Research Project

Effect of Asclepias syriaca herbivory on development rates and fitness in Danaus plexippus

Mary Beth Carpenter, Autumn White, Gary Smith, Anna Carpenter
Heart of the Earth Center for American Indian Education
Minneapolis, MN

Abstract

We tested whether Danaus plexippus larvae and pupae matured faster on Asclepias syriaca milkweed with herbivory or without herbivory. We found no difference in either the rate of development, adult mass or wing length between those raised on herbivorized and those raised on non-herbivorized milkweed. However, results may be inconclusive because of high loss of larvae from one treatment.

Introduction

In our experiment we wanted to determine whether the maturation rate of monarch butterflies (Danaus plexippus) varied depending on whether the larvae ate common milkweed (Asclepias syriaca) with herbivory or not. We suspected that ovipositing females might choose one plant over another based on the degree of herbivory of the plants. We knew that latex flows differently in herbivorized milkweed. We suspected that perhaps larvae raised on herbivorized milkweed might mature more quickly, giving them an advantage in survival, since larvae are so vulnerable during this stage of their life cycle.

We were also curious about the size of monarch adults raised on herbivorized versus non-herbivorized common milkweed. The wing length and weight of the adults is a measure of fitness and we thought a bigger butterfly might be a better butterfly, with a greater chance of withstanding a flight to Mexico or of successfully mating and laying eggs.

Questions

Is there a difference in the development rate of monarch (Danaus plexippus) larvae raised on common milkweed plants (Asclepias syriaca) with herbivory or without herbivory?
Is there a difference in the mass and wing size between adult monarchs raised on common milkweed with herbivory and those raised on common milkweed without herbivory?

Hypotheses

Question 1:

H₀=There is no difference in the maturation rate of larvae (Danaus plexippus) raised on common milkweed (Asclepias syriaca) with herbivory versus those raised on common milkweed without herbivory.
H_A1=Larvae raised on common milkweed with herbivory will mature faster than those raised on common milkweed without herbivory.
H_A2=Larvae raised on common milkweed without herbivory will mature faster than those raised on common milkweed with herbivory.

Question 2:

H₀=There is no difference in the mass and wing size between adult monarchs raised on common milkweed with or without herbivory.
H_A1=Adult monarchs raised on common milkweed with herbivory will have greater mass and greater wing size than those raised on common milkweed without herbivory.
H_A2=Adult monarchs raised on common milkweed without herbivory will have greater mass and greater wing size than those raised on common milkweed with herbivory.

Methods

Materials

2 small cages (2’x2’) to hold 4 potted common milkweed plants for each treatment
1 large walk-in cage to hold 60 potted milkweed plants
30 monarch (Danaus plexippus) mature eggs
8 third and fourth instars of Danaus plexippus
60 common milkweed plants (Asclepias syriaca)
Small paint brush from child’s watercolor set to transfer larvae

Procedure

To test for the effect of herbivory on monarch development, we set up our experiment with two treatments: larvae reared on plants with recent herbivory and larvae reared on plants with no herbivory (see Picture 1). In each treatment, a 0.75 x 0.75 x 0.75 meter cage was set up to house 3 potted plants and 15 monarchs, starting as eggs. Since not all eggs hatched on the same day, we placed the first 15 instars in the treatment without herbivory and the remaining 15 instars in the cage with herbivory. Cages were placed outdoors in an area shaded between 11:00 am and 2:00 pm. Larvae were fed fresh milkweed daily and kept in these cages until adult emergence.

Picture 1. Small cages, one containing plants with herbivory and one containing plants without herbivory.

Picture 2. Large walk-in cage for all milkweed plants.

Since the effect of herbivory on a plant may vary depending on the age and size of the herbivore, we needed to establish a method for generating a consistant amount of damage to our plants. To generate a relatively consistent degree of herbivory, we allowed separate third and fourth instar larvae (not used in either treatment) to feed on potted milkweed (Asclepias syriaca) plants for one day and then used these plants in the herbivory treatment. We kept other potted milkweed plants, intended for the non-herbivory treatment, in a large screened tent to eliminate unintended herbivory (see Picture 2).

Once larvae in each treatment were of sufficient size to cause noticeable herbivory, we used these larvae as our "herbivores," reusing the potted plants from the non-herbivory treatment a second day in the herbivory treatment. The larvae were removed from the potted plants in the non-herbivory treatment and the plants were transferred to the herbivory treatment. This was done to reduce the total number of plants used in this experiment. In all cases, plants placed in the herbivory treatment were subjected to only one day of herbivory prior to placement in the treatment.

To minimize an effects of handling, we transferred larvae from old plants to new plants using a small paintbrush, attempting to place the larvae in about the same place on the new plant as it was on the old. We transferred larvae inside each cage since larvae often roll off the plants when disturbed and are easily lost. We recorded obvious changes in larval development; however, we did not measure instar length as this often decreases after each molt.

To determine the effect of herbivory on adult fitness, we measured adult mass, right and left forewing length, and presence of disease. We took 1 day old adults to the University of Minnesota monarch lab to weigh, measure and spore-check (checking for the presence of disease) and then release them.

Results

We lost a significant number of larvae in our experiment due to extreme weather, parasitism and escape. Because of the high loss of larvae in the cage without herbivory, it was very difficult to draw authentic conclusions from our data. Out of 15 larvae in the treatment without herbivory, only three survived to become adults (see Table 1), primarily due to escape through small holes in the cage netting. We lost some of our larvae to extreme weather conditions on June 22, from both cages, although we attempted to remedy this situation by placing plywood over the cages for protection. We also lost two larvae to parasites that must have come in with the milkweed. Although our preference was to conduct this experiment under semi-natural conditions, we would recommend, whenever possible, conducting experiments of this nature in the lab where conditions can be more easily controlled.

Table 1: Mortality among larvae and adult monarchs. *Because the milkweed was kept in a large walk-in cage that was not completely enclosed, parasites and predators to the larvae were able to get on the milkweed, which was taken directly from the large tent and placed in the small cage.
		Reasons for Mortality
Larvae raised on plants…	With Herbivory	2 died making their chrysalis 1 died after emerging when it fell
Larvae raised on plants…	Without Herbivory	1 died as a first instar in the milkweed sap 1 lost in grass during transfer to knew plant 1 parasitized as fifth instar in the J position* 1 parasitized in chrysalis* 8 escaped through holes in the net of the cage

Table 2 lists the number of days to emergence for larvae in each treatment. In the treatment with herbivory, the average number of days to emerge was 27.3 days. The average number of days to emerge in the treatment without herbivory was 27.7 days. Table 3 lists the adult fitness data in chronological order. For a comparison of fitness data, see Table 4. On average, the mass of adults reared on plants with herbivory was 0.537 grams while the mass of adults reared on plants without herbivory was 0.562 grams. Average left forewing lengths were 51.6 mm and 52.4 mm in treatments with herbivory and without herbivory respectively.

Table 2: Days it took larvae from hatching to emerging. Note that individuals were not followed, but were grouped together in the cages.
Days from hatching to emerging		25	26	27	28	29	30	31
Number of larvae raised on plants…	With Herbivory	1	4	2	4	1	0	1
Number of larvae raised on plants…	Without Herbivory	0	0	1	2	0	0	0

Table 3: Measurement data on adult Monarchs (Danaus plexippus).
*Measured on the second day after it emerged.
Date Emerged	Treatment	Spores	Sex	mass (grams)	left forewing (mm)	Right forewing (mm)
7/14/99	With herb.	no	F	.485	51.61	51.87
7/15/99	With herb.	no	M	.434	49.81	50.30
7/15/99	With herb.	no	F	.399	47.78	47.60
7/15/99	With herb.	no	F	.480	51.84	51.28
7/15/99	With herb.	no	M	.578	53.14	52.46
7/15/99	Without herb.	no	M	.549	50.38	50.48
7/16/99	With herb.	no	M	.471	51.07	50.63
7/16/99	Without herb.	no	M	.626	54.87	54.58
7/16/99	Without herb.	no	F	.510	52.09	51.77
7/16/99	With herb.	no	F	.452	48.64	49.04
7/17/99	With herb.	no	F	.544	51.28	51.30
7/17/99	With herb.	no	M	.685	53.92	53.62
7/17/99	With herb.	no	F	.553	50.38	50.97
7/18/99	With herb.	no	M	.718	55.52	55.90
7/18/99*	With herb.	no	M	.645	53.79	54.51
7/20/99	With herb.	Died after emerging when it fell to the floor of the cage and was caught between the netting and the frame.

Table 4: Days to maturation, mass and left forewing length for larvae raised on plants with and without herbivory.
Averages	Days from hatching to emerging	Adult mass (grams)	Left forewing length (mm)
With herbivory	27.3 days	.537 grams	51.6 mm
Without herbivory	27.7 days	.562 grams	52.4 mm

Table 5: T-test results indicate we cannot reject our null hypotheses.
	t_calculated	t_{critical at the 95% confidence level}
Days from hatching to emerging	2.65	2.92
Mass	0.529	2.92
Left forewing length	0.598	2.92

Discussion

Table 5 shows the results of our statistical analysis. In all measures, there was not a significant difference between treatments. We therefore cannot reject either of our null hypotheses. First, there is no difference in the maturation rate of larvae raised on milkweed with herbivory versus those raised on milkweed without herbivory.

Secondly, there is no difference in the mass and wing size between adult monarchs raised on common milkweed with or without herbivory. However, because of the high loss of larvae from the cage with no herbivory and the low sample size of adults from this cage, these results are inconclusive.

Further investigation needs to be done. If we were to do this again, we would conduct the experiment inside to reduce weather effects. We would also track individual larvae, in separate cages, to determine individual development rates. This would eliminate the problem of not having thirty eggs hatch on the same day. Lastly, we would also like to include artificially created herbivory to insure uniformity in the herbivory.

Further Questions

Are bigger butterflies more successful at mating?
Do bigger butterflies stand a greater chance of reaching Mexico?
How far do larvae travel to find milkweed?
Are males generally bigger than females?
Are there sex differences of adults raised on milkweed with herbivory or without?

Acknowledgments

The Heart of the Earth team would like to gratefully acknowledge the dedication and cooperation of Liz Goehring. She was unwavering in her support of our effort, and demonstrated a great deal of patience. Our utmost thanks go to Dr. Karen Oberhauser, whose vision, dedication and energy have made this program possible. It was of tremendous benefit to all members of our team.