aphid watery saliva counteracts sieve-tube occlusion: a ... · 3305 introduction sieve tubes –...

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INTRODUCTIONSieve tubes – arrays of longitudinally arranged sieve elements (SEs)– are the transport conduits for an assortment of metabolites suchas carbohydrates, amino acids, phytohormones and vitamins(reviewed by van Bel, 2003). SEs are elongated cells that lack anucleus and vacuole and contain only an intact plasma membrane,phloem plastids, a SE endoplasmic reticulum (Sjölund and Shih,1983), a few mitochondria, and an extensive set of phloem-specificproteins (reviewed by Hayashi et al., 2000). During SE development,the terminal walls transform into sieve plates, perforated by sievepores (Evert, 1990) to connect adjacent SEs end to end.

Following production in the mesophyll and loading into sievetubes, plant nutrients are translocated through the transport phloemtowards the sinks, e.g. growing leaves, roots, stem or storage organs(van Bel, 1996). Mass flow through sieve tubes is driven by a turgor-pressure gradient between sources (high pressure) and sinks (lowpressure) (Münch, 1930; Gould et al., 2005).

Aphids (Homoptera: Aphididae: Aphidinae) puncture sieve tubeswith their stylets and passively ingest the nutrient-rich sieve-tubecontent owing to the high endogenous pressure. During styletpenetration, aphids are confronted with sieve-tube occlusionmechanisms (Sjölund, 1997; Knoblauch and van Bel, 1998; Ehlerset al., 2000). When damage occurs, occlusion of sieve poresprevents the loss of sieve-tube sap (Evert, 1982; Schulz, 1998) andthe invasion of pathogens through the wound site (van Bel, 2003).

Sieve tubes are occluded by proteins and callose in response toinjury. The nature of protein-occlusion mechanisms depends uponthe plant family. Fabaceae possess parietal proteins, protein networksand reversibly dispersive protein bodies, the so-called forisomes(Knoblauch and van Bel, 1998; Ehlers et al., 2000; Knoblauch etal., 2001). Cucurbitaceae possess parietal proteins and solubilizedphloem proteins (PP1) that form protein filaments after oxidation(Leineweber et al., 2000). In electron-microscopic images ofBrassicaceae phloem, SEs show merely protein networks that span

the SE lumen and are attached to the cell periphery (Sjölund, 1997).Sieve tubes of grasses appear virtually empty but may have anocclusion mechanism based on solidification of soluble proteins(Will and van Bel, 2006). To complement protein plugging, sievepores are occluded by callose deposition (McNairn and Currier,1968), a universal mechanism in all families studied so far[summarized by Behnke and Sjölund (Behnke and Sjölund, 1990)].

Induction of callose synthesis (Kauss et al., 1983), coagulationof soluble proteins (Will and van Bel, 2006) and dispersion offorisomes (Knoblauch et al., 2001) appear to be Ca2+ dependent.Occlusion is triggered by Ca2+ influx induced by damage(Knoblauch and van Bel, 1998). Aphids seem to have developedstrategies to prevent sieve-tube occlusion during stylet penetration,one of which is the secretion of Ca2+-binding watery saliva(Tjallingii, 2006; Will and van Bel, 2006; Will et al., 2007).

As shown by the electrical penetration graph (EPG) technique[for an overview, see Walker (Walker, 2000)], watery saliva ismassively secreted into the SE lumen at the beginning of sieve-tubepenetration (Prado and Tjallingii, 1994). Some watery-salivaproteins of the aphid species Megoura viciae (Buckton) have Ca2+-binding properties (Will et al., 2007). This was demonstrated byconfronting forisomes with salivary proteins in vitro. Forisomedispersal (which occludes sieve plates) can be triggered in vitro bysupply of Ca2+, and dispersal can subsequently be reversed by addingCa2+ chelators (Knoblauch et al., 2001) or concentrated aphid saliva(Will et al., 2007).

While feeding on SEs, aphids switch from phloem sap ingestion(EPG waveform E2) to secretion of watery saliva (EPG waveformE1) upon leaf-tip burning (Will et al., 2007). Leaf-tip burning wasobserved to induce an electropotential wave, accompanied by Ca2+

influx into the sieve-tube lumen resulting in sieve-tube occlusion(Fromm and Bauer, 1994; Furch et al., 2007). As demonstrated byGould and colleagues (Gould et al., 2004), sieve-tube occlusion isaccompanied by a decrease of sieve-tube pressure (Gould et al.,

The Journal of Experimental Biology 212, 3305-3312Published by The Company of Biologists 2009doi:10.1242/jeb.028514

Aphid watery saliva counteracts sieve-tube occlusion: a universal phenomenon?

Torsten Will1, Sarah R. Kornemann1, Alexandra C. U. Furch1, W. Fred Tjallingii2 and Aart J. E. van Bel1,*1Plant Cell Biology Research Group, Department of General Botany, Justus-Liebig-University, Senckenbergstrasse 17-21, D-35390

Giessen, Germany and 2Department of Entomology, Agricultural University Wageningen, Binnenhaven 7, NL-6700 EH,Wageningen, The Netherlands

*Author for correspondence ([email protected])

Accepted 14 July 2009

SUMMARYCa2+-binding proteins in the watery saliva of Megoura viciae counteract Ca2+-dependent occlusion of sieve plates in Vicia faba andso prevent the shut-down of food supply in response to stylet penetration. The question arises whether this interaction betweenaphid saliva and sieve-element proteins is a universal phenomenon as inferred by the coincidence between sieve-tube occlusionand salivation. For this purpose, leaf tips were burnt in a number of plant species from four different families to induce remotesieve-plate occlusion. Resultant sieve-plate occlusion in these plant species was counteracted by an abrupt switch of aphidbehaviour. Each of the seven aphid species tested interrupted its feeding behaviour and started secreting watery saliva. Theprotein composition of watery saliva appeared strikingly different between aphid species with less than 50% overlap. Secretion ofwatery saliva seems to be a universal means to suppress sieve-plate occlusion, although the protein composition of watery salivaseems to diverge between species.

Key words: aphids, plant defence, sieve-tube occlusion, watery saliva.

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2004). This pressure decrease inside the sieve-tube lumen most likelytriggers the switch in aphid behaviour (Will et al., 2008).

We investigated whether watery saliva acts as a universal tool inaphid–plant interactions through suppression of sieve-tube occlusion.We used the behavioural switch from phloem-sap ingestion tosecretion of watery saliva (Will et al., 2007) as an indicator forsuppression of sieve-tube occlusion by watery saliva in severalaphid–plant combinations. Watery saliva from different aphid specieswas collected to compare their protein composition and investigatepotential overlap with regard to Ca2+-binding proteins.

MATERIALS AND METHODSAphid and plant breeding

Aphid species were reared on their host plants (Table1) in Perspexcages with large gauze-covered windows in a controlled roomenvironment with a 17 h:7 h light (L):dark (D) regime at atemperature of 25°C. The same aphid/host plant combinations forrearing were also used for experiments.

Plants (Table1) were grown in a greenhouse at 20°C with naturallighting plus additional lamp light (SONT Agro 400W; Phillips,Eindhoven, The Netherlands) with a 14h:10h L:D period and wereused in a vegetative phase.

Sieve-tube occlusion visualised by confocal laser scanningmicroscopy

In order to document sieve-tube occlusion in response to leaf-tipburning, Brassica napus and Hordeum vulgare plants were preparedas described previously (Knoblauch and van Bel, 1998). A fewcortical cell layers were locally removed down to the phloem fromthe lower side of the main vein of a mature leaf that remainedattached to the plant during the entire experiment. The layers wereremoved by manual paradermal slicing with a new razor blade, whileavoiding damage to the phloem. Immediately after cutting a shallowwindow (~10mm long, 2mm wide and 6cm away from the leaftip), we bathed the free-lying phloem tissue in a buffer solution(Hafke et al., 2005) and the plant was allowed to recover for30–60min. The leaf was fixed in an upside-down position withdouble-sided adhesive tape onto a small Perspex table. The state ofthe tissue was checked under the microscope. If the phloemappeared to be undamaged (indicated by thin sieve plates), a loadingarea (5mm�5mm) was made by gently rubbing the lower side ofthe leaf vein, located 2–3cm upstream from the observation window,with fine sandpaper (Fig.1A).

A stock solution of the phloem-mobile fluorochrome 5(6)carboxyfluorescein diacetate (CFDA)-mixed isomers (Invitrogen,Karlsruhe, Germany) was prepared by solubilisation of 1mg CFDAin 1ml DMSO. A working solution of 1μl stock solution in 2ml buffersolution (Hafke et al., 2005) was applied at the loading site. CFDA

permeates the plasma membrane in the non-fluorescent acetate formand is cleaved by cytosolic enzymes producing membrane-impermeant fluorescent carboxyfluorescein (CF) (handbook fromMolecular Probes, Eugene, OR, USA). CF trapped inside SEs istransported by mass flow in the sieve tubes as observed by confocallaser scanning microscopy (CLSM; Leica TCS 4D; LeicaMicrosystems, Heidelberg, Germany) (Knoblauch and van Bel,1998). After CF had reached the observation window, the leaf tipwas burnt for 3s (Fig.1A,B; maximum area 1cm2) to induce blockageof SEs. To visualize stoppage of mass flow in SEs, CF wasphotobleached at the observation window for approximately 3min.If phloem transport was blocked, the photobleached SEs would remainnon-fluorescent because of the lack of fluorochrome supply (Fig.1C).

After burning and subsequent photobleaching, a time course ofthe events in the SEs was recorded. The experiment was repeatedfour times for each plant species with new individuals. Digital imageprocessing was done with Corel PHOTO-PAINT®10 to optimizebrightness, contrast and colouring.

Aphid behaviour monitored by EPGsAphid behaviour was monitored using the EPG technique modifiedby Tjallingii (Tjallingii, 1988). A gold wire electrode (1cm lengthand 20μm diameter) was attached to the dorsum of an adult apterousaphid using electrically conductive silver glue (Electrolube,Swadlincote, Derbyshire, UK). A vacuum device was used toimmobilize aphids during electrode attachment (van Helden andTjallingii, 2000). The aphid electrode was connected to a DC EPGGiga-8 (Tjallingii, 1978; Tjallingii, 1988) and the EPG output wasrecorded with PROBE 3.5 (hardware and software from EPG-Systems, Wageningen, The Netherlands, www.epgsystems.eu). Asecond electrode (plant electrode) was inserted into the soil of pottedplants. In this electrical circuit, the ‘living’ components, representedby the aphid and plant, act as a variable resistor, and plant cellmembrane potentials present additional voltage sources (reviewedby Walker, 2000). The whole setup was placed in a Faraday cageto shield it from electromagnetic influence.

Randomly selected adult and apterous aphids were placed on theirparticular host plant species (Table1) on the midrib of the lowerside of a mature leaf 6±0.5cm away from the leaf tip. When aphidsshowed ingestion of phloem sap for longer than 30min, recognizedby an E2 waveform in the EPG (Tjallingii, 1988), the leaf tip (ofmaximum area 1cm2) was carefully burned for 3s to trigger aphloem-mediated electrical long-distance signal (Furch et al., 2007).In EPG recordings, the time point of burning was marked bygenerating three –50mV pulses by pressing the EPG amplifiercalibration button. EPG recordings of control insects (no leaf-tipburning) were made under identical experimental conditions,including the three –50mV pulses. Burning experiments and controls

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Table 1. Aphid species reared for experimental use in electrical penetration graph (EPG) and protein studies

EPG data Protein composition Aphid species Host plant Host specificity (Table 1; Figs 2, 3) of watery saliva (Fig. 5)

Acyrtosiphon pisum (Harris) (green variant) Vicia faba cv. Witkiem major O + +Acyrtosiphon pisum (Harris) (red variant) Vicia faba cv. Witkiem major O – +Megoura viciae (Buckton) Vicia faba cv. Witkiem major M + +Aphis fabae (Scopoli) Vicia faba cv. Witkiem major P + +Myzus persicae (Sulzer) Brassica napus cv. unspecified P + –Macrosiphum euphorbiae (Thomas) Cucurbita maxima cv. Gele Reuzen P + +Rhopalosiphum padi (Linnaeus) Hordeum vulgare cv. unspecified O + –Schizaphis graminum (Rondani) Hordeum vulgare cv. unspecified O + +

M, monophagous; O, oligophagous; P, polyphagous.


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were repeated 12 times in most cases (except for Rhopalosiphumpadi, burning N=13; Macrosiphum euphorbiae, control N=7) andrecording time was set to 1h.

EPG waveforms were analyzed by waveform pattern and autopower spectra (APS) (example waveforms and APS are presentedin Fig.2) according to Prado and Tjallingii (Prado and Tjallingii,1994), using the PROBE analysis module. APS present thewaveform frequency (Hz) vs the relative magnitude with a maximumof 1, which provides a major characteristic of waveform identity.

Recorded EPGs were analyzed with regard to switches inbehaviour expressed as transitions from one waveform to another,e.g. from E2 (ingestion of sieve-tube sap) to E1 (secretion of waterysaliva into sieve-tube lumen) as observed for M. viciae (Will et al.,2007). EPGs from each aphid were scored as a ‘reaction’ if theychanged from E2 to E1 and as a ‘non-reaction’ if they remained inE2 for 10min after leaf-tip burning, or for controls, within 10minof the three –50mV pulses.

Statistical analysisStatistical analysis was done with Fisher’s exact test using SigmaStat3.0 software (SPSS, Chicago, IL, USA). Each treatment wascompared against its control group. The level for significance wasset to P=0.05.

Aphid saliva collection and saliva concentrationWatery saliva secreted by the aphid species M. viciae, Acyrtosiphonpisum (red and green variant), M. euphorbiae, Aphis fabae and

Schizaphis graminum was collected and concentrated according toa previous method (Will et al., 2007). Saliva from 100 aphids in1μl of saliva/diet-concentrate was used as a benchmark forconcentrating the diet/saliva mixture of each aphid species. The finalsample was partitioned in aliquots of 10μl and kept frozen at –80°C.

1-D SDS-PAGE1-D SDS-PAGE of the saliva concentrate (sample of 1000 aphidsper lane) was carried out according to Laemmli (Laemmli, 1970)using a 4% stacking gel and a 10% separation gel in a MiniProtean3 Electrophoresis System (Bio-Rad Laboratories, Hercules, CA,USA). Precision Plus Protein All Blue standards (Bio-Rad) was usedas a protein size marker. Fourfold concentrated reducing samplebuffer (Roti-Load 1; Carl-Roth, Karlsruhe, Germany) was added toeach saliva sample in the proportion 1:3. Protein gels were silverstained (Switzer and Merril, 1979; Schoenle et al., 1984). Similaritiesof protein band patterns were calculated using Quantity One® 1-DAnalysis Software (Bio-Rad) including all detected bands and settingthe band match tolerance to 1%. Protein band intensity was notweighted because of the varying amounts of protein in the respectivesamples.

RESULTSTime course of events in sieve tubes after leaf-tip burning

CFDA was applied to the loading site on B. napus leaves andtransported downstream in the CF form through the sieve tubes.Following phloem transport of CF in B. napus leaves, the leaf tip

Fig. 1. Transient stoppage of mass flow insieve tubes of Brassica napus induced byleaf-tip burning. (A) Phloem transport ofcarboxyfluorescein (CF) was observed byconfocal laser scanning microscopy(CLSM) at a tissue window 6 cm from theburning site. 5(6)Carboxyfluoresceindiacetate (CFDA) was applied onto aloading area located 2–3 cm upstreamfrom the observation window. (B,E) Themembrane-impermeable CF wastransported by mass flow through sievetubes (marked with an asterisk). (C,F) CFwas photobleached at the observationarea immediately after leaf-tip burning,resulting in a decrease of fluorescence.(C,F–I) Upon burning, reducedfluorescence remained stable for morethan 30 min, which indicates stoppage ofmass flow due to sieve-plate occlusion.(D,J) An increase in fluorescence between30 and 60 min implies recovery of CFsupply from the upstream loading areadue to release of sieve-tube blockage andhence resumption of translocation. Timepoints of observation are given in thelower right corner of each image (leaf tipwas burnt at t=0). Scale bars in E–J,10μm.


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was burnt (Fig. 1A,B) in order to induce sieve-plate occlusion.Subsequently, CF was photobleached at the observation window(Fig. 1C). The rationale for these treatments is that fluorescencewill not recover at the site of observation until the sieve plates areunblocked (Fig. 1D). Fluorescence inside the observed SEs(marked with an asterisk in Fig. 1) was reduced by photobleaching(Fig. 1E,F). A continued bleached appearance (Fig. 1F–I)demonstrates the absence of fluorochrome supply and is evidenceof stoppage of mass flow over a period of 30 min (Fig. 1C).Between 30 and 60 min after burning (Fig. 1J) fluorescencerecovered, indicating that mass flow had resumed (Fig. 1D).Similar results were obtained for all replicates (N=4) and for bothplant species (data for H. vulgare not shown).

EPG and leaf-tip burningAs with V. faba (Will et al., 2007), leaf-tip burning was adoptedto observe whether food deprivation caused by sieve-tube

occlusion triggers behavioural responses of other aphid species.Each aphid species (Table1) switched its behaviour from ingestion(E2: Fig. 2B; Fig. 3) to secretion of watery saliva (E1: Fig. 2C;Fig. 3), as shown in detail for the aphid species M. euphorbiae(Fig. 2A–D) and in 1 h overviews for other tested aphid species(Fig. 3). The behavioural switch was observed for all testedaphid–plant combinations (Table 2) shortly after leaf-tip burning(Figs 2 and 3) (M. persicae: 6 s; M. euphorbiae: 10 s; A. fabae:13 s; S. graminum: 17 s; R. padi: 18 s; A. pisum: 23 s). The responseis significant with P-values that range from


behaviour of 12 M. viciae individuals in reaction to SE occlusiondemonstrates the variability of the responses (Fig.4). Ten of 12aphids reacted to leaf-tip burning by secretion of watery salivafollowed by a mixed E1/E2 behaviour. The return from mixed E1/E2behaviour to ingestion occurred most frequently (5/10; Fig.4).Another frequent switch (4/10) was that from mixed behaviour backto secretion of watery saliva (E1), followed by a broad variety ofbehavioural profiles (Fig.4). Other aphid species showed a similardiversity of behaviour.

Comparison of watery saliva from six aphid species by 1-DSDS-PAGE

Protein band patterns of the different aphid species/biotypes (Fig.5)show only partial overlap (Fig.5, red bands) when matched to M.viciae as the reference species (Fig.5, green bands). Even if aphidsare reared and fed on the same host plant [M. viciae, A. pisum (bothvariants) and A. fabae on V. faba], the similarity is low. Acalculation for similarity shows values of 19.2–43.1% [43.1% S.graminum; 34.1% A. pisum (green variant); 32.9% A. pisum (red

Fig. 3. EPG for different species ofingesting aphids on their hostplants in response to heat-shocktriggered sieve-tube occlusion.Overviews of 1 h duration arepresented for different aphidspecies–host plant combinations.Horizontal arrows indicate thestart and end of a particularbehaviour. Response to leaf-tipburning (red arrow) occurs withinonly a few seconds [in thesemeasurements: M. persicae: 6 s;A. fabae: 13 s; S. graminum: 17 s;Rhopalosiphum padi: 18 s; A.pisum (green variant): 23 s]. Allaphid species show a changefrom ingestion (E2) to secretion ofwatery saliva (E1). Subsequent tothe immediate response, which isuniversal, further behaviour isquite diverse. A mixedingestion/salivation behaviour(mixed E1/E2) is observed for R.padi and S. graminum. Mixedbehaviour is followed by ingestion(E2) for R. padi. SE penetrationcan also be interrupted, asdemonstrated by pathwayactivities (path), observed for A.pisum, A. fabae, M. persicae andS. graminum. The shortoccurrence of F in the EPG of S.graminum reflects penetrationproblems inside the apoplast.


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variant); 19.2% A. fabae; 30.1% M. euphorbiae] when comparedwith M. viciae. The protein pattern varies not only between speciesbut also between variants of A. pisum (green and red). Watery salivaproteins of most species are distributed over the whole proteinmarker range (Fig.5).

Protein concentration varies strongly between species (Fig.5),although each sample was collected from 1000 aphids. Roughly, theamount of secreted saliva seems to be positively correlated with thesize of the aphid species. The total protein amount in watery-salivasamples can affect the number of detectable protein bands (Will etal., 2007). Thus, a comparison of samples with differing proteinconcentrations is not conclusive for determining differences in proteinbands (e.g. Fig.5, red and green biotype of A. pisum). Nevertheless,samples with comparable protein concentrations (e.g. Fig.5, M.viciae, A. fabae and M. euphorbiae) show a low similarity as well,which suggests that dissimilarity is not due to a difference in theamount of protein at least in the comparison among the samples ofM. viciae, S. graminum, A. fabae and M. euphorbiae in Fig.5.

DISCUSSIONSieve-tube occlusion mechanisms are presumably involved in plantdefence against phloem-feeding insects by blockage of nutrient

supply (Caillaud and Niemeyer, 1996). Aphids appear to be able tocounteract sieve-tube occlusion by secretion of watery saliva(Tjallingii, 2006; Will and van Bel, 2006; Will et al., 2007). Thisnotion is based on the following observations: (i) aphids startsecreting watery saliva (E1) at the beginning of each SE penetration(Prado and Tjallingii, 1994), (ii) M. viciae switches to secretion ofwatery saliva in response to sieve-tube occlusion triggered by distantleaf-tip burning (Will et al., 2007), (iii) watery saliva concentrateof M. viciae inhibits Ca2+-induced dispersion of forisomes (legumeproteins involved in sieve-plate plugging) (Will et al., 2007), and(iv) several watery saliva proteins of M. viciae are able to bind Ca2+

in biochemical assays (Will et al., 2007). To answer the questionof whether suppression of sieve-tube occlusion by aphid waterysaliva is a general phenomenon, the leaf-burning test (Will et al.,2007) was extended to a variety of aphid–host plant combinations(Table1). Aphid behavioural responses were monitored with EPGrecordings (Figs2 and 3; Table2) and protein composition of waterysaliva was compared among different aphid species/biotypes (Fig.5).

Callose-mediated sieve-tube occlusion induced by remote leaf-tip burning has been visualized for broad bean, tomato (Furch etal., 2007) and cucurbits (A.C.U.F., unpublished). Prior to callosedeposition, sieve tubes are plugged by proteins such as giant protein

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Fig. 4. Behaviour succession tree – individual behaviour afterdistant leaf-tip burning exemplified by M. viciae. Sequence ofbehaviour during a 1 h recording period of 12 aphids followingleaf-tip burning (time course from top to bottom). Green numbersin brackets represent the number of individuals that exhibit aparticular behaviour. The thickness of the arrows correspondswith the behavioural frequency. Most frequently, E1 (secretion ofwatery saliva) is followed by a mixed E1/E2 behaviour and E2(ingestion). Another frequently noted behaviour succession ismixed E1/E2 behaviour, E1, mixed E1/E2 behaviour again andthen E2. While seven aphids stay inside the penetrated SEs andreturn to ingestion before the end of the 1 h observation period,only 3 of 10 aphids stop SE penetration, indicated by pathwayactivities (path), of which two return to ingestion. Four groups canbe distinguished: aphids that (i) show no reaction, (ii) penetrate anew SE, (iii) stay inside the original SE or (iv) stop feeding duringthe 1 h recording period.

Table 2. Aphid behaviour change induced by leaf-tip burning experiments

Leaf-tip burning Control Fisher’s exact test, P-valuePlant Aphid species (reaction/non-reaction) (reaction/non-reaction) (significance level P=0.05)

Vicia faba Megoura viciae 10/2 0/12


bodies (forisomes) in V. faba (Knoblauch et al., 2001) or probablystructural phloem proteins in cucurbits (Leineweber et al., 2000).Sieve-plate plugging by forisomes was reversed by the time(15–20 min after burning) that callose deposition reached itsmaximum level (Furch et al., 2007). Thus, sieve-plate occlusionseems to be a two-step event in which protein plugging precedescallose deposition (Will and van Bel, 2006).

Both Rosopsida and Liliopsida employ callose deposition on sieveplates for occlusion (Eleftheriou, 1990; Evert, 1990). In addition,Rosopsida also employs structural proteins to occlude SEs (Evert,1990), whilst we (Will and van Bel, 2006) speculated about theexistence of soluble proteins in phloem sap of Liliopsida whichcoagulate in response to wounding. All in all, the rapidity of theobserved sieve-tube occlusion in B. napus (Rosopsida) and H.vulgare (Liliopsida) (Fig.1) and the speed of aphid reaction to leaf-tip burning (Figs2 and 3) suggest sieve-plate plugging by proteinsprior to callose deposition, because the latter is a slower responsethat requires several minutes for synthesis (Furch et al., 2007).

Activation of occlusion seems to depend on increasing Ca2+

concentration in SEs (Kauss et al., 1983; Knoblauch and van Bel,1998; Knoblauch et al., 2001). Induction of sieve-plate occlusionby electropotential waves (Fromm and Bauer, 1994; Furch et al.,2007) and turgor shocks (Knoblauch et al., 2001; Hafke et al., 2007)triggers a release of Ca2+ into the SE lumen which then inducesocclusion. Furch and colleagues (Furch et al., 2007) demonstrated

that sieve-tube occlusion is reversible probably through activeremoval of Ca2+ ions.

The crucial role of Ca2+ in sieve-tube occlusion makes it anoutstanding target for insects and pathogens in order to interferewith occlusion mechanisms. Therefore, the switch from ingestionto secretion of watery saliva, provoked by leaf-tip burning (Figs2and 3; Table2), has been interpreted as an attempt to suppress sieve-plate occlusion (Will et al., 2007). In addition to the Ca2+-bindingproteins that were described for M. viciae, one was recentlyidentified in the saliva of A. pisum (Carolan et al., 2009). Aphidsmay use proteins in watery saliva as a tool for Ca2+ binding (Willand van Bel, 2006; Will et al., 2007). Thus, secretion of waterysaliva into the SE lumen can be regarded as a method to counteractsieve-plate occlusion, although it does not seem to succeed in allcases (Figs3 and 4).

This study indicates that the involvement of watery salivation inthe suppression of plant defence is independent of aphid species[N=7; monophagous (1), oligophagous (3) and polyphagous (3)],host plant species (N=4) and host family (Fabaceae, Brassicaceae,Cucurbitaceae and Poaceae) (Figs2 and 3; Table2).

Detailed behavioural analysis of the aphid species M. viciae(Fig.4) shows that 10 of 12 aphids reacted to leaf-tip burning andthat seven of these returned to ingestion without interruption of sieve-tube penetration. The mixed E1/E2 behaviour appears to expresstesting of the penetrated sieve tube for its further suitability as asource of nutrition. A change of turgor pressure in sieve tubes causedby occlusion (Gould et al., 2004) represents a relevant signal foraphids to initiate the behaviour switch from ingestion to salivation,observed in vitro for M. persicae (Will et al., 2008).

Aphid behaviour that has been observed by EPG thus far [e.g.Brevicoryne brassicae, M. viciae and M. euphorbiae (Tjallingii,1985); R. padi (Prado and Tjallingii, 1994); Aphis gossypii and M.persicae (Martin et al., 1997); A. pisum (Tjallingii and Gabrys,1999)] suggests that aphids possess only a small and conservedrepertoire of behaviour which hardly varies among aphid speciesand host plants. The key for adaptation to the respective host plant(s)may lie in the watery saliva, which is astonishingly variable inquantity and protein composition, even if aphid species feed on thesame host plant (Fig.5). The variable composition may reflect anevolutionary adaptation to a variety of defence mechanisms of hostplant(s) (e.g. phenols, hydrogen peroxide) other than Ca2+-inducedocclusion. Several watery-saliva proteins which are not involved inCa2+ binding may function in the detoxification of such compounds(Miles and Oertli, 1993; Urbanska et al., 1998).

LIST OF ABBREVIATIONSAPS auto power spectrumCF carboxyfluoresceinCFDA 5(6)carboxyfluorescein diacetateCLSM confocal laser scanning microscopyDC direct currentEPG electrical penetration graphSE sieve element

We thank Gregory P. Walker (Department of Entomology, University of California,Riverside, USA) for critical reading of the manuscript. We are grateful to EdgarSchliephake (Julius Kühn-Institut, Institut für Resistenzforschung undStresstoleranz, Quedlinburg, Germany) for supplying a starter colony of M. persicae.

REFERENCESBehnke, H. D. and Sjolund, R. D. (1990). Sieve Elements: Comparative Structure,

Induction and Development (ed. H. D. Behnke and R. D. Sjolund). Berlin: Springer.Caillaud, C. M. and Niemeyer, H. M. (1996). Possible involvement of the phloem

sealing system in the acceptance of a plant as host by an aphid. Cell Mol. Life Sci.52, 927-931.

Fig. 5. Comparison of watery saliva from six aphid species by 1-D SDS-PAGE. Lane 1 contains protein standard (molecular masses in kDa givenon the left side of the gel). Lanes 2–7 [Megoura viciae on V. faba (N=10);S. graminum on H. vulgare (N=1); A. pisum (green variant) on V. faba(N=3); A. pisum (red variant) on V. faba (N=1); A. fabae on V. faba (N=1);M. euphorbiae on C. maxima (N=2)] contain watery saliva concentrateobtained from 1000 aphids each, separated on a 10% gel. Lanes 3–7 werematched with a tolerance of 1% to lane 2 (M. viciae, marked green) byQuantity One® software (Bio-Rad). Matched protein bands were markedred and numbered by protein band numbers of M. viciae, while non-matching protein bands were labelled yellow.


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