Stable isotope labelling of earthworms can help deciphering belowground–aboveground interactions involving earthworms, mycorrhizal fungi, plants and aphids

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Pedobiologia 57 (2014) 197–203

Contents lists available at ScienceDirect

Pedobiologia - Journal of Soil Ecology

j ourna l homepage: www.elsev ier .de /pedobi

table isotope labelling of earthworms can help decipheringelowground–aboveground interactions involving earthworms,ycorrhizal fungi, plants and aphids

ndrea Grabmaiera, Florian Heigla, Nico Eisenhauerb,c, Marcel G.A. van der Heijdend,ohann G. Zallera,∗

Institute of Zoology, University of Natural Resources and Life Sciences Vienna, Gregor Mendel Straße 33, A-1180 Vienna, AustriaGerman Centre for Integrative Biodiversity Research, University of Leipzig, Deutscher Platz 5e, 04103 Leipzig, GermanyInstitute for Biology, University of Leipzig, Johannisallee 21, 04103 Leipzig, GermanyAgroscope, Reckenholzstrasse 191, CH-8049 Zurich, Switzerland

r t i c l e i n f o

rticle history:eceived 27 May 2014eceived in revised form 20 October 2014ccepted 20 October 2014

eywords:boveground–belowground interactionsphidsrbuscular mycorrhizal fungiarthwormsultitrophic interactions

table isotopes

a b s t r a c t

Functional relationships between belowground detritivores and/or symbionts and aboveground primaryproducers and their herbivores are not well studied. In a factorial greenhouse experiment we studiedinteractions between earthworms (addition/no addition of Lumbricus terrestris; Clitellata: Lumbricidae)and arbuscular-mycorrhizal fungi (AMF; with/without inoculation of Glomus mosseae; Glomerales: Glom-eraceae) on the leguminous herb Trifolium repens (Fabales: Fabaceae) and associated plant aphids (Aphisgossypii, A. craccivora; Hemiptera: Aphidoidea). In order to be able to trace organismic interactions, earth-worms were dual-labelled with stable isotopes (15N-ammonium nitrate and 13C-glucose). We specificallywanted to investigate whether (i) isotopic signals can be traced from the labelled earthworms via surfacecastings, plant roots and leaves to plant aphids and (ii) these compartments differ in their incorporationof stable isotopes. Our results show that the tested organismic compartments differed significantly intheir 15N isotope enrichments measured seven days after the introduction of earthworms. 15N isotopeincorporation was highest in casts followed by earthworm tissue, roots and leaves, with lowest 15N sig-nature in aphids. The 13C signal in roots, leaves and aphids was similar across all treatments and is for thisreason not recommendable for tracing short-term interactions over multitrophic levels. AMF symbiosisaffected stable isotope incorporation differently in different subsystems: the 15N isotope signature washigher below ground (in roots) but lower above ground (leaves and aphids) in AMF-inoculated meso-cosms compared to AMF-free mesocosms (significant subsystem × AMF interaction). Aphid infestation

was unaffected by AMF and/or earthworms. Generally, these results demonstrate that plants utilize nutri-ents excreted by earthworms and incorporate these nutrients into their roots, leaf tissue and phloem sapfrom where aphids suck. Hence, these results show that earthworms and plant aphids are functionallyinterlinked. Further, 15N-labelling earthworms may represent a promising tool to investigate nutrientuptake by plants and consequences for belowground-aboveground multitrophic interactions.

ntroduction

In the last decade it has increasingly been recognized that aombined aboveground–belowground approach is necessary tonderstand the functioning of terrestrial ecosystems (Wardle et al.,

004; van der Putten et al., 2009; Bardgett and Wardle, 2010;isenhauer, 2012). Plants thereby play an essential role as theynterlink above- and belowground subsystems. Factors above the

∗ Corresponding author. Tel.: +43 1 47654 3205; fax: +43 1 47654 3203.E-mail address: [email protected] (J.G. Zaller).

ttp://dx.doi.org/10.1016/j.pedobi.2014.10.002031-4056/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

soil surface can directly or indirectly influence the plant itself,but can also affect soil processes and soil organisms that can feedback to plants (Bardgett and Wardle, 2003; Porazinska et al., 2003;Schröter et al., 2004; Wardle et al., 2004; van der Putten et al.,2009). Several studies investigating the functional diversity andmultitrophic interactions in terrestrial ecosystems have shown thataboveground–belowground interactions can have consequences atthe ecosystem level (Scheu, 2001; Wardle et al., 2004; Megías and

Müller, 2010; Eisenhauer and Schädler, 2011; Zaller et al., 2011b;Eisenhauer, 2012; Arnone et al., 2013). However, so far only a fewstudies have focussed on the effects of belowground detritivoresand symbionts on aboveground herbivory (e.g. Poveda et al., 2003;

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egías and Müller, 2010; Wurst, 2010; Wurst and Rillig, 2011;rouvé, 2013).

Earthworms make up the majority of the soil faunal biomassn temperate grasslands (Lee, 1985; Curry, 1994) and are consid-red as ecosystem engineers (e.g. Jones et al., 1994). They alterhe quality of resource inputs either directly, through the returnf up to 45 ton ha−1 a−1 of nutrient rich casts (Bohlen et al., 1997),r indirectly by altering soil processes through bioturbation, accel-ration of decomposition of organic materials and an increase inicrobial activity and nutrient mineralization, which results in

ncreased plant nutrient uptake and plant growth (Brussaard, 1999;onkowski et al., 2001). Despite the plethora of information on theffects of earthworms on soil structure, nutrient availability andlant growth (Bohlen et al., 1997; Scheu, 2003), little is knownbout their effects on aboveground herbivores (Wurst, 2010). A fewtudies report earthworm-stimulated herbivory (Scheu et al., 1999;

urst and Jones, 2003; Poveda et al., 2005), but it appears that noffects or negative effects of earthworms on aboveground herbi-ores are more often found (Bonkowski et al., 2001; Wurst et al.,003; Wurst et al., 2004; Trouvé, 2013; Zaller et al., 2013a).

Arbuscular mycorrhizal fungi (AMF) form a widespread mutu-lism between the plant roots of over 80% of all families of landlants (Smith and Read, 2008). In most environmental conditions,hese fungi are beneficial to their host plants, by providing accesso limiting soil nutrients, increasing photosynthetic rates and resis-ance to drought, insect herbivores and fungal pathogens (reviewedn van der Heijden and Sanders, 2002; Smith and Read, 2008). Stud-es on the effects of AMF on herbivores show either positive or noffects of AMF on phloem feeding insects (Koricheva et al., 2009;eidinger et al., 2012).

In most terrestrial ecosystems of the temperate region,arthworms and arbuscular mycorrhizal fungi are commonlyo-occurring and interacting with each other, however our under-tanding of these functional interactions is still very rudimentary.enerally, earthworms are thought to affect AMF populations by (i)

ngesting fungal species (Dash et al., 1979; Cooke, 1983; Edwardsnd Fletcher, 1988; Morgan, 1988; Kristufek et al., 1992; Bonkowskit al., 2000) thus affecting the germination of ingested spores (Parle,963; Hoffmann and Purdy, 1964; Keogh and Christensen, 1976;triganova, 1988) and (ii) dispersal of AMF spores (Coûteaux, 1994;attinson et al., 1997; Wurst et al., 2004). Even less is known abouthe effects of earthworms and AMF on aboveground herbivores,specially on sap-sucking herbivores such as aphids.

In both natural and agricultural ecosystems aphids are knowno affect plant growth, biomass production, phenology and chem-stry (Dixon, 1998). The presence of both earthworms and AMF

as shown to accelerate the development of an aphid speciesMyzus persicae), while aphids were delayed when only AMF orarthworms were present (Wurst et al., 2004). In another studyphid abundance on plants decreased in treatments with earth-orms, but this was independent of the presence of AMF (Wurst

nd Forstreuter, 2010). Despite these important contributions,he mode of interaction between earthworms, AMF, plants andap-feeding insects remains uncertain and mainly conceptual asraditional ecological methods provided only limited informationn nutrient fluxes among organisms involved in these trophic rela-ionships.

To better understand potential multitrophic interactionsetween earthworms and other organisms in terrestrial ecosys-ems we for the first time used 15N–13C dual stable isotope labellingf earthworms (Schmidt et al., 2004; Heiner et al., 2011) in exper-mental ecosystems comprising AMF, plants and aphids. We used

5N ammonium-nitrate and 13C glucose as both elements can beaken up by plant roots either directly or when incorporated inmino acids or other organic sources (Marschner, 1995). For theresent study, we hypothesized that due to functional interactions

gia 57 (2014) 197–203

the isotopic label can be detected in different parts of this modelecosystem and that AMF would increase stable isotope uptake intoplants and aphids. In particular we investigated whether (i) isotopicsignals would be passed on from labelled earthworms to surfacecastings, plants and aphids, and (ii) these compartments differ intheir incorporation of stable isotopes.

These objectives were tested in a factorial experiment wherewe manipulated the factors earthworms (Lumbricus terrestris) andAMF (Glomus mosseae) and studied their single or interactive effectson the legume Trifolium repens and associated aphids (Aphis spp.).The chosen species are commonly co-occurring and interacting intemperate grassland ecosystems throughout Europe.

Materials and methods

Experimental setup and treatments

The experiment was carried out in the research greenhouseof the University of Natural Resources and Life Sciences, Vienna(BOKU) between March and November 2011. Using a factorialdesign we manipulated earthworms (two levels – addition of L.terrestris, +EW, without earthworms, −EW) and AMF inoculation(two levels – inoculation with G. mosseae, +AMF, no AMF inoc-ulation, −AMF) to investigate interactions between earthworms,AMF, plants and aphids. We set up six replicates of each treatmenttotalling 24 experimental units.

Experimental units consisted of 20 L plastic planting pots(diameter: 31 cm, height: 30 cm; further called mesocosms)filled with 18 L sterilized field soil (Haplic Chernozem, siltyloam; steamed at 100 ◦C for 15 h) and quartz sand (grain size1.4–2.2 mm) in a ratio of 40:60 (v/v) (bulk density 1.4 g cm−3,pH = 7.6, Corg = 22.0 g kg−1, Ntot = 0.92 g kg−1, P-CAL = 64.5 mg kg−1,K-CAL = 113.6 mg kg−1). We successfully used this substrate mix-ture in other experiments involving the same earthworm, plantand AMF taxa (e.g. Putz et al., 2011; Zaller et al., 2011c; Zaller et al.,2013b). All mesocosms were lined at the bottom with two layers ofgarden fleece material to prevent earthworms from escaping whilestill allowing water drainage.

The uppermost 10 cm soil layer (approximately 6 L) was inoc-ulated with AM fungal propagules [Glomus mosseae (T.H. Nicolson& Gerdemann) Gerdemann & Trappe (La Banque Européenne desGlomales – BEG 198) (Glomeraceae)] by mixing 25 g L−1 of commer-cial AMF inoculum (Symbion, Landskroun, Czech Republic) to thesubstrate (treatment +AMF). AMF-free control treatments (−AMF)received the same amount of sterilized, inactive AMF inoculum(steamed at 110 ◦C for 2 h). In addition, each mesocosm received100 ml microbial wash from active AMF inoculum and 300 mlmicrobial wash of field soil. This microbial wash corrects for pos-sible differences in microbial communities between the differenttreatments (Koide and Li, 1989). It was prepared with a total of0.5 kg AMF inoculum and 3.5 kg field soil that was wet-sievedthrough a cascade of sieves, where the finest sieve had a mesh sizeof 25 �m to receive a filtered non-mycorrhizal microbial inoculum.

Furthermore, Trifolium repens L. (Fabaceae) seeds from an agri-cultural seed supplier (Lagerhaus, Groß-Enzersdorf, Austria) weregerminated in commercial steam-sterilized (105 ◦C for 20 h) pot-ting soil on a greenhouse bench. Eight days after germination, 18seedlings (approx. 2 cm high) were transplanted into each meso-cosm in a consistent hexagonal pattern in equal distance of eachplant individual of 5 cm (equals 240 plants m−2). The greenhousecontaining the mesocosms was not temperature-controlled, arti-

ficial light was only provided during the last 38 days (14 h lightday−1 in October and November). Mean air temperature duringthe course of the experiment was 21.7 ◦C at 51.6% mean relativehumidity. The mesocosms were irrigated daily by adding 500 ml of

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ap water mesocosm−1 and were randomly rearranged every twoeeks to avoid a bias of potential environmental gradients within

he greenhouse. No fertilizers were applied during the course of thexperiment.

sotopic labelling of earthworms

In order to be able to trace the interactions between earth-orms, AMF, plants and aphids, earthworms were labelled with

5N and 13C stable isotopes prior to introduction into the respec-ive mesocosms. Adult L. terrestris (Linnaeus 1758) were obtainedrom a fishing bait shop (Anglertreff E. Lux, Vienna, Austria). Prioro the labelling, all earthworms were cultivated in sterile soil anded with ground oat flakes for four days. Following Heiner et al.2011) we added 100 mg of 13C6H12O6 (99 at.% 13C6-glucose; Cam-ro Scientific, Berlin, Germany) and 100 mg 15NH4NO3 (98 at.%5N-ammonium nitrate; Campro Scientific, Berlin, Germany) to00 g of dry field soil and stored it in a climate chamber at 15 ◦C forleven days. After this incubation time earthworms from sterile soilere carefully rinsed with water, transferred into the labelled soilixture and kept there for seven days to allow proper incorporation

f 15N and 13C into their tissue and gut. We placed two adult earth-orm individuals per 100 g of soil-isotope mixture; earthwormsere not fed during this labelling period.

Two months prior to the end of the experiment, four labelleddult specimens of L. terrestris with a mean initial biomass of.89 ± 0.14 g per individual (mean ± SE) were added to each meso-osm of the +EW treatment, resembling a density of 204.8 g m−2,hich is slightly higher than the ambient density in permanent

rasslands (Decaens et al., 1997). The upper rim of all mesocosmsas extended with a transparent plastic barrier (10 cm height) torevent earthworms from escaping.

phids

Aphids (Aphididae) coming into the greenhouse through theermanently open side windows colonized the mesocosms startingbout eleven weeks prior to the insertion of the labelled earth-orms. Aphids comprised a mixed abundance of 50% Aphis gossypii

Glover 1877) and 50% Aphis craccivora (Koch 1854; E. Koschier,ersonal communication). Aphid abundance was estimated once aeek over a period of 10 weeks. The investigation of freely moving

phids has been proven successful in exploring aphid selectivityor host plants and reproduction in response to manipulation ofoil organisms (Eisenhauer and Scheu, 2008).

ampling and analytical measurements

Aboveground plant growth was assessed on days 71, 115 and89 after germination by measuring plant height from the soil sur-ace to the uppermost end of the leaves. Plant biomass productionas assessed by cutting the vegetation 1 cm above the soil sur-

ace on the dates mentioned above. For the statistical analyseshe cumulated height growth and cumulated biomass productionsums of the three measurements and harvests) were used. Plant

aterial was dried at 50 ◦C for two days and weighed to determinery mass production.

Samples for 13C and 15N analyses were taken one week after thentroduction of the labelled earthworms by randomly collectingwo surface casts per mesocosm, clipping five Trifolium leaves per

esocosm and collecting approximately 80 aphids per mesocosm;hree root samples were taken using a corer (diameter: 3 cm, depth:

0 cm) inserted near Trifolium plants three weeks after earthworm

ntroduction. Plant leaves and earthworm casts were dried at 60 ◦Cor 24 h, aphids and root samples were freeze-dried for 48 h. Sub-equently all samples were ground using a ball mill. Samples for

gia 57 (2014) 197–203 199

13C and 15N analyses in earthworms were taken 8 weeks after theirintroduction by collecting one earthworm per mesocosm. This wasdone at night when earthworms were searching for food on thesoil surface. Collected earthworms were carefully rinsed in distilledwater and weighed. For isotopic analysis of the earthworm tissueonly the anterior 15 segments of the earthworms without gut con-tent were used, this tissue part was freeze-dried and homogenizedmanually using a mortar and pestle.

For isotopic analysis 1.0 mg of aphids, 1.8 mg of leaf material,1.8 mg of root material, 5.0 mg of earthworm casts, and 1.0 mgof earthworm tissue samples were weighed into tin capsulesand analysed for N and C by continuous flow isotope ratio massspectrometry (CF-IRMS) at the SILVER lab of the Department ofTerrestrial Ecosystem Ecology at the University of Vienna, Austria.Ratios (R) of 15N: 14N and 13C: 12C were expressed relative to theinternational standards (atmospheric air for 15N and Vienna PeeDee Belemnite for 13C) in parts per thousand (�‰) using the formulaRsample × Rstandard

−1 × 1000. The analytical precision was 0.15‰ forN and 0.1‰ for C isotopes, respectively. Natural abundance val-ues of 15N and 13C of earthworm tissue and casts were obtainedfrom earthworms that were cultivated in the same way as describedabove, except no isotopic labels were added to the substrate.

To quantify AMF root colonization rates fresh root subsampleswere cleared with boiling KOH and vinegar for 4 min and stainedfor 1 min with Shaeffer® black ink (Vierheilig et al. 1998). Stainedroots were analysed under a dissecting microscope (100× mag-nification) and the percentage of colonized roots with AMF wascalculated using the gridline intersection method by counting 200intersections per sample (Giovanetti and Mosse, 1980).

Statistical analysis

Normal distribution (Shapiro–Wilk test) and homogeneity ofvariances (Levene’s test) were tested and the data were trans-formed if necessary to match the prerequisites for analysis ofvariance (ANOVA). Multiple samples per mesocosm were averagedto create one value per mesocosm. One-way ANOVAs were per-formed to test for effects of earthworms on root mycorrhization andfor effects of AMF on earthworm numbers and biomass, on cumu-lated plant height growth, on cumulated aboveground biomassproduction and on aphid abundance. Two way ANOVAs with AMFand earthworms as factors were performed to test for treatmenteffects on 13C and 15N in earthworm tissue, earthworm casts, roots,leaves and aphids. Two way ANOVAs were also performed to testfor effects of earthworms and AMF on 13C and 15N signatures ofthe aboveground subsystem (comprising leaves and aphids) vs. thebelowground subsystem comprising roots. Significance of differ-ences between means of two or more groups was analysed usingTukey’s HSD post hoc tests. Linear regressions were performed toexplore relationships between isotopic enrichments of the differentcompartments. All statistical analyses were conducted using thesoftware IBM SPPS Statistics 20 (SPSS Inc. Headquarters, Chicago,Illinois, USA). Values given throughout the text are means ± SD.

Results

Mycorrhiza and earthworms

Across the AMF-inoculated mesocosms 32.3 ± 1.6% of the rootsof T. repens were colonized by AMF; 3.6 ± 2.3% of the roots of theAMF-free treatments were colonized by AMF. Percent root length

colonized by AMF was significantly decreased in the presenceof earthworms (−EW = 34.0 ± 1.6%, +EW = 29.0 ± 2.0%; F1,22 = 4.74,P = 0.042). At the end of the experiment, the biomass of therecaptured earthworms was 3.53 ± 0.23 g earthworm−1; a decrease

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200 A. Grabmaier et al. / Pedobiologia 57 (2014) 197–203

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ig. 1. Isotopic enrichment of �15N in (a) earthworm surface casts, (b) earthworm tind with (+AMF) inoculation with arbuscular mycorrhizal fungi. Note different scal

f 9.2% compared with the initial weights. Neither earthwormumbers at the end of the experiment (F1,8 = 0.39, P = 0.552) norecaptured earthworm biomass were affected by AMF (F1,12 = 0.01,

= 0.911).

lant performance and aphid infestation

Both cumulated height growth (+21%) and cumulated above-round biomass (+19%) production of T. repens was significantlyncreased in the presence of AMF (height: −AMF = 12.9 ±.9 cm; +AMF = 15.4 ± 1.0 cm: F1,22 = 50.68, P < 0.001; biomass:AMF = 9.3 ± 1.4 g; +AMF = 11.0 ± 2.3 g: F1,22 = 5.01, P = 0.036). Thebundance of aphids was not significantly affected by AMF treat-ents (F1,22 = 2.18, P = 0.153; total number of aphids mesocosm−1:AMF = 663.3 ± 75.0, +AMF = 781.7 ± 59.5). No statements aboutarthworms effects on plant growth, plant production and aphidbundance can be made, because earthworms were inserted intohe mesocosms after the third biomass harvest and aphid abun-ance estimations were not continued during this time.

sotopic enrichments and nutrient concentrations

Mean �15N natural abundance values of treatments containingon-labelled earthworms varied between different compartments,owever was not significantly affected by AMF (F4,52 = 1.023,

= 0.336; earthworms: 6.39 ± 0.4‰, casts: 24.38 ± 0.7‰, roots:.64 ± 0.5‰, leaves: −0.62 ± 0.1‰, aphids: 0.45 ± 0.1‰). Mean15N values of earthworms, casts, roots, leaves, and aphidsf treatments containing labelled earthworms differed signifi-antly from each other (F4,52 = 116.08, P = 0.001), but were notffected by AMF (F1,55 = 0.315, P = 0.577; Fig. 1). Across AMF

reatments, the highest 15N enrichment levels were measuredn casts (11,751.21 ± 1405.2‰), followed by earthworm tissue454.68 ± 59.4‰), roots (62.65 ± 21.8‰), leaves (50.94 ± 16.9‰)nd aphids (37.75 ± 14.3‰; Fig. 1).

c) aphids, (d) leaves and (e) roots of Trifolium repens in mesocosms without (−AMF)y-axes. Means ± SD, n = 5–6.

Regarding �13C, only earthworms and casts showed significantlyincreased values relative to −EW control treatments (Table 1).Delta 13C values did not differ significantly between compartments(roots, leaves and aphids; F2,53 = 4.79, P = 0.543). With the excep-tion of �13C in casts (−AMF: 39.54 ± 9.9‰, +AMF: 99.21 ± 18.3‰;F1,5 = 10.13, P = 0.007) � 13C values were not significantly affectedby AMF treatments. Although �15N values for earthworms, castsand roots seemed to be positively affected by the presence ofAMF, this was only marginally statistically significant (Fig. 1;Table 1).

When considering �15N of leaves and aphids as abovegroundsubsystem and �15N of roots representing the belowground sub-system, while excluding earthworm tissue and casts as theyform the vector of isotopic dispersal, a significant interactionbetween AMF and these subsystems was found (F1,32 = 4.61,P = 0.039). Mycorrhiza decreased 15N signatures in the above-ground subsystem, but increased the signature in the belowgroundsubsystem (Fig. 1). The same analysis with �13C did not showa significant interaction between AMF and the subsystems(F1,32 = 0.70, P = 0.415). Neither in the 15N nor in the 13C signa-ture was there a significant earthworm × AMF interaction effect(P > 0.05).

Aphid �15N was significantly positively correlated with leaf�15N, however 15N signals of the other compartments investi-gated appeared to be unrelated to each other (Table 2). Aphid�13C was significantly positively correlated with leaf �13C, also�13C of roots and casts as well as �13C of roots and leaves weresignificantly positively related (Table 2). The initial earthwormbiomass was positively correlated with the 15N of casts (r = 0.65,P = 0.029).

Total N and C in leaves and aphids were significantly lowerin +EW than in −EW treatments, but were unaffected by AMF(Table 3). At the end of the experiment, total N and C in leaves

and aphids were affected by EW but not by AMF treatments(Table 3).

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A. Grabmaier et al. / Pedobiologia 57 (2014) 197–203 201

Table 1Summary of ANOVA results for the effects of earthworms (EW) or arbuscular mycorrhizal fungi (AMF) and their interactions on log �15N (‰) and log �13C signature inearthworm tissue, earthworm casts, roots, leaves and aphids. Significant effects (P < 0.05) are in boldface. Model degrees of freedom (df) are represented by the numeratordf and the denominator df, respectively.

Variable Experimental factors

Earthworms (EW) AMF EW × AMF

df F P df F P df F P

Log �15NEarthworms – – – 1,12 4.144 0.064 – – –Casts – – – 1,13 4.134 0.063 – – –Roots 1,20 63.204 <0.001 1,20 2.443 0.134 1,20 0.883 0.359Leaves 1,20 239.220 <0.001 1,20 0.973 0.336 1,20 0.335 0.569Aphids 1,20 171,658 <0.001 1,20 1.935 0.179 1,20 1.077 0.312

Log �13CEarthworms – – – 1,12 2.151 0.168 – – –Casts – – – 1,13 10.127 0.007 – – –Roots 1,20 0.434 0.517 1,20 1.511 0.233 1,20 1.956 0.177Leaves 1,19 0.504 0.486 1,19

Aphids 1,20 2.787 0.111 1,20

–, not applicable as -EW contained no earthworms or casts.

Table 2Pearson correlation coefficients between log �15N and log �15C (‰) signatures ofdifferent compartments investigated in the present experiment. Only mesocosmsthat contained labelled earthworms were considered.

Variable Earthworms Casts Roots Leaves

Log �15NCasts 0.352Roots −0.439 0.245Leaves −0.150 −0.519 0.237Aphids −0.180 −0.389 0.125 0.781**

Log �13CCasts 0.539Roots 0.203 0.639*

Leaves −0.137 0.351 0.717**

Aphids −0.129 0.185 0.299 0.704*

Asterisks denote significant deviations from horizontal line:

D

T

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bution of 13C tracer in different compartments could also be that

TCfM(

–

* P < 0.10.** P < 0.05.

iscussion

racking functional relationships with stable isotopes

This study demonstrates for the first time that plants utilizeitrogen originated from earthworms and incorporate this nitro-en into roots, leaf tissue and phloem from where it is taken up

able 3oncentrations of nitrogen and carbon (means ± SD) of earthworm tissue, earthworm casts

ungi (AMF). Summaries of two-way ANOVAs testing treatment effects in different compartodel degrees of freedom (df) are represented by the numerator df and the denominator d

P < 0.05; n = 5–6) based on Tukey HSD posthoc mean comparisons.

Variable Treatments

−EW−AMF −EW+AMF +EW−AMF +EW+AMF

Means ± SD

N concentration (%)Earthworms – – 11.55 ± 0.45a 12.28 ±Casts – – 0.17 ± 0.00a 0.16 ±Roots 1.40 ± 0.12a 1.11 ± 0.14a 1.21 ± 0.08a 1.06 ±Leaves 6.18 ± 0.14a 6.10 ± 0.14a 5.48 ± 0.17b 5.78 ±Aphids 6.53 ± 0.24ab 6.58 ± 0.08a 5.82 ± 0.18b 5.88 ±

C concentration (%)Earthworms – – 44.69 ± 1.27a 46.07 ±Casts – – 5.05 ± 0.21a 4.99 ±Roots 39.89 ± 1.28a 41.10 ± 0.85a 38.91 ± 0.91a 37.80 ±Leaves 44.66 ± 0.65ab 43.68 ± 0.16b 41.65 ± 0.17cd 41.72 ±Aphids 51.91 ± 0.24a 51.90 ± 0.22a 51.17 ± 0.33abc 50.47 ±

, not applicable as -EW contained no earthworms or casts.

0.258 0.617 1,19 0.589 0.4520.444 0.513 1,20 0.469 0.501

by sap-sucking herbivores. Even across three trophic levels includ-ing detritivores, plants and herbivores the 15N labelling signaturewas substantially stronger than natural abundance levels. It isconcluded that a one-time addition of 15NH4NO3 to earthwormsubstrate can adequately label earthworms and associated orga-nisms. We took most of our samples one week after introducingthe labelled earthworms to mesocosms and it remains to be investi-gated for how long and for which sampling scale isotopic signaturesare effective. A report that isotopically labelled earthworm castslose little of their 15N and 13C signature even when stored in fieldsoil for over three months (Heiner et al., 2011) suggests that trophicinteractions can be studied for much longer periods than in thepresent study. Overall, this approach will be useful when furtherinvestigating previously unknown functional relations betweenabove- and belowground organisms also under field conditions.Contrary to our assumption, that 13C could be taken up by plantroots via amino acids and other organic substances (Chapin et al.,1993; Marschner, 1995; Jones et al., 2004), the amounts used in thisexperiment seem to be insufficient for tracing trophic interactionsas 13C enrichment in roots, leaves and aphids was in the range ofnatural abundance levels. An explanation for the more even distri-

13C cycling likely involved mineralization and uptake of 13CO2.Consistent with our expectations, AMF altered the nutrient

uptake of plants resulting in an increased root 15N signature, but

, roots, leaves and aphids in response to earthworms (EW) or arbuscular mycorrhizalments are mentioned on the right side of the table; significant effects are in boldface.f, respectively. Different letters within each column represent significant differences

ANOVA results

EW AMF EW × AMF

df F P df F P df F P

0.50a – – – 1,12 1.612 0.228 – – – 0.00a – – – 1,13 0.124 0.730 – – – 0.06a 1,20 1.190 0.288 1,20 4.313 0.050 1,20 0.458 0.506 0.13ab 1,20 12.602 0.002 1,20 0.580 0.455 1,20 1.782 0.197 0.18ab 1,20 15.344 0.001 1,20 0.118 0.735 1,20 0.001 0.976

1.37a – – – 1,12 0.770 0.398 – – – 0.19a – – – 1,13 0.068 0.798 – – – 1.22a 1,20 3.923 0.062 1,20 0.002 0.965 1,20 1.142 0.298 0.31d 1,20 42.398 <0.001 1,20 1.442 0.244 1,20 1.893 0.184 0.29b 1,20 15.795 0.001 1,20 1.684 0.209 1,20 1.579 0.223

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decreased leaf and aphid 15N (significant subsystem–AMF inter-ction). We assume that this is because AMF facilitated nitrogenptake by plants (Smith and Read, 2008), which resulted in a higherboveground plant biomass and consequently diluted (decreased)5N levels in leaves and aphids.

arthworm–AMF interactions

In the current study, effects of earthworms and interactions withMF on growth and biomass production of the T. repens stands wereot explored. The enhanced growth and biomass production of T.epens in the presence of AMF in the present study is in line witheveral previous studies indicating that AMF increased the growthf legumes (van der Heijden and Sanders, 2002; Smith and Read,008; Wagg et al., 2011a, 2011b; Zaller et al., 2011a; Trouvé, 2013).

Only a few studies have investigated interactions betweenarthworms and AMF. In the present study, root mycorrhizationates were decreased by earthworms, indicating that earthwormsay have grazed on the mycelium and possibly disrupted the con-

act of the external hyphae from the roots. Contrastingly, sometudies reported increased mycorrhization rates in the presence ofarthworms (Gormsen et al., 2004; Zarea et al., 2009; Trouvé, 2013),hile in several other studies no significant effects of earthworms

n AMF colonization were found (Eisenhauer et al., 2009; Wurstnd Rillig, 2011; Zaller et al., 2011c). Presumably, these interac-ions are species-specific and/or context-dependent and no generalattern can be seen. In several greenhouse experiments no interac-ive effects of earthworms and AMF on the performance of plantsere found (Eisenhauer et al., 2009; Wurst and Rillig, 2011; Zaller

t al., 2011c; Trouvé, 2013; Zaller et al., 2013b). Another study onombined effects of earthworms and AMF on plant communitynd diversity showed that seedling emergence and diversity waseduced by anecic earthworms in the presence of AMF (Zaller et al.,011b). More studies on the interactions between earthworms andMF are needed, which may allow subsequent meta-analyses and

he exploration of biotic and abiotic factors shaping such below-round interactions.

arthworm–AMF–aphid interactions

Both positive and negative effects of AMF on the performancend abundance of invertebrate herbivores have been reported inrevious studies (e.g. Koricheva et al., 2009; Trouvé, 2013; Zallert al., 2013a). Earthworms have been shown to have consider-ble effects on nutrient cycling in soil affecting nutrient uptakend growth of plants (Brown, 1995; Curry and Schmidt, 2007;utenschoen et al., 2009). Furthermore, earthworms can indirectlyffect sap-sucking insects through altering primary and secondaryetabolites and the expression of stress-responsive genes of plants

Bonkowski et al., 2001; Scheu, 2001; Blouin et al., 2005; Wurst,010; Trouvé, 2013). In the present experiment, AMF had no sig-ificant effects on leaf N or C concentrations and hence did notlter the performance of the aphids. Although the effect of earth-orms on aphid performance was not quantitatively tested in theresent study, other reports have shown that earthworms cani) increase (Scheu et al., 1999; Wurst and Jones, 2003; Povedat al., 2005; Eisenhauer and Scheu, 2008; Eisenhauer et al., 2010),ii) decrease (Wurst et al., 2003; Ke and Scheu, 2008) or (iii) notffect aphid infestations (Bonkowski et al., 2001). These inconsis-ent results suggest that effects are context- and species-specific;

ombined individual effects of different soil organisms have alsoeen reported to cancel each other out (Bradford et al., 2002; Wurstt al., 2008) or have synergistic effects on plant and herbivore per-ormance (Eisenhauer et al., 2010).

gia 57 (2014) 197–203

Surprisingly, the concentrations of N and C in aphid and leaftissue were decreased in the presence of earthworms in our exper-iment. This negative earthworm effect can be explained by the factthat this legume species is less responsive to soil improvementsprovided by earthworms through its association with N-fixing rhi-zobia. One other explanation may be that due to enhanced biomassproduction nitrogen was diluted and therefore N concentrationsare only marginally affected by the amount of N bound in planttissue.

Conclusions

The results of this work provide first experimental evidencethat 15N-labelled earthworms can be a powerful tool to investigateaboveground-belowground interactions across multiple trophiclevels. Given the poor knowledge on how stable isotopes mightbe incorporated in other organisms, the next challenge would be toquantify isotopic pools and fluxes and to implement this approachin more complex, multi-species systems in the laboratory and in thefield. Future research aiming at a better understanding of temporaland spatial interactions between aboveground and belowgroundbiota might benefit from employing this methodological approach.

Acknowledgments

We are grateful to Norbert Schuller for help in the greenhouseand laboratory and to Wolfgang Wanek for advice on stable isotopeanalyses. The staff from the Department of Applied Plant Sciencesand Plant Biotechnology at the University of Natural Resourcesand Life Sciences Vienna provided logistical support. This researchwas partly funded by the Austrian Science Fund (FWF project no.P20171-B16).

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