plants and herbivorous insects share a long and complex evolutionary history ( labandeira 2007 ) , where plants have developed multi - leveled defense strategies , and insects have evolved equally complex counter - strategies . the perception of herbivory is crucial for the plant to differentiate insect attack from , for example , wounding to prevent unnecessary activation of costly or autotoxic defense mechanisms . grazing insects such as caterpillars can be recognized by their continuous wounding ( mithfer et al .
2005 ) , and/or by deposition of salivary fluids on the wounded plant interface ( musser et al . 2005 ) .
components in these fluids can act as an elicitor and specifically activate plant defense responses . a number of elicitors have been identified in different herbivores .
these are compounds , often peptides , isolated from the oral secretions ( os ) of insects ( alborn et al .
2006 ) , or fluids deposited during oviposition , giving the plant an early warning ( doss et al .
herbivores however , have evolved mechanisms to evade plant defenses for example by manipulating part of the plant s defense pathway to their own benefit , also through components in the herbivore s os ( hogenhout and bos 2011 ; kant et al .
2008 ; musser et al . 2005 ; sarmento et al . 2011 ; weech et al .
the sweetpotato whitefly bemisia tabaci ( homoptera : aleyrodidae ) is a highly polyphageous insect that has been reported to feed from 420 plant species and 74 families worldwide ( greathead 1986 ) , although more recent country - specific studies suggest this number is likely an underestimation ( erdogan et al .
b. tabaci also poses an economic challenge to crops such as cotton , bell pepper , and tomato , mostly because b. tabaci is a vector of begomo viruses , which cause major economic damage .
b. tabaci is a phloem - feeder , and causes minimal damage to plant cells during feeding , unlike aphids that puncture cells during probing ( janssen et al .
aphids release proteins into the phloem that prevent calcium - regulated blockage of the vessel , thus protecting their feeding sites ( will et al . 2007 ) . in whiteflies , a number of enzymes
have been identified that could play an important role in dealing with the plant s defense system ( peng et al .
thus , it is likely that there are complex interactions between plants and phloem feeders , that find their origin in the phloem .
this intimate relationship between phloem - feeders and plants has resulted in plant - defense mechanisms that are similar to the mechanisms found in plant - pathogen interactions , where recognition - based genes ( such as nbs - lrrs ) can confer a high level of resistance ( klingler et al . 2005 ; nombela et al .
however , the interaction between phloem - feeders and plants is not limited to the feeding mechanism alone since many phloem feeders secrete phloem - derived honeydew , a sticky deposit containing mostly sugars .
phloem concentrations of sugars are estimated as high as 0.50.8  m ( cernusak et al .
this honeydew ( hd ) , due to its high sugar content , facilitates secondary infections with ( black ) molds , but it also presents an opportunity for recognition .
it previously has been shown that whiteflies are susceptible to jasmonic acid ( ja)-mediated defenses ( zarate et al .
2007 ) , and it has been suggested that whiteflies activate the salicylic acid ( sa ) pathway , thus preventing the activation of the ja pathway ( zarate et al .
2007 ) since cross - talk between sa and ja will prevent the ja pathway from being activated ( koornneef and pieterse 2008 ) .
here , we studied the role of whitefly honeydew in the tomato - whitefly interaction , particularly with respect to the presence of sa in the phloem , the metabolism of sa in whiteflies , and the implications of this for the plant s defense response .
whiteflies ( b. tabaci biotype b ) were reared in a growth chamber as previously described ( bleeker et al .
2011 ) , on a diet of both cucumber ( cucumis sativus ) and tomato ( lycopersicum esculentum cultivar moneymaker ) plants . for hd collection ,
petri dishes were placed under tomato leaflets in the insect rearing chamber for 24  h. honeydew was subsequently collected with a pipette by adding 500  l of ddh2o to the petri dishes . to the adaxial side of leaflets of intact plants 20  l hd ( containing 0.02  % tween-20 to avoid running - off of the liquid and to promote absorption ) or 20  l ddh2o ( containing 0.02  % tween-20 )
these leaflets were subsequently harvested after 1  h and extracted for sa analysis according to ( vandoorn et al .
briefly , samples were extracted in ethyl acetate containing deuterated salicylic acid ( d4- sa ) ( c / d / n isotopes inc .
, pointe - claire , quebec , canada ) as internal standard , evaporated and reconstituted in 70  % ( v / v ) meoh , and analyzed by lc - ms / ms using instrument parameters as described previously ( scala et al .
phloem sap was collected using the  edta method ( king and zeevaart 1974 ) .
tomato leaflets were excised from the plant and their petioles put in a petri dish containing 5  mm edta ( ph  8) for 1  min , after which they were transferred to a 50  ml tube containing fresh 5  mm edta .
the tubes were covered with a plastic tray lined with wet paper towels to increase the humidity , and thus minimizing transpiration .
after 1  h , the solution was replaced with fresh 5  mm edta ( 30  ml ) , and subsequently the phloem sap was collected for 10  h. four samples were collected , each sample from 5 to 6 leaves , representing ~15  g fw . for analysis of sag , the protocol of ( van den burg et al .
2010 ) was adapted as follows : 200  l of hd or phloem sap were added to 200  l na - acetate solution ( 0.2  mm ph  4.5 ) containing 1  mg -glucosidase ( sigma - alrich ) , or 200  l na - acetate ( control ) . after overnight incubation at 37  c , the samples were acidified to ph  1 with 20  l 37  % hcl , and immediately extracted twice with 700  l ethyl acetate / pentane/2-propanol ( 50/50/1  v / v / v ) .
the combined extracts were evaporated , reconstituted in 100  % meoh , and analyzed by lc - ms / ms as described above .
the water phase was used for sugar analysis by lc - ms / ms according to ( clarke et al .
sugars were separated over a luna nh2-column ( phenomenex ) using 50  % acetonitril ( containing 0.05  % formic acid ) and 50  % ddh2o ( containing 0.05  % formic acid ) with a flow of 0.5  ml  min for 5  min .
a 20-ml plastic container with a soft plastic lid was used as a feeding chamber for whiteflies . a hole ( approximately 5    5  mm ) was cut in the lid , and parafilm stretched over this .
the parafilm had to be stretched maximally for flies to be able to feed through .
a drop of artificial diet was placed on the parafilm , and a second layer of parafilm was stretched over this . the artificial diet consisted of a 20  % ( w / v ) sucrose solution containing 2  mg  ml threonine , and 100  g  ml d4-sa .
since sa is only slightly soluble in water , it was first dissolved in a small volume of meoh and subsequently diluted with ddh2o to 1  % meoh ( v / v ) .
after 2  days , containers were placed at 80  c to kill the whiteflies , and hd was collected in ddh2o and analyzed as above .
four containers , each containing approximately 30 whiteflies had to be pooled in order to detect d4-sa in the hd . for the experiment with sa - laced artificial diet , sa artificial diet containing 100 
g  ml sa and control diet was prepared as above . in order to get enough hd , 2000 flies per treatment had to be used , with approximately 40 flies per cage .
after 3  days , flies were killed at 80  c and honeydew collected . to dissolve the hd ,
100  l were added to the first cage , and transferred to the next to maximize the hd concentration .
plants were treated with honeydew as described above , and samples were taken 24  h later .
one g rna was transcribed into cdna , and rt - qpcr reactions were carried out according to spyropoulou et al .
primers were tggtggttcatttcttgcaactac and atcaatccgatccacttatcatttta for pr-1a ( gb : aj011520 ) , gtactgcatcttcttgtttcca and tagataagtgcttgatgtcca for pr - p6 ( gb : m69248.1 ) , gacaaggtactagtaatcaattatcc and gggcatatcccgaacccaaga for pi - ii ( gb : ay129402.1 ) . for normalization , actin ( gb : xm_004235020.1 )
in order to determine the levels of sa and sag in phloem sap and honeydew using lc / ms , we first collected phloem sap from tomato plants ( cultivar moneymaker ) according to a well - established method ( king and zeevaart 1974 ) and collected honeydew by simply putting a petri dish under a leaflet infested with whiteflies .
since the methods of collecting phloem sap and honeydew do not allow for a precise determination of their volumes , the levels of sa and sag were normalized according to their sugar ( hexose and dihexose ) content .
the data show that the ratio of sag to sa in honeydew is approximately 8 ( fig . 
1a ) but that the levels of sag and sa in phloem are approximately equal ( fig . 
this indicates that conversion of sa to sag happens in the whitefly or its honeydew , unless whiteflies metabolize sa and thereby skew the ratio.fig .
honeydew ( a ) and phloem sap ( b ) were collected , treated with and without -glucosidase , and analyzed by lc - ms / ms for sa and sugar ( hexose  +  dihexose ) content .
n  =  4 ; * indicates a significant difference ( student s t - test , p  <  0.05 ) ; error bars represent standard errors relative salicylic acid ( sa ) levels in phloem and honeydew .
honeydew ( a ) and phloem sap ( b ) were collected , treated with and without -glucosidase , and analyzed by lc - ms / ms for sa and sugar ( hexose  +  dihexose ) content .
n  =  4 ; * indicates a significant difference ( student s t - test , p  <  0.05 ) ; error bars represent standard errors to investigate the possibility that whiteflies can convert sa to sag , we fed whiteflies an artificial diet containing d4-labeled sa and sucrose .
subsequently we collected the honeydew derived from this artificial diet , which we retrieved from the bottom of the feeding chambers and analyzed it for sa and sag levels .
the results show that the honeydew indeed contained sag , i.e. , more free d4-sa after -glucosidase treatment ( fig . 
2deuterated salicylic acid ( d4-sa ) in honeydew after feeding on artificial diet laced with d4-sa .
whiteflies were fed an artificial diet containing d4-sa for 2 d , honeydew ( hd ) was collected , -glucosidase treatment performed and subsequently analyzed for d4-sa by lc - ms / ms .
the solid , black line shows d4-sa in honeydew treated with beta - glucosidase , the dashed grey line without deuterated salicylic acid ( d4-sa ) in honeydew after feeding on artificial diet laced with d4-sa .
whiteflies were fed an artificial diet containing d4-sa for 2 d , honeydew ( hd ) was collected , -glucosidase treatment performed and subsequently analyzed for d4-sa by lc - ms / ms .
the solid , black line shows d4-sa in honeydew treated with beta - glucosidase , the dashed grey line without to test the effect of honeydew deposition on sa levels , hd ( containing approximately 2  ng sa ) was applied to undamaged leaves , and the levels of free sa determined after 1  h. the results ( fig . 
3a ) show that honeydew increased free sa levels to 50  ng  g fw , while the control , water treated , leaves only show a level of 19  ng  g-1 fw . to test
if applied sa was fully recoverable after application on the leaf surface , 50  ng d4-labeled sa were applied to the leaf surface and extracted . a comparison with the same amount spiked in extraction solution showed that the recovery rate was 42  % ( fig  3b).fig .
3salicylic acid ( sa ) levels after honeydew ( hd ) treatment and recovery of sa . a induction of sa after honeydew treatment .
20  l honeydew or ddh2o ( both containing 0.02  % tween-20 ) were pipetted onto the adaxial side of a tomato leaf , the leaves were harvested and analyzed for sa and quantified with a deuterated salicylic acid ( d4-sa ) internal standard .
50  ng sa were applied to the leaf surface and extracted after 1  h. as a control , a non - treated leaf was extracted with the extraction solution containing 50n g d4-sa .
n  =  4 * indicates a significant difference ( student s t - test , p  <  0.05 ) ; error bars represent standard errors salicylic acid ( sa ) levels after honeydew ( hd ) treatment and recovery of sa . a induction of sa after honeydew treatment .
20  l honeydew or ddh2o ( both containing 0.02  % tween-20 ) were pipetted onto the adaxial side of a tomato leaf , the leaves were harvested and analyzed for sa and quantified with a deuterated salicylic acid ( d4-sa ) internal standard .
50  ng sa were applied to the leaf surface and extracted after 1  h. as a control , a non - treated leaf was extracted with the extraction solution containing 50n g d4-sa .
n  =  4 * indicates a significant difference ( student s t - test , p  <  0.05 ) ; error bars represent standard errors the increase in sa levels prompted us to study the effects of hd on gene expression , using the pr-1a and pr - p6 genes , well - established sa - markers ( van kan et al .
1992 ) , and the pi - ii gene , a ja marker ( graham et al . 1985 ) , as read outs .
figure  4 shows that hd application resulted in a moderate increase of pr1 and prp6 transcripts levels over the control treatment , and in a decrease of the pi - ii transcript levels.fig .
plants were treated with 20  l honeydew or ddh2o ( both containing 0.02  % tween-20 ) and after 24  h the leaf material was harvested for rt - qpcr analyses .
n  =  3 , * indicates a statistical difference ( student t - test , p  <  0.05 ) ; error bars represent standard errors transcript levels of defense - related genes after honeydew treatment .
plants were treated with 20  l honeydew or ddh2o ( both containing 0.02  % tween-20 ) and after 24  h the leaf material was harvested for rt - qpcr analyses .
n  =  3 , * indicates a statistical difference ( student t - test , p  <  0.05 ) ; error bars represent standard errors honeydew from sa - fed whiteflies induces more sa in planta . to test
if sa fed through artificial diet influences the sa elicitation in plants , artificial diet was laced with sa .
honeydew from sa - free artificial diet and the solvent for honeydew ( ddh2o ) were used .
the results show that lacing whitefly diet with sa leads to increased sa - elicitation in planta .
honeydew resulting from artificial diet containing no sa induced sa levels to approximately 60  ng  g fw , whereas sa - laced diet resulted in the elicitation of sa levels to 130  ng  g fw ( fig . 
. 5honeydew from artificial medium laced with salicylic acid ( sa ) increases sa levels in planta .
two thousand whiteflies were fed artificial diet  ( ad ) with or without 100  g / ml sa for 72  h. the honeydew was subsequently collected , and 20  l applied to undamaged leaves for 1  h. sa levels were subsequently measured by lc - ms / ms .
asterisk indicates a significant difference ( anova , followed by scheff posthoc test p  <  0.01 ) ; error bars represent standard errors ( n  =  4 ) honeydew from artificial medium laced with salicylic acid ( sa ) increases sa levels in planta .
two thousand whiteflies were fed artificial diet  ( ad ) with or without 100  g / ml sa for 72  h. the honeydew was subsequently collected , and 20  l applied to undamaged leaves for 1  h. sa levels were subsequently measured by lc - ms / ms .
asterisk indicates a significant difference ( anova , followed by scheff posthoc test p  <  0.01 ) ; error bars represent standard errors ( n  =  4 )
when herbivores feed , they leave chemical traces behind , in and on plant tissues .
plants can sense and process some of such herbivore - derived signals to activate defense responses .
self recognition , where plants recognize ( parts of ) their own molecules and/or herbivore - specific compounds , sometimes after they have been metabolized by either the plant or the insect ( alborn et al .
because phloem feeders excrete a large portion of the fluids they ingest , mostly in the form of small droplets , this provides a 
when we assayed the honeydew of whiteflies as well as their natural diet , tomato phloem sap , for phytohormones , we detected free sa in both ( but no ja , data not shown ) .
the presence of sa in phloem had been shown previously ( smith - becker et al .
1998 ) , while the honeydew of aphids also is know to contain it ( cleland and ajami 1974 ) . in the honeydew of whiteflies , however , we observed that most of this sa was converted to sag , a much more polar compound ( figs .  1 and 2 ) .
since depositing free sa on plants will likely elicit a relatively strong defense response , it is possible that the glycosylation of sa to sag serves as a mechanism to prevent such a strong activation of defense responses in the distal parts of the plant , since this may consume valuable resources for defense or for early flowering , and thus negatively affect resource flows ( agtuca et al .
2012 ; donovan et al . 2013 ) and may hence facilitate defense priming rather than direct induction as has been suggested .
salicyclic acid glycoside itself is inactive in planta and needs to be converted back to sa to induce sa - related defenses ( for a review see e.g. , vlot et al .
2009 ) an alternative explanation of our results would be direct toxicity of sa to the whiteflies , with glycosylation necessary to detoxify the sa .
when , after honeydew treatment , sa levels in planta were analyzed , it appeared that whitefly honeydew slightly induced sa levels ( fig . 
the level of induction is relatively small , approximately 30  ng  g fw after 1  h , but this sa could not be contributed to the small levels of sa that are present in honeydew , which are approximately 2  ng in the 20 ul hd applied .
our results are consistent with those of ( schwartzberg and tumlinson 2014 ) , which showed induction of sa production after application of aphid ( acyrthosipon pisum ) honeydew to bean ( vica faba ) plants .
however , they did not analyze sag levels in aphid honeydew , so it is unclear whether aphids also can convert sa to sag .
moreover , when we applied honeydew resulting from diet with or without sa to leaves , it was clear that honeydew from sa - containing diet induced more sa in planta then sa - free diet ( fig .  5 ) .
this probably is due to the fact that the sa - containing diet resulted in the presence of sa and sag in the honeydew , which induced higher sa levels in the plant .
when gene expression after hd application was analyzed , there was a trend towards the induction of genes activated by sa signaling , and towards the suppression of genes activated by ja signaling ( fig . 
4 ) , indicating that the small amount of free sa present in the hd is biologically relevant .
these results are in line with the vast amount of literature concerning the antagonism of sa on ja signaling in dicots ( koornneef and pieterse 2008 ; spoel and dong 2008 ) . moreover , our results also are consistent with the facts that whiteflies feeding on arabidopsis induce sa - related genes and repress ja - related genes ( zarate et al .
2007 ) , and that whiteflies increase sa - levels when feeding from lima bean plants ( phaseolus lunatus ) and repress ja - related genes ( zhang et al . , 2009 ) .
typically , the density of feeding whiteflies on leaves in these previous experiments was high , up to 200 individuals per arabidopsis leaf ( zarate et al .
2007 ) or 50 per bean leaf ( zhang et al . , 2009 ) . with these densities ,
the amount of honeydew deposited will be significant , and thus we argue that the sa and sag from honeydew of whiteflies could augment salivary factors in relaying defensive processes .
it recently was discovered that aphid ( acyrthosiphon pisum ) honeydew contains many proteins ( from the aphid and associated microbes ) that may act as mediators in the plant - aphid interaction ( sabri et al . , 2013 ) .
this , together with the fact that honeydew of acyrthosipon pisum can attract natural enemies of the aphid ( leroy et al . , 2009 ) , and
the classical example that ants can harvest the aphid honeydew and in return provide the aphids with protection against predators in some systems ( styrsky and eubanks 2007 ; vlkl et al .
1999 ; way 1963 ) , all point to a myriad of functions for honeydew in multitrophic systems . our results , indicating that whiteflies can modulate the sa levels in the honeydew , add to this complexity .
hence , the deposition of honeydew on plants can be an important factor in fine - tuning the interaction between whiteflies and plants , which so far has been overlooked .
whiteflies can feed on a large number of host plants , and the mechanism to modulate sa signaling by glycosylation might be a broad - spectrum mechanism .
further studies will be needed to determine the role of sag in plant - insect interactions .
 during insect feeding , a complex interaction takes place at the feeding site , with plants deciphering molecular information associated with the feeding herbivore , resulting in the upregulation of the appropriate defenses , and the herbivore avoiding or preventing these defenses from taking effect . 
 whiteflies can feed on plants without causing significant damage to mesophyll cells , making their detection extra challenging for the plant . 
 however , whiteflies secrete honeydew that ends up on the plant surface at the feeding site and on distal plant parts below the feeding site . 
 we reasoned that this honeydew , since it is largely of plant origin , may contain molecular information that alerts the plant , and we focused on the defense hormone salicylic acid ( sa ) . 
 first , we analyzed phloem sap from tomato plants , on which the whiteflies are feeding , and found that it contained salicylic acid ( sa ) . 
 subsequently , we determined that in honeydew more than 80  % of sa was converted to its glycoside ( sag ) . when whiteflies were allowed to feed from an artificial diet spiked with labeled sa , labeled sag also was produced . 
 however , manually depositing honeydew on undamaged plants resulted still in a significant increase in endogenous free sa . 
 accordingly , transcript levels of pr1a , an sa marker gene , increased whereas those of pi - ii , a jasmonate marker gene , decreased . 
 our results indicate that whiteflies manipulate the sa levels within their secretions , thus influencing the defense responses in those plant parts that come into contact with honeydew . 
Introduction
Methods and Materials
Results
Discussion