The Biology and Ecology of Parasitoid Wasps of Predatory Arthropods.

Parasitoid wasps are important components of insect food chains and have played a central role in biological control programs for over a century. Although the vast majority of parasitoids exploit insect herbivores as hosts, others parasitize predatory insects and arthropods, such as ladybird beetles, hoverflies, lacewings, ground beetles, and spiders, or are hyperparasitoids. Much of the research on the biology and ecology of parasitoids of predators has focused on ladybird beetles, whose parasitoids may interfere with the control of insect pests like aphids by reducing ladybird abundance. Alternatively, parasitoids of the invasive ladybird Harmonia axyridis may reduce its harmful impact on native ladybird populations. Different life stages of predatory insects and spiders are susceptible to parasitism to different degrees. Many parasitoids of predators exhibit intricate physiological interrelationships with their hosts, adaptively manipulating host behavior, biology, and ecology in ways that increase parasitoid survival and fitness.

Indirect plant-mediated interactions between heterospecific parasitoids that develop in different caterpillar species.

Parasitoids induce physiological changes in their herbivorous hosts that affect how plants respond to herbivory. The signature of parasitoids on induced plant responses to feeding by parasitized herbivores indirectly impacts insect communities interacting with the plant. The effect may extend to parasitoids and cause indirect interaction between parasitoids that develop inside different herbivore hosts sharing the food plant. However, this type of interactions among parasitoid larvae has received very little attention. In this study, we investigated sequential and simultaneous plant-mediated interactions among two host-parasitoid systems feeding on Brassica oleracea plants: Mamestra brassicae parasitized by Microplitis mediator and Pieris rapae parasitized by Cotesia rubecula. We measured the mortality, development time, and weight of unparasitized herbivores and performance of parasitoids that had developed inside the two herbivore species when sharing the food plant either simultaneously or sequentially. Plant induction by parasitized or unparasitized hosts had no significant effect on the performance of the two herbivore host species. In contrast, the two parasitoid species had asymmetrical indirect plant-mediated effects on each other's performance. Cotesia rubecula weight was 15% higher on plants induced by M. mediator-parasitized hosts, compared to control plants. In addition, M. mediator development time was reduced by 30% on plants induced by conspecific but not heterospecific parasitoids, compared to plants induced by its unparasitized host. Contrary to sequential feeding, parasitoids had no effect on each other's performance when feeding simultaneously. These results reveal that indirect plant-mediated interactions among parasitoid larvae could involve any parasitoid species whose hosts share a food plant.

Specialist root herbivore modulates plant transcriptome and downregulates defensive secondary metabolites in a brassicaceous plant.

Plants face attackers aboveground and belowground. Insect root herbivores can lead to severe crop losses, yet the underlying transcriptomic responses have rarely been studied. We studied the dynamics of the transcriptomic response of Brussels sprouts (Brassica oleracea var. gemmifera) primary roots to feeding damage by cabbage root fly larvae (Delia radicum), alone or in combination with aboveground herbivory by cabbage aphids (Brevicoryne brassicae) or diamondback moth caterpillars (Plutella xylostella). This was supplemented with analyses of phytohormones and the main classes of secondary metabolites; aromatic, indole and aliphatic glucosinolates. Root herbivory leads to major transcriptomic rearrangement that is modulated by aboveground feeding caterpillars, but not aphids, through priming soon after root feeding starts. The root herbivore downregulates aliphatic glucosinolates. Knocking out aliphatic glucosinolate biosynthesis with CRISPR-Cas9 results in enhanced performance of the specialist root herbivore, indicating that the herbivore downregulates an effective defence. This study advances our understanding of how plants cope with root herbivory and highlights several novel aspects of insect-plant interactions for future research. Further, our findings may help breeders develop a sustainable solution to a devastating root pest.

Insect egg-killing: a new front on the evolutionary arms-race between brassicaceous plants and pierid butterflies.

Evolutionary arms-races between plants and insect herbivores have long been proposed to generate key innovations such as plant toxins and detoxification mechanisms that can drive diversification of the interacting species. A novel front-line of plant defence is the killing of herbivorous insect eggs. We test whether an egg-killing plant trait has an evolutionary basis in such a plant-insect arms-race. Within the crucifer family (Brassicaceae), some species express a hypersensitive response (HR)-like necrosis underneath butterfly eggs (Pieridae) that leads to eggs desiccating or falling off the plant. We studied the phylogenetic distribution of this trait, its egg-killing effect on and elicitation by butterflies, by screening 31 Brassicales species, and nine Pieridae species. We show a clade-specific induction of strong, egg-killing HR-like necrosis mainly in species of the Brassiceae tribe including Brassica crops and close relatives. The necrosis is strongly elicited by pierid butterflies that are specialists of crucifers. Furthermore, HR-like necrosis is linked to PR1 defence gene expression, accumulation of reactive oxygen species and cell death, eventually leading to egg-killing. Our findings suggest that the plants' egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied plant toxins that have evolved against their caterpillars.

Reprotoxic effects of the systemic insecticide fipronil on the butterfly Pieris brassicae.

In addition to controlling pest organisms, the systemic neurotoxic pesticide fipronil can also have adverse effects on beneficial insects and other non-target organisms. Here, we report on the sublethal effects of fipronil on the farmland butterfly Pieris brassicae. Caterpillars were reared on plants that had been grown from seeds coated with fipronil or on leaf discs topically treated with a range of fipronil dosages (1-32 µg kg-1 on dry mass basis). Females that had developed on fipronil plants laid ca half the number of eggs than females that had developed on control plants. In the bioassay with leaf discs, longevity and lifetime egg production declined with increasing fipronil dosage. Remarkably, exposure to fipronil during larval development primarily affected the adult stage. Chemical analyses of leaf tissues collected from seed-treated plants revealed concentrations of fipronil and its degradation products close to the analytical limit of detection (less than or equal to 1 µg kg-1). The effective dosage was fivefold higher in the leaf-disc than in the whole-plant experiment. In the whole plant, degradation of fipronil to products that are more toxic than fipronil may explain this discrepancy. Neurotoxicity of insecticides at the level of detection decreases the probability of pinpointing insecticides as the causal agent of harmful effects on non-target organisms.

Detoxification of plant defensive glucosinolates by an herbivorous caterpillar is beneficial to its endoparasitic wasp.

Plant chemical defences impact not only herbivores, but also organisms in higher trophic levels that prey on or parasitize herbivores. While herbivorous insects can often detoxify plant chemicals ingested from suitable host plants, how such detoxification affects endoparasitoids that use these herbivores as hosts is largely unknown. Here, we used transformed plants to experimentally manipulate the major detoxification reaction used by Plutella xylostella (diamondback moth) to deactivate the glucosinolate defences of its Brassicaceae host plants. We then assessed the developmental, metabolic, immune, and reproductive consequences of this genetic manipulation on the herbivore as well as its hymenopteran endoparasitoid Diadegma semiclausum. Inhibition of P. xylostella glucosinolate metabolism by plant-mediated RNA interference increased the accumulation of the principal glucosinolate activation products, the toxic isothiocyanates, in the herbivore, with negative effects on its growth. Although the endoparasitoid manipulated the excretion of toxins by its insect host to its own advantage, the inhibition of herbivore glucosinolate detoxification slowed endoparasitoid development, impaired its reproduction, and suppressed the expression of genes of a parasitoid-symbiotic polydnavirus that aids parasitism. Therefore, the detoxification of plant glucosinolates by an herbivore lowers its toxicity as a host and benefits the parasitoid D. semiclausum at multiple levels.

Climate change-mediated temperature extremes and insects: From outbreaks to breakdowns.

Insects are among the most diverse and widespread animals across the biosphere and are well-known for their contributions to ecosystem functioning and services. Recent increases in the frequency and magnitude of climatic extremes (CE), in particular temperature extremes (TE) owing to anthropogenic climate change, are exposing insect populations and communities to unprecedented stresses. However, a major problem in understanding insect responses to TE is that they are still highly unpredictable both spatially and temporally, which reduces frequency- or direction-dependent selective responses by insects. Moreover, how species interactions and community structure may change in response to stresses imposed by TE is still poorly understood. Here we provide an overview of how terrestrial insects respond to TE by integrating their organismal physiology, multitrophic, and community-level interactions, and building that up to explore scenarios for population explosions and crashes that have ecosystem-level consequences. We argue that TE can push insect herbivores and their natural enemies to and even beyond their adaptive limits, which may differ among species intimately involved in trophic interactions, leading to phenological disruptions and the structural reorganization of food webs. TE may ultimately lead to outbreak-breakdown cycles in insect communities with detrimental consequences for ecosystem functioning and resilience. Lastly, we suggest new research lines that will help achieve a better understanding of insect and community responses to a wide range of CE.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

With or without you: Effects of the concurrent range expansion of an herbivore and its natural enemy on native species interactions.

Global climatic changes may lead to the arrival of multiple range-expanding species from different trophic levels into new habitats, either simultaneously or in quick succession, potentially causing the introduction of manifold novel interactions into native food webs. Unraveling the complex biotic interactions between native and range-expanding species is critical to understand the impact of climate change on community ecology, but experimental evidence is lacking. In a series of laboratory experiments that simulated direct and indirect species interactions, we investigated the effects of the concurrent arrival of a range-expanding insect herbivore in Europe, Spodoptera littoralis, and its associated parasitoid Microplitis rufiventris, on the native herbivore Mamestra brassicae, and its associated parasitoid Microplitis mediator, when co-occurring on a native plant, Brassica rapa. Overall, direct interactions between the herbivores were beneficial for the exotic herbivore (higher pupal weight than the native herbivore), and negative for the native herbivore (higher mortality than the exotic herbivore). At the third trophic level, both parasitoids were unable to parasitize the herbivore they did not coexist with, but the presence of the exotic parasitoid still negatively affected the native herbivore (increased mortality) and the native parasitoid (decreased parasitism rate), through failed parasitism attempts and interference effects. Our results suggest different interaction scenarios depending on whether S. littoralis and its parasitoid arrive to the native tritrophic system separately or concurrently, as the negative effects associated with the presence of the parasitoid were dependent on the presence of the exotic herbivore. These findings illustrate the complexity and interconnectedness of multitrophic changes resulting from concurrent species arrival to new environments, and the need for integrating the ecological effects of such arrivals into the general theoretical framework of global invasion patterns driven by climatic change.

Responses of insect herbivores and their food plants to wind exposure and the importance of predation risk.

Wind is an important abiotic factor that influences an array of biological processes, but it is rarely considered in studies on plant-herbivore interactions. Here, we tested whether wind exposure could directly or indirectly affect the performance of two insect herbivores, Plutella xylostella and Pieris brassicae, feeding on Brassica nigra plants. In a greenhouse study using a factorial design, B. nigra plants were exposed to different wind regimes generated by fans before and after caterpillars were introduced on plants in an attempt to separate the effects of direct and indirect wind exposure on herbivores. Wind exposure delayed flowering, decreased plant height and increased leaf concentrations of amino acids and glucosinolates. Plant-mediated effects of wind on herbivores, that is effects of exposure of plants to wind prior to herbivore feeding, were generally small. However, development time of both herbivores was extended and adult body mass of P. xylostella was reduced when they were directly exposed to wind. By contrast, wind-exposed adult P. brassicae butterflies were significantly larger, revealing a trade-off between development time and adult size. Based on these results, we conducted a behavioural experiment to study preference by an avian predator, the great tit (Parus major) for last instar P. brassicae caterpillars on plants that were exposed to either control (no wind) or wind (fan-exposed) treatments. Tits captured significantly more caterpillars on still than on wind-exposed plants. Our results suggest that P. brassicae caterpillars are able to perceive the abiotic environment and to trade off the costs of extended development time against the benefits of increased size depending on the perceived risk of predation mediated by wind exposure. Such adaptive phenotypic plasticity in insects has not yet been described in response to wind exposure.

Ant-like Traits in Wingless Parasitoids Repel Attack from Wolf Spiders.

A recent study showed that a wingless parasitoid, Gelis agilis, exhibits a suite of ant-like traits that repels attack from wolf spiders. When agitated, G. agilis secreted 6-methyl-5-hepten-2-one (sulcatone), which a small number of ant species produce as an alarm/panic pheromone. Here, we tested four Gelis parasitoid species, occurring in the same food chain and microhabitats, for the presence of sulcatone and conducted two-species choice bioassays with wolf spiders to determine their degree of susceptibility to attack. All four Gelis species, including both winged and wingless species, produced sulcatone, whereas a closely related species, Acrolyta nens, and the more distantly related Cotesia glomerata, did not. In two-choice bioassays, spiders overwhelmingly rejected the wingless Gelis species, preferring A. nens and C. glomerata. However, spiders exhibited no preference for either A. nens or G. areator, both of which are winged. Wingless gelines exhibited several ant-like traits, perhaps accounting for the reluctance of spiders to attack them. On the other hand, despite producing sulcatone, the winged G. areator more closely resembles other winged cryptines like A. nens, making it harder for spiders to distinguish between these two species. C. glomerata was also preferred by spiders over A. nens, suggesting that other non-sulcatone producing cryptines nevertheless possess traits that make them less attractive as prey. Phylogenetic reconstruction of the Cryptinae reveals that G. hortensis and G. proximus are 'sister'species, with G. agilis, and G.areator in particular evolving along more distant trajectories. We discuss the possibility that wingless Gelis species have evolved a suite of ant-like traits as a form, of mimicry to repel predators on the ground.

Intraspecific chemical diversity among neighbouring plants correlates positively with plant size and herbivore load but negatively with herbivore damage.

Intraspecific plant diversity can modify the properties of associated arthropod communities and plant fitness. However, it is not well understood which plant traits determine these ecological effects. We explored the effect of intraspecific chemical diversity among neighbouring plants on the associated invertebrate community and plant traits. In a common garden experiment, intraspecific diversity among neighbouring plants was manipulated using three plant populations of wild cabbage that differ in foliar glucosinolates. Plants were larger, harboured more herbivores, but were less damaged when plant diversity was increased. Glucosinolate concentration differentially correlated with generalist and specialist herbivore abundance. Glucosinolate composition correlated with plant damage, while in polycultures, variation in glucosinolate concentrations among neighbouring plants correlated positively with herbivore diversity and negatively with plant damage levels. The results suggest that intraspecific variation in secondary chemistry among neighbouring plants is important in determining the structure of the associated insect community and positively affects plant performance.

Combined biotic stresses trigger similar transcriptomic responses but contrasting resistance against a chewing herbivore in Brassica nigra.

BACKGROUND: In nature, plants are frequently exposed to simultaneous biotic stresses that activate distinct and often antagonistic defense signaling pathways. How plants integrate this information and whether they prioritize one stress over the other is not well understood. RESULTS: We investigated the transcriptome signature of the wild annual crucifer, Brassica nigra, in response to eggs and caterpillars of Pieris brassicae butterflies, Brevicoryne brassicae aphids and the bacterial phytopathogen Xanthomonas campestris pv. raphani (Xcr). Pretreatment with egg extract, aphids, or Xcr had a weak impact on the subsequent transcriptome profile of plants challenged with caterpillars, suggesting that the second stress dominates the transcriptional response. Nevertheless, P. brassicae larval performance was strongly affected by egg extract or Xcr pretreatment and depended on the site where the initial stress was applied. Although egg extract and Xcr pretreatments inhibited insect-induced defense gene expression, suggesting salicylic acid (SA)/jasmonic acid (JA) pathway cross talk, this was not strictly correlated with larval performance. CONCLUSION: These results emphasize the need to better integrate plant responses at different levels of biological organization and to consider localized effects in order to predict the consequence of multiple stresses on plant resistance.

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.

Does Aphid Infestation Interfere with Indirect Plant Defense against Lepidopteran Caterpillars in Wild Cabbage?

Attraction of parasitoids to plant volatiles induced by multiple herbivory depends on the specific combinations of attacking herbivore species, especially when their feeding modes activate different defense signalling pathways as has been reported for phloem feeding aphids and tissue feeding caterpillars. We studied the effects of pre-infestation with non-host aphids (Brevicoryne brassicae) for two different time periods on the ability of two parasitoid species to discriminate between volatiles emitted by plants infested by host caterpillars alone and those emitted by plants infested with host caterpillars plus aphids. Using plants originating from three chemically distinct wild cabbage (Brassica oleracea) populations, Diadegma semiclausum switched preference for dually infested plants to preference for plants infested with Plutella xylostella hosts alone when the duration of pre-aphid infestation doubled from 7 to 14 days. Microplitis mediator, a parasitoid of Mamestra brassicae caterpillars, preferred dually-infested plants irrespective of aphid-infestation duration. Separation of the volatile blends emitted by plants infested with hosts plus aphids or with hosts only was poor, based on multivariate statistics. However, emission rates of individual compounds were often reduced in plants infested with aphids plus hosts compared to those emitted by plants infested with hosts alone. This effect depended on host caterpillar species and plant population and was little affected by aphid infestation duration. Thus, the interactive effect of aphids and hosts on plant volatile production and parasitoid attraction can be dynamic and parasitoid specific. The characteristics of the multi-component volatile blends that determine parasitoid attraction are too complex to be deduced from simple correlative statistical analyses.

Oviposition Preference for Young Plants by the Large Cabbage Butterfly (Pieris brassicae ) Does not Strongly Correlate with Caterpillar Performance.

The effects of temporal variation in the quality of short-lived annual plants on oviposition preference and larval performance of insect herbivores has thus far received little attention. This study examines the effects of plant age on female oviposition preference and offspring performance in the large cabbage white butterfly Pieris brassicae. Adult female butterflies lay variable clusters of eggs on the underside of short-lived annual species in the family Brassicaceae, including the short-lived annuals Brassica nigra and Sinapis arvensis, which are important food plants for P. brassicae in The Netherlands. Here, we compared oviposition preference and larval performance of P. brassicae on three age classes (young, mature, and pre-senescing) of B. nigra and S. arvensis plants. Oviposition preference of P. brassicae declined with plant age in both plant species. Whereas larvae performed similarly on all three age classes in B. nigra, preference and performance were weakly correlated in S. arvensis. Analysis of primary (sugars and amino acids) and secondary (glucosinolates) chemistry in the plant shoots revealed that differences in their quality and quantity were more pronounced with respect to tissue type (leaves vs. flowers) than among different developmental stages of both plant species. Butterflies of P. brassicae may prefer younger and smaller plants for oviposition anticipating that future plant growth and size is optimally synchronized with the final larval instar, which contributes >80% of larval growth before pupation.

Defensive insect symbiont leads to cascading extinctions and community collapse.

Animals often engage in mutualistic associations with microorganisms that protect them from predation, parasitism or pathogen infection. Studies of these interactions in insects have mostly focussed on the direct effects of symbiont infection on natural enemies without studying community-wide effects. Here, we explore the effect of a defensive symbiont on population dynamics and species extinctions in an experimental community composed of three aphid species and their associated specialist parasitoids. We found that introducing a bacterial symbiont with a protective (but not a non-protective) phenotype into one aphid species led to it being able to escape from its natural enemy and increase in density. This changed the relative density of the three aphid species which resulted in the extinction of the two other parasitoid species. Our results show that defensive symbionts can cause extinction cascades in experimental communities and so may play a significant role in the stability of consumer-herbivore communities in the field.

Interactive Effects of Cabbage Aphid and Caterpillar Herbivory on Transcription of Plant Genes Associated with Phytohormonal Signalling in Wild Cabbage.

Plants are commonly attacked by a variety of insect herbivores and have developed specific defenses against different types of attackers. At the molecular level, herbivore-specific signalling pathways are activated by plants in response to attackers with different feeding strategies. Feeding by leaf-chewing herbivores predominantly activates jasmonic acid (JA)-regulated defenses, whereas feeding by phloem-sucking herbivores generally activates salicylic acid (SA)-regulated defenses. When challenged sequentially by both phloem-sucking and leaf-chewing herbivores, SA-JA antagonism may constrain the plant's ability to timely and adequately divert defense to the second herbivore that requires activation of a different defensive pathway. We investigated the effect of the temporal sequence of infestation by the aphid Brevicoryne brassicae and three caterpillar species, Plutella xylostella, Pieris brassicae, and Mamestra brassicae, on the interaction between JA and SA signal-transduction pathways in three wild cabbage populations. We found no support for SA-JA antagonism, irrespective of the temporal sequence of herbivore introduction or the identity of the caterpillar species based on the transcript levels of the JA- and SA-regulated marker genes LOX and PR-1, respectively, at the examined time points, 6, 24, and 48 h. In general, infestation with aphids alone had little effect on the transcript levels of the two marker genes, whereas the three caterpillar species upregulated not only LOX but also PR-1. Transcriptional changes were different for plants from the three different natural cabbage populations.

Integrating Insect Life History and Food Plant Phenology: Flexible Maternal Choice Is Adaptive.

Experience of insect herbivores and their natural enemies in the natal habitat is considered to affect their likelihood of accepting a similar habitat or plant/host during dispersal. Growing phenology of food plants and the number of generations in the insects further determines lability of insect behavioural responses at eclosion. We studied the effect of rearing history on oviposition preference in a multivoltine herbivore (Pieris brassicae), and foraging behaviour in the endoparasitoid wasp (Cotesia glomerata) a specialist enemy of P. brassicae. Different generations of the insects are obligatorily associated with different plants in the Brassicaceae, e.g., Brassica rapa, Brassica nigra and Sinapis arvensis, exhibiting different seasonal phenologies in The Netherlands. Food plant preference of adults was examined when the insects had been reared on each of the three plant species for one generation. Rearing history only marginally affected oviposition preference of P. brassicae butterflies, but they never preferred the plant on which they had been reared. C. glomerata had a clear preference for host-infested B. rapa plants, irrespective of rearing history. Higher levels of the glucosinolate breakdown product 3-butenyl isothiocyanate in the headspace of B. rapa plants could explain enhanced attractiveness. Our results reveal the potential importance of flexible plant choice for female multivoltine insects in nature.

Crop domestication and its impact on naturally selected trophic interactions.

Crop domestication is the process of artificially selecting plants to increase their suitability to human requirements: taste, yield, storage, and cultivation practices. There is increasing evidence that crop domestication can profoundly alter interactions among plants, herbivores, and their natural enemies. Overall, little is known about how these interactions are affected by domestication in the geographical ranges where these crops originate, where they are sympatric with the ancestral plant and share the associated arthropod community. In general, domestication consistently has reduced chemical resistance against herbivorous insects, improving herbivore and natural enemy performance on crop plants. More studies are needed to understand how changes in morphology and resistance-related traits arising from domestication may interact with environmental variation to affect species interactions across multiple scales in agroecosystems and natural ecosystems.