DOI: 10.5061/dryad.1zcrjdg0n
title: Toxin sequestration of milkweed insects and inhibition of their sodium pump constructs

description: Repeatable macroevolutionary patterns provide hope for rules in biology,
 especially when we can decipher the underlying mechanisms. Here we
 synthesize natural history, genetic adaptations, and toxin sequestration
 in herbivorous insects that specialize on plants with cardiac glycoside
 defenses. Work on the monarch butterfly provided a model for evolution of
 the sequestering specialist syndrome, where specific amino acid
 substitutions in the insect’s Na+/K+-ATPase are associated with 1) high
 toxin-resistance (target site insensitivity, TSI), 2) sequestration of
 toxins, and 3) aposematic coloration. We evaluate convergence for these
 traits within and between Lepidoptera, Coleoptera, Diptera, Hemiptera,
 Hymenoptera and Orthoptera, encompassing hundreds of toxin-adapted
 species. Using new and existing data on ~28 origins of specialization, we
 show that the monarch model evolved independently in five taxonomic orders
 (but not Diptera). An additional syndrome occurs in five orders:
 aposematic sequesterers with modest to medium TSI (all but Hymenoptera).
 Indeed, all sequestering species were aposematic and all but one had at
 least modest TSI. Additionally, several species were aposematic
 non-sequesterers (potential Batesian mimics), and this combination evolved
 in species with a range of TSI levels. Finally, we identified some biases
 among these strategies within taxonomic orders. Biodiversity in this
 microcosm of life evolved repeatedly with a high degree of similarity
 across six taxonomic orders, yet we identified alternative trait
 combinations as well as lineage-specific outcomes.
description: Three types of data are presented: 1) cardenolide concentrations
 in insects measured by high performance liquid chromatography, 2)
 cardenolide concentrations in insects measured by mass spectroscopy, and
 3) sodium pump enzyme inhibition values (IC50s) measured on several
 different constructs. Raw data used in statistical analyses are provided. 
   
description: # Raw data on sequestration and sodium pump constructs
 [https://doi.org/10.5061/dryad.1zcrjdg0n](https://doi.org/10.5061/dryad.1zcrjdg0n) Three types of data are presented: 1) cardenolide concentrations in insects measured by high performance liquid chromatography, 2) cardenolide concentrations in insects measured by mass spectroscopy, 3) sodium pump enzyme inhibition values (IC50s) measured on several different constructs. Raw data used in statistical analyses are provided.   Analyses of these data is presented in Convergence and divergence among herbivorous insects specialized on toxic plants: revealing syndromes among the cardenolide-feeders across the insect tree of life. To be published in American Naturalist. ## Description of the data and file structure 3 files: sequestration raw HPLC data: Concentration (µg cardenolide/mg dry plant tissue) estimated from HPLC peak area, sample mass, and an internal standard. Insect species, sample ID, and concentrations are provided. Each cardenolide peak is identified by a retention time on the HPLC column. sequestration raw mass spec data: Concentrations of cardenolides from individual samples of Diabrotica and Liriomyza analyzed by mass spectroscopy. Ion counts were converted to ng/ml, to ng, to ng/mg of dry insect tissue. Each step is shown and the means and SE per species is also calculated. Sequestration data is provided for all specific compounds identified by mass spec. For Diabrotica, insect sample #1 was split into two technical replicates (one elytra was used in each). sod pump constructs: Resistance of engineered Drosophila sodium pumps (Na+/K+-ATPase) into which the amino acids of interest (at positions 111 and 122) were introduced and the enzymes produced in cell culture. By this approach all substitutions were introduced into the same genetic background, thus allowing an assessment of the effect of individual substitutions. Here we report the concentration of the standard cardenolide, ouabain, causing 50% inhibition of the enzyme (IC50) *in vitro*. Columns: test = the experimental block that analyses were conducted in (a-e), AA11 = the amino acid engineered at site 111, AA122 = the amino acid engineered at site 122, IC50 µM = concentration causing 50% inhibition of the enzyme, Means and standard errors of each combination are also provided. Cells that are blank were not observed and can be considered "zero" values. ## How figures were generated Figure 1: Digital photographs arranged in PowerPoint. Figure 2: Generated with means and standard errors of previously published data as indicated in the figure legend: Y-axis data from Petschenka and Agrawal (2015), X axis data from Karageorgi et al. (2019); arrows and phylogeny hand-drawn in PowerPoint. Figure 3: Generated with Mesquite and edited in PowerPoint. Figure 4: Hand-drawn in PowerPoint. Figure 5: Generated with means of raw data in Excel file "sequestration raw HPLC data" based on the main datasheet and "total" column for each species (which are in separated cells labeled for each species), taking the average of the replicates and calculating the standard errors; additions to graphs added in PowerPoint. Figure S1: Bar graphs are the raw data (single replicates); arrows and phylogeny hand drawn in PowerPoint. UV spectra are outputted by HPLC ChemStation software. Figure S2: Bar graphs generated with means of raw data in Excel file "sequestration raw HPLC data" based on the main datasheet and "total" column for *Lerina incarnata* and *Asclepias linaria* leaves, taking the average of the replicates and calculating the standard errors; Figure S3: Digital photographs arranged in PowerPoint. Figure S4: Bar graphs generated with means of raw data in Excel file "Sequestration raw mass spec data" based on the main datasheet and yellow highlighted cells in rows 66-73.  Figures S5-S6: Digital photographs arranged in PowerPoint.