DOI: 10.5061/dryad.r4xgxd29h
title: ABCB transporters in a leaf beetle respond to sequestered plant toxins

description: Phytophagous insects can tolerate and detoxify toxic compounds present in
 their host plants and have evolved intricate adaptations to this end. Some
 insects even sequester the toxins for their defense. This necessitates
 specific mechanisms, especially carrier proteins that regulate uptake and
 transport to specific storage sites or protect sensitive tissues from
 noxious compounds. We identified three ATP-binding cassette subfamily B
 (ABCB) transporters from the transcriptome of the cardenolide-sequestering
 leaf beetle Chrysochus auratus and analyzed their functional role in the
 sequestration process. These were heterologously expressed and tested for
 their ability to interact with various potential substrates: verapamil
 (standard ABCB substrate), the cardenolides digoxin (commonly used),
 cymarin (present in the species’ host plant) and calotropin (present in
 the ancestral host plants). Verapamil stimulated all three ABCBs and each
 was activated by at least one cardenolide, however, they differed as to
 which they were activated by. While the expression of the most versatile
 transporter fits with a protective role in the blood-brain-barrier, the
 one specific for cymarin shows an extreme abundance in the elytra,
 coinciding with the location of the defensive glands. Our data thus
 suggest a key role of ABCBs in the transport network needed for
 cardenolide sequestration.
description: <b><i>Ca_</i>ABCB ATPase activity assays</b> Crude <i>Sf</i>9 cell membranes overexpressing <i>Ca_</i>ABCBs were isolated to determine the substrate-dependent ATPase activity by measuring the release of inorganic phosphate following the method described by Sarkadi<i> et al</i>. [27]<i> </i>with slight modifications. We examined the interaction of the <i>Ca_</i>ABCB transporters with verapamil (Merck; Darmstadt, Germany), a well-known stimulator of Pgp ATPase activity, and three different cardenolides, cymarin (Phytolab; Vestenbergsgreuth, Germany), digoxin (Merck) and calotropin (isolated by preparative high performance liquid chromatography from monarch caterpillars raised on <i>Asclepias curassavica</i>, following the protocol in Petschenka <i>et al</i>. 2018,[28]). All cardenolides were first dissolved in dimethyl sulfoxide that was further diluted to a final concentration of 2%. Assays were run in 96-well plates in duplicates. Finally, we produced 12 to 32 technical replicates for each <i>Ca_</i>ABCB transporter-cardenolide combination. <i>Ca_</i>ABCB membrane vesicle preparations (5 µg protein/ well) were incubated (20 min, 27°C) in 30 µl assay buffer (final concentration 50 mM MOPS-Tris, pH 7.0, 50 mM KCL, 5 mM Na-acid, 100 mM EGTA-Tris, pH 7.0) with different cardenolide concentrations in the presence and the absence of 500 µM sodium orthovanadate (Merck), a non-competitive P-type ATPase inhibitor. The reaction was started by adding 20 µl Mg-ATP (Merck; final concentration 3 mM) and stopped (after 20 min, 27°C) by adding 20 µl 5% sodium dodecyl sulfate. To reveal the amount of phosphate released the following reagents were added:  50 µl P-reagent (2.5 M H<sub>2</sub>SO<sub>4 </sub>+ 1% ammonium molybdate (Carl Roth) + 0,014% antimony potassium tartrate (Merck), 20 µl 20% acetic acid, 25 µl 1% ascorbic acid). The colorimetric reaction took place at room temperature (15 min) and was measured in a microplate reader at 655 nM (Model 680, Bio-Rad). Based on the estimated raw data and phosphate standard curves of K<sub>2</sub>HPO<sub>4</sub> we calculated the release of inorganic phosphate and the ATPase activity (nmol Pi/mg protein/min) in each well. Dose-dependent effects of cardenolide treatment (0-800 µM) on <i>Ca_</i>ABCB ATPase activities were calculated as the difference between ATPase activity (nmol Pi/mg protein/min) in the presence and absence of sodium orthovanadate. Vanadate-sensitive ATPase activity of membrane vesicles of uninfected <i>Sf</i>9 cells in the absence of cardenolides was defined as baseline activity.   <b>Kinetics/ statistics</b> Experimental data were fitted to the expanded Michaelis-Menten equation including Hill coefficients (OriginPro 2016G; <i>OriginLab</i> Corporation):   Where V = velocity (nmol pi/mg protein/min), V<sub>min</sub> = minimal velocity, V<sub>max</sub> = maximal velocity, EC<sub>50 </sub>= ligand concentration producing 50% of maximal response, c = the actual test drug concentration, and n = Hill slope, the parameter characterizing the degree of substrate binding site cooperativity. <i>Ca_</i>ABCB ATPase transporter-specific activities at 800 µM cardenolide treatment were tested for statistically significant differences by two-way ANOVA (OriginPro 2016G) followed by Tukey post-hoc tests.   <b>qRT-PCR of <i>C. auratus</i> tissues</b> To investigate the tissue distribution of<i> Ca</i>_ABCB transporters eight different tissues of nine <i>C. auratus</i> beetles (collected in Ithaca, New York, USA) were dissected: crop to midgut, midgut to rectum, Malpighian tubules, reproductive organs, muscles, tracheae, nervous tissue, and elytra. Tissue samples of three beetles were pooled (resulting in one biological replicate) before RNA extraction (RNeasy Mini Kit; Qiagen). RNA quality was checked spectrophotometrically (Nanodrop 2000, Thermo Fisher Scientific) and concentrations equilibrated before cDNA synthesis (ProtoScript II Synthesis Kit, New England Biolabs). Exon-spanning gene-specific primers (Supplement Table 2) were designed based on the <i>Ca</i>_ABCB sequences and the Ensembl Metazoa database. Quantification of the transcript levels was based on a standard curve of linearized pFastBacDual plasmids containing the entire coding region of<i> Ca</i>_ABCB1,2 or 3. Threshold cycle (Ct) values were estimated on a StepOne Real-Time PCR System and pre-analyzed with the StepOne Software v2.3 (Thermo Fisher Scientific). cDNA products were amplified in triplicates using the EvaGreen qPCR Mix II (ROX) (Bio-Budget Technologies, Germany) in a volume of 20 µl with a final cDNA amount of 30 µg total RNA by following the manufacturer's protocol. Amplification conditions were 90°C for 15 min followed by 40 cycles consisting of 15 sec at 95°C, 20 sec at 60°C, and 20 sec at 70°C. Control samples without cDNA were loaded on each 48-well plate to check for unspecific amplification products via melting curve analysis. Specificity and amplification efficiencies of the qPCR-primers were first evaluated by duplicate standard curve reactions with 10-fold serial dilutions (from 10<sup>8</sup> to 10<sup>2</sup>) of recombinant plasmids [29]. Means of three biological replicates were estimated and statistically analyzed using one-way ANOVA (OriginPro 2016G, OriginLab Corporation).