1There is increasing evidence that many above-ground and below-ground processes are tightly linked through plant-mediated mechanisms, including indirect interactions between foliar and root herbivores underpinned by changes in host plant chemistry (e.g. defensive or nutritional status). To date, studies addressing interactions involving nutritional mechanisms have relied on rudimentary chemical measurements (e.g. total/soluble nitrogen), overlooking other nutrients such as minerals and specific amino acids.
2This study investigated how the nutrient composition of barley (Hordeum vulgare) responded to separate and simultaneous attack by root-feeding wireworms (Agriotes spp.) and foliar-feeding aphids (Rhopalosiphum padi) after short and long exposures to herbivory, and determined whether the two insects indirectly interacted with each other.
3Wireworms significantly reduced total plant mass and leaf dry mass by up to 25%, but had little impact on nutritional chemistry compared with aphids. Aphids did not affect plant growth but caused significant reductions in most leaf minerals: nitrogen (N), sulphur (S), calcium (Ca) and phosphorus (P). In contrast, aphids increased root mineral concentrations: N, S, Ca and potassium (K). Aphid-induced changes occurred in both short and long exposure experiments, with the exception of leaf Ca, and root N and K which were only significantly affected in the short exposure experiment.
4Above-ground and below-ground herbivory had several interactive (often reversible) effects on plant nutrients. For example, wireworms strengthened the aphid-driven increases in root S by 35% after short exposure, but reduced aphid-induced increases by 10% after longer exposure.
5There was evidence for positive bi-directional interactions between above-ground and below-ground herbivores, with wireworms promoting aphid numbers by 30% and aphids increasing wireworm mass by 25%.
6Contrary to previous mechanistic explanations, below-ground herbivory had little impact on foliar amino acid concentrations, whereas aphids caused significant (approximately 50%) reductions in essential amino acids. However, after longer exposure to wireworm herbivory, aphid-driven reductions in essential amino acids were attenuated. Aphids caused significant increases in root mineral concentrations (especially root S) which potentially promoted wireworm performance.
7The significance of these findings is discussed in the context of current hypotheses of above-ground × below-ground herbivore interactions. In particular, examining specific plant nutrients could be critical to understanding conditional outcomes of insect × plant interactions.
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Root feeding herbivores can detrimentally affect foliar herbivores, and vice versa, by systemically inducing plant defence compounds (Bezemer & Van Dam 2005). Indeed, the emphasis has shifted towards investigating defensive compounds as the principal mechanism underpinning interactions between above-ground and below-ground herbivores (most recently reviewed by Kaplan et al. 2008). This shift probably reflects an increasing interest in herbivore-induced plant defences generally (Karban & Baldwin 1997; Agrawal et al. 1999), but also the advances in techniques for studying defensive chemistry (Bezemer & Van Dam 2005). Nutritional mechanisms underpinning above-ground × below-ground interactions have therefore received comparatively less attention lately. In one of the original papers addressing nutritional interactions between above-ground and below-ground herbivores, Masters et al. (1993) argued that root herbivory impairs the plant's ability to take up water and nutrients from the soil. This results in a reduction in the relative water content of foliage and an increase in soluble nitrogen (e.g. amino acids) and carbohydrates, which subsequently promotes insect, especially aphid, performance. A similar effect is observed when plants experience prolonged water stress that impairs protein metabolism and amino acid synthesis (Brodbeck & Strong 1987), leading to hydrolysis of existing proteins and increases in free amino acids in the foliage (Huberty & Denno 2004).
Despite these nutritional mechanisms being proposed 16 years ago (Masters et al. 1993), surprisingly few studies have experimentally quantified nutritional changes in the plant in the context of multiple attack by above-ground and below-ground herbivores (reviewed by Johnson et al. 2008b). Moreover, for pragmatic reasons these studies tend to make rudimentary measurements such as total (e.g. Gerber et al. 2007) or soluble (e.g. Gange & Brown 1989) nitrogen concentrations, which could overlook many important and complex changes in plant nutritional quality. For example, root herbivores have been shown to affect foliar minerals such as potassium, phosphorus and other micro-elements (Coale & Cherry 1989; Borowicz et al. 2005). An additional complexity that has yet to be fully examined is that many aspects of plant chemistry change with plant phenology and duration of insect attack, so any ‘outcome’ of interactions between above-ground and below-ground herbivores could be highly dependent on when experimental measurements are made.
The aim of this study was: first, to investigate in detail the response of tissue nutrient composition in barley (Hordeum vulgare) to individual and simultaneous attack by different insect types feeding at spatially separate locations, that is, above-ground and below-ground; second, to ascertain whether each insect type influenced the performance of the other; and finally, to explore the possibility that any observed above-ground × below-ground interactions in herbivore performance were mediated by changes in plant nutrients. The system comprised wireworms (Agriotes spp. L. Coleoptera: Elateridae) as the below-ground insect herbivore and the bird cherry oat aphid (Rhopalosiphum padi L. Homoptera: Aphididae) as the above-ground insect herbivore. It has become increasingly common for these two insects to simultaneously attack cereals in the UK, due to increasing frequencies of wireworms on arable sites previously under grass (Johnson et al. 2008a). In particular, this study focused on several aspects of plant nutritional quality including total carbon (C), total nitrogen (N) and soluble amino acids, together with plant mineral nutrients identified as having potentially significant impacts on insect herbivore fitness (reviewed by Awmack & Leather 2002). Specifically, these were phosphorus (P) (e.g. Clancy & King 1993; Huberty & Denno 2006), potassium (K) (e.g. Stamp 1994), calcium (Ca) (e.g. Scutareanu & Loxdale 2006) and sulphur (S) (e.g. Marazzi & Städler 2004, 2005). Compared to plant N, these minerals have received less attention in terms of the nutritional ecology of herbivorous insects but some studies have shown that they can have either positive or negative impacts on herbivores depending on concentrations and herbivore taxa (Awmack & Leather 2002). Phosphorus, in particular, has been identified as occurring in much higher concentrations in insect herbivores than plants and consequently may become a major limiting nutritional factor (Elser et al. 2000). Indeed, Hubety & Denno (2006) suggest phosphorus may play an important role in plant-mediated interactions between herbivores. However, like the other nutrients quantified in this study, phosphorus has not, to our knowledge, been investigated in the context of above-ground × below-ground herbivory.
We hypothesized that plant nutritional quality would change with the duration and type of insect herbivory and that root or shoot herbivory would influence insect performance at spatially-separate locations. In particular, following the hypothesis of Masters et al. (1993) we predicted that aphid performance was likely to improve in the presence of root-feeding wireworms.
Materials and methods
Wireworms were recovered from a site in Drumsturdy, Angus, UK (56°29′25 N, 2°53′19 W) during May 2006 (see Johnson et al. 2008a for details). Forty individuals were selected randomly from the population and identified according to Van Emden (1945) as being Agriotes obscurus (22%) and A. lineatus (78%). Since these species are functionally very similar and only reliably distinguished by dissection, they were treated as a single population during this study, as has been reported elsewhere (Parker & Howard 2001). A clonal culture of Rhopalosiphum padi was maintained at 18 °C with 18 h daylight on caged 2–3-week-old barley plants (Hordeum vulgare cv Optic).
Two experiments were conducted in a glasshouse between 19 June and 2 August 2006, with a short exposure to herbivory (17 days of wireworm and 10 days of aphid infestation) and a long exposure to herbivory (27 days of wireworm and 20 days of aphid infestation). For each of the two experiments approximately 100 barley seeds were soaked in sterilized water for 3 h and then in calcium hypochlorite for 15 min before being germinated on damp filter paper at 15 °C for 3 days. Forty seedlings of equivalent mass were sown in 150 mm diameter plant pots containing 1·17 kg of compost (peat–sand–perlite mix containing 17N : 10P : 15 K; William Sinclair Horticulture Ltd, Lincoln, UK) then enclosed in mesh cages (0·7 × 0·25 m; height × diameter). The cages were designed to maximise free air movement and allow good light transmittance (air temperature increased by < 1 °C and photosynthetic rate reduced by < 16% inside cages). Experiments were performed under glass (16 : 8 h light : dark, 20 ± 5 οC) and plants were irrigated at a rate of approximately 0·06 L water day−1, maintaining compost water content at approximately 15% which was monitored with a profile probe and moisture meter (Delta-T Devices, Cambridge, UK). After 15 days, 80 mature wireworms were weighed (in groups of four) and applied to 20 plants (four on each plant). Following a further seven day period to allow wireworm establishment, eight teneral adult apterous aphids were weighed and applied to 10 plants with wireworms and 10 insect-free plants. Each experiment comprised 40 plants: 10 control insect-free plants; ten plants with wireworms; 10 plants with aphids; and 10 plants with wireworms and aphids. At harvest, the short exposure plants were 35 days old and at the stem elongation stage and the long exposure plants were 45 days old and at the ear emergence stage.
For each harvest, aphids were removed from the aphid-treated plants, counted and a sub-sample of five adult aphids were selected at random and weighed to assess aphid performance. Once free of aphids, the number of tillers and leaves and plant height were recorded. Root material was rinsed free of compost, maximum root depth recorded and all wireworms recovered and weighed. Fresh mass of root and shoot material was recorded and roots and leaves were snap frozen in liquid nitrogen and stored at –80 °C before freeze-drying for dry mass determination and chemical analysis (see below).
Freeze-dried shoot material was ball-milled to a fine powder for all extractions and chemical analyses. The N and C concentrations of 1 mg samples were determined by continuous flow Dumas combustion using a Europa Scientific (Crewe, UK) ANCA-SL sample converter and mass spectrometric detection (of N2 and CO2) using a Europa Scientific 20–20 mass spectrometer, as described by Scrimgeour and Robinson (2003). The percentage of C and N in the sample was calculated by comparison with known standards.
For measurement of mineral elements other than carbon and nitrogen, powdered leaf and root samples (0·05 g) were subjected to acid digestion for 20 min at 180 °C in 3 mL of 15·8 m HNO3 (Aristar grade, VWR International, Poole, UK) followed by oxidation for 20 min at 180 °C with 1 mL of H2O2 in closed vessels using a MARS-Xpress microwave oven (CEM, Buckingham, UK). Digested samples were diluted to a final volume of 50 mL with de-ionised water. Total mineral contents of P, Ca, S, and K in the digested plant material were quantified by inductively-coupled plasma-mass spectrometry (Elan DRC-e, Perkin-Elmer, Beaconsfield, Bucks, UK).
Barley leaf soluble amino acids were used as a reliable indicator of phloem amino acid composition (Winter et al. 1992) as phloem is the primary source of dietary nitrogen for aphids. Soluble amino acids were extracted from 0·05 g of powdered leaf material by gentle agitation with 1 mL of 80% methanol (HPLC-grade) at 4 °C. Following 15 min centrifugation at 10 000 g and 4 °C to sediment particulate matter, the supernatant was transferred to a clean tube and the pellet re-extracted, as described above, with a further 1 mL of 80% methanol. The supernatants from each extraction were pooled for each sample. The pooled supernatant was centrifuged again at 10 000 g and 4 °C and filtered through a 0·2 µm nylon filter; the final filtrate was analysed in 10 µL aliquots. Leaf amino acids extracted by this method were separated by reverse-phase HPLC following derivatization with o-phthaldialdehyde (Jones et al. 1981) using a Hewlett-Packard HP1100 series autosampling LC system with ZORBAXTM Eclipse XDB-C8 column and fluorescence detection. Amino acids were quantified by comparison with the AA-S-18 (Sigma-Aldrich, Poole, UK) reference amino acid mixture supplemented with asparagine, glutamine and tryptophan. All protein amino acids except proline and cysteine could be detected with this method, with a detection limit of approximately 0·5 pmol, including the nine essential amino acids that cannot be synthesized de novo by animals: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine (Morris 1990).
Parametric statistical tests were applied to insect response variables and plant trait data that were confirmed to be normally-distributed (Ryan–Joiner one-sample test) with homogeneous variance (Bartlett's test). Some insect, amino acid and mineral concentration data required natural-log transformation to meet assumptions of normality. Plant traits were analysed using two-way anova to examine main effects of aphid or wireworm herbivory and determine any interactions between the two treatments in plant response variables. Insect response variables were analysed by one-way anova. For wireworm mass, initial body mass was fitted as a covariate to account for individual variation in starting weight. Generalized linear regression was applied to test the strength of association between different plant traits. Step-wise deletion of non-significant terms (treatment and each measured plant trait) was used to achieve parsimony for each measure of insect performance (aphid number, mean aphid adult mass, wireworm mass and wireworm survival).
plant growth and resource allocation
Wireworms were more influential in shaping plant growth than aphids, with significant reductions in total plant biomass (Fig. 1a,b) and leaf dry mass (Fig. 1c,d). While wireworm or aphid herbivory alone had no effect on root growth, there was an interactive effect in the short exposure experiment; wireworms stimulated root growth in the absence of aphids, but caused a decline in root mass when aphids were present (Fig. 1e). Both herbivores reduced water content in the tissues they were feeding on after short exposure (Fig. 1f,g) but these effects were absent in the long exposure experiment (data not shown).
plant mineral nutrient composition
Aphids altered plant mineral composition to a much greater extent than wireworms. Aphids significantly increased carbon : nitrogen (C : N) ratios in leaves after both short (Fig. 1h) and long exposures (Fig. 1i), due to reductions in tissue N concentration (data not shown). The increase in leaf C : N was strengthened by wireworm herbivory in the presence of aphids, although the interaction was not quite statistically significant (P = 0·064) (Fig. 1h). Conversely, root C : N concentrations decreased in response to aphids (due to increased tissue N concentration; data not shown) in the short exposure experiment, and the effect of aphids was enhanced where wireworms were present (Fig. 1j).
Aphids significantly affected concentrations of S, Ca, P and K in either leaves or roots, or both, in a least one of the herbivore exposure periods (Fig. 2). Aphids reduced concentrations of minerals in leaves, with significant declines in S (Fig. 2a,b) in both exposure experiments, and leaf concentrations of Ca (Fig. 2c) and P (Fig. 2d) in the short exposure experiments. Aphids also reduced leaf P in the long exposure experiment, and this reduction was enhanced when wireworms were also present (Fig. 2e).
The opposite trend was seen in the roots, with aphids causing significant increases in root concentrations of minerals such as S (Fig. 2f,g) and Ca (Fig. 2 h,i) after either exposure period, and in root concentrations of K in the short exposure period (Fig. 2j). After long exposure, aphids only increased root K in the absence of wireworms: in the presence of wireworms, the opposite effect was seen (Fig. 2k). Indeed, there were a number of interactive effects of wireworm herbivory on root mineral concentrations. For example, of all the minerals quantified, aphids caused the biggest increase in root S concentrations which could be interactively strengthened or weakened by the presence of wireworms depending on the duration of herbivore exposure (Fig. 2f,g, respectively). Likewise, aphid-driven increases in root Ca were greater when wireworms were absent during the long exposure experiment (Fig. 2i).
Wireworms had far fewer impacts on plant mineral concentrations, except in the short exposure experiment where they increased leaf Ca (Fig. 2c) and root S (Fig. 2f), with the latter being enhanced as an interactive effect of the presence of aphids.
leaf amino acid content and composition
Aphids dominated changes in leaf amino acid content and composition (Fig. 3), with wireworms having no independent (i.e. without aphid presence) impact on this aspect of plant chemistry. In both experiments, aphids significantly reduced leaf total amino acids (Fig. 3a,b and also essential amino acids, in terms of both absolute (Fig. 3c,d) and relative (Fig. 3e,f) concentrations. When analysed individually, most amino acids showed similar trends to the changes in total amino acid content, except for the non-essentials glutamine, glycine and alanine, which all showed significant increases in response to aphid feeding (data not shown). While wireworms had no independent effect on amino acid concentrations, they did have an interactive effect in the long exposure experiment by significantly mitigating the aphid-driven decreases in essential amino acids, whether measured as absolute (Fig. 3d) or relative (Fig. 3f) concentrations.
insect performance and interactions
Aphid numbers increased with infestation period (Fig. 4a). Aphid numbers were significantly higher on wireworm-infested plants compared to wireworm-free plants in the long exposure experiment (with mean populations of 897 and 1172, respectively; F1,18 = 8·26; P = 0·01) but there was no significant difference after short exposure (F1,18 = 0·11; P = 0·75). The presence of wireworms had no impact on aphid mass in either experiment (F1,18 = 1·48; P = 0·24; F1,18 = 0·67; P = 0·39, respectively) (Fig. 4b). Wireworm survival was generally lower in the long exposure experiment (Fig. 4c), but not to the extent that the difference was statistically significant (P > 0·05). Survival was unaffected by the presence of aphids whether in the short exposure (F1,18 = 1·53; P = 0·23) or long exposure (F1,18 = 0·67; P = 0·42) experiment. Wireworm mass, however, was significantly greater when aphids were feeding on the plant in both the short exposure (F1,17 = 4·79; P = 0·04) and long exposure (F1,17 = 4·69; P = 0·04) experiments (Fig. 4d) when initial wireworm mass was included as a covariate.
plant nutrients in relation to insect performance
The relation between insect performance parameters and each of the measured plant-traits was assessed by generalized linear regression. Of all the possible plant trait-insect performance parameter combinations, only one showed a significant association: wireworm mass was negatively related to the P concentration of leaves and, to a lesser extent, of the roots across both exposure times (analysis not shown).
plant responses to above-ground and below-ground herbivory
This study investigated how two spatially segregated herbivore types on a common host plant shaped aspects of plant growth and nutritional chemistry and whether they affected the performances of one another through plant-mediated mechanisms. Wireworms predominantly affected plant growth allocation patterns, whereas aphids affected plant chemistry to a much greater extent, with divergent outcomes for above-ground and below-ground plant tissues. In particular, aphids caused significant decreases in most of the leaf minerals quantified in this study (N, S, Ca, P), but significant increases in root minerals (N, S, Ca and K). A key finding of this study is that many of these herbivore induced plant changes can be modified or even reversed by a spatially separated herbivore. For instance, aphid driven increases in root S were initially strengthened by wireworm presence but subsequently ameliorated after sustained root herbivory. In other words, above-ground or below-ground herbivores experiencing induced changes in the plant do not passively respond to these changes but have the capacity to modify them. We suggest that such interactive effects on the plant should be incorporated into existing models of above-ground × below-ground interactions between herbivores (e.g. Masters et al. 1993), which hypothesize that simple induced changes in the plant underpin interactions between the two herbivores.
impacts of root feeders on aphids
Each herbivore type promoted some aspect of the performance of the other herbivore, presumably through changes in plant physiology that altered plant quality for herbivory. Positive effects of root herbivory on aphids have been reported elsewhere (see Johnson et al. 2008b) but the hypothesis that root herbivory leads to mobilization of soluble nitrogen in foliage due to water stress (e.g. Gange & Brown 1989; Masters et al. 1993) was not supported by this study. Wireworm herbivory did not increase foliar water stress, and there was no accompanying increase in soluble nitrogen, measured here as leaf soluble amino acid concentration (a reliable indication of phloem content in barley; Winter et al. 1992). It could be conjectured that wireworms acting alone did not affect amino acids, but the interactive effect of root herbivory was to increase the relative supply of essential amino acids to aphids which were subsequently taken up by the aphids prior to chemical analysis.
impacts of aphids on root-feeders
Although positive, negative and neutral effects have been reported for root-feeders on shoot-feeding insects, the impacts of shoot-feeders on root herbivores are predominantly neutral or negative (reviewed by Johnson et al. 2008b), and this study is one of the few reports that are starting to emerge of bi-directional positive effects (see also Kaplan et al. 2008). Indeed, the prediction that above-ground insects generally have negative impacts on below-ground insects by causing plants to allocate resources away from the roots to compensate for foliar damage (Masters et al. 1993) was not supported by this study. In their review, Blossey and Hunt-Joshi (2003) also speculated that such mechanisms were uncommon and this study provides empirical support for this viewpoint. In contrast, aphids caused very large increases in root minerals which may have contributed to the positive effects on wireworm mass. In particular, aphids caused extremely large increases in root S which is known to promote the performance of some root-feeding insects (Marazzi & Städler 2005). When aphid-driven increases in root mineral concentrations were compared with increases in wireworm mass, significant relations were not detected. However, this may reflect the fact that wireworm mass can be highly variable, for instance by taking on water before moulting (Evans & Gough 1942), which could make any direct relationship with root chemical concentrations difficult to detect. Interestingly, there was a weakly negative correlation generally between root P and wireworm mass (see also Clancy & King 1993), but this was not affected by aphid presence, so is unlikely to underpin the positive interaction between the two insects.
Understanding the linkages between above-ground and below-ground communities is essential for determining the underlying mechanisms that drive an array of ecosystem processes, including plant succession and diversity (De Deyn & Van der Putten 2005), food web complexity (Van der Putten et al. 2001) and soil microbial processes (Bardgett et al. 1998). Many of these processes will be influenced by the dynamic and complex interplay between plant nutritional quality and induced plant defences (Wardle et al. 2004). In reappraising the role of plant nutrients in above-ground × below-ground interactions, we hope to stimulate further interest in assessing the interplay between these two aspects of plant quality for herbivores. Unravelling the complex interactions that shape ecosystems will rely on a combination of both approaches to truly characterize the fundamental plant-mediated mechanisms that link organisms above and below the soil surface.
We thank Carolyn Mitchell, Fiona Falconer, Charlie Scrimgeour, Steven Gellatly and Alistair Chalmers at SCRI for technical assistance, Mr Jim McNicol and editorial reviewers for statistical advice, and Dr Jeremy Pritchard at the University of Birmingham for providing a culture of aphids. The work was funded by RERAD workpackages 0171 and 0172.