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.

Ca_ABCB ATPase activity assays

Crude Sf9 cell membranes overexpressing Ca_ABCBs were isolated to determine the substrate-dependent ATPase activity by measuring the release of inorganic phosphate following the method described by Sarkadi et al. [27] with slight modifications. We examined the interaction of the Ca_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 Asclepias curassavica, following the protocol in Petschenka et al. 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 Ca_ABCB transporter-cardenolide combination. Ca_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 H₂SO₄ + 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 K₂HPO₄ 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 Ca_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 Sf9 cells in the absence of cardenolides was defined as baseline activity.

Kinetics/ statistics

Experimental data were fitted to the expanded Michaelis-Menten equation including Hill coefficients (OriginPro 2016G; OriginLab Corporation):

Where V = velocity (nmol pi/mg protein/min), V_min = minimal velocity, V_max = maximal velocity, EC₅₀ = 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.

Ca_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.

qRT-PCR of C. auratus tissues

To investigate the tissue distribution of Ca_ABCB transporters eight different tissues of nine C. auratus 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 Ca_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 Ca_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 10⁸ to 10²) of recombinant plasmids [29].

Means of three biological replicates were estimated and statistically analyzed using one-way ANOVA (OriginPro 2016G, OriginLab Corporation).