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

Abstract

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.

Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo

“…Substitutions at sites 111, 119 and 122 increase adult survival upon exposure to CGs. D. melanogaster do not normally consume CG-containing plants and consumption of CGs results in increased mortality (Groen et al 2017). Given that substitutions at sites 111 and 122 decrease sensitivity to CG-inhibition of NKA, such substitutions should confer an advantage upon exposure of D. melanogaster to CGs.…”

The ABCB Multidrug Resistance Proteins Do Not Contribute to Ivermectin Detoxification in the Colorado Potato Beetle, Leptinotarsa decemlineata (Say)

“…LedMDR3 could have a more general detoxification role, as its gene was expressed in both the head and MT tissue. Expression of MDR genes in the MTs of two flies, D. melanogaster and Mayetiola destructor, has been linked to detoxification of PSMs [60,61]. Excretory mechanisms in the MT tissues also protect against nicotine and vinblastine in M. sexta [62] and nicotine in the hemipteran Rhodnius prolixus [63].…”

Insect adaptations toward plant toxins in milkweed–herbivores systems – a review

“…Two lepidopteran species, Daphnis nerii (L.) (cardenolide adapted) and Manduca sexta (L.) (not cardenolide adapted), lack the protective Na + /K + ATPase a-subunit substitutions and express efflux carrier p-glycoproteins that protect sensitive nerve tissues against cardenolides (Petschenka & Dobler, 2009;Petschenka et al, 2013b). Using RNAi and mutant stock lines, multidrug transporters and organic anion-transporting polypeptides have also been shown to be important in protecting Drosophila melanogaster Meigen from dietary cardenolides (Torrie et al, 2004;Groen et al, 2017). Analyses comparing cardenolide uptake and composition across tissues in various cardenolide-adapted insect species suggest both passive and selective uptake models of both non-polar and polar cardenolide compounds (Brower et al, 1972;Yoder et al, 1976;Duffey et al, 1978;Scudder & Meredith, 1982;von Nickisch-Rosenegk et al, 1990;Detzel & Wink, 1995;Frick & Wink, 1995).…”

“…Such 'target site insensitivity', which protects the Na + /K + pumps from the inhibitory effects of cardenolides, has evolved convergently in diverse insect taxa in the Lepidoptera, Heteroptera, Coleoptera, Diptera, and Hymenoptera (Dobler et al, 2012(Dobler et al, , 2015Zhen et al, 2012). Using RNAi and mutant stock lines, multidrug transporters and organic anion-transporting polypeptides have also been shown to be important in protecting Drosophila melanogaster Meigen from dietary cardenolides (Torrie et al, 2004;Groen et al, 2017). Two lepidopteran species, Daphnis nerii (L.) (cardenolide adapted) and Manduca sexta (L.) (not cardenolide adapted), lack the protective Na + /K + ATPase a-subunit substitutions and express efflux carrier p-glycoproteins that protect sensitive nerve tissues against cardenolides (Petschenka & Dobler, 2009;Petschenka et al, 2013b).…”