Blood-feeding arthropods secrete specialsalivary proteins that suppress thedefensive reaction they induce in their

hosts1,2. This is in contrast to herbivores,which are thought to be helpless victims of plant defences elicited by their oralsecretions3,4. On the basis of the findingthat caterpillar regurgitant can reduce theamount of toxic nicotine released by thetobacco plant Nicotiana tabacum5, weinvestigate here whether specific salivarycomponents from the caterpillar Helico-verpa zea might be responsible for this suppression. We find that the enzyme glucose oxidase counteracts the productionof nicotine induced by the caterpillar feeding on the plant.

Spinnerets are the principal secretorystructures of the labial salivary glands of H. zea. To determine whether saliva fromthis caterpillar affects the induced defencesof the tobacco plant in situ, we preventedsalivation by ablating their spinnerets with aheated probe (Fig. 1a, b). This cauterizationinhibits salivation, and hence the secretionof salivary enzymes, as we demonstrated byfeeding glucose-soaked fibre discs to thesecaterpillars and then staining the discs todetect hydrogen peroxide (a product of theaction of glucose oxidase)6. The ablationprocedure prevented the release of glucoseoxidase without affecting feeding rate.

Caterpillars with ablated spinnerets andcaterpillars with intact spinnerets were indi-

vidually caged on the second uppermostfully expanded leaf of an N. tabacum plant(one caterpillar per plant) for 24 hours.Three days after feeding, we analysed thedamaged leaf for nicotine, an inducibledefence compound7. Feeding by caterpillarswith intact spinnerets reduced foliar nico-tine levels by over 26% compared with feeding by caterpillars with ablated spin-nerets (P*0.05, Tukey–Kramer test; fordetails of nicotine quantification, see ref. 7).

We removed circular sections from theleaves of the caterpillar-exposed plants andfed them to H. zea neonates. Neonates thatwere fed on leaves that had been ‘treated’with caterpillars with intact spinneretsshowed significantly increased survival andmean body weights relative to neonates that

were fed on leaves exposed to caterpillarswithout spinnerets (Mann–Whitney U-test;Fig. 2a, b).

Glucose oxidase is the principal salivaryenzyme in H. zea, and converts D-glucoseand molecular oxygen to D-gluconic acidand hydrogen peroxide1,6. To determinewhether this protein could be the salivarycomponent that is responsible for suppres-sing the plant’s herbivore-induced resistance,we cut one attached leaf per plant with a1.5-cm-diameter cork borer in order tosimulate insect damage. Six holes were uniformly distributed on the second-uppermost expanded leaf.

We then treated individual leaves withone of four preparations: purified, activeglucose oxidase (prepared as in ref. 6);unpurified salivary-gland extract; inactivated(autoclaved) purified glucose oxidase; orwater. Leaves treated with salivary extractreceived 20 mg protein in total; eachwound received about 10 ml water. Threedays later, leaves treated with enzyme orsalivary extract contained significantly lessnicotine than plants that were treated withwater or inactive enzyme (Tukey–Kramertest; Fig. 2c).

We also analysed the survival andgrowth of second-instar caterpillars onplants treated with either purified glucoseoxidase or water. Survival and mean larvalweights were significantly greater for cater-pillars fed on enzyme-treated leaves thanfor caterpillars fed on leaves treated withwater (P*0.05; Tukey–Kramer test). Wealso applied the individual reaction prod-ucts H2O2 and gluconic acid to leaf wounds(10 ml 40 mM solution) and found thatthese products reduced nicotine levels inthe leaf relative to a water control by 43.6%and 29.3%, respectively (Tukey–Kramertest; results not shown).

The saliva of herbivorous insects hasbeen overlooked as a factor in overcoming

brief communications

NATURE | VOL 416 | 11 APRIL 2002 | www.nature.com 599

Caterpillar saliva beats plant defencesA new weapon emerges in the evolutionary arms race between plants and herbivores.

Figure 2 Effect of caterpillar saliva on induced resistance in tobacco plants (Nicotiana tabacum). a, Proportion of neonates surviving after

feeding on leaves previously damaged by sixth-instar caterpillars. b, Weights of surviving neonates fed on leaves damaged by sixth-instar

caterpillars. c, Glucose oxidase (GOX) and salivary-gland extracts suppress nicotine production in leaves that have been wounded to

mimic insect damage. Wounded leaves were treated with water, inactive GOX, active GOX, or salivary-gland extracts containing active

GOX; the nicotine content of treated leaves was analysed by high-performance liquid chromatography7. Asterisks represent significant

differences (P*0.05) between treatments; error bars represent mean5s.e.

Figure 1 Ablation of the caterpillar (Helicoverpa zea) spinneret to prevent production of saliva. a,b, Scanning electron micrographs

showing the caterpillar labium (arrows) with the spinneret intact (a) and ablated (b). Scale bars, 100 mm.

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University of Arkansas, Fayetteville, Arkansas 72701, USA‡Department of Entomology, Pennsylvania State University, University Park,Pennsylvania 16802, USAe-mail: gwf10@psu.edu

1. Felton, G. W. & Eichenseer, H. Induced Plant Defences Against

Pathogens and Herbivores (Am. Phytopathol. Soc., St Paul,

Minnesota, 1999).

2. Ribeiro, J. M. C. Regulatory Mechanisms in Insect Feeding

(Chapman & Hall, New York, 1995).

3. Mattiacci, L., Dicke, M. & Posthumus, M. A. Proc. Natl Acad.

Sci. USA 92, 2036–2040 (1995).

4. Alborn, H. T. et al. Science 276, 945–949 (1997).

5. Kahl, J. et al. Planta 210, 336–342 (2000).

6. Eichenseer, H., Mathews, M. C., Bi, J. L., Murphy, J. B. &

Felton, G. W. Arch. Insect Biochem. Physiol. 42, 99–109 (1999).

7. Saunders, J. A. & Blume, D. E. J. Chromatogr. 205,

147–154 (1981).

transgenic constructs, in the authors’ sam-ples is more consistent with F1 hybridiza-tion than introgression. In introgression, asmall, polymorphic genomic region is bredinto a given variety through repeated back-crossing, so all progeny (or kernels) froman individual in which a (trans)gene hasbeen introgressed will possess that gene.Quist and Chapela report, however, that on the basis of “low amplification” by the polymerase chain reaction (PCR), thetransgene is evident only in a small percent-age of kernels in each cob, citing a pressrelease that reported a 3–10% abundance oftransgenes in similar samples to supporttheir claim.

The authors interpret their inverse PCR(i-PCR) results as evidence of a high frequency of transgene insertion into arange of genomic contexts, inferring fromthis that introgression events are relativelycommon and well maintained. However,their i-PCR products all seem to be arte-facts of the methodology used. We exam-ined the sequences of the reported i-PCRproducts (Fig. 1) and found that none contains a reasonable number of the featuresthat would be expected in a legitimateproduct of amplified genomic DNA flank-ing the anchor sequence. An i-PCR productderived from the circularization of a singlepiece of DNA should contain the sequence

600 NATURE | VOL 416 | 11 APRIL 2002 | www.nature.com

host defences1,2. Our results show that glucose oxidase, one of the principal com-ponents of H. zea saliva, is responsible for suppressing induced resistance in N.tabacum. This enzyme may prevent theinduction of nicotine by directly inhibitingthe wound-signalling molecule jasmonicacid and/or by antagonizing its interactionwith other signalling pathways. As glucoseoxidase is produced by a wide variety ofcaterpillar species1,6, we may have discov-ered a new feature of the evolutionary armsrace between plants and herbivores.Richard O. Musser*, Sue M. Hum-Musser†,Herb Eichenseer*, Michelle Peiffer‡, GaryErvin*, J. Brad Murphy†, Gary W. Felton‡Departments of *Entomology and †Horticulture,

brief communications

concluding that reassortment of integratedtransgenic DNA occurs during transforma-tion or recombination.

The discovery of transgenes fragmentingand promiscuously scattering throughoutgenomes would be unprecedented and isnot supported by Quist and Chapela’s data1

— the incorrectly cited work2 merely claimsthat multiple transgenes or transgene fragments can integrate into geneticallylinked regions of the genome during trans-formation, and not that they can movearound the genome by recombination afterintegration (W. Pawlowski, personal com-munication).

The discovery of cauliflower mosaicvirus promoter sequences, an element of

COMMUNICATIONS ARISING

Biodiversity

Suspect evidence oftransgenic contamination

Quist and Chapela1 claim that trans-genic DNA constructs have beenintrogressed into a traditional maize

variety in Mexico, and furthermore suggestthat these constructs have been reassortedand introduced into different genomicbackgrounds. However, we show here thattheir evidence for such introgression isbased on the artefactual results of a flawedassay; in addition, the authors misinterpreta key reference2 to explain their results,

Upstream

Downstream

Figure 1 Alignment of primers with cauliflower mosaic virus 35S promoter sequences and the ends of inverse polymerase chain

reaction (i-PCR) products. The upstream and downstream orientations with respect to the 35S promoter were confused in the original

publication1 and are corrected here. In parentheses: primer name, or GenBank accession number and fragment size. Shaded regions

represent the maize genomic sequences with the highest homology (BLAST e-value shown) to the preceding i-PCR fragment. The

reverse complements of even-numbered primers are shown for alignment. The footprint of the EcoRV restriction enzyme is shown in

bold and is italicized if in alignment with the site on the 35S promoter. Capitals denote bases that match 35S sequences and are

underlined in the regions of the primers; a dashed underline denotes these respective sequences in cases in which they appear at the

wrong end and in the wrong orientation on the product. For the hypothetical legitimate i-PCR products, plus signs indicate areas

where further transgene DNA would be found.

Editorial noteIn our 29 November issue, we publishedthe paper “Transgenic DNA introgressedinto traditional maize landraces in Oaxaca, Mexico” by David Quist andIgnacio Chapela. Subsequently, wereceived several criticisms of the paper,to which we obtained responses from the authors and consulted referees overthe exchanges. In the meantime, theauthors agreed to obtain further data, on a timetable agreed with us, that might prove beyond reasonable doubtthat transgenes have indeed become integrated into the maize genome. The authors have now obtained someadditional data, but there is disagree-ment between them and a referee as towhether these results significantly bolster their argument.

In light of these discussions and thediverse advice received, Nature has concluded that the evidence available isnot sufficient to justify the publication ofthe original paper. As the authors never-theless wish to stand by the available evidence for their conclusions, we feel itbest simply to make these circumstancesclear, to publish the criticisms, theauthors’ response and new data, and toallow our readers to judge the science for themselves.Editor, Nature

© 2002 Macmillan Magazines Ltd