Research Project

Comparison of Larval Behavior in Danaus plexippus

Brooklyn Park team

Sarah Austing, Jenny Bradley, Tanner Courrier, Lori Funderburk, Kirsten Kulcsar, and Margaret Russell
North View Junior High School
Brooklyn Park, MN
ISD #279

Abstract

Our purpose was to discover if the age of the instar of monarch larva had any bearing on the amount of time the larva spent doing certain activities. We accomplished this by raising larvae and using scan sampling to observe and record the behaviors of the larvae. We would scan every 30 seconds to one minute and tally how many larvae were doing each of our cataloged behaviors during that scan. Our results showed no significant difference between old and young instars for any behaviors except resting and eating. The older the instar, the more time it spent eating and less it spent resting. These results suggest that as the caterpillar grew, it required more food to sustain its mass. Our results also showed that old and young instars have differences in where on the milkweed plant they prefer to eat. Younger instars seemed to prefer the inner leaves, whereas the older instars preferred the outer leaf edges.

Introduction

Question 1: What proportion of time do instars spend doing the following behaviors – resting, head moving, crawling, eating, and twitching?
Question 2: On what part of the milkweed plant do different instars prefer to eat?

Our project investigated the behaviors of monarch larvae during different instars. For our study we developed a catalog of larval behaviors. We did this by raising a single generation from egg to adult and observing their behaviors at different times over the course of their development. We compiled our data into 5 basic behaviors: resting, crawling, eating, head moving (only), and twitching. Each team member then raised a second generation of eight larvae and recorded their behaviors during each instar. Instars were divided into "young" (first through third instars) and "old" (fourth and fifth instars) categories. We focused on two questions. First, what proportion of time do instars spend doing each of our cataloged behaviors? Our hypotheses for this question were as follows:

H₀ - There are no differences in the proportion of time different instars spend resting, crawling, eating, head moving, and twitching.
H_A1 - Different instars spend different proportions of time doing different behaviors.
Our second question investigated where on the milkweed plant each instar preferred to feed. We divided the plant into three areas: the edge of the leaf, the middle of the leaf, or the stem. Our hypotheses for this question were as follows:
H₀ - There will be no difference in the proportion of time different instars spend eating on the edge of the leaf, middle of the leaf, or the stem of a milkweed plant.
H_A1 - Young instars will spend a greater proportion of time eating on the edge of a leaf than older instars.
H_A2 - Young instars will spend a greater proportion of time eating in the middle of a leaf than older instars.
H_A3 - Young instars will spend a greater proportion of time eating on the stem of a leaf than older instars.

Methods

For our project, we studied the amount of time different instars spend doing different daily activities. We first raised a generation of monarchs to observe and catalogue their behaviors. We studied them for 15 to 30 minutes per day. We summarized all observed behaviors into five main categories: resting (totally still), crawling (whole body moving forward), head moving (if only head and/or upper body was in motion), eating (when mandibles were moving), and twitching (if body or antenna made quick, sharp movements).

Once these behaviors were defined, we reared a second generation to observe the amount of time each instar spent doing these behaviors. First, we placed a fresh milkweed plant upright in a bottle of water to best simulate natural conditions. We then placed the larva on the milkweed plant and allowed it to adjust to its new setting for about 30 minutes. We used the scan sampling method to observe all the larvae in 30 second to 1 minute intervals for at least 30 minutes each day.

To record our data, we designed a data sheet that included a place to record the time of day, date, number of molting larva (not included as resting behavior), room temperature, noise level of room, observer, type of light (natural, artificial, or both), and our five behaviors. With each scan, we would tally how many of our visible larvae were observed doing each behavior.

Results

We found that monarch larvae spent more time resting than doing any other behavior (Figure 1). They also ate frequently, while twitching and head moving were much less common behaviors.

Figure 1. Lifetime percent of larval behaviors.

Our results showed that there was not a great difference in time spent crawling, twitching, or head moving between young and old instars (Figure 2). However, eating and resting times drastically changed from young to old instars (Table 1). We found that young larvae spent much more time resting (64%) than the old larvae (37%). The difference in time is significant at a 98% confidence level (t =3.5705; p =0.019). The old larvae spent more time eating (37%) than the younger instars (16%). While this difference is not significant at a 95% confidence level, we are at least 90% confident that there is a difference (t =1.8811, p =0.078). This information shows that as young instars, caterpillars spend much more time resting than they do eating. By the time they reach 4th and 5th instar status, the time spent eating vs. resting has evened out.

Figure 2. Percent of time spent in different activities between young and old monarch larvae.

With respect to our second question, our results showed significant differences in the relationships between the ages of the instars and the location they prefer to eat on the milkweed plant (Figure 3). Young larvae ate more in the middle of the leaf than the old larvae did. (t =3.5534, p =0.019) (Table 2). The old larvae preferred the edge of the leaf more than the young larvae did(t =2.2139, p =0.057).

Figure 3. Larval feeding location preferences.

Table 1. Proportion of time the larvae spent resting and eating (mean ± standard error).
	Young	Old	t-Test Statistics
Resting	0.64 ± 0.05	0.40 ± 0.04	t = 3.57, 3 df, p = 0.019
Eating	0.17 ± 0.05	0.34 ± 0.08	t = 1.88, 3 df, p = 0.078

Table 2. Proportion of time spent eating from different locations on the milkweed plant (mean ± standard error).
	Young	Old	t-Test Statistics
Middle	0.77 ± 0.13	0.18 ± 0.10	t = 3.55, 3 df, p = 0.019
Edge	0.23 ± 0.13	0.72 ± 0.18	t = 2.21, 3 df, p = 0.057

Discussion

Our results show that while all larvae participate in the same types of activities, the amount of time spent doing those activities vary by age of the larvae. We grouped 1st-3rd instars and 4th-5th instars together because the time variance was seemed marginal within both groups, but very distinct between the two groups. The only difference in activity type between the groups was their defense behaviors. Instars 1-3 would drop off the plant and hang by a silk thread, while instars 4-5 would curl into a ball.

The two behaviors that consume most of the larvae's time are eating and resting. Resting seemed to be the single most important activity for a larva, consuming a full 51% of its entire life. Second to that was eating, consuming 27% of the larvae’s entire life. Between the two groups of instars, young (1st-3rd) and old (4th-5th), these two categories showed the greatest differences.

Young caterpillars spent the largest amount (64%) of their time resting and 16% of their time eating. The older caterpillars spent about the same amount of time resting (37%) and eating (37%). The younger caterpillars spent 27% more time resting than their older counterparts and older caterpillars spent twice as much time eating than their younger counterparts (17% more time). We were able to reject our null hypothesis on resting time differences and state with a 98% confidence level that younger instars spend more time resting than older instars. While our data does not support rejecting our null hypothesis on the eating differences at a 95% confidence level, there is a strong trend that does suggest older instars spend more time eating than younger instars at a 92% confidence level. This trend seems to suggest that the older larvae increase their food intake to support their larger body mass and to store energy for their approaching pupation. You will notice also that the resting vs. eating ratio for the older larvae is very close (Figure 2). This seems to indicate that rest is just as important to the older larvae as eating is and that rest allows them to store more of their food energy for the pupal stage.

With regard to the plant eating location preference for the larvae, we found the younger instars preferred the middle of the leaf to the edge of the leaf or the stem. We can reject our null hypothesis at a 98% confidence level and state that younger instars prefer eating the middle of the milkweed leaf. The older instars preferred the edge of the leaf to the middle of the leaf or the stem. We are able to reject our null hypothesis at the 94% confidence level and state that older instars prefer eating the edge of the milkweed leaf.

We did find it difficult to compile some of our data because each team member observed a group of larva independent from the other team members. This resulted in a few differences of opinion as to the type of behavior observed. We also noted that each group of larvae was observed under different environmental conditions. Things such as temperature, type of light, time of day, noise level, or other environmental disturbances could have affected our results. The next time, we would all observe together to be in agreement with each other and allow for complete consistency throughout our data. We would also be able to equalize the environmental factors for every larva.

Acknowledgements

We would like to thank our friends and families for the help and support during our research. We especially thank the parents of the student members: Duane and Beth Austing, Kevin and Paulette Kulcsar, Brian and Jackie Courrier, and Mike and Sue Bradley.

Thanks to Dr. Boddie and Mr. Ben Zachary for backing the North View team and arranging the finances needed to support the time off for Ms. Russell and Ms. Funderburk.

A very big thanks to Michelle Solensky for her time, ideas, and math skills and to David Astin for his help and support.

Research was supported by the National Science Foundation (ESI-9731429) and the Monarchs in the Classroom program at the University of MN.