Dale Pulis, April Schmitz, Christine Steckling,
Holly R., Matt Kometz and Ruth Naber

Mississippi Heights Middle School
Sauk Rapids, Minnesota

Abstract  |  Introduction  |  Methods  |  Results  |  Discussion  |  Acknowledgements  |  Literature Cited  |   Research Projects

Abstract

We tested the effect of monarch larvae (Danaus plexippus) eating their chorion after hatching. Data were collected on growth rates (both length and mass), length of time in instars, and survival. Two treatments consisted of larvae which were allowed to eat their chorion completely and those that were prevented from eating their chorion. Larvae were housed in individual containers and fed daily with common milkweed (Asclepias syriaca). On a daily basis measurements were recorded on length, mass, and instar stage. The data suggest that larvae that do not eat their chorion remain longer in the first instar than those that do. Also larvae took longer in the 5th instar when they did eat the chorion. There was a significant difference in monarch survival rates in the homes of different members of our research team, but not on whether or not the larvae ate their chorion.

Introduction

After observing hatching monarch larvae (Danaus plexippus) eating their chorion, our team of six designed an experiment to determine if monarch instars that were allowed to eat the chorion would grow better than the instars that were not allowed to eat the chorion. Although we were able to find little research on nutritional value of chorion material in butterflies, we did find an article on the eggshell material of Drosophila. The article reveals that the eggshell of this insect consists of three layers of a protein material (Pascucci et al. 1996). We think that added nutritional value if present in the chorion, might affect larvae growth. We also examined survival rates for those that were and were not allowed to eat their chorion.

Hypotheses::

Ha₁  Monarch larvae that eat the chorion will be more likely to survive than those that don't.
Ha₂ Monarch larvae that don't eat the chorion will be more likely to survive than those that don't.

Ha₃  Monarch larvae that eat the chorion will grow larger than those that don't.
Ha₄  Monarch larvae that don't eat the chorion will grow larger than those that don't.

Ha₅ Monarch larvae that eat the chorion will grow faster in the early instars than those that don't.
Ha₆  Monarch larvae don't eat the chorion will grow faster in the early instars than those that don't.

H_o   Not eating the chorion will have no effect on the monarch larvae.

Methods

We reared enough monarch eggs to supply at least 30 larvae for each member of our six person team. While waiting for the eggs to hatch each person labeled containers with a number, tester's initials, date of hatching, and "C" or "NC". Each container was lined with slightly moist paper towel and had a cover that would keep the larva from escaping. Petri dishes were used during the first two instars. During the experiment caterpillars in their containers were given enough Asclepias syriaca (common milkweed) so they would always have ample food. Each day as we worked with the caterpillars their containers were cleaned and they were fed.

To begin the experiment we observed our eggs and, as the caterpillars emerged, we carefully removed half of the newly hatched instars before they had a chance to turn around and eat the chorion. We rejected larvae that we either did not observe emerging from the egg or see finish eating the chorion to avoid non-random assignment of larvae to treatments. We tried to get fifteen instars and we placed them in the "NC" containers.  We recorded on our data sheets the label of the containers (in this case "NC" for no chorion) and the date and time that they hatched.

We also collected approximately fifteen more instars per person. These instars had finished eating the chorion. They were given the label "C" and the same information was recorded for them.

We started recording data on the second day. Through the entire experiment we recorded daily length measurements for each larva as well as its instar stage. We also recorded extra notes of interest. When each larva reached the third instar we also recorded mass measurements which were taken with an electronic balance which measured to the nearest hundredth of a gram. We continued to monitor and record data until the larvae pupated. When larvae reached the fourth instar, they were transferred to clear plastic cups with covers for larvae to pupate on .

Results

Mass Measurements

Mass measurements were taken daily beginning with the first day of the 3rd instar. Our electronic balances were not able to get readings on younger instars. Analysis was done using the mass on the final day of the 3rd and 4th instars and the third day of the 5th instar. We did not wish to disturb the larvae for measurements as they neared the pupation stage. See the graph in figure 1 to examine results. A t-test was run on this data. Average masses for both treatments in the 3rd instar were 0.07 grams resulting in a t-value of 0. In the 4th instar, the average was 0.27 grams for chorion eaters and 0.31 grams for non-chorion eaters resulting in a t-value of 1.00. The 5th instars showed an average mass at the end of the third day of 1.19 grams for the chorion eaters and 1.24 grams for the non-chorion eaters resulting in a t-value of 1.04. With a critical t-value of 1.671 and values of p>0.100 for all comparisons, no significant difference was found in relation to mass for chorion versus non-chorion eaters.

Figure 1.  This graph shows the average mass increase for 3rd to 5th instars of chorion eaters and non-chorion eaters.

Length Measurements

Length measurements were taken daily beginning with the second day. Analysis was done using the length measurements on the final day of each of the 1st through 4th instars and the third day of the 5th instar. See the graph in figure 2 to compare the results. The length at the end of the 1st instar averaged 4.72 mm for chorion eaters versus 4.76 mm for non-chorion eaters. The resulting t-value was 0.4106. Averages for the 2nd instar were 8.2 mm for chorion eaters and 8.2 mm for non-chorion eaters resulting in a t-value of 0.17733. The average length of the 3rd instars was 13.8 mm for chorion eaters and 13.8 mm for non-chorion eaters. The resulting t-value was 0.087. Averages for the 4th instar were 24.0 mm for chorion eaters and 24.3 mm for non-chorion eaters with a t-value of 0.49. After the third day of the 5th instar, average length for chorion eaters was 41.4 mm and for non-chorion eaters was 40.0 mm. This generated a t- value of 1.565. With a critical value of 1.671 and p>0.100 for all comparisons, we found no significant difference in length for chorion versus non-chorion eaters.

Figure 2.  This graph gives the comparison in average length of larvae from the day after hatching to the 3rd day of the 5th instar.

Time Spent in Instars (see graph in figure 3)

Each day the instar was recorded and averages were later calculated to determine the average time spent in each instar. Sample sizes ranged from 74 to 88 larvae for various test groups. T-tests were run on the data for each instar. The 1st instar had averages of 2.46 days for chorion eaters and 2.89 days for non-chorion eaters resulting in a t-value of 2.3 with 0.010<p<0.025. We can be about 98% confident that there is a significant difference in the amount of time spent in the 1st instar. We found that the non-chorion eaters spent about a half day more in the first instar. We can thus reject our null hypothesis that there is no difference in development time between the two treatments.

In the 2nd instar the average time spent was 2.22 days for chorion eaters and 2.24 days for non-chorion eaters with a t-value of 0.11. Averages for the 3rd instar were 2.17 days for chorion eaters and 2.19 days for non-chorion eaters resulting in a t-value of 0.087. The average time spent in the 4th instar was 2.58 days for chorion eaters and 2.69 days for non-chorion eaters resulting in a t-value of 0.49. Since the critical value of t would be 1.671, p>0.100 , no significant difference was found for the time spent in the 2nd to 4th instars between chorion and non-chorion eaters.

In the 5th instar, averages were 4.93 days for chorion eaters and 4.64 days for non-chorion eaters. The t-value was 1.81 with 0.025<p<0.05. We can be 95% confident that there is a significant difference in the amount of time spent in the 5th instar. The chorion eaters spent a bit longer in the 5th instar also but the difference was not quite as great as in the 1st instar.

Figure 3.  This graph depicts the average number of days our larvae spent in each instar.  In compares chorion eaters to non-chorion eaters.

Survival Rates

We collected data on survival rates in two areas and ran two Chi-square tests. For survival rates by individual testers, a Chi-square value of 21.01 with five degrees of freedom, this was highly significant with a p-value < 0.001. We are 99.9% confident that some testers kept their larvae alive better than others (see graph in figure 4).

Figure 4.  This graph shows the result of comparing survival rates of larvae among individual testers.

(a)

(b)

Table 1.  Chi-square tables for (a) observed and (b) expected survival of larvae among individual testers.

For survival rates for chorion versus non-chorion eaters, the Chi-square value was 0.0902 with one degree of freedom (see table 1). Since the p-value for this is greater than 0.100, we cannot reject the null hypothesis that there is no significant difference in survival for chorion versus non-chorion eaters. (see graphs in figure 6 and 7)

Figure 5.  Percent survival of chorion eaters and non-chorion eaters.

(a) 

(b) 

Table 2.  Chi-square tables for (a) observed and (b) expected survival of chorion eaters versus non-chorion eaters.

Discussion

After evaluating our data on chorion eaters versus non-chorion eaters, we found significant differences in the amount of time spent in 1st and 5th instars between chorion eaters and non-chorion eaters. Differences also existed in terms of survival rates by individual testers but not between our two experimental treatments.

In both length and mass we found no significant difference between chorion eaters and non-chorion eaters. We cannot reject our null hypotheses of no difference in relation to length and mass. During our daily recordings, we found accurate length measurements were difficult to obtain since larvae were very active. Length varied within a few seconds by five or more millimeters in the later instars. In addition, the larvae when nearing molting seemed to contract and decrease in length.

In our experiment we found the most significant differences existed in relation to the length of time larvae spent in the 1st and 5th instars. We are 98% confident of the significant difference in the 1st instar. Chorion eaters spent about a half day less in the 1st instar. This would give the 1st instar larvae that ate its chorion a survival advantage over those that didn’t. Organisms that develop or grow more slowly could suffer more predation.

Our statistical results on the 5th instar larvae indicate that we can be 95% confident that there is a significant difference in time spent as a 5th instar. This difference is not what we might expect; chorion eaters spent more time in the 5th instar. However, we are not sure of the mechanism that could cause this difference, and would like to see further studies on the time spent in each instar in the future.

Acknowledgments

We would like to thank those members of our families who were patient and supportive and helped us in many ways to complete this research project. A special thanks goes to Jane Pulis and Steve Naber for their assistance in building larvae and adult cages so that we could rear adults to lay enough eggs for our research. Thank you also to friends who helped monitor and care for the larvae at times, and our colleagues who helped provide us with supplies. Our gratitude goes to Karen Oberhauser and her daughter, Leah. They spent many hours helping us crunch numbers and analyze other data. Karen was our source of encouragement and inspiration when things got tougher. We are very appreciative of the support given us by our school district and especially our building principal at Mississippi Heights Middle School, Bonnie Strobbe.

We are grateful that the Monarchs in the Classroom program at the University of Minnesota, the Science Museum of Minnesota, and the National Science Foundation combined their efforts to provide this wonderful program for teachers and students. In addition to their financial support, the staff that they provided were truly dedicated individuals.

Literature Cited

Pascucci, T., J. Perrino, A. P. Mahowald and G. L. Waring.   1996.  Eggshell assembly in Drosophila: processing and localization of vitelline membrane and chorion     proteins.  Developmental Biology 177(2): 590-598.