DOI: 10.5061/dryad.pzgmsbcs5
title: A nutrition-defense tradeoff drives diet choice in a toxic plant generalist

description: Plant toxicity shapes the dietary choices of herbivores. Especially when
 herbivores sequester plant toxins, they may experience a tradeoff between
 gaining protection from natural enemies and avoiding toxicity. The
 availability of toxins for sequestration may additionally trade off with
 the nutritional quality of a potential food source for sequestering
 herbivores. We hypothesized that diet mixing might allow a sequestering
 herbivore to balance nutrition and defense (via sequestration of plant
 toxins). Accordingly, here we address diet mixing and sequestration of
 large milkweed bugs (Oncopeltus fasciatus) when they have differential
 access to toxins (cardenolides) in their diet. In the absence of toxins
 from a preferred food (milkweed seeds), large milkweed bugs fed on
 nutritionally adequate non-toxic seeds, but supplemented their diet by
 feeding on nutritionally poor, but cardenolide-rich milkweed leaf and stem
 tissues. This dietary shift corresponded to reduced insect growth but
 facilitated sequestration of defensive toxins. Plant production of
 cardenolides was also substantially induced by bug feeding on leaf and
 stem tissues, perhaps benefitting this cardenolide-resistant herbivore.
 Thus, sequestration appears to drive diet mixing in this toxic plant
 generalist, even at the cost of feeding on nutritionally poor plant
 tissue.
description: <strong>Performance and sequestration of <em>O.
 fasciatus</em> on non-preferred food
 sources</strong> We examined performance and
 sequestration of seed bugs on the tropical milkweed (<em>Asclepias
 curassavica</em>), an important host plant in the southern end of
 the bug’s range. All bugs used in this experiment were from a colony (30
 adult bugs) of wild-collected <em>O. fasciatus</em> from
 Tompkins County, NY, USA. The colony had been reared for several
 generations on common milkweed (<em>Asclepias syriaca</em>)
 seeds. First instar bugs (from a pool of clutches laid by several females)
 were divided into five experimental treatment groups within twenty-four
 hours after hatching: bugs with access to milkweed seeds (nutritionally
 adequate, toxic), bugs with access to milkweed seeds and milkweed plant
 tissue (nutritionally adequate, toxic + nutritionally inadequate, toxic),
 bugs with access to sunflower seeds (nutritionally adequate, nontoxic),
 bugs with access to sunflower seeds and milkweed plant tissue
 (nutritionally adequate, nontoxic + nutritionally inadequate, toxic), and
 bugs with access to milkweed plant tissue with no seeds (nutritionally
 inadequate, toxic). These treatments were chosen to test all factorial
 combinations of nutritionally adequate toxic and nontoxic foods (both seed
 types), and nutritionally inadequate toxic foods (plant tissue) (Fig. 1).
 Plants were reared following the method outlined in Appendix I.
 We conducted ten replicates of each treatment, and each replicate
 received five bugs. All trials were conducted in 10 cm diameter pots
 filled with potting soil. After bugs were added, all pots were covered
 with semi-transparent mesh bags to prevent bugs from escaping. We also
 conducted a “no herbivory” treatment, which consisted of a milkweed plant
 that was bagged, but received no bugs or additional seeds (n=9; one plant
 was removed due to infestation with thrips). For seed treatments, 0.5 g of
 seed was presented on the soil surface and replenished with another 0.5 g
 of seeds once per week. This amount of seed was determined to be well in
 excess of the seed required for the growth and development of five
 <em>O. fasciatus</em> nymphs. Seeds that germinated over the
 course of the experiment were removed and replaced with fresh,
 ungerminated seeds. A. curassavica seeds were used for milkweed seed
 treatments and organic shelled sunflower seeds from a local supermarket
 were used as a cardenolide-free food source. All pots were watered
 approximately once every two days, regardless of the presence of a
 milkweed plant. The experiment consisted of 5 blocks,
 each consisting of two replicates of each experimental treatment (10 pots
 per block). Bugs were allowed to feed and develop to adulthood. Bugs were
 collected from pots within 24 hours of reaching adulthood, starved alone
 in a petri dish for 24 hours (with access to water), frozen at -80°C, and
 freeze-dried. n= 31 bugs (15 males and 16 females) survived to adulthood
 on milkweed seeds alone, n = 18 bugs (7 males and 11 females) survived to
 adulthood on sunflower seeds alone, n = 35 bugs (19 males and 16 females)
 survived to adulthood that had access to both milkweed seeds and plants,
 and n = 36 bugs (23 males and 13 females) survived to adulthood that had
 access to sunflower seeds and milkweed plants. These samples were used in
 analyses of development time to adulthood and adult dry mass, while a
 smaller subset was used for chemical analyses. The sex of adult bugs was
 noted, as <em>O. fasciatus</em> bugs are known to be sexually
 dimorphic in terms of adult dry mass and cardenolide sequestration (with
 females growing to larger sizes and sequestering higher concentrations of
 cardenolides) (Isman, 1977). For treatments in which
 bugs had access to a milkweed plant, all aboveground biomass was collected
 once all bugs had reached adulthood and the crown of the plant (the top
 six fully expanded leaves and all intervening stem) was freeze-dried for
 chemical analysis. The remaining aboveground biomass was dried for 3-5
 days at 45 oC in a drying-oven. All bugs with access to milkweed plants
 but without seeds died before reaching adulthood. For plants with no adult
 bugs (i.e., the “no herbivory” treatment, which received no bugs and the
 treatment where bugs had access to milkweed plants only), collection of
 the plants of a given treatment was timed with the collection of the first
 and last plants in the same block on which all bugs reached
 adulthood. <strong>Impacts of <em>O.
 fasciatus</em> feeding on milkweed performance and defensive
 chemistry </strong> Due to low bug survivorship
 when feeding on milkweed plant tissue alone, a separate experiment was
 conducted to assess the impacts of bug feeding on milkweed growth and
 defensive chemistry. Eighteen milkweed plants were placed in bags at the
 10-12 leaf stage as above and separated into three blocks of six plants
 each. Half the plants within each block received five first-instar
 milkweed bugs, and control plants received no bugs. One control and one
 herbivore-damaged plant was collected from each block at one, two, and
 three weeks after the start of treatments. As in the previous experiment,
 the crown of each plant was freeze-dried for chemical analysis, and the
 remaining aboveground biomass was dried in a drying oven. Height,
 aboveground dry mass, number of leaves damaged by bugs, and total number
 of leaves were recorded for all plants.
 <strong>Analysis of plant and insect
 cardenolides</strong> To analyze cardenolides, we
 used high performance liquid chromatography (HPLC) on freeze-dried leaves,
 adult bugs (n=10 per treatment, 5 male and 5 female; except for the
 treatment which only received milkweed seeds, for which 3 male and 3
 female bugs were run), and seeds (n=5 sunflower and A. curassavica).
 Cardenolides were extracted from samples and analyzed following the method
 established by Züst et al. (2019). For seeds and leaves, 50 mg (+/- 1 mg)
 of powdered sample was used (except for two small leaf samples with 35
 mg), while whole single adults were used for bug samples. To extract
 cardenolides, we ground each sample to a powder and added 1.6 mL of
 methanol, spiked with 20 μg of the cardenolide digitoxin (Sigma-Aldrich,
 St. Louis, MO), as an internal standard, along with approximately 30
 FastPrep beads. A separate vial (containing only methanol and digitoxin)
 was run to generate a standard curve. Samples were agitated twice for 45s
 at 6.5 m/s (FastPrep-24 homogenizer), centrifugated at 12,000 rpm for 15
 min, and solids were discarded. Bug and seed samples were defatted by
 adding 1.5 mL of hexanes and vortexing three times for 10s. The top
 (hexane) layer was removed and discarded. Supernatant was dried in a
 centrifugal concentrator at 35oC, resuspended in methanol (250 μL), and
 filtered with a 0.45 μm low-binding hydrophobic filter plate. Following
 the method established by Züst et al. (2019), 15 μL of each sample was
 injected into an Agilent 1100 series HPLC equipped with a diode-array
 detector and a Gemini-NX C18 reversed-phase column 3 μm, 150 mm × 4.6 mm
 (Phenomenex, Torrance, CA, USA). The injected sample was eluted at 0.7
 mL/min across an acetonitrile-water gradient (0-2 min 16% acetonitrile,
 2-25 min 16-70% acetonitrile, 25-30 min 70-95% acetonitrile, 30-35 min 95%
 acetonitrile, and a 10 min post-sample run with 16%
 acetonitrile). The commercially-available cardenolide
 digitoxin, which is not found in milkweed, was used as an internal
 standard. HPLC chromatogram peaks with a single absorption maximum between
 214 and 222 nm were considered to be cardenolides (Malcolm &
 Zalucki, 1996), and cardenolide concentrations were calculated from peak
 areas at 218 nm and were standardized by the concentration of the internal
 standard digitoxin in each sample and sample dry mass.
 <strong>Statistical analyses</strong>
 For each sample, the Shannon-Wiener diversity index (H’) was
 calculated by taking the natural logarithm of the proportion each
 cardenolide comprised of the total cardenolides in a sample and summing
 this across all cardenolides in the sample. Additionally, a polarity index
 was calculated by expressing each cardenolide as a proportion of the total
 cardenolides in a sample, weighting this by the cardenolide’s retention
 time, and summing this across all cardenolides in the sample (Jones et
 al., 2019). Higher polarity index corresponds to a more nonpolar
 assemblage of cardenolides. Statistics were performed in R version 4.1.2. 
 Total cardenolide concentration, polarity, diversity, and metrics of bug
 performance were compared among bugs using fixed effects ANOVAs (response
 variable ~ seed type*plant presence + bug sex). Bugs that only fed on
 sunflower seeds were excluded from polarity and diversity analyses, as
 these bugs contained only trace amounts of cardenolides. Plant growth and
 cardenolide diversity in the second experiment (examining plant induction
 over time) were also analyzed using ANOVAs (response variable ~ week*bug
 presence). Pairwise comparisons were performed using Student’s t-tests.
 The relationships between bug feeding and plant cardenolide concentration,
 polarity, and diversity were evaluated using Pearson
 correlations.
description: Raw data are attached as .csv files. Analyses were performed in R
 version 4.1.2.