Multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of cardenolides

In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant cardenolides.We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-cardenolide-level diets. Our study further establishes Drosophila as a model for the study of cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary cardenolides.     

Physiological screening for target site insensitivity and localization of Na(+)/K(+)-ATPase in cardenolide-adapted Lepidoptera

Cardenolides are toxic plant compounds which specifically inhibit Na(+)/K(+)-ATPase, an animal enzyme which is essential for many physiological processes, such as the generation of action potentials. Several adapted insects feeding on cardenolide-containing plants sequester these toxins for their own defence. Some of these insects were shown to possess Na(+)/K(+)-ATPases with a reduced sensitivity towards cardenolides (target site insensitivity). In the present study we screened five species of arctiid moths feeding on cardenolide-containing plants for target site insensitivity towards cardenolides using an in vitro enzyme assay. The derived dose response curves of the respective Na(+)/K(+)-ATPases were compared to the insensitive Na(+)/K(+)-ATPase of the monarch butterfly (Danaus plexippus). Na(+)/K(+)-ATPases of all arctiid species tested were highly sensitive to ouabain, a water-soluble cardenolide which is most widely used in laboratory studies. Nevertheless, we detected substantial amounts of cardenolides in the haemolymph of two of the arctiid species. In caterpillars of the sequestering arctiid Empyreuma pugione and of D. plexippus we localized Na(+)/K(+)-ATPase by immunohistochemistry and western blot (in D. plexippus). Both techniques revealed strong expression of the enzyme in the nervous tissue and indicated weak expression or even absence in other tissues tested. We conclude that instead of target site insensitivity the investigated arctiid species use a different strategy to tolerate cardenolides. Most plausibly, the perineurium surrounding the nervous tissue functions as a barrier which prevents cardenolides from reaching Na(+)/K(+)-ATPase in the ventral nerve cord.     

Molecular adaptation of Chrysochus leaf beetles to toxic compounds in their food plants

Herbivores that feed on toxic plants must overcome plant defenses and occasionally may even benefit from them. The current challenge is to understand how herbivores evolve the necessary physiological adaptations and which changes at the molecular level are involved. In this context we studied the leaf beetles genus Chrysochus (Coleoptera, Chrysomelidae). Two species of this genus, C. auratus and C. cobaltinus, feed on plants that contain toxic cardenolides. These beetles not only avoid poisoning by the toxin but also use it for their own defense against predators. All other Chrysochus species feed on plants that are devoid of cardenolides. The most important active principle of cardenolides is their capacity to bind to and thereby block the ubiquitous Na(+)/K(+)-ATPase responsible for maintaining cellular potentials. By analyzing the DNA sequence of the putative ouabain-binding site of the alpha-subunit of the Na(+)/K(+)-ATPase gene of Chrysochus and its close relatives feeding on plants with or without cardenolides, we here trace the evolution of cardenolide insensitivity in this group of beetles. The most interesting difference among the sequences involves the amino acid at position 122. Whereas all species that do not encounter cardenolides have an asparagine in this position, both Chrysochus species that feed on cardenolide plants have a histidine instead. This single amino acid substitution has already been shown to confer cardenolide insensitivity in the monarch butterfly. A mtDNA-based phylogeny corroborates the hypothesis that the asparagine at position 122 of the alpha-subunit of the Na(+)/K(+)-ATPase gene as observed in Drosophila and other insects is the plesiomorphic condition in this group of leaf beetles. The later host-plant switch to cardenolide-containing plants in the common ancestor of C. auratus and C. cobaltinus coincides with the exchange of the asparagine for a histidine in the ouabain binding site.     

PHYLOGENETIC TRENDS IN PHENOLIC METABOLISM OF MILKWEEDS (ASCLEPIAS): EVIDENCE FOR ESCALATION

Although plant-defense theory has long predicted patterns of chemical defense across taxa, we know remarkably little about the evolution of defense, especially in the context of directional phylogenetic trends. Here we contrast the production of phenolics and cardenolides in 35 species of milkweeds ( Asclepias and Gomphocarpus ). Maximum-likelihood analyses of character evolution revealed three major patterns. First, consistent with the defense-escalation hypothesis, the diversification of the milkweeds was associated with a trend for increasing phenolic production; this pattern was reversed (a declining evolutionary trend) for cardenolides, toxins sequestered by specialist herbivores. Second, phylogenetically independent correlations existed among phenolic classes across species. For example, coumaric acid derivatives showed negatively correlated evolution with caffeic acid derivatives, and this was likely driven by the fact that the former are used as precursors for the latter. In contrast, coumaric acid derivatives were positively correlated with flavonoids, consistent with competition for the precursor p- coumaric acid. Finally, of the phenolic classes, only flavonoids showed correlated evolution (positive) with cardenolides, consistent with a physiological and evolutionary link between the two via malonate. Thus, this study presents a rigorous test of the defense-escalation hypothesis and a novel phylogenetic approach to understanding the long-term persistence of physiological constraints on secondary metabolism.     

Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias): a phylogenetic test of plant defense escalation

A tremendous diversity of plants exude sticky and toxic latex upon tissue damage, and its production has been widely studied as a defensive adaptation against insect herbivores. Here, we address variation in latex production and its constituent chemical properties (cardenolides and cysteine proteases) in 53 milkweeds [Asclepias spp. (Apocynaceae)], employing a phylogenetic approach to test macroevolutionary hypotheses of defense evolution. Species were highly variable for all three traits, and they showed little evidence for strong phylogenetic conservatism. Latex production and the constituent chemical defenses are thus evolutionarily labile and may evolve rapidly. Nonetheless, in phylogenetically independent analyses, we show that the three traits show some correlations (and thus share a correlated evolutionary history), including a positive correlation between latex exudation and cysteine protease activity. Conversely, latex exudation and cysteine protease activity both showed a trade‐off with cardenolide concentrations in latex. We also tested whether these traits have increased in their phenotypic values as the milkweeds diversified, as predicted by plant defense escalation theory. Alternative methods of testing this prediction gave conflicting results – there was an overall negative correlation between amount of evolutionary change and amount of latex exudation; however, ancestral state reconstructions indicated that most speciation events were associated with increases in latex. We conclude by (i) summarizing the evidence of milkweed latex itself as a multivariate defense including the amount exuded and toxin concentrations within, (ii) assessing the coordinated evolution of latex traits and how this fits with our previous notion of ‘plant defense syndromes’, and finally, (iii) proposing a novel hypothesis that includes an ‘evolving community of herbivores’ that may promote the escalation or decline of particular defensive strategies as plant lineages diversify.     

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core)

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na⁺/K⁺-ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.     

Gut microbes of mammalian herbivores facilitate intake of plant toxins

The foraging ecology of mammalian herbivores is strongly shaped by plant secondary compounds (PSCs) that defend plants against herbivory. Conventional wisdom holds that gut microbes facilitate the ingestion of toxic plants; however, this notion lacks empirical evidence. We investigated the gut microbiota of desert woodrats (Neotoma lepida), some populations of which specialise on highly toxic creosote bush (Larrea tridentata). Here, we demonstrate that gut microbes are crucial in allowing herbivores to consume toxic plants. Creosote toxins altered the population structure of the gut microbiome to facilitate an increase in abundance of genes that metabolise toxic compounds. In addition, woodrats were unable to consume creosote toxins after the microbiota was disrupted with antibiotics. Last, ingestion of toxins by naïve hosts was increased through microbial transplants from experienced donors. These results demonstrate that microbes can enhance the ability of hosts to consume PSCs and therefore expand the dietary niche breadth of mammalian herbivores.     

Coexisting congeners: demography, competition, and interactions with cardenolides for two milkweed-feeding aphids

Explaining the coexistence of closely related species sharing a single resource has been a long-standing challenge in ecology. Here we report on studies comparing the aphids Aphis nerii and A. asclepiadis that feed sympatrically on the milkweed Asclepias syriaca in northeastern North America. We sought to identify tradeoffs among species' attributes that might promote coexistence, but in most instances A. nerii was superior to A. asclepiadis . Aphis nerii was 84% more fecund, fed upon 880% more phloem sap, and was affected 70% less by intraspecific competition as compared to A. asclepiadis . In interspecific competition, A. nerii reduced A. asclepiadis abundance by 77%, whereas A. asclepiadis did not affect A. nerii . In dispersal trials, 10% of winged A. nerii but only 1% of A. asclepiadis successfully moved from non-host plants to A. syriaca . We also investigated whether there were differences in aphid interactions with milkweed cardenolides. Jasmonic acid increased milkweed cardenolides by 33%, a realistic amount similar to that induced by several leaf-chewing herbivores. Nevertheless, jasmonate-induced cardenolides failed to affect aphid performance and aphid feeding had no effect on milkweed cardenolide concentration. Yet cardenolides were important for aphid resistance to predators; A. nerii sequestered 25% more cardenolides and was preyed upon 50% less than A. asclepiadis . Interactions with cardenolides thus again favored A. nerii over A. asclepiadis . Given that A. nerii and A. asclepiadis are decidedly not equivalent in their demography, competitive ability, defense and dispersal, our results strongly refute the notion that neutral processes can explain coexistence of these aphids. Based on field observations, we propose two tradeoffs – timing of milkweed colonization and relationships with ants – as putative mechanisms for the coexistence of these congeners.     

Coping with toxic plant compounds--the insect's perspective on iridoid glycosides and cardenolides

Specializing on host plants with toxic secondary compounds enforces specific adaptation in insect herbivores. In this review, we focus on two compound classes, iridoid glycosides and cardenolides, which can be found in the food plants of a large number of insect species that display various degrees of adaptation to them. These secondary compounds have very different modes of action: Iridoid glycosides are usually activated in the gut of the herbivores by β-glucosidases that may either stem from the food plant or be present in the gut as standard digestive enzymes. Upon cleaving, the unstable aglycone is released that unspecifically acts by crosslinking proteins and inhibiting enzymes. Cardenolides, on the other hand, are highly specific inhibitors of an essential ion carrier, the sodium pump. In insects exposed to both kinds of toxins, carriers either enabling the safe storage of the compounds away from the activating enzymes or excluding the toxins from sensitive tissues, play an important role that deserves further analysis. To avoid toxicity of iridoid glycosides, repression of activating enzymes emerges as a possible alternative strategy. Cardenolides, on the other hand, may lose their toxicity if their target site is modified and this strategy has evolved multiple times independently in cardenolide-adapted insects.     

When do herbivores affect plant invasion? Evidence for the natural enemies and biotic resistance hypotheses

Two venerable hypotheses, widely cited as explanations for either the success or failure of introduced species in recipient communities, are the natural enemies hypothesis and the biotic resistance hypothesis. The natural enemies hypothesis posits that introduced organisms spread rapidly because they are liberated from their co‐evolved predators, pathogens and herbivores. The biotic resistance hypothesis asserts that introduced species often fail to invade communities because strong biotic interactions with native species hinder their establishment and spread. We reviewed the evidence for both of these hypotheses as they relate to the importance of non‐domesticated herbivores in affecting the success or failure of plant invasion.
To evaluate the natural enemies hypothesis, one must determine how commonly native herbivores have population‐level impacts on native plants. If native herbivores seldom limit native plant abundance, then there is little reason to think that introduced plants benefit from escape from these enemies. Studies of native herbivore‐native plant interactions reveal that plant life‐history greatly mediates the strength with which specialist herbivores suppress plant abundance. Relatively short‐lived plants that rely on current seed production for regeneration are most vulnerable to herbivory that reduces seed production. As such, these plants may gain the greatest advantage from escaping their specialist enemies in recipient communities. In contrast, native plants that are long lived or that possess long‐lived seedbanks may not be kept “in check” by native herbivores. For these species, escape from native enemies may have little to do with their success as exotics; they are abundant both where they are native and introduced.
Evidence for native herbivores providing biotic resistance to invasion by exotics is conflicting. Our review reveals that: 1) introduced plants can attract a diverse assemblage of native herbivores and that 2) native herbivores can reduce introduced plant growth, seed set and survival. However, the generality of these impacts is unclear, and evidence that herbivory actually limits or reduces introduced plant spread is scarce. The degree to which native herbivores provide biotic resistance to either exotic plant establishment or spread may be greatly determined by their functional and numerical responses to exotic plants, which we know little about. Generalist herbivores, through their direct effects on seed dispersal and their indirect effects in altering the outcome of native–non‐native plant competitive interactions, may have more of a facilitative than negative effect on exotic plant abundance.     

Insect-resistant transgenic plants in a multi-trophic context

So far, genetic engineering of plants in the context of insect pest control has involved insertion of genes that code for toxins, and may be characterized as the incorporation of biopesticides into classical plant breeding. In the context of pesticide usage in pest control, natural enemies of herbivores have received increasing attention, because carnivorous arthropods are an important component of insect pest control. However, in plant breeding programmes, natural enemies of herbivores have largely been ignored, although there are many examples that show that plant breeding affects the effectiveness of biological control. Negative influences of modified plant characteristics on carnivorous arthropods may induce population growth of new, even more harmful pest species that had no pest status prior to the pesticide treatment. Sustainable pest management will only be possible when negative effects on non-target, beneficial arthropods are minimized. In this review, we summarize the effects of insect-resistant crops and insect-resistant transgenic crops, especially Bt crops, from a food web perspective. As food web components, we distinguish target herbivores, non-target herbivores, pollinators, parasitoids and predators. Below-ground organisms such as Collembola, nematodes and earthworms should also be included in risk assessment studies, but have received little attention. The toxins produced in Bt plants retain their toxicity when bound to the soil, so accumulation of these toxins is likely to occur. Earthworms ingest the bound toxins but are not affected by them. However, earthworms may function as intermediaries through which the toxins are passed on to other trophic levels. In studies where effects of insect-resistant (Bt) plants on natural enemies were considered, positive, negative and no effects have been found. So far, most studies have concentrated on natural enemies of target herbivores. However, Bt toxins are structurally rearranged when they bind to midgut receptors, so that they are likely to lose their toxicity inside target herbivores. What happens to the toxins in non-target herbivores, and whether these herbivores may act as intermediaries through which the toxins may be passed on to the natural enemies, remains to be studied.     

Functional evidence for physiological mechanisms to circumvent neurotoxicity of cardenolides in an adapted and a non-adapted hawk-moth species

Because cardenolides specifically inhibit the Na⁺K⁺-ATPase, insects feeding on cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii, possess highly sensitive Na⁺K⁺-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar cardenolide (ouabain). By contrast, for non-polar cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar cardenolides and provides an active barrier for non-polar cardenolides. This may explain the high in vivo resistance to cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na⁺K⁺-ATPases.     

Chemical ecology of host-plant selection by herbivorous arthropods: a multitrophic perspective

Most herbivorous arthropods are specialists that feed on one or a few related plant species. To understand why this is so, both mechanistic and functional studies have been carried out, predominantly restricted to bitrophic aspects. Host-selection behaviour of herbivorous arthropods has been intensively studied and this has provided ample evidence for the role of secondary plant chemicals as source of information in behavioural decisions of herbivores. Many evolutionary studies have regarded co-evolution between plants and herbivores to explain the diversity of secondary plant chemicals and host specialisation of herbivores. However, many cases remain unexplained where herbivores select host plants that are suboptimal in terms of fitness returns. A stimulating paper by Bernays and Graham [(1988) Ecology 69, 886-892)] has initiated a discussion on the need of a multitrophic perspective to understand the evolution of host-plant specialisation by herbivorous arthropods. However, this has hardly resulted in ecological studies on host-selection behaviour that take a multitrophic perspective. Yet, evidence is accumulating that constitutive and induced infochemicals from natural enemies and competitors can affect herbivore behaviour. These cues may constitute important information on fitness prospects, just as plant cues can do. In this paper I selectively review how information from organisms at different trophic levels varies in space and time and how herbivores can integratively exploit this information during host selection. In doing so, research areas are identified that are likely to provide important new insights to explain several of the questions in herbivore host selection that remain unanswered so far. These research areas are at the interface of evolutionary ecology, behavioural ecology and chemical ecology.     

TROPHIC INTERACTIONS IN THE SEA: AN ECOLOGICAL ROLE FOR CLIMATE RELEVANT VOLATILES?1

When attacked by herbivores, land plants can produce a variety of volatile compounds that attract carnivorous mutualists. Plants and carnivores can benefit from this symbiotic relationship, because the induced defensive interaction increases foraging success of the carnivores, while reducing the grazing pressure exerted by the herbivores on the plants. Here, we examine whether aquatic phytoplankton use volatile chemical cues in analogous tritrophic interactions. Marine algae produce several classes of biogenic gases such as non‐methane hydrocarbons, organohalogens, ammonia and methylamines, and dimethylsulfide. The grazing‐induced release of marine biogenic volatiles is poorly understood, however, and its effect on the chemical ecology of plankton and the foraging behavior of predators is essentially unknown. We outline grazing‐induced defenses in algae and highlight the biogenic production of volatiles when phytoplankton are attacked by herbivores. The role of chemical signaling in marine ecology presents several possible avenues for future research, and we believe that progress in this area will result in better understanding of species competition, bloom development, and the structuring of food webs in the sea. This has further implications for biogeochemical cycles, because several key compounds are emitted that influence the chemistry of the atmosphere and global climate.     

Determinants of growth rate on chemically heterogeneous host plants by specialist insects

Nine Passiflora species growing sympatrically in a Costa Rican rain forest were screened for the presence of alkaloids, tannins, cyanogenic compounds, saponins and cardenolides. The first three chemical classes were found to be present and the Passiflora species could be classified into five groups according to the presence/absence of each chemical class. In order to assess the effects of this chemical variability on specialist herbivores, larvae of Heliconius ismenius and H. melpomane were experimentally introduced to seven species of Passiflora host plants growing naturally in the rain forest. Survival and growth rates were subsequently monitored at 2 day intervals and were found to be independent of each other. Larval growth rate and larval survivorship were not significantly affected by cyanogenesis in the host plants, nor by the presence of alkaloids, tannins and non-tanning phenolic compounds. Larval growth rate was found to be correlated to percentage nitrogen in the host plants, but was independent of host water content and non-structural carbohydrate content. Although these insects would be classified as host-specific by most authors, the results indicate the presence of a sufficiently general detoxication apparatus to consume many different classes of potential toxins with no apparent ill effects. We conclude that Passiflora natural product diversity does not cause major deleterious effects in Heliconius growth characteristics and that these effects are not the major component maintaining the current pattern of larval feeding specialization in this plant-herbivore system.     

Assessing the role of large herbivores in the structuring and functioning of freshwater and marine angiosperm ecosystems

While large herbivores can have strong impacts on terrestrial ecosystems, much less is known of their role in aquatic systems. We reviewed the literature to determine: 1) which large herbivores (> 10 kg) have a (semi‐)aquatic lifestyle and are important consumers of submerged vascular plants, 2) their impact on submerged plant abundance and species composition, and 3) their ecosystem functions. We grouped herbivores according to diet, habitat selection and movement ecology: 1) Fully aquatic species, either resident or migratory (manatees, dugongs, turtles), 2) Semi‐aquatic species that live both in water and on land, either resident or migratory (swans), 3) Resident semi‐aquatic species that live in water and forage mainly on land (hippopotamuses, beavers, capybara), 4) Resident terrestrial species with relatively large home ranges that frequent aquatic habitats (cervids, water buffalo, lowland tapir). Fully aquatic species and swans have the strongest impact on submerged plant abundance and species composition. They may maintain grazing lawns. Because they sometimes target belowground parts, their activity can result in local collapse of plant beds. Semi‐aquatic species and turtles serve as important aquatic–terrestrial linkages, by transporting nutrients across ecosystem boundaries. Hippopotamuses and beavers are important geomorphological engineers, capable of altering the land and hydrology at landscape scales. Migratory species and terrestrial species with large home ranges are potentially important dispersal vectors of plant propagules and nutrients. Clearly, large aquatic herbivores have strong impacts on associated species and can be critical ecosystem engineers of aquatic systems, with the ability to modify direct and indirect functional pathways in ecosystems. While global populations of large aquatic herbivores are declining, some show remarkable local recoveries with dramatic consequences for the systems they inhabit. A better understanding of these functional roles will help set priorities for the effective management of large aquatic herbivores along with the plant habitats they rely on.     

Assessing the biosecurity risk from pathogens and herbivores to indigenous plants: lessons from weed biological control

Some potentially invasive herbivores/pathogens in their home range may attack plants originating from another geographic area. Methods are required to assess the risk these herbivores/pathogens pose to these plants in their indigenous ecosystems. The processes and criteria used by weed biological control researchers to assess the impact of potential biological control agents on a plant species in its non-native range provide a possible framework for assessing risks to indigenous plants. While there are similarities between these criteria such as the need for clear objectives, studies in the native range of the herbivore/pathogen, good knowledge of the ecology of the target plant and taxonomy of the plant and herbivore/pathogen, and modelling of the interaction between the two organisms, there are some important differences in approach. These include the need to consider the threat classification of the plant, the likely greater risk from polyphagous herbivores/pathogens than oligophagous or monophagous species, and the need to consider the impact of an additional natural enemy in conjunction with a suite of existing natural enemies. The costs of conducting a risk assessment of a herbivore/pathogen in another country that damages plants indigenous to another geographic area means that criteria will be needed for deciding which foreign herbivore/pathogen species should be assessed. These criteria could include the threat classification of the plant, the amount of damage to the particular plant organs affected, and the importance in key ecosystems.     

Evolution of specialization: a phylogenetic study of host range in the red milkweed beetle (Tetraopes tetraophthalmus)

Specialization is common in most lineages of insect herbivores, one of the most diverse groups of organisms on earth. To address how and why specialization is maintained over evolutionary time, we hypothesized that plant defense and other ecological attributes of potential host plants would predict the performance of a specialist root-feeding herbivore (the red milkweed beetle, Tetraopes tetraophthalmus). Using a comparative phylogenetic and functional trait approach, we assessed the determinants of insect host range across 18 species of Asclepias. Larval survivorship decreased with increasing phylogenetic distance from the true host, Asclepias syriaca, suggesting that adaptation to plant traits drives specialization. Among several root traits measured, only cardenolides (toxic defense chemicals) correlated with larval survival, and cardenolides also explained the phylogenetic distance effect in phylogenetically controlled multiple regression analyses. Additionally, milkweed species having a known association with other Tetraopes beetles were better hosts than species lacking Tetraopes herbivores, and milkweeds with specific leaf area values (a trait related to leaf function and habitat affiliation) similar to those of A. syriaca were better hosts than species having divergent values. We thus conclude that phylogenetic distance is an integrated measure of phenotypic and ecological attributes of Asclepias species, especially defensive cardenolides, which can be used to explain specialization and constraints on host shifts over evolutionary time.     

Start making scents: the challenge of integrating chemistry into pollination ecology

Although research on plant volatiles and pollination ecology has grown explosively over the past 15 years, there remains little dialogue between these fields. Here I examine the historical and cultural reasons for this impasse, focusing on the ways that questions in each field are addressed, and the potential for productive cross-talk. The specialization–generalization debate in pollination has cast doubt on the importance of sensory biology in mediating plant–pollinator interactions on the community scale. However, chemical 'filters' of volatile or nectar-borne repellents are likely to explain the absence of specific interactions in plant–pollinator webs. In addition, the omission of plant volatiles from path analyses measuring the relative impacts of herbivores and pollinators on plant fitness may be one reason for large unexplained variance terms in such models. Floral scent functions in concert with visual and gustatory cues by attracting pollinators from a distance, increasing approaches and landings, and mediating outcrossing rates through changes in visitation frequency and duration. All dimensions of floral chemistry, including ontogenetic and diel variation in scent emissions, have the potential to respond to balancing selection between herbivores and pollinators. The available data reveal that chemical aspects of floral phenotypes are important across the specialization–generalization spectrum, and thus are widely applicable to mainstream pollination ecology.