TY - JOUR
T1 - Multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of cardenolides
AU - Groen, Simon C.
AU - LaPlante, Erika R.
AU - Alexandre, Nicolas M.
AU - Agrawal, Anurag A.
AU - Dobler, Susanne
AU - Whiteman, Noah K.
N1 - Funding Information:
We thank Tobias Z?st for verifying cardenolide concentrations in Drosophila food solutions, Julianne Ray and Joseph Hernandez for assistance with CAFE assays, and Jennifer Koop and Andrew Gloss for advice on assay design and fly stock maintenance. We are grateful to Safaa Dalla, Amy Hastings, Jennifer Lohr, Georg Petschenka, Sophie Zaaijer, Tobias Z?st, and members of the Dobler and Whiteman laboratories for useful discussions, and to Sophie Zaaijer for providing images. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and the Exelixis Collection at the Harvard Medical School were used in this study. This work was supported by the John Templeton Foundation (SCG, ERL, AAA, SD and NKW; Grant ID #41855 to AAA, SD, and NKW). SD was supported by the German Science Foundation (DFG, grant Do527/5-1 to SD). NKW was funded by the National Geographic Society (Grant 9097-12 to NKW), the University of Arizona (Faculty Seed Grant, Center for Insect Science Seed Grant, and laboratory set-up grant to NKW), the National Science Foundation (grant DEB-1256758 to NKW) and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM119816. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding sources had no involvement in the study design; the collection, analysis and interpretation of data; the writing of the report; or in the decision to submit the article for publication. Data accessibility: all raw data will be deposited in the Dryad data repository (doi # pending).
PY - 2017/2/1
Y1 - 2017/2/1
N2 - 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.
AB - 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.
KW - Cardenolide
KW - Drosophila melanogaster
KW - Herbivore
KW - Multidrug transporter/P-glycoprotein
KW - Na/K-ATPase
KW - Organic anion transporting polypeptide
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UR - http://www.scopus.com/inward/citedby.url?scp=85007145635&partnerID=8YFLogxK
U2 - 10.1016/j.ibmb.2016.12.008
DO - 10.1016/j.ibmb.2016.12.008
M3 - Article
C2 - 28011348
AN - SCOPUS:85007145635
VL - 81
SP - 51
EP - 61
JO - Insect Biochemistry and Molecular Biology
JF - Insect Biochemistry and Molecular Biology
SN - 0965-1748
ER -