Georg Petschenka

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.

Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+ -ATPase of milkweed butterflies (lepidoptera: Danaini).

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na+/K+‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na+/K+‐ATPase gene and by enzymatic assays with extracted Na+/K+‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.

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.

Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera).

Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs) seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera) indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST) data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3). Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs) are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects and the functional characterization of a further member of this enzyme family shed light on this almost uncharacterized detoxification system in insects.

Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet.

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium–potassium pump (Na+/K+-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na+/K+-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na+/K+-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na+/K+-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na+/K+-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na+/K+-ATPase, but had systematically reduced effects on Na+/K+-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na+/K+-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.

Target-site sensitivity in a specialized herbivore towards major toxic compounds of its host plant: the Na+K+-ATPase of the oleander hawk moth (Daphnisnerii) is highly susceptible to cardenolides.

The caterpillars of the oleander hawk moth, Daphnis nerii (Linnaeus, 1758) (Lepidoptera: Sphingidae) feed primarily on oleander (Nerium oleander). This plant is rich in cardenolides, which specifically inhibit the Na+K+-ATPase. Since some insects feeding on cardenolide plants possess cardenolide-resistant Na+K+-ATPases, we tested whether D. nerii also possesses this strategy for circumventing cardenolide toxicity. To do so, we established a physiological assay, which allowed direct measurement of Na+K+-ATPase cardenolide sensitivity. Using Schistocerca gregaria, as a cardenolide-sensitive reference species, we showed that D. nerii Na+K+-ATPase was extremely sensitive to the cardenolide ouabain. Surprisingly, its sensitivity is even higher than that of the cardenolide-sensitive generalist, S. gregaria. The presence or absence of cardenolides in the diet of D. nerii did not influence the enzyme’s cardenolide sensitivity, indicating that target-site insensitivity is not inducible in this species. However, despite the sensitivity of their Na+K+-ATPase, caterpillars of D. nerii quickly recovered from an injection of an excessive amount of ouabain into their haemocoel. We conclude that D. nerii possesses adaptations, which enable it to feed on a cardenolide-rich diet other than that previously described in cardenolide specialized insects, and discuss other potential resistance mechanisms.

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na+/K+-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na+/K+-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na+/K+-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.

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.

Convergent adaptive evolution – how insects master the challenge of cardiac glycoside‐containing host plants

Cardiac glycosides are a prime example of highly toxic plant secondary compounds, which block an essential transmembrane carrier in animals, the Na,K‐ATPase. Nevertheless, over 100 insect species from diverse orders are known to feed on plants containing these compounds and in many cases these toxins are additionally sequestered without ill effect. We investigated whether the insects’ adaptations for handling cardiac glycosides are based on a single physiological mechanism or whether various strategies have evolved across groups. We analyzed gene sequences of the Na,K‐ATPase α‐subunit from cardiac glycoside‐adapted insects and screened for amino‐acid substitutions which could alter the affinity of the enzyme toward cardiac glycosides. In representatives from five insect orders, separated by over 300 million years of evolutionary divergence, we uncovered amino‐acid substitutions at identical positions. Especially striking is the convergent substitution of a histidine for the conserved asparagine at position 122, which we report here for the first time in a sawfly, Monophadnus latus Costa (Hymenoptera: Tenthredinidae), and which was previously observed in the orders Lepidoptera, Coleoptera, Hemiptera, and Diptera. Prior in vitro expression and enzyme assays indicated that this substitution as well as combined substitutions with other residues result in a strongly increased cardenolide resistance of the Na,K‐ATPase. The substitutions to threonine111 and histidine122 observed in M. latus are highly effective and were previously known only in lygaeid bugs. However, not all insects dealing with dietary cardenolides rely on target‐site insensitivity as a mechanism of resistance. An impermeable gut or the exclusion of cardenolides from the nervous tissue with the greatest expression of Na,K‐ATPase by the perineurium, the insect blood brain barrier, apparently represent alternative strategies. Immuno‐histochemical data presented here support the existence of P‐glycoprotein‐like efflux transporters in insect gut membranes that might prevent the uptake of allelochemicals like cardenolides.

Convergently Evolved Toxic Secondary Metabolites in Plants Drive the Parallel Molecular Evolution of Insect Resistance

Natural selection imposed by natural toxins has led to striking levels of convergent evolution at the molecular level. Cardiac glycosides represent a group of plant toxins that block the Na,K-ATPase, a vital membrane protein in animals. Several herbivorous insects have convergently evolved resistant Na,K-ATPases, and in some species, convergent gene duplications have also arisen, likely to cope with pleiotropic costs of resistance. To understand the genetic basis and predictability of these adaptations, we studied five independent lineages of leaf-mining flies (Diptera: Agromyzidae). These flies have colonized host plants in four botanical families that convergently evolved cardiac glycosides of two structural types: cardenolides and bufadienolides. We compared each of six fly species feeding on such plants to a phylogenetically related but nonadapted species. Irrespective of the type of cardiac glycoside in the host plant, five out of six exposed species displayed substitutions in the cardiac glycoside–binding site of the Na,K-ATPase that were previously described in other insect orders; in only one species was the gene duplicated. In vitro assays of nervous tissue extractions confirmed that the substitutions lead to increased resistance of the Na,K-ATPase. Our results demonstrate that target site insensitivity of Na,K-ATPase is a common response to dietary cardiac glycosides leading to highly predictable amino acid changes; nonetheless, convergent evolution of gene duplication for this multifunctional enzyme appears more constrained.