title: 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.
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA. Electronic address: sg189@nyu.edu.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Groen
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA; Department of Integrative Biology, University of California, Berkeley, 3040 Valley Life Sciences Building, Berkeley, CA 94720, USA.
authors.forename: Erika R
authors.initials: ER
authors.lastname: LaPlante
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA; Department of Integrative Biology, University of California, Berkeley, 3040 Valley Life Sciences Building, Berkeley, CA 94720, USA.
authors.forename: Nicolas M
authors.initials: NM
authors.lastname: Alexandre
authors.affiliation: Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA; Department of Entomology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Anurag A
authors.initials: AA
authors.lastname: Agrawal
authors.affiliation: Molecular Evolutionary Biology, Zoological Institute, Biocenter Grindel, Universität Hamburg, Martin-Luther-King Pl. 3, 20146 Hamburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Dobler
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA; Department of Integrative Biology, University of California, Berkeley, 3040 Valley Life Sciences Building, Berkeley, CA 94720, USA. Electronic address: whiteman@berkeley.edu.
authors.forename: Noah K
authors.initials: NK
authors.lastname: Whiteman
mesh_terms: D000818:Animals; D002298:Cardenolides / Q000633:toxicity*; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D008297:Male; D027361:Organic Anion Transporters / Q000378:metabolism*
chemical_list: D002298:Cardenolides; D029721:Drosophila Proteins; D027361:Organic Anion Transporters
keywords: Cardenolide; Drosophila melanogaster; Herbivore; Multidrug transporter/P-glycoprotein; Na/K-ATPase; Organic anion transporting polypeptide