An Aphis nerii colony was established from a single individual captured near Ann Arbor, Michigan, and maintained on Asclepias tuberosa in butterfly nets at the University of Michigan Biological Station (Pellston, Michigan).
Both locally collected and commercially obtained milkweed seeds were germinated and grown in a glasshouse. In late June, when plants were 71 old, one individual of each species was placed in one of 40 open-top controlled atmosphere chambers at UMBS, half of which were kept at ambient (400.753 ppm) or elevated (739.01 ppm, dawn until dusk) atmospheric CO2 concentrations. CO2 concentrations in chambers were monitored using a LI-COR LI-6262 IRGA (LI-COR, Lincoln, NE, USA) and were adjusted multiple times daily.
After 4 weeks under either elevated or ambient CO2, physical and chemical traits of each plant were measured. Plant height, leaf number, and stem diameter were recorded before and after growth in the chamber array to determine change in plant biomass. To sample plant chemistry, one leaf was harvested from each plant; six leaf punches were taken using a paper hole punch from one side of the leaf and were stored in cold methanol (1 mL). Extracted leaf disks were kept at -10° C for cardenolide analysis. Six corresponding leaf disks from the same leaf were punched, weighed (wet weight), dried, and re-weighed (dry weight). Percent foliar water content was calculated by taking the difference between wet and dry weight and dividing by wet weight. In a subset of 48 plants, the opposite leaf was frozen in liquid nitrogen and stored at -80° C for amino acid analysis. The remainder of both leaves were collected, dried, and ground into a powder to determine C:N ratios and foliar phosphorus. To investigate plant mechanical defenses, average trichome density for each plant was determined by counting the number of trichomes on each dried leaf disk using a dissecting microscope (4x magnification). Latex exudation was quantified by collecting exuded latex from 6 punched holes on preweighed, dried, 1 cm cellulose disks, which were then dried and re-weighed.
Following chemistry sampling, one reproductive aphid was placed on each plant and secured with a net (approximately 45 cm tall) made of fine mesh. After 24 hours, all but 10 aphids on each plant were removed. Aphids were allowed to reproduce undisturbed for 14 days. Population growth rate was measured by counting total number of aphids per plant every 3 days (4 temporal blocks of 20 plants). After 2 weeks, aphids were removed from their associated plant and the numbers of alate and apterous individuals were counted. Each plant’s aphid population was stored in cold methanol at -10° C for subsequent chemical analysis.
Chemical Analysis
Foliar cardenolides were analyzed as described in Vannette and Hunter (2011); a ball mill was used to grind leaf disks or aphids suspended in methanol, followed by sonication at 60° C for 1 h. Samples were evaporated to dryness and resuspended in 150 μL methanol containing 0.15 mg/mL of digitoxin as an internal standard. Samples were then analyzed using reverse phase high performance liquid chromatography (UPLC, Waters Inc., Milford, MA, USA) with a 9-minute water: acetonitrile gradient. Peak detection was conducted using a diode ray detector recording absorbance from 200 to 300 nm. Symmetric peaks with absorption maxima between 216 and 222 were accepted as cardenolides. Total cardenolides were calculated through the summation of each individual cardenolide peak and corrected by the amount of internal standard, digitoxin. Foliar carbon and nitrogen ratios were determined using a CHN analyzer (Costech, Valencia, CA, USA).
A colorimetric assay was utilized to determine total foliar phosphorus. Dried, ground tissue samples (0.05 g) were refluxed at 90° C with concentrated sulfuric acid (0.5 mL) and concentrated nitric acid (2.0 mL). The heat was raised to 120°C and allowed to reflux for 30 minutes. The nitric acid was driven off by raising the heat to 150-170° C. The resultant solution was allowed to cool to room temperature and pipetted into a volumetric flask (100 mL) with de-ionized water (30 mL). To bring the diluted solution to pH 1.5, 6 N NaOH was added (1.5 mL). Absorption of resultant solution was determined using an autoanalyzer.
Foliar amino acid concentration was determined utilizing a colorimetric ninhydrin assay. Lyophilized, homogenized leaf sample was extracted in DMSO (1 mL) and water (1 mL). Extract was sonicated on ice, mixed with ninhydrin reagent, and incubated at 100° C for 15 minutes. After cooling to room temperature, ethanol was added (10 mL, 50%) and samples were centrifuged. The absorbance of the supernatant was measured at 570 nm using a spectrometer.