Mandy Atteln, Donna Kemp, & Jenni Kemp

Sparta High School
Sparta, WI

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

Abstract

We investigated the relationship between the age of a female Monarch butterfly (Danaus plexippus) and the number and sex ratio of the offspring. Females of the same age mated on the same day and were then kept in separate cages. Their eggs were collected for 18 days, counted and then reared to adulthood to determine if the age of the female had any effect on the sex of her offspring. Using a Chi-square analysis comparing the data from the first and last 6 days of oviposition, we found a trend for an aging female to have more female offspring (p<0.10). There was not a statistically significant association between the age of the female and the sex ratio of her offspring when the data were grouped into the first and last 9 days ( p>0.10 ) or the first, second, and third six days (p>0.10). This question warrants further investigation.

Introduction

Most outbreeding populations maintain an even ratio of males to females, but certain species of insects can adjust the sex ratio of their offspring (Thornhill & Alcock, 1983). A female in poor condition would tend to produce daughters rather than competitively disadvantaged males, because female mates are always in demand and therefore are likely to have some reproductive success. A small or sickly male would usually fail to reproduce at all (Thornhill & Alcock, 1983). Therefore we decided to investigate this phenomenon in monarch butterflies. We wondered if the adult female would have more male or female offspring as she aged and was less healthy? It is also known that as females age and lose mass they lay smaller eggs (Oberhauser, 1997). There might be a reason that it is more costly for one sex than the other to come from a smaller egg, so this could cause a change in sex ratio with female age. We came up with three hypotheses to test.

H_A1: Young female Monarch butterflies produce proportionately more female offspring than older females produce. This may happen because female offspring are less risky and almost always have reproductive success. The female is assured of her reproductive success.

H_A2: Young female Monarch butterflies produce proportionately more male offspring than older females produce. This may happen because it may physiologically be more costly to produce males versus females. As the female ages she has less energy for reproduction and therefore has the males first.

H₀: The age of the female Monarch butterfly has no effect on the sex of her offspring.

Methods

We conducted the experiment indoors at a residence in Sparta, Wisconsin. Three butterfly cages made with PVC pipe and netting with dimensions of 2 ft x 2 ft x 2ft housed the fertilized female butterflies. The cages were positioned along an east wall of the house, approximately two feet from patio doors. We attached 60-watt plant grow lights to the right top of each cage. The three lights were on a timer, which was on from 7am to 8pm daily.

The three females were in the third generation of a lab population. Their grandparents were collected from the wild in Texas in April. Butterflies #1 and #3 eclosed July 8 while Butterfly #8 eclosed July 9. The three butterflies mated on the evening of July 9 and were expressed mailed from the University of Minnesota to Sparta on July 11. Upon their arrival on July 12, we weighed them and released them into their cages. We provided the butterflies with 40 ml of a 20 % honey-water solution placed on a yellow non-germ resistant sponge in the bottom half of a petri dish.

A female monarch (top)  in her cage.

We began the experiment on the morning of July 13, 1999. Each day we collected three stalks of milkweed from an unmowed lot. We chose milkweed that was not flowering and which displayed little herbivory. We pulled it close to the base of the plant with some of the root still intact and approximately 10 minutes after picking, placed the root end in a sink of water and washed the leaves off with cool water. We cut the root off with a scissors while we held it underwater, and then placed the milkweed stalk in a 20 oz clear plastic bottle containing water and some gravel. The bottles containing the milkweed were each placed on the left side of the cages at 7:30 am. The milkweed extended from the bottle approximately 40-45 cm and touched the top of the netting. We rinsed the sponges daily and added 40 ml of fresh honey solution to each sponge. The butterflies were placed on the sponges every morning to ensure feeding. A needle was used to unroll the proboscis if the butterfly did not feed upon being placed on the sponge. Two attempts were made to unroll the proboscis and get the butterfly to feed. After the two attempts the butterfly was released. At 7:30 am each day, we removed and labeled the bottles containing the milkweed stalks and placed a new bottle and stalk in each cage. We added water to the bottles containing the milkweed stalks when needed.

Milkweed stalks after females have laid eggs.

On Sunday, July 18 the top of the milkweed stalks from Day 1 were cut off and placed in flower water vials, which were transferred to 30 cm x 16 cm x 12 cm larva cages. The cage lids had a screen covering a 15 cm x 6 cm opening. We placed a fresh milkweed stalk in a water vial in each cage and lined the bottom with damp paper towels. Any larvae not located on the top of the plant were transferred to the fresh milkweed stalk with a paintbrush. We labeled the containers with the dates the eggs were laid and the date that the larvae were transferred, along with the number of the butterfly. The larvae from each butterfly were given a separate container each day. On Monday, July 19 we divided the larvae into cages so approximately 10-12 larvae were in each cage. Again the containers were labeled with all the information. This procedure was followed every day thereafter.

We cleaned larva cages and added fresh milkweed daily. Stalks were always placed in water vials. Until Monday, August 2 when we decided to use only leaves once the larvae were third or fourth instars because the larvae were pupating on the branches of the milkweed. The pupae on the branches were transferred to the top of the cage by tying a piece of string to the top of the pupa and then taping the string to the lid.

Larvae & milkweed in cages.

We concluded the collecting of eggs and switching of the milkweed in the cages on Tuesday, August 3, when Butterfly 1 died. Butterflies 3 and 8 were then released.

Friday, August 6 we discovered that a few of the black pupa in the cages were diseased and we removed them from all of the cages. Eclosion also began on August 6. New butterflies were numbered and their sex was recorded each day.

Results

Mass:
          7/12/99  Butterfly 1: 0.52 grams, Butterfly 3:  0.53 grams, Butterfly 8:  0.51 grams

Observed Chi-Square – first 6 days vs last 6 days

Calculated Chi-Square – first 6 days vs last 6 days

Calculated x²=3.05, Critical x²=3.84 @ 95%, 2.70 @ 90%.

Using the Chi-square test we were able to reject our null hypothesis (p<0.10) when we grouped the data using the first 6 days and the last 6 days of data. This supported the hypothesis that as a female Monarch butterfly ages she has more female offspring. We also grouped the data by the first 6 days, second 6 days, and third 6 days and also by the first nine days versus the last nine days. Chi-square results with either of these groupings were not statistically significant (p>0.10).

Discussion

Overall, there was not a statistically significant association between the age of the female and the sex ratio of her offspring. When comparing the first and last thirds of their reproductive life, we found that as the females got older, they had more female offspring than expected. This may happen because it takes more energy to have males and the females will have more available energy for reproduction at the beginning of their reproductive life cycle than at the end. Males may mate with many females, therefore the female may have the males first to ensure the possibility of more offspring.

In this experiment the number of eggs laid by an adult female monarch were counted and then reared to adulthood. The sex of the butterflies was recorded to determine if the age of the female influenced the sex of the offspring. Butterfly 1 laid 58 eggs, Butterfly 3 laid 701, and Butterfly 8 laid 629. Butterfly 1 had 3 male and 2 female adult butterflies emerge. Butterfly 3 had 200 females and 200 males emerge and Butterfly 8 had 166 females and 181 males emerge. Butterfly 1 did not lay many eggs and therefore resulted in less data being collected. More data would give more conclusive results.

In this experiment there were several problems which led to a high mortality rate. First we had a disease which infested our larvae and pupae. This may be avoided in a future experiment because now we know what to look for and would quickly remove infested organisms and containers would then be sterilized. Not all the eggs survived to adulthood. A few of the milkweed plants became so dried out that we may have lost larvae on the plants. If we had used potted plants or placed the eggs on filter paper or a water source this problem may have been avoided.

Other sources of error would include the possibility that latex emitted from the plant when injury has occurred may have been counted as an egg. Some eggs may have been brought in with the new stalks of milkweed. Placing the lights on one side of the cage resulted in many eggs being deposited on a very few number of milkweed leaves. By changing the lighting, the egg distribution may be more equal and result in less mortality and better results. The butterflies did not all feed the same and a variance in food quantity may have affected the results as well.

The results of this experiment are intriguing and worth more investigation. It would also be interesting to investigate the possibility of a relationship between egg size and sex of offspring in a future investigation.

Acknowledgements

We would like to thank Karen Oberhauser, Michelle Prysby, and Bill Calvert for their help and support during this project. Research was supported by the National Science Foundation (ESI-9731429) and the Monarchs in the Classroom program at the University of MN.

Literature Cited

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 Alcock, J. 1983. The Evolution of Insect Mating Systems. Harvard University Press.

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