RAPID COMMUNICATION

Hormaphis hamamelidis Fundatrices Benefit by ManipulatingPhenolic Metabolism of Their Host

Brian J. Rehill & Jack C. Schultz

Received: 16 January 2012 /Revised: 26 March 2012 /Accepted: 4 April 2012 /Published online: 25 April 2012# Springer Science+Business Media, LLC (outside the USA) 2012

Abstract We investigated the pattern and potential adaptivevalue of phenolic concentrations in galls induced by theaphid Hormaphis hamamelidis on leaves of Hamamelisvirginiana. By the time that founding females began repro-duction, galls had higher concentrations of condensed tan-nins and lower concentrations of hydrolyzable tannins thanleaves. Galled and ungalled leaf laminas never differedsignificantly in any phenolic measure. Condensed tanninconcentrations also were positively related to the numberof offspring per gall when gall dry weight, another impor-tant correlate of fecundity, was accounted for. This couldindicate the prior sink strength of the gall. Polyphenols mayact as a repository for excess carbon drawn to the gall byincreased sink strength, or be an indication of the fundatrix’ability to manipulate host physiology. This study is the firstto demonstrate a tangible, quantitative association betweenphenolic accumulation in galls and gall-former reproductiveperformance, and illustrates that condensed tannins mayplay roles other than plant defense.

Keywords Gall-former . Herbivory . Host manipulation .

Polyphenols . Source-sink metabolism . Condensed tannin

Introduction

Gall-formers manipulate their hosts (Flaherty and Quiring,2008; Detoni et al., 2010) and many galls have altered levelsof phenolic metabolites (Ikai and Hijii, 2007), but the adap-tive value of these chemical alterations is uncertain (Janzen,1977; Inbar et al., 2009). Speculation regarding the role ofaltered phenolics in galls has included a potential defensiverole against natural enemies (Schultz, 1992) or as a correlateof gall-former success at manipulating host quality (Nymanand Julkunen-Tiitto, 2000).

We tested two hypotheses in this study. H1: Galls formedby the aphid Hormaphis hamamelidis have altered levels ofphenolic metabolites. Our findings of elevated condensedtannins in galls led to the second hypothesis. H2: Aphidfecundity is positively associated with concentrations ofcondensed tannins in galls.

Methods and Materials

Research System Thewitch hazel cone gall aphid,Hormaphishamamelidis (Fitch), forms 7–10 mm tall conical galls onwitch hazel leaves. The fundatrix bears the second generationparthenogenetically within the gall; all the aphids feed fromphloem inside the gall. The number of second-generationaphids provides a fecundity measure for each fundatrix.

Study Site Witch hazel trees from a single site in Pennsylvania,USA, (centered at 40° 45′ 22″ N latitude and 77° 44′ 10″ Wlongitude) were used.

B. J. Rehill : J. C. SchultzPesticide Research Laboratory, Department of Entomology,The Pennsylvania State University,University Park, PA 16802, USA

Present Address:B. J. Rehill (*)Department of Chemistry, United States Naval Academy,572M Holloway Road,Annapolis, MD 21402, USAe-mail: rehill@usna.edu

Present Address:J. C. Schultz105 Bond Life Sciences Center, University of Missouri,Columbia, MO 65211, USA

J Chem Ecol (2012) 38:496–498DOI 10.1007/s10886-012-0115-9

Chemistry Time Course: Material Collection Leaves werecollected on 17 May, 28 May, 6 June, and 8 July 1997.Respectively, these dates corresponded to fundatrices being1st, 2nd, and 3rd instar nymphs, then adults just beginningreproduction in fully grown galls. Six to twelve single-galled and ungalled leaves each were taken from each of 3trees on the first three collection dates and from 6 trees onthe last collection date. Our sample size was relatively smallbecause we sought only a tissue level measure of phenolicconcentrations. Each gall was opened with a sterile scalpel,and the fundatrix was removed by suction with a Pasteurpipette. Leaves and galls were flash frozen in liquid nitrogenand kept on dry ice until storage at −80 °C.

Chemistry Time Course: Phenolic Assays Tissues werelyophilized, ground in a mortar and pestle under liquid N2,then extracted as in Adams et al. (2009). Methods used wereFolin-Denis, potassium iodate, and n-butanol-HCl for totalphenolics, hydrolyzable tannins, and condensed tannins,respectively, and quantification was based on standardcurves generated with purified witch hazel tannins (Adamset al., 2009).

Gall Dry Weight, Fecundity and Phenolic Content On 25July 1999, 120 galls were collected (30/tree × 4 trees). Themean number of offspring per gall was at its peak on thatdate (Rehill and Schultz, 2001). Galls were collected, stored,and handled as above. Only galls without signs of attackfrom natural enemies were used (N070).

Statistical Analyses All statistical analyses were performedwith SAS Version 8 (SAS Institute, Cary, NC, USA).

Results

Chemistry Time Course Folin-reactive phenolics decreasedin leaves between 6/6/97 and 7/8/97, and concentrations ingalls exceeded those in leaves on 7/8/97 (Fig. 1a; date:F3,609.76; P00.010; tissue: F2,406.71; P00.053; date bytissue: F4,801.67; P00.248).

Galls had higher concentrations of condensed tanninsthan leaves, and condensed tannins increased over time inall tissues (date: F3,60133.5; P<0.001; tissue: F2,4079.79;P<0.001; date by tissue: F4,805.47; P00.020). The con-centrations in galls increased between the last two dateswhereas those in leaves did not (Fig. 1b).

Hydrolyzable tannin concentrations started the same inall tissues (Fig. 1c), but diverged between 6/6/97 and 7/8/97when galls contained lower concentrations than galledleaves due to an increase in the galled leaves (date: F3,608.81; P00.013; tissue: F2,401.68; P0.295; date by tissue:F4,804.61; P00.032). For all three phenolic measures, there

were never significant differences between galled andungalled leaves.

Gall Dry Weight, Fecundity and Phenolic Content Phenolicconcentrations in individual galls were largely independentof one another and gall dry weight. Condensed tanninconcentrations were not significantly correlated with Folin-reactive phenolic or hydrolyzable tannin concentrations(both: P>0.2), but Folin-reactive phenolics and hydrolyz-able tannins were correlated (rp00.56; P<0.001).

Considered together with gall dry weight, condensedtannin concentrations positively affected reproductiveoutput. In a stepwise regression, the effects of dry weight(P<0.001) and condensed tannin concentration on fecunditywere significant (P00.005), but Folin-reactive phenolicswere not (P00.230). In a regression that included onlythe significant factors, condensed tannin concentrationexplained 7.4 % of the variation of and was positivelyrelated to offspring per gall [#offspring0−14.6+3.00(dryweight (mg)]+0.52 [condensed tannin concentration

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Fig. 1 Phenolic concentrations in the chemistry time course study: aFolin-reactive phenolics, b condensed tannins, and c hydrolyzabletannins

J Chem Ecol (2012) 38:496–498 497

(% tannin equivalents)]; R200.380; F2, 67020.52; P<0.001;coefficients for dry weight and condensed tannins; P<0.01).Similar to prior experiments, dry weight explained 30.6 %of the variation in offspring per gall. Thus, for a gall of agiven dry weight, fecundity increased with increasingcondensed tannin concentrations.

Discussion

Galls induced by Hormaphis hamamelidis fundatrices havemarkedly different phenolic concentrations from the adja-cent leaf laminas. When galls reach full size and the funda-trices begin to reproduce parthenogenetically, galls havenearly twice the concentration of condensed tannins, buttwo thirds the hydrolyzable tannins of leaves. Furthermore,galled and ungalled leaves never differ in any of the mea-sured phenolic metabolites, and thus, the changes seen ingall tissue are consistent with manipulation by the aphid, notthe choice of leaf. Furthermore, these drastic alterations inphenolic metabolism occur due to the interaction of thefundatrix with the host tissue, as opposed to her offspring,since the changes begin long before they appear. Thus, theevidence suggests that Hormaphis hamamelidis fundatricesmanipulate phenolic metabolism of their galls.

Hormaphis hamamelidis galls accumulated substantialconcentrations of condensed tannins, like many other arthro-pod galls. Interestingly, we found a positive relationshipbetween condensed tannin concentration and the reproductiveperformance of fundatrices. Condensed tannins, typicallythought of as defenses, could in fact reflect nutritional enhance-ment. Galls are physiological sinks, and Hormaphis hamame-lidis galls have upregulated sink metabolism (Rehill andSchultz, 2003). Increased sink strength draws additional pho-tosynthate to a site, thus, condensed tannins might accumulatein these galls as enhanced sink strength—providing increasednutrient flow to the aphids—draws additional substrates forphenolic metabolism to the gall. Alternatively, by first inducinggreater condensed tannin synthesis, fundatrices could inducesink enzyme activity to meet this demand. Enhanced nutritioncould, thus, be a secondary effect of tannin induction.

Other explanations for phenolic accumulation in gallsinclude defense for the gall inhabitants. Caterpillars rarelyeat Hormaphis galls; folivory often extends up to or evensurrounds the uneaten gall (Schultz, 1992, B. Rehill,pers.obs.). Since a gall dies once the fundatrix perishes,

we only measured phenolic concentrations of inhabitedgalls, thus we cannot determine if gall-former survival wasrelated to gall phenolic content.

Regardless of mechanism(s), the positive relationshipbetween condensed tannins and gall-former performancedemonstrates adaptive value other than plant defense. Toour knowledge, this study is the first to demonstrate arelationship between condensed tannin concentrations ininsect gall tissues (vs. galled leaves) and gall-former repro-ductive performance. Thus, these data suggest that regard-ing condensed tannins only as defenses in plant–animalinteractions may be misleading in some cases.

Acknowledgments We thank Annie Rehill, Moriah Szpara, OlivierGautreau, Alice Arrighi, Heidi Appel, Mike Grove, Bryan Severyn,and Tom Arnold for assistance and NSF grants DIB-9413204 andDEB-9902198 for support.

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