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Food preferences of Danaus plexippus larvae

Roxanne Kuerschner, Jessie Peterson, Nash Pherson, Nicole Schliemann, Robert Shoemaker, and Dan Wunderlich
St. Peter High School
St. Peter, MN 56082

St Peter team

from left to right: Roxanne (teacher), Dan, Jessie, Nash, Shoe (teacher), Nicole

Abstract | Introduction | Hypotheses | Methods | Results | Discussion | Research Projects

Abstract

We tested the food preference of the Monarch butterfly (Danaus plexippus) on the two primary species of milkweed in Minnesota: swamp milkweed (Asclepias incarnata) and common milkweed (Asclepias syriaca). Individual leaves were cut into 100 mm2 pieces. One square of each variety of milkweed was placed in a container at a distance of 5 centimeters (cm) from each other. An individual third instar larva was entered into each chamber facing in a neutral position. The caterpillars in each treatment were allowed to feed undisturbed for a period of one hour and then removed. We then measured the percent of eaten leaf material by placing the partially eaten leaf on millimeter-sized graph paper. The treatments tested the larval preference for old leaves versus new leaves in each of the two varieties of milkweed, as well as their preference for common versus swamp milkweed. The data suggest that young leaf material is preferred over old in each of the two varieties, and common milkweed is preferred over swamp milkweed.

Introduction

In our experiment, the objective was to determine if Danaus plexippus larvae have a preference in food given the choices of common and swamp milkweed. Our experimental set-up consisted of three groups each with twenty third instar larvae. Each larva had an individual container in which there were two pieces of milkweed. The condition and species of the milkweed varied in the three groups. The larvae were given the choices of common milkweed (Asclepias syriaca) versus swamp milkweed (Asclepias incarnata), young common milkweed versus old common milkweed, and young swamp milkweed versus old swamp milkweed.

Hypotheses

  • Ho= There will be no difference in preference for milkweed age or species.
  • HA1= Larvae will prefer young plants to old plants.
  • HA2= Larvae will prefer common milkweed to swamp milkweed.

Methods

For the research project, our team designed a food preference test. In the experiment, we tested which type of milkweed the larvae preferred, either common or swamp milkweed. In addition, we tested how plant age affected preference. We collected all leaves on the day of the experiment from wild plants. "Old plants" were partially yellowed, and were beginning to senesce. "Young plants" had green leaves and appeared to be in better condition than old plants.

For the experiment, we placed sixty third instar larvae into individual plastic cups with two food choices. There were a total of three groups with twenty larvae in each group. One group was given a choice of young and old common milkweed. Another was given young and old swamp milkweed, and the last group was given common and swamp milkweed. To make the milkweed samples, we used a 100 mm2 paper punch to obtain samples of equal area. We handled the samples with latex gloves to avoid biases due to handling. The food choices were pinned with insect pins on both sides of the larva, five centimeters from each other. The larva was initially placed in the plastic cup facing away from both leaf samples to remove any bias.

Preparing leaves

Jessie and Nicole preparing leaves

Punching out leaf squares

Jessie punching out leaf squares

To determine their preference, we gave the larva one hour to eat. We then collected two types of data. We observed which of the food choices was first eaten by the larva. To do this we would simply watch the cup and mark down which type of plant was the first to be eaten. We then covered up the container with a petri dish lid for the remainder of the sixty minutes and let the larva eat undisturbed. At the end of this period, we measured the area of each leaf that had been consumed.

Cups with leaf squares

Plastic cups with leaf squares pinned in them

Third instars

Third instar larva ready to be tested

Results

We defined preference as the plant that each individual larva consumed the most of. In most cases, the larva only ate from one plant type. We excluded larvae that didn’t eat anything from the analysis. For each two way comparison we answered three questions:

  1. Did the larvae eat more of (prefer) the plant that they first contacted?
  2. Which plant did they prefer?
  3. Once the preferred plant had been chosen, how much of it was eaten?

We used contingency tables and Chi2 tests to determine the significance of fidelity to the first plant contacted and preference, and t-tests to determine the significance of differences in the area of leaf material consumed.

Young Common versus Old Common

  1. Ten larvae initially contacted old common (OC) milkweed, and 90% of these stayed on that plant. Ten larvae initially contacted young common (YC) milkweed, and of these 100% stayed on that plant. This difference was not significant. (Fig. 1 YC vs. OC, Chi-square=1.06, p > 0.05)
  2. Eleven larvae preferred young common where only nine preferred old common milkweed. This difference was not significant. (Fig. 2 YC vs. OC, Chi-square=0.2, p > 0.05)
  3. Larva consumed a greater area of young common than old common milkweed. The results of this test were significant. (Fig. 3 YC vs. OC, t=7.2, p < 0.005)

Figure 1

Figure 1. The proportion of the larvae contacting each plant type first that showed a preference for that type. Sample sizes are shown on the bars. Only larvae that ended up showing a preference are shown.

Young Swamp versus Old Swamp

  1. Six larvae initially contacted young swamp (YS) milkweed and also showed a preference (two additional larvae contacted young swamp milkweed, but then did not eat any leaf material). Of these six, 83% stayed on that plant. Seven larvae initially contacted old swamp (OS) milkweed (7), and 29% of these stayed on that plant. This difference was not significant. (Fig. 1 YS vs. OS, Chi-square=4.03, p > 0.05)
  2. Ten larvae preferred young swamp where only 3 preferred old swamp milkweed. This difference was significant. (Fig. 2 YS vs. OS, Chi-square=3.8, p < 0.05)
  3. Larvae consumed a greater area, on average, of young swamp than old swamp milkweed. The results of this test were significant. (Fig. 3 YS vs. OS, t=5.0, p < 0.005)

Seven of the twenty larvae tested in this group did not consume any milkweed, and five of these did not make any contact with either leaf.

Figure 2

Figure 2. The proportion of larvae that preferred (ate more of) each type of plant after a 60 minute test period.

Young Swamp versus Young Common

  1. Seven larvae initially contacted young swamp milkweed and also showed a preference (one additional larva contacted young swamp milkweed, but then did not eat any leaf material). 43% of these seven stayed on that plant. Seven larvae initially contacted young common milkweed and also showed a preference (three additional larvae contacted young common milkweed, but then did not eat any leaf material). Of these, 100% stayed on that plant. This difference was significant. (Fig. 1 YC vs. YS, Chi-square=8, p < 0.05)
  2. Eleven larvae preferred young common where only three preferred young swamp milkweed. This difference was significant. (Fig. 2 YC vs. YS, Chi-square=4, p < 0.05)
  3. Larva ate more young common than swamp milkweed, on average. The results of this test were significant. (Fig. 3 YC vs. YS, t=7.24, p < 0.005)

Six of the larvae in this test group did not consume any milkweed, and two of these did not make any contact with either leaf.

Figure 3

Figure 3. The mean number of mm2 of each plant type consumed in 60 minutes.

Discussion

In the common versus swamp milkweed tests, we found that the common milkweed was preferred. This was not simply because they were more likely to contact common milkweed first. Seven larvae contacted each leaf type first, but while all seven stayed on the common milkweed, four of those that initially contacted swamp milkweed ended up preferring (eating more) common milkweed. Not only did the larvae prefer common to swamp milkweed, but those that preferred common milkweed consumed a larger area than those that preferred swamp milkweed, on average. Thus we can reject the null hypothesis with respect to milkweed species.

When given the choice of an old or young leaf of the same species, we did not see significant differences in fidelity to either age. 83% of those that contacted young swamp milkweed ended up preferring this leaf, while only 29% of those that first contact old swamp milkweed preferred the old leaf, but this difference was not significant at the 95% confidence level. Values for young and old common milkweed were 100% and 90%, respectively. There was also no difference in preference for young vs. old common, but larvae preferred young to old swamp milkweed. After making their choices, larvae tended to eat a larger area of the younger leaves of both species. We cannot reject the null hypothesis with respect to age in the case of common milkweed, even though the larvae tended to eat a larger area of young leaves. We can, however, reject the null hypothesis with respect to age of swamp milkweed plants; larvae tended to prefer young swamp milkweed plant to old.

It does not appear that larval preference is determined simply by being able to sense one plant more easily than the other one. In all tests, there were no differences in the proportion of larvae that first contacted either plant.

Roxanne

Roxanne after being asked if she’d
prefer old or young swamp milkweed

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