DOI: 10.5061/dryad.wpzgmsbtv
title: Chemical profiling of milkweed and monarch butterfly wing extracts via mass spectrometry

description: Herbivores that sequester toxins are thought to have cracked the code of
 plant defenses. Nonetheless, coevolutionary theory predicts that
 plants should evolve toxic variants that also negatively impact
 specialists. We propose and test the selective sequestration hypothesis,
 that specialists preferentially sequester compounds that are less toxic to
 themselves, while maintaining toxicity to enemies. Using chemically
 distinct plants, we show that monarch butterflies sequester only a subset
 of cardenolides from milkweed leaves that are less potent against their
 target enzyme (Na+/K+-ATPase) compared to several dominant cardenolides
 from leaves. However, sequestered compounds remain highly potent against
 sensitive Na+/K+-ATPases found in most predators. We confirmed this
 differential toxicity with mixtures of purified cardenolides from leaves
 and butterflies. The genetic basis of monarch adaptation to sequestered
 cardenolides was also confirmed with transgenic Drosophila that were
 CRISPR-edited with the monarch’s Na+/K+-ATPase. Thus, the monarch’s
 selective sequestration appears to reduce self-harm while maintaining
 protection from enemies.
description: We also used high resolution mass spectroscopy to characterize
 and quantify the specific chemical composition of leaves and monarch wings
 focusing on the two primary host plant species (A. syriaca and A.
 curassavica) (n=3 replicates per tissue type per plant species). We follow
 the protocol described in Agrawal et al. (2022). Briefly we used
 reversed-phase chromatography in a Dionex 3000 LC coupled to an Orbitrap
 Q-Exactive mass spectrometer controlled by Xcalibur software (ThermoFisher
 Scientific). Methanolic extracts were separated on an Agilent Zorbax
 Eclipse XDB-C18 column (150 mm x 2.1 mm, particle siz1.8 µm) maintained at
 40 °C with a flow rate of 0.5 mL/min. Each sample was analyzed in positive
 electrospray ionization mode with m/z ranges 70-1000. MS2 spectra were
 obtained via Excalibur software (ThermoFisher Scientific). LC-MS data were
 analyzed using MZmine software (Pluskal et al. 2010). The acquired LC-MS
 data files were converted to mzXML files using the ProteoWizard MSconvert
 tool. LC-MS data was then pre-processed with the open-source MZmine 2
 software and consisted of peak detection, removal of isotopes, alignment,
 filtering, and peak filling. We mined the generated feature table to
 retrieve cardenolide ion adducts known to be present in A. syriaca and A.
 curassavica and confirmed their structure by comparing MS2 fragmentation
 spectra and retention time with pure isolated standards if available in
 our in-house library. The list of cardenolides can be found in Table S1
 and their corresponding chemical structures in Figure S2. The relative
 concentration (semi-quantification) based on ion counts for all
 cardenolides was determined using the calibration curve of
 aspecioside.
description: # Chemical profiling of milkweed and monarch butterfly wing extracts via
 mass spectrometry
 [https://doi.org/10.5061/dryad.wpzgmsbtv](https://doi.org/10.5061/dryad.wpzgmsbtv) We reared monarchs on four North American *Asclepias* spp. that represent diversity in cardenolide concentration, composition, and are used as host plants by the monarch butterfly: *A. syriaca* (low cardenolides, a main host), *A. curassavica* (higher cardenolides, a main host). Milkweed seeds were obtained from the following sources: *A. curassavica*, commercially purchased from Everwilde Farms, Fallbrook, California (USA), and *A. syriaca* collected from Dryden, Tompkins County, New York (USA). Plant growth and feeding trials were conducted separately for each plant species. Seeds were surface sterilized with 10% bleach, rinsed, and then nicked to encourage germination. Scarified seeds were sandwiched between layers of moist paper towels and housed in sealed petri dishes at 30C until germination (3-5 days). Seedlings were planted into 500 ml pots filled with soil and kept in a growth chamber with 400 microeinsteins of photosynthetically active radiation at 27C day /24 °C night with a 14h daylength. After plants had grown for at least one month, freshly hatched monarch caterpillars from a laboratory colony (reared from wild caught butterflies in NY, USA, and in the lab for less than 5 generations reared on the main host, *A. syriaca*), were added to plants (typically at low density, up to a few individuals per plant). Most plants had extensive damage; we sampled leaves for chemistry from at least five plants of each species (pooled), from young fully expanded undamaged leaves. Caterpillars were allowed to pupate and emerging adults were collected (both males and females). Milkweed leaf tissue and butterflies were frozen at -80 °C and then freeze-dried. Wings were clipped from butterfly bodies, and wing and leaf samples were ground to a fine powder, using a coffee grinder (leaf tissue), and 10 mL stainless steel grinding jars with 9 mm stainless steel balls on a Retsch Mixer Mill (MM300; wing tissue). For each leaf and wing sample type, 20-70 mg ground tissue was then extracted with methanol that had been spiked with hydrocortisone (25 mg/mL as an internal standard), and processed in a FastPrep-24 homogenizer (MP Biomedicals, Irvine, CA, USA). Samples were centrifuged at 14,000 rpm for 12 min to remove particulates and the supernatant was taken to dryness in a rotary evaporator (Labconco CentriVap). Wing extracts were defatted twice by dissolving residues in 250 mL methanol, adding 750 mL hexane, vortexing 3 times for 30 sec, centrifuging for 10 min at 13,200 rpm and pipetting off the hexane layer. Defatted samples were then taken to dryness and brought back, along with leaf samples, in 300 mL methanol. These samples were filtered (0.2 um Millipore syringe filter) and 200 mL was set aside for HPLC analysis. We used high resolution mass spectroscopy to characterize and quantify the specific chemical composition of leaves and monarch wings focusing on the two primary host plant species (A. syriaca and A. curassavica) (n=3 replicates per tissue type per plant species). We follow the protocol described in Agrawal et al. (2022) (https://doi.org/10.1073/pnas.2205073119). Briefly we used reversed-phase chromatography in a Dionex 3000 LC coupled to an Orbitrap Q-Exactive mass spectrometer controlled by Xcalibur software (ThermoFisher Scientific). Methanolic extracts were separated on an Agilent Zorbax Eclipse XDB-C18 column (150 mm x 2.1 mm, particle siz1.8 µm) maintained at 40 °C with a flow rate of 0.5 mL/min. Each sample was analyzed in positive electrospray ionization mode with m/z ranges 70-1000. MS2 spectra were obtained via Excalibur software (ThermoFisher Scientific). The acquired LC-MS data files were converted to mzXMLfiles using theProteoWizard MSconvert tool. This data contains raw MS2 data files in mzXML format. ## Description of the data and file structure There are three replicates per sample type and these are named 1, 2, and 3. MS2 data files of A. syriaca leaf Asclepias\_syriaca-1.mzXML Asclepias\_syriaca-2.mzXML Asclepias\_syriaca-3.mzXML MS2 data files of A. curassavica leaf Asclepias\_curassavica-1.mzXML Asclepias\_curassavica-2.mzXML Asclepias\_curassavica-3.mzXML MS2 data files of monarch wings fed on A. syriaca leaf Monarch\_wing\_on\_Asclepias\_syriaca-1.mzXML Monarch\_wing\_on\_Asclepias\_syriaca-2.mzXML Monarch\_wing\_on\_Asclepias\_syriaca-3.mzXML MS2 data files of monarch wings fed on A. curassavica leaf Monarch\_wing\_on\_Asclepias\_curassavica-1.mzXML Monarch\_wing\_on\_Asclepias\_curassavica-2.mzXML Monarch\_wing\_on\_Asclepias\_curassavica-3.mzXML