aphid-plant interactions: Populations to
molecules,August 12-17, 1990.Stillwater Oklahoma U.S.A.
Helden, M. van, Thijssen, M.H. and W.F. Tjallingii 1992. The behaviour of
Nasonovia ribisnigri on resistant and susceptible lettuce lines.
Proceedings 8th international symposium on Insect-Plant Relationships,
March 9-13, 1992. Wageningen, The Netherlands. Kluwer academic
publishers:286-288
Helden, M. van & W.F. Tjallingii, 1994. The use of electrical penetration
graphs in plant resistance research. In: E.A. Backus & G. Walker,
Homopteran feeding behaviour: Recent research advances and experimentaltechniques. Special issues Ent.Soc.Am.:inpress
Reese, J.C., Tjallingii, W.F., Helden, M. van & E. Prado, 1994. Waveform
comparison among ACand DCsystems for electronic monitoring of aphid
feeding behaviour. In: E.A. Backus & G. Walker, Homopteran feeding
behaviour: Recent research advances and experimental techniques.
Special issues Ent. Soc.Am.:in press
120
PUBLICATIONS
NAWOORD
Toen ik in september 1988, niet geremd door al te veel kennis van
bladluis-plant interacties, begon met mijn werk, wist ik niet wat voor
moeilijkheden mij nog te wachten stonden. Het onderzoeksvoorstel droeg de
optimistische titel "De rol van secundaire plantestoffen in de resistentie van
sla tegen bladluizen", maar dat bleek niet zo eenvoudig te onderzoeken. Het
idee dat een systematische chemische vergelijking van vatbare en resistente
planten verschillen aan het licht zou brengen, bleek iets te optimistisch. Er
zijn, in samenwerking met de vakgroep Organische Chemie, vele chemische
analyses van totaalextracten en floëemmonsters uitgevoerd die slechts lieten
zien dat er geen verschil was in inhoudsstoffen tussen resistente en vatbare
planten.
Je zou kunnen zeggen dat ik er in die jaren slechts in ben geslaagd om
de uitgangspunten van het onderzoeksvoorstel te bevestigen, namelijk dat
secundaire plantestoffen inderdaad een rol lijken te spelen. Die voorstelling
van zaken zou echter veel te negatief zijn, ik denk dat het onderzoek zeker
wel succesvol kan worden genoemd. Via een systematische benadering is
een groot aantal mogelijke factoren onderzocht, veelal door een nieuwe
combinatie van bestaande technieken. Zo is duidelijk aangetoond dat de
bladluis de resistente plant pas afkeurt als de stiletten tot in het floëem zijn
doorgedrongen. De ontwikkeling van de biotoets is een doorbraak voor het
onderzoek naar bladluis-plant relaties. Via deze toets hebben we aangetoond
dat de resistentie zeer waarschijnlijk op een chemische stof in het floëemsap
NAWOORD
ÜT
berust. Met deze biotoets hebben we bovendien ook meteen een instrument
in handen gekregen waarmee de isolatie en identificatie van actieve stoffen
uit het floëemsap mogelijk moet zijn.
Het proefschrift is dan wel gereed, het echte werk moet dus eigenlijk nog
beginnen. Het afgelopen jaar heb ik mij bezig gehouden met het schrijven
van dit proefschrift, gecombineerd met enkele niet entomologische hobby's
en geduldig wachten op de uitslag van enige beursaanvragen. Onverwacht is
het dan toch nog gelukt en in de komende jaren heb ik de mogelijkheid om,
via een STW beurs, als post-doc verder te werken aan dit onderzoek.
Hopelijk kunnen we het project succesvol voortzetten.
Hierbij wil ik natuurlijk alle mensen die actief betrokken zijn geweest bij dit
onderzoek van harte bedanken: mijn begeleiders Freddy Tjallingii
(Entomologie) en Teris van Beek (Organische Chemie); mijn promotor Louis
Schoonhoven, de andere leden van de denktank: Frans Dieleman, Paul
Harrewijn en Kees Reinink; Hanneke van Heest voor haar prima assistentie
bij de experimenten en bladluizenkweek, Paul Piron voor de opkweek van de
planten; mijn kamergenoten Jan Janssen en Theo Jetten voor hun aan- en
afwezigheid; de instrumentenmakerij, fotoafdeling,tekenkamer en alle andere
onderdelen van gecombineerde diensten binnenhaven voor hun onmisbare
hulp; alle (sla)zaaddonoren en alle overige medewerkers van entomologie en
organische chemie. Ik heb al die jaren met veel plezier onderzoek verricht.
122
nawoord
CURRICULUM VITAE
Indejaren nazijn geboorte op 24september 1963 doorliep Maartenvan
Helden metsucces kleuterschool,basisschool enV.W.O.inzijn
geboorteplaats Ermelo. Hoeweleenberoepskeuzetest hemgeschikt achtte
voor de Hogere Hotelschool, Hogere Laboratoriumschool,Academie voor
Lichamelijke Opvoeding of Fysiotherapie,verkoos hijtoch de
Landbouwuniversiteit Wageningen,geïnspireerd doorvakantiebaantjes bij
familieleden inde land- entuinbouw. In1981begon hijzijnstudie
Planteziektenkunde. In 1985/86 bracht hij 7maanden door in Ivoorkust
(Afrika) met onderzoek naarvirusoverdracht door dewitte vliegBemisia
tabaciincassave. Metafstudeervakken in de Entomologie (insektenkunde),
Virologie enTheoretische Produktie Ecologie rondde hij inseptember 1988
zijnstudie af. Indiezelfde maandwas hijtoen als Assistent InOpleiding
(AIO) begonnen bij devakgroep Entomologie met het indit proefschrift
beschreven onderzoek naar deachtergrondvande resistentievanslategen
de bladluis Nasonovia ribisnigri, de eerstetweejaren full-time,later als 0,8
deeltijdbaan. Naast zijnwerk houdt hijzichbezig met andere hobby's als
doe-het-zelven,tuinieren,sporten enterraristiek.
Momenteel is hij,dankzij een NWO/STW beurs,weerterug inhet
onderzoek met een project dat voort bouwt opde resultatenvanhet
promotieonderzoek. Dekomendejaren zal hij bijdevakgroepen Entomologie
enOrganische Chemie proberenom hetvraagstukvan de resistentievansla
tegen Nasonovia ribisnigriverderteontrafelen.