Appendix A. Methods for assessing cardiac glycosides, latex, and leaf carbon and nitrogen content.
Cardiac glycoside concentrations were measured as digitoxin equivalents per 50 mg dry leaf tissue; I employed a spectrophotometric assay modified from Nelson (1993). The assay was adapted for rapid high throughput sampling using a microplate reader (PowerWave X, Bio-Tek Instruments, Winooski, Vermont, USA). Briefly, the leaf that was collected was kept on ice, frozen, and freeze-dried. Although latex exuded from the wound site on the stem, most of the latex contained within the leaf remained in the leaf, thus preserving the cardenolides in the latex for the assay. Leaf material was then ground in a Wiley mill, and weighed in 2 mL boil-proof tubes (MCT 200-C, AXYGEN Scientific, Union City, California, USA). 1.9 mL of 95% ethanol was added to each tube, they were each vortexed, and the tubes were floated in a sonicating water bath (65 ºC) for 10 minutes. Tubes were then centrifuged at 5000 rpm for 5 minutes at room temperature. Two 45 mL aliquots of the supernatant from each tube were then pipetted into wells of a 96 well-plate, one above the other (active sample and blank). Each plate also contained six samples for a standard curve generated using digitoxin (Sigma Chemical Co., 0.125 mg/mL to 3 mg/mL). I then added 90 mL of ethanol to the blanks and 90 mL of 0.15% 2,2'4,4'-tetranitrodiphenyl (TNDP) in ethanol to the active samples. Finally, 70 mL of 0.1 M aqueous NaOH was added to all wells to make the solutions basic and catalyze the colorimetric reaction. After 18 minutes, all wells in the plate were read at 620 nm on the microplate reader.
I measured latex by cutting the tip (0.5 cm) off of the intact leaf in the field and collecting the exuding latex onto a 1cm disc of filter paper. Latex stopped flowing after ca.10 seconds, all latex was absorbed on the filter paper, and this disc was placed on top of another dry filter paper disc in a 24 well plate. The discs were dried at 60 °C and then weighed to the microgram. This is a repeatable method for determining latex exudation; I have found positive phenotypic and genetic correlations of latex exudation across two years when plants were measured in a neighboring common garden (AAA, unpublished data). In addition, this method of assessing latex production likely reflects what feeding insects must contend with, and has been shown to negatively correlate with the growth of other milkweed herbivores (AAA, unpublished data, Van Zandt and Agrawal 2003). Total leaf nitrogen and carbon concentration were measured by microcombustion, using 5 mg of dried ground leaf material in an Elemental Combustion System 4010, CHNS-O analyzer (Costech Analytical Technologies, Valencia, California, USA).
Nelson, C. J. 1993. Sequestration and storage of cardenolides and cardenolide glycosides by Danaus plexippus and D. chrysippus petila when reared on Asclepias fruticosa: With a review of some factors that influence sequestration. Pages 95105 in S. B. Malcolm and M. P. Zalucki, editors. Biology and conservation of the monarch butterfly. Natural History Museum of Los Angeles County, Los Angeles, California, USA.
Van Zandt, P. A., and A. A. Agrawal. 2004. Specificity of induced plant responses to specialist herbivores of the common milkweed, Asclepias syriaca. Oikos 104:401409.
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