Exogenous application of RNA for the eco-friendly control of insect pests

In Anbetracht der hohen gesellschaftlichen Forderungen nach nachhaltigen und umweltschonenden Pflanzenschutzverfahren, stellt das Sprühen von RNA-basierten Wirkstoffen eine innovative und vielversprechende Alternative zu konventionellen chemisch-synthetischen Pflanzenschutzmitteln dar. Dabei vermitteln die sequenzspezifischen und somit maßgeschneiderten RNAs nicht nur eine hohe Selektivität, sondern lassen sich zudem sehr schnell adaptieren. Dadurch sind sie, verglichen mit der langwierigen, herkömmlichen Wirkstoff­entwicklung, schneller verfügbar. Trotz der vielfältigen Vorteile und des dringenden Bedarfs an Alternativen, stehen wir erst am Anfang des Transfers von RNA Sprays ins Freiland. Hier diskutieren wir die damit verknüpften wissenschaftlich-technischen, gesellschaftlichen und wirtschaftlichen Herausforderungen. Zudem zeigen wir die offenen Forschungsfragen auf, die es zu adressieren gilt, um RNA-basierte Pflanzenschutzmittel zeitnah zu etablieren. Zuletzt führen wir aktuelle Beispiele zu innovativen Lösungsansätzen aus der Forschung an, die die Weiterentwicklung von RNA Sprays für die Kontrolle von Schadinsekten in der Pflanzenproduktion zum Ziel haben. Teile dieses Artikels wurden bereits in einem breiter angelegten englischsprachigen Übersichtsartikel (Rank & Koch 2021) dargestellt und werden hier in einen detaillierteren Kontext eingebettet. Durch die Übersetzung ins Deutsche sollen sie zudem einem erweiterten Leserkreis zugänglich gemacht werden.Under the perspective of high social demands for sustainable and environmentally friendly crop protection, RNA sprays represent an innovative and promising alternative to conventional synthetic pesticides. Due to sequence specificity, custom-made RNAs not only provide high selectivity but can also be easily adapted to target different species. Consequently, they can be available more quickly compared to the time-consuming development of conventional synthetic pesticides. Despite the multiple benefits and the desperate need for alternatives, field application of RNA sprays is still in its infancy. Here, we discuss the challenges resulting from scientific-technical, social and economic demands. In addition, we identify open research questions that need to be addressed to establish RNAi-based products in a timely manner. Moreover, we highlight recent examples of innovative solutions, which could inspire further optimization of RNA sprays for the control of insect pests in the field. Parts of this article have already been presented in a broader review article (Rank & Koch 2021) and are embedded here into a more detailed context. The presentation in German should also make them accessible to a broader readership

Relative Selectivity of Plant Cardenolides for Na+/K+-ATPases From the Monarch Butterfly and Non-resistant Insects

This work is licensed under a Creative Commons Attribution 4.0 International License.A major prediction of coevolutionary theory is that plants may target particular herbivores with secondary compounds that are selectively defensive. The highly specialized monarch butterfly (Danaus plexippus) copes well with cardiac glycosides (inhibitors of animal Na+/K+-ATPases) from its milkweed host plants, but selective inhibition of its Na+/K+-ATPase by different compounds has not been previously tested. We applied 17 cardiac glycosides to the D. plexippus-Na+/K+-ATPase and to the more susceptible Na+/K+-ATPases of two non-adapted insects (Euploea core and Schistocerca gregaria). Structural features (e.g., sugar residues) predicted in vitro inhibitory activity and comparison of insect Na+/K+-ATPases revealed that the monarch has evolved a highly resistant enzyme overall. Nonetheless, we found evidence for relative selectivity of individual cardiac glycosides reaching from 4- to 94-fold differences of inhibition between non-adapted Na+/K+-ATPase and D. plexippus-Na+/K+-ATPase. This toxin receptor specificity suggests a mechanism how plants could target herbivores selectively and thus provides a strong basis for pairwise coevolutionary interactions between plants and herbivorous insects.German Research Foundation (GP, PE 2059/1-1)US National Science Foundation (AA, DEB-1619885

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

Relative Selectivity of Plant Cardenolides for Na+/K+-ATPases From the Monarch Butterfly and Non-resistant Insects

This work is licensed under a Creative Commons Attribution 4.0 International License.A major prediction of coevolutionary theory is that plants may target particular herbivores with secondary compounds that are selectively defensive. The highly specialized monarch butterfly (Danaus plexippus) copes well with cardiac glycosides (inhibitors of animal Na+/K+-ATPases) from its milkweed host plants, but selective inhibition of its Na+/K+-ATPase by different compounds has not been previously tested. We applied 17 cardiac glycosides to the D. plexippus-Na+/K+-ATPase and to the more susceptible Na+/K+-ATPases of two non-adapted insects (Euploea core and Schistocerca gregaria). Structural features (e.g., sugar residues) predicted in vitro inhibitory activity and comparison of insect Na+/K+-ATPases revealed that the monarch has evolved a highly resistant enzyme overall. Nonetheless, we found evidence for relative selectivity of individual cardiac glycosides reaching from 4- to 94-fold differences of inhibition between non-adapted Na+/K+-ATPase and D. plexippus-Na+/K+-ATPase. This toxin receptor specificity suggests a mechanism how plants could target herbivores selectively and thus provides a strong basis for pairwise coevolutionary interactions between plants and herbivorous insects.German Research Foundation (GP, PE 2059/1-1)US National Science Foundation (AA, DEB-1619885

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches.

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions

Antioxidant availability trades off with warning signals and toxin sequestration in the large milkweed bug (Oncopeltus fasciatus)

In some aposematic species the conspicuousness of an individual's warning signal and the concentration of its chemical defense are positively correlated. Several mechanisms have been proposed to explain this phenomenon, including resource allocation trade-offs where the same limiting resource is needed to produce both the warning signal and chemical defense. Here, the large milkweed bug (Oncopeltus fasciatus: Heteroptera, Lygaeinae) was used to test whether allocation of antioxidants, that can impart color, trade against their availability to prevent self-damage caused by toxin sequestration. We investigated if (i) the sequestration of cardenolides is associated with costs in the form of changes in oxidative state; and (ii) oxidative state can affect the capacity of individuals to produce warning signals. We reared milkweed bugs on artificial diets with increasing quantities of cardenolides and examined how this affected signal quality (brightness and chroma) across different instars. We then related the expression of warning colors to the quantity of sequestered cardenolides and indicators of oxidative state-oxidative lipid damage (malondialdehyde), and two antioxidants: total superoxide dismutase and total glutathione. Bugs that sequestered more cardenolides had significantly lower levels of the antioxidant glutathione, and bugs with less total glutathione had less luminant orange warning signals and reduced chroma of their black patches compared to bugs with more glutathione. Bugs that sequestered more cardenolides also had reduced red-green chroma of their black patches that was unrelated to oxidative state. Our results give tentative support for a physiological cost of sequestration in milkweed bugs and a mechanistic link between antioxidant availability, sequestration, and warning signals

Sequestration of Defenses against Predators Drives Specialized Host Plant Associations in Preadapted Milkweed Bugs (Heteroptera: Lygaeinae)

AbstractHost plant specialization across herbivorous insects varies dramatically, but while the molecular mechanisms of host plant adaptations are increasingly known, we often lack a comprehensive understanding of the selective forces that favor specialization. The milkweed bugs (Heteroptera: Lygaeinae) are ancestrally associated with plants of the Apocynaceae from which they commonly sequester cardiac glycosides for defense, facilitated by resistant NaNa+/K+-ATPases and adaptations for transport, storage, and discharge of toxins. Here, we show that three Lygaeinae species independently colonized four novel nonapocynaceous hosts that convergently produce cardiac glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three milkweed bug species tested, feeding on seeds containing toxins did not improve growth or speed of development and even impaired growth and development in two species, but sequestration mediated protection of milkweed bugs against two natural predators: lacewing larvae and passerine birds. We conclude that physiological preadaptations and convergent phytochemistry facilitated novel specialized host associations. Since toxic seeds did not improve growth but either impaired growth or, at most, had neutral effects, selection by predators on sequestration of defenses, rather than the exploitation of additional profitable dietary resources, can lead to obligatory specialized host associations in otherwise generalist insects

Independent evolution of ancestral and novel defenses in a genus of toxic plants (Erysimum, Brassicaceae)

Phytochemical diversity is thought to result from coevolutionary cycles as specialization in herbivores imposes diversifying selection on plant chemical defenses. Plants in the speciose genus Erysimum (Brassicaceae) produce both ancestral glucosinolates and evolutionarily novel cardenolides as defenses. Here we test macroevolutionary hypotheses on co-expression, co-regulation, and diversification of these potentially redundant defenses across this genus. We sequenced and assembled the genome of E. cheiranthoides and foliar transcriptomes of 47 additional Erysimum species to construct a phylogeny from 9868 orthologous genes, revealing several geographic clades but also high levels of gene discordance. Concentrations, inducibility, and diversity of the two defenses varied independently among species, with no evidence for trade-offs. Closely related, geographically co-occurring species shared similar cardenolide traits, but not glucosinolate traits, likely as a result of specific selective pressures acting on each defense. Ancestral and novel chemical defenses in Erysimum thus appear to provide complementary rather than redundant functions.Austrian Science Fund (FWF) PZ00P3-161472National Science Foundation (NSF) 1811965 1645256Triad FoundationGerman Research Foundation (DFG) DFG-PE 2059/3-1Agencia Estatal de Investigacion CGL2017-86626-C2-2-PLOEWE Program Insect Biotechnology and BioresourcesJunta de Andalucía A-RNM505-UGR1

Data from: 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. In contrast, sequestration of cardenolides in the body (as a defense 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 nonsequestering 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