Monarchs in the Classroom

Beth Lavoie
University Scientist
University of Minnesota
St. Paul MN

Overview of Lavoie's Research | Research Projects

Abstract

I characterized common milkweed (Asclepias syriaca) leaf nitrogen content and condition in unfertilized nonagricultural and fertilized agricultural habitats. Old field milkweed ramets varied such that top and new leaves, leaves from non-senescing plants, leaves from flowering plants, and leaves from plants with high aphid and stem herbivory damage had higher leaf nitrogen content than bottom leaves, leaves from senescing plants, leaves from non-flowering plants, and leaves from plants with high leaf herbivory damage, respectively. When pooled by type of habitat, ramets in the three non-Bt cornfields had significantly higher leaf nitrogen content than those in the four roadsides. These results raise questions about how milkweed leaf nitrogen in the wild affects monarch larvae

While monarch butterflies in the Midwestern US originate on milkweed that grows in a variety of habitats, most of these monarchs come from agricultural habitats (Oberhauser et al. 2001). Monarch per plant densities in agricultural fields are as high as or higher than densities in nonagricultural habitats, and even though milkweed densities are higher in nonagricultural than in agricultural habitats, more monarchs originate in agricultural habitats because agricultural fields constitute such a large portion of the landscape (Oberhauser et al. 2001).

In these different habitats, monarchs probably experience varying levels of nitrogen. Fertilizing plants with nitrogen tends to increase total leaf nitrogen content (Mattson 1980), and leaf nitrogen content tends to decrease over the growing season (Mattson 1980, Slansky 1993). Young, new shoots contain the most nitrogen, followed by leaves from plants with flowers and seedpods, and then by leaves from old, senescing plants.

Plant protein yield and leaf nitrogen content usually increase when plants are damaged by pathogens or herbivores because damage stimulates compensatory plant growth and the saliva of some herbivores contains chemicals that promote plant growth (Mattson 1980, Hamilton et al. 1998). While terrestrial forbs generally contain 1.6-5.0% leaf nitrogen content by dry weight (dw) (Mattson 1980), the variation in leaf nitrogen content for common milkweed between and within plants in fertilized agricultural and unfertilized nonagricultural habitats has not been determined.

Nitrogen is important for caterpillar growth (Scriber 1984, Slansky and Scriber 1985), and insects are usually considered to be nitrogen limited (McNeil and Southwood 1978, Mattson 1980, White 1993). While animal tissue generally consists of 7-14% nitrogen (dw), plants consist of 0.03-7.0% nitrogen (dw) (Mattson 1980), and herbivores must therefore consume large quantities of their host plants in order to accumulate enough nitrogen for growth and development.

The monarch butterfly obtains its nitrogen requirements for growth, development, and reproduction from plants in the family Asclepiadaceae (milkweeds). Milkweeds defend themselves from herbivores with a secretory canal system made of specialized plant cells (laticifers) that run throughout each plant and contain latex (Dussourd 1993). Latex provides both mechanical and chemical protection to milkweeds. It is a sticky plant secretion released upon injury that contains cardenolides, or cardiac glycosides, heart poisons with potent effects on vertebrates (Dussourd 1993). Zalucki and Brower (1992) found that milkweed latex glued some first instar monarch larvae to the plant while the cardenolides in the latex and the leaf tissue often immobilized them. Monarch larvae combat milkweed defenses through feeding behaviors such as petiole cutting and leaf trenching to slow the flow of latex (Zalucki and Brower 1992, Malcolm et al. 1999), and metabolic processes such as sequestering the cardenolides in their exoskeletons (Malcolm et al. 1999).

In this study I characterized milkweed leaf nitrogen content within and between common milkweed (Ascelpias syriaca) ramets in an old field and characterized milkweed leaf nitrogen content and condition in unfertilized nonagricultural and fertilized agricultural habitats. This study addresses two questions. 1) How does leaf nitrogen content vary within and between milkweed plants in a Wisconsin old field? 2) How does leaf nitrogen content vary between plants in fertilized agricultural and unfertilized nonagricultural habitats in Hennepin County, Minnesota?

Methods

In 2001, I determined the natural variability in leaf nitrogen content within and between common milkweed ramets (aboveground stems) in a west central Wisconsin nonagricultural area. The site contained over 1000 milkweed ramets and is an old field last used for agriculture approximately 20 years ago and burned 2 years before the study to promote native plant growth. On two different dates, I collected milkweed leaf samples from ramets along randomly selected transects; ramets needed to be at least 2 m apart to be included in the sample.

I collected samples on 26 July to characterize the leaf nitrogen levels within ramets (top, bottom or new leaves) and between ramets with varying reproductive status and varying types and levels of damage from herbivory. For each sample ramet, I identified whether or not it was flowering, its level of stem and leaf damage from herbivory, and the number of aphids on the ramet. Holes in the stem indicated stem damage from herbivory (probably from the milkweed weevil, Rhyssomatus lineaticollis), and the length of the stem with holes divided by the entire length of the stem provided an estimate of the

percentage of stem damage from herbivory (low: <6% or high: 6-25%). Holes or missing portions of leaves divided by total leaf area on the plant provided a visual estimate of leaf damage from herbivory (low: <6% or high: 6-25%). I did not record higher percentages of damage from herbivory because they did not occur. I estimated aphid herbivory using the number of aphids on the ramet (low: <100 aphids or high: >100 aphids).

After categorizing the ramet, I randomly selected a new, top, or bottom leaf from the ramet. To be included in the sample, ramets needed to have high levels of damage from one type of herbivory and low levels from the other types. This process may have forced biological interactions between the three types of herbivory. I repeated this process using many random transects through the old field until I obtained samples from up to four ramets for each combination of variables shown in Table 1a.

On 12 September I collected samples from the same old field to characterize leaf nitrogen levels in senescing and not senescing ramets with varying reproductive status. For each sample ramet, I identified whether or not it had reproductive seedpods and whether or not it was senescing. I defined senescence using leaf yellowing. On senescing ramets greater than 40% of the leaves were 100% yellow; ramets that were not senescing had less than 10% yellowing on all leaves. I collected the topmost mature leaf on each sample ramet. I repeated this process using many random transects until I obtained samples from six ramets for each combination of variables shown in Table 1b.

Table 1a. Milkweed leaves collected by herbivory type and level, leaf location, and flowering status. X's indicate combinations of variables for which samples were collected. No samples were collected for the combinations of variables in the shaded cells, because I did not find these in the field.

Table 1b. Milkweed leaves collected from senescing and not senescing ramets with and without seedpods. X's indicate combinations of variables for which samples were collected.

	Senescing	Not senescing
Plant with seedpods	X	X
Plant without seedpods	X	X

I determined how leaf nitrogen content varied between common milkweed (Asclepias syriaca) plants in fertilized agricultural and unfertilized nonagricultural habitats. The sites included three cornfields and four nonagricultural roadside areas. I selected cornfields and roadsides in Hennepin County, Minnesota within 15 km of each other to minimize differences in climate, parent material, and relief at the sites. I minimized the effect of fertilizer runoff for roadside sites by selecting sites separated from nearby agricultural fields and residential lawns by roads or parking lots. The roadside sites represented different types of roadside habitats where common milkweed grows in the Minneapolis-St. Paul area, including mowed and unmowed sites with 100 to over 1000 milkweed ramets (aboveground stems) along wetlands and woods, near interstate ramps, and in highway medians. At the cornfield sites, I used milkweed ramets along the field edge (within 0.5 m of the outer row of corn). The farmers fertilized the three cornfields before planting the corn and sprayed herbicide targeting dicot plants once during the last week of June. At each site, I collected the topmost mature milkweed leaf from 10 ramets along randomly selected transects; ramets needed to be at least 2 m apart to be included in the sample. I collected all leaf samples on 28-29 July.

I determined leaf nitrogen content (% dw) of milkweed leaf samples harvested from the old field, cornfield, and roadside sites. As I collected leaf samples, I stored them in paper envelopes in a cooler with an ice pack for up to 8 hours. Upon returning to the lab, I immediately dried the leaves at 65°C for 48 hours in a drying oven. Then I milled the leaves using a Wiley mill with a 20 mesh screen and determined the nitrogen content using a Perkin Elmer Series II CHNS/O Analyzer 2400.

I analyzed the leaf nitrogen content of milkweed plants in the old field, cornfield and roadside sites using ANOVA. To compare the leaf nitrogen content of senescing and not senescing milkweed ramets with and without pods, I used two-way ANOVA. I used one-way ANOVA to compare leaf nitrogen content of top, mature leaves among plants with different types of damage due to herbivory and used Tukey's HSD to make all possible pairwise comparisons. To compare the leaf nitrogen content of leaves from different locations on the ramets, with different types of herbivory, and with and without flowers, I used three-way ANOVA and Tukey's HSD for all possible pairwise comparisons. I pooled data from the top leaves of the three cornfields and four roadsides, respectively, and used an independent samples t-test to compare the leaf nitrogen content of cornfields and roadsides because these data were independent observations, were normally distributed (Kolmogorov-Smirnov with a Lilliefors significance correction and Shapiro-Wilk tests) and had homogeneous variances (Levene test).

Results

A. syriaca ramets and leaves varied in leaf nitrogen content. Senescing ramets in the old field had significantly lower leaf nitrogen content than non-senescing ramets, and there was a significant interaction between senescence and presence of pods such that senescing plants with no pods had the lowest nitrogen levels (Table 3 and Figure 1a).

For top, mature leaves of old field ramets without flowers, ramets with aphid herbivory and stem damage from herbivory had significantly higher leaf nitrogen content than ramets with leaf damage from herbivory (Fig 1b). Old field ramets with flowers had marginally higher leaf nitrogen content than ramets without flowers, and top and new leaves had significantly higher leaf nitrogen content than bottom leaves (Tables 3a & b and Figure 2).

Table 3a. Old field A. syriaca leaf nitrogen content (% dry weight) group means for top, new, and bottom leaves on ramets with differing reproductive status (flowering or not) and levels of damage from herbivory on July 26, 2001. Herbivory categories were low damage from herbivory (< 6% stem and leaf damage from herbivory and < 100 aphids) and high stem damage from herbivory (6-25%) with < 100 aphids and < 6% leaf damage from herbivory. Analysis with ANOVA (Table 3b). Means followed by the same letter are not significantly different at the 0.05 level of confidence (Tukey HSD comparisons).

Table 3b. Three-way ANOVA for A. syriaca leaf nitrogen content in top, new, and bottom leaves of ramets with and without flowers and with low and high stem herbivory.

When pooled by type of habitat, ramets in the cornfields had significantly higher leaf nitrogen content than those in the roadsides (Figure 3).

Discussion

The variation in leaf nitrogen for milkweed plants in the field approximates leaf nitrogen variation in other herbaceous plants. Forb leaves tend to range from 1.6% to 5.0% nitrogen (dw) with top leaves containing more nitrogen than bottom leaves (Mattson 1980). Old field milkweed leaves ranged from 1.14 ± 0.36% to 3.36 ± 0.33% nitrogen (dw), and top and new leaves had significantly higher leaf nitrogen content than bottom leaves. Leaf nitrogen content of leaves also increases with fertilization (Mattson 1980), and fertilized cornfields, pooled, had milkweed with higher leaf nitrogen content than unfertilized roadsides.

Leaf nitrogen content tends to decrease over the growing season (Slansky 1993, Mattson 1980). As plants invest in reproduction, they allocate more nitrogen to the reproductive structures and less to their leaves (Mattson 1980). Young, actively growing tissues require higher nitrogen concentrations to support the rapid protein synthesis needed for growth, while senescing plants do not need to synthesize proteins (Mattson 1980). Old field milkweed leaves partially followed this pattern; senescing ramets had significantly lower leaf nitrogen content than ramets not senescing. Reproductive ramets (pods or flowers) did not have lower leaf nitrogen content than non-reproductive ramets, possibly because comparisons were made between ramets at one time point instead of within the same ramets over the growing season. Perhaps only those ramets with high enough nitrogen levels develop reproductive structures.

Actual and simulated herbivory tend to increase the protein yield (Mattson 1980) and leaf nitrogen content of plants (Hamilton et al. 1998), but sometimes plants concentrate this additional nitrogen in the damaged tissue (de Nooij et al. 1992). Old field milkweed leaves on plants with higher levels of herbivory contained similar leaf nitrogen content to those with low levels of herbivory, possibly because they concentrated any additional nitrogen in the damaged tissues and not in the leaves I sampled.

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