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.
Notes
Funding provided by: National Science Foundation
Crossref Funder Registry ID: https://ror.org/021nxhr62
Award Number: 2209762
Methods
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.
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