Research Project

Relationships between Female Age and Offspring Sex Ratios in the Monarch Butterfly

Hamilton Team

Trey Phillips, Sharon Wilder, and Marci Charpentier

Hamilton Middle School
Cypress-Fairbanks ISD
Cypress, TX

Abstract

In this experiment we looked for a relationship between the age of female monarch butterflies (Danaus plexippus) and the sex ratio of their offspring. Three mated females of the same age were placed in a cage. We collected the eggs laid by these females on three days corresponding with when they were seven, fourteen, and twenty-one days old. We then raised the larvae from these collected eggs and recorded the sex of each resulting adult. Using a Chi-square analysis, we did not find a statistically significant association between the age of the females and the sex ratio of the offspring when we examined the data from the three days.

Introduction

The monarch butterfly (Danaus plexippus) is easily identified by its distinctive orange and black coloration and can be found throughout the Midwestern states during the summer months. This butterfly is best known for its ability to migrate long distances each fall to reach sites in Mexico where it spends the winter before returning to the United States the following spring (Grace 1997). While much research has been completed on various aspects of monarch biology and ecology, many aspects of its population ecology remain understudied. For example, although it is known that overall sex ratios in monarch populations are approximately 50/50, it is not known what factors might influence sex ratios in monarchs, or whether these ratios ever vary (K. Oberhauser, pers. comm.). We decided to look for a relationship between the age of female monarchs and the sex ratio of their offspring. Research has indicated that certain species of insects are able to vary the sex ratio of their offspring (Thornhill & Alcock 1983). Research has also revealed that in the monarch female, as the female ages and loses body mass, the female lays smaller eggs (Oberhauser 1997). We wondered if the ratio of male to female offspring would change as the female monarch aged. We developed the following three hypotheses to test.

H_A-1: The younger the female monarch, the more female offspring she will produce.
H_A-2: The younger the female monarch, the more male offspring she will produce.
H_O: The age of the female monarch will have no effect on the sex ratio of the offspring.

Methods

We conducted our experiment on the campus of Hamilton Middle School in Cypress, Texas. Our equipment included one hanging butterfly cage made of bridal net used for the egg-laying females, forty tote boxes with screen tops, five aquariums with screen tops, fifty plastic florist water vials, twenty-five four-inch pots of Asclepias curassavica, and over fifty one-gallon pots of A. curassavica. Fluorescent lights in the classroom were left on for twelve hours and off for twelve hours. The temperature remained constant at approximately 78 degrees Fahrenheit.

We initially received two mated females from the University of Minnesota. We placed the females in individual cages and fed them a 20% honey/water solution placed on sponges. Unfortunately, these butterflies died after several days without laying any eggs. We then decided to use butterflies that we had raised in our classroom. The eggs for these butterflies came from a teacher in Victoria, Texas. Since we were concerned with our ability to keep butterflies alive in cages long enough to retrieve eggs for the experiment, we decided to mate five butterflies that had eclosed on the same day in our room. We placed three of the mated female butterflies together in one hanging cage and did not attempt to keep records of each individual butterfly but instead used the total number of eggs layed each day. Since we intended to combine the numbers per day rather than per butterfly for our analysis at the end of the experiment, we did not believe it was necessary to separate the females during the egg laying process.

We first placed three mated females in a hanging cage with one four-inch plant of A. curassavica. The butterflies were fed hummingbird nectar prepared from a commercial brand of instant hummingbird food. We put the nectar on sponges placed on the bottom half of petri dishes in the bottom of the cage. We also sprayed the cage occasionally with water from a spray bottle. Spraying the butterflies with water seemed to encourage them to eat. When we noticed eggs on the milkweed, we began the experiment. At 8:00AM on Wednesday, September 15, 1999, we removed one plant of milkweed and replaced it with a new plant. This new plant of milkweed was left in the hanging cage for twenty-four hours until 8:00AM on Thursday, September 16, 1999. The milkweed was removed from the cage and the eggs were counted. A fresh plant of milkweed was placed in the cage each day at 8:00AM but we only sampled three days, September 16, 23, and 30. We left the eggs on the milkweed until they hatched. When the eggs on each of these milkweed plants hatched, we placed the larvae in tote boxes with fresh cuttings of A. curassavica. The milkweed cuttings were put in plastic florist vials filled with water to keep the milkweed fresh for the larvae. As the larvae grew, we separated them into additional tote boxes to prevent crowding. Some of the larvae were placed in aquariums so students could more easily observe the development. Each tote box and aquarium was labeled with the date the eggs were laid. Tote boxes were cleaned and larvae were fed fresh milkweed on a daily basis. The larvae pupated on the screen tops of the tote boxes and aquariums. We had originally intended to raise all the eggs laid by the three females, not just those from our three sampling days, but we soon ran out of room and began giving the excess to neighboring classrooms. As the adult butterflies emerged, we recorded the sex of the butterfly. The butterflies were numbered and then released. The sex of each offspring was recorded based on the date of the egg was laid, not the date of emergence.

Results

The results from our experiment are recorded in Table 1. We used a Chi-square test to analyze the data.

Table 1. Sex ratios in offspring from 3 female monarchs. These are the observed values for the Chi-square test.
Date	Age in days	# Females	# Males	Totals
16-Sep	7	45	42	87
23-Sep	14	142	153	295
30-Sep	21	75	59	134
Totals	--	262	254	516

Table 2. Expected sex ratio values for Chi-square test.
Date	Age in days	# Females	# Males	Totals
16-Sep	7	44	43	87
23-Sep	14	150	145	295
30-Sep	21	68	66	134
Totals	--	262	254	516

Since the calculated chi-square value was not greater than the critical value, we were unable to reject our null hypothesis (Calculated Chi-square= 2.37, Critical Chi-square=5.99 @ 95%, 4.61 @ 90%, d.f.=2).

Discussion

Overall, we did not find a statistically significant relationship between the age of the female and the sex ratio of her offspring. However, during the experiment we lost approximately fifty percent of our pupae to an infestation of Ophryocystis elektroscirrha and this loss may have distorted our data. As soon as we became aware of the problem, we examined each of our adult butterflies by taking a sample of scales from the abdomen area and examining the scales under a microscope. We froze and disposed of any infected adults, sterilized all of our equipment with a 20% bleach solution, and cut back all of the milkweed. Since we believe there is a possibility that such a high incidence of infected pupae may have influenced the outcome of the experiment, we would like to see the experiment repeated with healthy females. We also would be interested in repeating this experiment to investigate the effect of changing the variables of daylight hours and temperature to mimic spring, summer, and fall weather conditions. We also wonder if the number of times a female is mated might influence the sex ratio of her offspring. Obviously, we have concluded that we still have many unanswered questions.

Acknowledgements

We thank Karen Oberhauser, Michelle Prysby, and Bill Calvert for their help and guidance during this project. We also thank teachers and students at Hamilton Middle School for help raising the monarch larvae and recording data. The National Science Foundation (ESI-9731429) and the Monarchs in the Classroom program at the University of Minnesota supported our research.

Literature Cited

Grace, E.S. 1997. The World of the Monarch Butterfly. Sierra Club Books.

Oberhauser, K. S. 1997. Fecundity, lifespan and egg mass in butterflies: effects of male- derived nutrients and female size. Functional Ecology. 11, 166-175.

Thornhill, R. and Allicock, J. 1983. The Evolution of Insect Mating Systems. Harvard University Press.