Assimilatory potential of Helicoverpa armigera reared on host (Chickpea) and nonhost (Cassia tora) diets

Adaptation to plant allelochemicals is a crucial aspect of herbivore chemical ecology. To understand an insect ecology, we studied an effect of nonhost Cassia tora seed-based diet (Ct) on growth, development, and molecular responses in Helicoverpa armigera. We employed a comparative approach to investigate the proteomic differences in gut, hemolymph, and frass of H. armigera reared on a normal (chickpea seed-based, Cp) and Ct diet. In this study, a total of 46 proteins were identified by nano-LC-MS(E). Among them, 17 proteins were up-regulated and 29 proteins were down-regulated when larvae were exposed to the Ct diet. Database searches combined with GO analysis revealed that gut proteases engrossed in digestion, proteins crucial for immunity, adaptive responses to stress, and detoxification were down-regulated in the Ct fed larvae. Proteins identified in H. armigera hemolymph were found to be involved in defense mechanisms. Moreover, proteins found in frass of the Ct fed larvae were observed to participate in energy metabolism. Biochemical and quantitative real-time PCR analysis of selected candidate proteins showed differential gene expression patterns and corroborated with the proteomic data. Our results suggest that the Ct diet could alter expression of proteins related to digestion, absorption of nutrients, adaptation, defense mechanisms, and energy metabolism in H. armigera.     

Xenobiotic absorption and binding by proteins in hemolymph of the weevil Diaprepes abbreviatus

A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1–5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 M_r. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 M_r, while the lower band (band II) yielded only one size subunit of 75,000 M_r on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a K_D of 1.5 μM.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

The role of neuropeptides in caterpillar nutritional ecology

Plant diet strongly impacts the fitness of insect herbivores. Immediately, we think of plant defensive compounds that may act as feeding deterrents or toxins. We are, probably, less aware that plants also influence insect growth and fecundity through their nutritional quality. However, most herbivores respond to their environment and select the diet which optimizes their growth and development. This regulation of nutritional balance may occur on many levels: through selecting and ingesting appropriate plant tissue and nutrient digestion, absorption and utilization. Here, we review evidence of how nutritional requirements, particularly leaf protein to digestible carbohydrate ratios, affect caterpillar herbivores. We propose a model where midgut endocrine cells assess and integrate hemolymph nutritional status and gut content and release peptides which influence digestive processes. Understanding the effects of diet on the insect herbivore is essential for the rational design and implementation of sustainable pest management practices.     

Increased temperature alters feeding behavior of a generalist herbivore

Temperature can regulate a number of important biological processes and species interactions. For example, environmental temperature can alter insect herbivore consumption, growth and survivorship, suggesting that temperature‐driven impacts on herbivory could influence plant community composition or nutrient cycling. However, few studies to date have examined whether rising temperature influences herbivore preference and performance among multiple plant species, which often dictates their impact at the community level. Here, we assessed the effects of temperature on the performance and preference of the generalist herbivore Popillia japonica among nine plant species. We show that, on average, consumption rates and herbivore performance increased at higher temperatures. However, there was considerable variation among plant species with consumption and performance increasing on some plant species at higher temperatures but decreasing on others. Plant nutritional quality appeared to influence these patterns as beetles increased feeding on high‐nitrogen plants with increasing temperature, suggesting stronger nitrogen limitation. In addition to changes in feeding rates, feeding preferences of P. japonica shifted among temperatures, a pattern that was largely explained by differential deterrence of plant chemical extracts at different temperatures. In fact, temperature‐induced changes in the efficacy of plant chemical extracts led P. japonica to reduce its diet breadth at higher temperatures. Our results indicate that rising temperatures will influence herbivore feeding behavior by altering the importance of plant nutritional and chemical traits, suggesting that climate change will alter the strength and sign of plant–insect interactions.     

Perception of insect feeding by plants

The recognition of phytophagous insects by plants induces a set of very specific responses aimed at deterring tissue consumption and reprogramming metabolism and development of the plant to tolerate the herbivore. The recognition of insects by plants requires the plant’s ability to perceive chemical cues generated by the insects and to distinguish a particular pattern of tissue disruption. Relatively little is known about the molecular basis of insect perception by plants and the signalling mechanisms directly associated with this perception. Importantly, the insect feeding behaviour (piercing‐sucking versus chewing) is a decisive determinant of the plant’s defence response, and the mechanisms used to perceive insects from different feeding guilds may be distinct. During insect feeding, components of the saliva of chewing or piercing‐sucking insects come into contact with plant cells, and elicitors or effectors present in this insect‐derived fluid are perceived by plant cells to initiate the activation of specific signalling cascades. Although receptor–ligand interactions controlling insect perception have yet not been molecularly described, a significant number of regulatory components acting downstream of receptors and involved in the activation of defence responses against insects has been reported. Some of these regulators mediate changes in the phytohormone network, while others directly control gene expression or the redox state of the cell. These processes are central in the orchestration of plant defence responses against insects.     

Protein mediated cholesterol absorption in locusts Schistocerca gregaria (Forskal) and Locusta migratoria (Linn)

Absorption and transport of 3H cholesterol from the midgut to hemolymph and other tissues was studied in the locusts Schistocerca gregaria and Locusta migratoria. S. gregaria are able to absorb dietary cholesterol in the midgut and release into the hemolymph in vivo and into the incubation medium in virto. Certain proteins of midgut origin are involved in the absorption and release of cholesterol. The proteins designated as cholesterol binding proteins (CBP's) were fractionated by gel filtration chromatography using Sepharose CL-6B-200 column. Presence of a protein and its binding with cholesterol is confirmed by TCA precipitation after subsequent incubation of midgut in the incubation medium. Cholesterol binding with the proteins was also confirmed in native polyacrylamide gel electrophoresis. Biosynthesis of this protein takes place in the midgut which is inhibited by a protein synthesis inhibitor, cycloheximide. It also inhibits absorption and release of cholesterol from the midgut. The cholesterol binding activity was associated with a peak containing proteins ranging from molecular weights of 17-32 kDa in SDS-PAGE gels. Treatment of midgut with cycloheximide resulted in reduced cholesterol binding activity. Dilipidation of mucin and transport in presence of bile salts yielded a higher cholesterol binding activity. Although the absorption and release of cholesterol was observed in the hemolymph of both sexes, the ovary exhibited higher cholesterol binding as compared to testis.     

虫害诱导的植物挥发物:植物与植食性昆虫及其天敌相互作用的进化产物

综述了植物、植食性昆虫及其天敌相互作用的进化过程。虫害诱导的植物挥发物的特征和功能是植物-植食性昆虫-天敌之间长期进化的结果。在植物、植食性昆虫与天敌相互作用的进化过程中,3个不同营养级,包括植物、植食性昆虫和天敌有着各自的调节和利用虫害诱导的植物挥发物的策略。但有一些问题,如通过实验研究得出的诱导防御在田间是否真正能起到保护作用等需进一步研究、阐明。     

No evidence of modulation of indirect plant resistance of Brassica rapa plants by volatiles from soil-borne fungi

1. Upon herbivory, plants emit specific herbivore-induced plant volatiles (HIPVs) that can attract natural enemies of the herbivore thus serving as indirect plant resistance. Not only insect herbivores, but microorganisms may also affect HIPV emission before or after plant colonisation, which in turn can affect behaviour of natural enemies of the herbivore. Yet, it remains elusive whether volatiles from microorganisms influence HIPV emission and indirect plant resistance.
2. In this study, we investigated whether exposure of Brassica rapa roots to volatiles from soil-borne fungi influence HIPV emission and the recruitment of natural enemies of Pieris brassicae larvae.
3. Using a two-compartment pot system, we performed greenhouse and common-garden experiments, and we profiled plant HIPV emission.
4. We found that exposure of plant roots to fungal volatiles did not affect the number of P. brassicae larvae recollected from the plants, suggesting a neutral effect of the fungal volatiles on natural predation. Likewise, in a greenhouse, similar numbers of larvae were parasitised by Cotesia glomerata wasps on control plants as on fungal volatile-exposed plants. Additionally, chemical analysis of HIPV profiles revealed no qualitative and quantitative differences between control plants and fungal volatile-exposed plants that were both infested with P. brassicae larvae.
5. Together, our data indicate that root exposure to fungal volatiles did not affect indirect plant resistance to an insect herbivore. These findings provide new insight into the influence of indirect plant resistance by fungal volatiles that are discussed together with the effects of fungal volatiles on direct plant resistance.

     

Iron metabolism in insect disease vectors: mining the Anopheles gambiae translated protein database

All animals require iron for survival. This requirement reflects the role of this mineral as a cofactor of numerous proteins. However, under physiological conditions, Fe(2+) oxidizes to Fe(3+) encouraging the formation of toxic free radicals. In mammals, the potential for oxidative damage from iron is minimized by binding iron to proteins. Mammalian iron metabolism is complex and numerous proteins are involved in iron absorption, transport, uptake and utilization. We have analyzed the Anopheles gambiae translated protein database for candidates that show identity to proteins involved in mammalian iron metabolism (Holt et al., 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129-149). Our results indicate that proteins involved in iron absorption and intracellular iron utilization are, for the most part, conserved in A. gambiae. In contrast, proteins involved in the pathways of iron export from the gut, transport in hemolymph and uptake at peripheral tissues in mosquitos differ from those for mammals.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Plant and insect genetic variation mediate the impact of arbuscular mycorrhizal fungi on a natural plant–herbivore interaction

1. While both arbuscular mycorrhizal (AM) fungi and plant and insect genotype are well known to influence plant and herbivore growth and performance, information is lacking on how these factors jointly influence the relationship between plants and their natural herbivores. 2. The aim of the present study was to investigate how a natural community of arbuscular mycorrhizal fungi affects the growth of the perennial herb Plantago lanceolata L. (Plantaginaceae), as well as its interaction with the Glanville fritillary butterfly [Melitaea cinxia L. (Nymphalidae)]. For this, a multifactorial experiment was conducted using plant lines originating from multiple plant populations in the Åland Islands, Finland, grown either with or without mycorrhizal fungi. For a subset of plant lines, the impact of mycorrhizal inoculation, plant line, and larval family on the performance of M. cinxia larvae were tested. 3. Arbuscular mycorrhizal inoculation did not have a consistently positive or negative impact on plant growth or herbivore performance. Instead, plant genetic variation mediated the impact of arbuscular mycorrhizal fungi on plant growth, and both plant genetic variation and herbivore genetic variation mediated the response of the herbivore. For both the plant and insect, the impact of the arbuscular mycorrhizal community ranged from mutualistic to antagonistic. Overall, the present findings illustrate that genetic variation in response to mycorrhizal fungi may play a key role in the ecology and evolution of plant–insect interactions.     

Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori

Abstract Two‐dimensional gel electrophoresis (2‐DE) followed by matrix‐assisted laser desorption ionization – time‐of‐flight/time‐of‐flight mass spectrometry (MS) analysis were used to charaterize the hemolymph proteomic profiles of the silkworm, Bombyx mori. At days 4 (V4) and 5 (V5) of the fifth (final) instar, when the larvae were at the fast‐growing stage, we found dramatic changes in spots representing proteins having an approximate molecular weight (MW) of 30 kDa. Of these spots, four 30K proteins were highly up‐regulated, implying a close association with the growth and development of B. mori larvae. To understand the molecular basis and underlying mechanisms involved in development and metamorphosis, the proteome of whole hemolymph at V5 was analyzed using shotgun liquid chromatography tandem mass spectrometry with an LTQ‐Orbitrap. A total of 108 proteins were identified without any false discovery hits. These proteins were involved in a variety of cellular functions, including metabolism, development, nutrient transport and reserve, and defense response. Gene ontology analysis showed that 3.4% of these proteins had nutrient reservoir activities and 5.7% were involved in the response to stimulus. Pathway analysis revealed that 22 proteins with common targets were involved in various cellular processes such as immunity, differentiation, proliferation and metamorphosis. These results suggested that some key factors such as the 30K proteins in hemolymph play important roles in B. mori growth and development. Moreover, the multiple functions of hemolymph may be operated by a complex biological network.     

Evidence for long-distance, chemical gall induction by an insect

Abstract We report that a chemical stimulus from a herbivore, a galling insect, changes plant morphology and physiology to benefit the herbivore. Previous studies could not determine whether insect galls are induced by mechanical or chemical stimuli because feeding and oviposition both occurred at the site of gall formation. We report that the mouthparts of a spruce‐galling insect, Adelges cooleyi, were inserted in stem phloem cells far from induced galls, that tissues between mouthparts and galls appeared normal, and that the ability to initiate galls was inversely correlated with distance from buds (potential gall sites). Thus the effects of chemical stimuli were unambiguously separated from any mechanical influence of probing stylets or ovipositors. Our results strongly suggest that galls were induced by a chemical stimulus transported to buds via vascular tissue and that its efficacy was dose‐dependent.     

Dual herbivore attack and herbivore density affect metabolic profiles of Brassica nigra leaves

Plant responses to dual herbivore attack are increasingly studied, but effects on the metabolome have largely been restricted to volatile metabolites and defence‐related non‐volatile metabolites. However, plants subjected to stress, such as herbivory, undergo major changes in both primary and secondary metabolism. Using a naturally occurring system, we investigated metabolome‐wide effects of single or dual herbivory on Brassica nigra plants by Brevicoryne brassicae aphids and Pieris brassicae caterpillars, while also considering the effect of aphid density. Metabolomic analysis of leaf material showed that single and dual herbivory had strong effects on the plant metabolome, with caterpillar feeding having the strongest influence. Additionally, aphid‐density‐dependent effects were found in both the single and dual infestation scenarios. Multivariate analysis revealed treatment‐specific metabolomic profiles, and effects were largely driven by alterations in the glucosinolate and sugar pools. Our work shows that analysing the plant metabolome as a single entity rather than as individual metabolites provides new insights into the subcellular processes underlying plant defence against multiple herbivore attackers. These processes appear to be importantly influenced by insect density.     

DIFFERENTIAL PROTEOME ANALYSIS OF THE MALE AND FEMALE ANTENNAE FROM Holotrichia parallela

To understand the olfactory mechanisms of Holotrichia parallela antennae in detecting volatile compounds in the environment, protein profiles of H. parallela antennae were analyzed using two‐dimensional electrophoresis followed by mass spectrometry and bioinformatics analyses. Approximately 1,100 protein spots in silver staining gel were detected. Quantitative image analysis revealed that in total 47 protein spots showed significant changes in different genders of adult antennae. Thirty‐five differentially expressed proteins were identified by Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF/TOF) tandem mass spectrometer, among which 65.7% are involved in carbohydrate and energy metabolism, antioxidant system, transport, and amino acid/nucleotide metabolism. Some proteins identified here have not been reported previously in insect antennae. Identified male‐biased proteins included odorant‐binding protein 4, pheromone‐binding protein‐related protein 2, odorant‐binding protein 14, prophenoloxidase‐I, acyl‐CoA dehydrogenase, aldo‐keto reductase‐like, carbamoyl phosphate synthetase, etc. whereas some proteins are female biased, such as antennae‐rich cytochrome P450, aldehyde dehydrogenase, and putative glutamine synthetase. Alterations in the levels of some proteins were further confirmed by real time polymerase chain reaction (RT‐PCR). The proteomic resources displayed here are valuable for the discovery of proteins from H. parallela antennae.     

Use of an exotic host plant shifts immunity,chemical defense,and viral burden in wild populations of a specialist insect herbivore

Defense against natural enemies constitutes an important driver of herbivore host range evolution in the wild. Populations of the Baltimore checkerspot butterfly, Euphydryas phaeton (Nymphalidae), have recently incorporated an exotic plant, Plantago lanceolata (Plantaginaceae), into their dietary range. To understand the tritrophic consequences of utilizing this exotic host plant, we examined immune performance, chemical defense, and interactions with a natural entomopathogen (Junonia coenia densovirus, Parvoviridae) across wild populations of this specialist herbivore. We measured three immune parameters, sequestration of defensive iridoid glycosides (IGs), and viral infection load in field‐collected caterpillars using either P. lanceolata or a native plant, Chelone glabra (Plantaginaceae). We found that larvae using the exotic plant exhibited reduced immunocompetence, compositional differences in IG sequestration, and higher in situ viral burdens compared to those using the native plant. On both host plants, high IG sequestration was associated with reduced hemocyte concentration in the larval hemolymph, providing the first evidence of incompatibility between sequestered chemical defenses and the immune response (i.e., the “vulnerable host” hypothesis) from a field‐based study. However, despite this negative relationship between IG sequestration and cellular immunity, caterpillars with greater sequestration harbored lower viral loads. While survival of virus‐infected individuals decreased with increasing viral burden, it ultimately did not differ between the exotic and native plants. These results provide evidence that: (1) phytochemical sequestration may contribute to defense against pathogens even when immunity is compromised and (2) herbivore persistence on exotic plant species may be facilitated by sequestration and its role in defense against natural enemies.     

Chemical interaction between undamaged plants--effects on herbivores and natural enemies

Most research on plant-plant chemical interactions has focussed on events following herbivore or pathogen attack. However, undamaged plants also interact chemically as a natural facet of their behaviour, and this may have consequences for insects that use the plants as hosts. In this review, the links between allelopathy and insect behaviour are outlined. Findings on how chemical interactions between different plant species and genotypes affect aphid herbivores and their natural enemies are reviewed, and the role of plant diversity and chemical interaction for trophic interactions in crops is discussed.     

Canopy insect herbivores in the Azorean Laurisilva forests: key host plant species in a highly generalist insect community

This article explores patterns of insect herbivore distribution in the canopy of the Laurisilva forests on seven islands in the Azores archipelago. To our knowledge, this is one of the first extensive study of this type in tree or shrub canopies of oceanic island ecosystems. One of the most frequently debated characteristics of such ecosystems is the likely prevalence of vague, ill‐defined niches due to taxonomic disharmony, which may have implications for insect‐plant interactions. For instance, an increase in ecological opportunities for generalist species is expected due to the lack of predator groups and reduced selection for chemical defence in host plants. The following two questions were addressed: 1) Are specialists species rare, and insect herbivore species randomly distributed among host plant species in the Azores? 2) Are the variances in insect herbivore species composition, frequency and richness explained by host plants or by regional island effects? We expect a proportional distribution of herbivore species between host plants, influenced by host frequency and distinct island effects; otherwise, deviation from expectation might suggest habitat preference for specific host tree crowns. Canopy beating tray samples were performed on seven islands, comprising 50 transects with 1 to 3 plant species each (10 replicates per species), giving 1320 samples from ten host species trees or shrubs in total. From a total of 129 insect herbivore species, a greater number of herbivore species was found on Juniperus brevifolia (s=65) and Erica azorica (s=53). However, the number of herbivore species per individual tree crown was higher for E. azorica than for any other host, on all islands, despite the fact that it was only the fourth more abundant plant. In addition, higher insect species richness and greater insect abundance were found on the trees of Santa Maria Island, the oldest in the archipelago. Insect species composition was strongly influenced by the presence of E. azorica, which was the only host plant with a characteristic fauna across the archipelago, whereas the fauna of other plant crowns was grouped by islands. The great insect occurrence on E. azorica reflects strong habitat fidelity, but only four species were clearly specialists. Our findings indicate a broadly generalist fauna. The simplicity of Azorean Laurisilva contributed to the understanding of insect‐plant mechanisms in canopy forest habitats.     

Effects of nitrogen deposition on the interaction between an aphid and its host plant

Abstract 1. Anthropogenic increases in nitrogen deposition are impacting terrestrial ecosystems worldwide. While some of the direct ecosystem‐level effects of nitrogen deposition are understood, the effects of nitrogen deposition on plant–insect interactions and on herbivore population dynamics have received less attention. 2. Nitrogen deposition will potentially influence both plant resource availability and herbivore population growth. If increases in herbivore population growth outstrip increases in resource availability, then increases in the strength of density dependence expressed within the herbivore population would be predicted. Alternatively, if plant resources respond more vigorously to nitrogen deposition than do herbivore populations, a decline in the strength of density dependence would be expected. No change in the strength of density dependence acting upon the herbivore population would suggest equivalent responses by herbivores and plants. 3. A density manipulation experiment was performed to examine the effect of nitrogen deposition on the interaction between a host plant, Asclepias tuberosa, and its herbivore, Aphis nerii. Aphid maximum per capita growth rate (R_max), carrying capacity (K), and the strength of density dependence were measured under three nitrogen deposition treatments. The effect of nitrogen deposition on the relationship among these three measures of insect population dynamics was explored. 4. Simulated nitrogen deposition increased aphid per capita population growth, plant foliar nitrogen concentrations, and plant biomass. Nitrogen deposition caused R_max and K to increase proportionally, leading to no overall change in the strength of density dependence. In this system, potential changes in the negative feedback processes operating on herbivore populations following nitrogen deposition appear to be buffered by concomitant changes in resource availability.