Bob Dunlap, Ashley Hannigan, and Cindy Petersen
St. Hubert School Chanhassen, MN

Carol Boettcher, Sarah Bradbury, Jesse Evans
Mance Park Middle School, Huntsville, TX

Alexis Keith, Stephanie Nelson, and Jennifer Warber
Minnetonka High School, Minnetonka, MN
Minnetonka Middle School East, Minnetonka, MN

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

Abstract

The purpose of this experiment was to see if humidity has an effect on the egg development and survivorship of monarch butterfly eggs. In 1997, the midwestern and central United States experienced record flood conditions. This season was followed in 1998 with a significant drought. The monarch butterfly populations increased in 1997 and decreased in 1998, possibly a function of weather conditions. Specifically, we tested whether moisture levels could have played a role in monarch butterfly egg mortality, which would then affect population sizes.

Three sites (Chanhassen, MN; Hunstville, TX; Minnetonka, MN) developed methods to test this question. Two sites (Chanhassen and Huntsville) constructed humidity chambers from 15-quart, plastic containers. Three levels of humidity were maintained using water and heating blankets. In Chanhassen, monarch butterfly eggs laid on Aesclepias syriaca stems were placed in low, medium, and high humidity levels (40%, 55%, and 69%). These were established with temperatures ranging from 73 °F to 83 °F. In Huntsville, monarch butterfly eggs were collected from the wild. These were placed on leaves of A. curissavica, rather than on the entire stem, and put into humidity chambers at low, medium, and high humidities (24-48%, 59-73% and 85-93%). Temperatures remained relatively constant at 88–98 °F for each treatment. In Minnetonka, monarch eggs laid on A. curissavica leaves were placed in a Biotron Environmental Chamber. Low and medium humidity levels (40% and 52%) were established with temperatures ranging from 88 °F to 90 °F. For one trial, the clipped leaves of A. curissavica were placed on damp filter paper in covered petri dishes. For another trial, an A. curissavica plant and stems of A. syriaca were placed in the chamber. Each group checked the eggs and plants three times daily until all the eggs either hatched or died.

It can be concluded that high humidities yield higher hatching success. Although the experiments were conducted using different equipment, in each case the highest humidity treatment yielded the greatest percent hatch. Huntsville and Minnetonka also found that temperature affects what effect humidity has on survival. High temperatures might stress the egg and lower humidity might exacerbate the problem. Furthermore, it was discovered that substrate has an effect on hatching success. Eggs placed on clipped leaves did not survive as well as eggs placed on live plants or whole stems.

Introduction

In 1997, the midwestern and central United States experienced record flood conditions. The following year, the same regions experienced a significant drought. Scientists studying Danaus plexippus noticed a large variation between the 1997 and 1998 populations. In 1997, the flood year, the D. plexippus population was quite high. During the drought conditions the following year, the population was noticeably smaller.

W. P. Morrison (1970) studied the responses of Lepidopteran eggs to different humidities and found that they exhibit a wide range of responses. Lepidopteran eggs, such as Plodia interpuntella, tolerated very low humidities whereas eggs of Oncopera intricata hatched only at high humidities. Other Lepidoptera, namely the Blue Grass Webworm, had eggs hatching successfully between 10% and 100% relative humidity with mortality increasing below 50% relative humidity.

What effect does the amount of moisture have on the Lepidopteran populations such as D. plexippus? Does a lack of moisture affect the D. plexippus egg? This topic was chosen to be investigated further. The question to be investigated was: How does humidity affect the egg development and survivorship of D. plexippus?

The hypotheses were as follows:

H_o     Humidity does not affect egg development and survivorship.
H₁     Higher humidity leads to faster development and higher survivorship.
H₂     Higher humidity leads to slower development and higher survivorship.
H₃     Higher humidity leads to faster development and lower survivorship.
H₄     Higher humidity leads to slower development and lower survivorship.
H_5-12  The remaining hypotheses incorporate similar comparisons in the rate of development and range of survivorship using medium and low humidities.

To aid in a better understanding of these hypotheses, the following graphs are provided.

Methods

Chanhassen

In our experiment, we created low (40%), medium (55%), and high (69%) humidity chambers using three, 15-quart plastic containers. We taped a hygrometer/thermometer to the inside lid of each container. To achieve a low level of humidity, we placed 2 cm of rice and a reptile heating stone in the bottom of the container. We covered the container and placed it on a heating pad set at high. To produce a medium-humidity chamber, we left the container empty and the lid halfway open. To make the high-humidity chamber we placed 5 cups of water in the bottom of the container and added a rock to hold the eggs on the plant material above the water. We covered the container and placed it on a heating pad set at high.

We used eggs laid on Aesclepias syriaca plants. Stems with eggs were cut the day after oviposition and were placed in water tubes to maintain moisture within the leaves. We placed 30 eggs in the high humidity chamber, 50 eggs in the medium humidity chamber, and 38 eggs in the low humidity chamber. Each morning, the stems were trimmed and placed in fresh water. The low and high humidity chambers were set away from windows in our classroom. In order to achieve a medium level of humidity, the medium humidity chamber was brought to a home and placed under the same light conditions as the chambers at school. We recorded temperature, humidity, and egg hatching data at 9:00 a.m. and 5:00 p.m. daily until all of the eggs had either hatched or died. We also recorded the condition of the milkweed (A. syriaca) as the experiment progressed. We used a Chi-square test to compare the egg hatching success of monarch butterflies in the three humidity treatments.

Huntsville

We collected monarch butterfly eggs from the wild, typically on the day that they were deposited. All eggs collected were found on A. curissavica plants. We clipped the leaves containing eggs, placed them in Tupperware shoeboxes, and exposed the eggs to one of three humidity treatments: high (85-93%), medium (59-73%), and low (24-48%). To create the different humidity environments, we used water and paper towels inside each box. Leaves in the low humidity treatment were placed on dry paper towels. Leaves in the medium humidity treatment were placed on wet paper towels. Leaves in the high humidity treatment were also placed on wet paper towels. In addition, the bottom of the box was filled with water. The eggs were placed atop the water on a small platform. All of the boxes were placed next to each other on a heating blanket.

We monitored temperature and humidity inside the boxes daily, at approximately 6-hour intervals, from the first day the eggs were placed in the treatments until the eggs hatched. We collected eggs over the course of 5 weeks, with each week constituting one trial. The number of eggs per trial varied.

In our analysis, we combined data between the 5 weeks and analyzed differences in hatching success using Chi-square tests to determine significance.

Minnetonka

In order to investigate our question, we ran two trials, each at a different humidity, in a controlled environment. To keep the conditions the same at all times, we used a Biotron Environmental Chamber, which can control photoperiod and temperature. In both of our trials the temperatures were kept at 88-92 °F with a photoperiod of 12:12 (L:D). Low humidity (40%) and medium humidity (52%) conditions were established.

For Trial 1, we obtained five, newly mated D. plexippus females from the University of Minnesota and placed them in a cage with two milkweed plants (A. curissavica). After eight hours, the females had laid 47 eggs. These leaves were then cut off the plant, placed in covered, glass petri dishes lined with damp filter paper, and subjected to a low humidity (40%) environment in the Biotron Chamber.

Clipped leaves tended to dry out quickly. To minimize the effect this might have had on egg hatch, we used whole plants of A. curissavica in the second trial. For Trial 2, we obtained five, newly mated D. plexippus females (different from those in Trial 1) from the University of Minnesota and placed them in a cage with an A. curissavica plant and a stem of A. syriaca. The stem of A. syriaca was in a jar of water. After eight hours, the females had laid 79 eggs. The plant and stem were then placed in the Biotron chamber and subjected to medium humidity (52%).

To create the specified humidities, a pan of water was placed in the Biotron and kept filled throughout the experiment. We recorded the following three times daily: number of eggs hatched, number of eggs unhatched, humidity (measured with a hygrometer that was kept in the Biotron), temperature, and any other observations. The experiment ran until all the eggs had either hatched or died.

To analyze our data, we used the chi-square test to see if there was a significant difference in egg survivorship between the low and medium humidity treatments. We also tested for a difference in the average number of days it took for the eggs to hatch.

Results

Chanhassen

In the Chanhassen experiment, where humidity treatments ranged from 40% to 69% and temperatures were moderate (73 °F - 83 °F), egg mortality in monarch butterflies was not affected by humidity. The results of our experiment show that at 40% relative humidity, there was a 92% hatching success rate. At 55% there was a 96% success rate in egg hatching, and at 69% relative humidity, 100% of the eggs hatched successfully (Table 1).

Table 1: This table includes the number of eggs, the average percent of humidity in each container, temperature in degrees Fahrenheit, and the percent of egg hatching success in each treatment.

Our study also showed that lower temperatures within our 73 °F - 83 °F range slowed the rate of egg hatching in monarch butterflies (Figure 1). In our low and high humidity chambers, where average temperatures were 80 °F and 83 °F respectively, all the eggs had either hatched or died after 4 days. The eggs in the medium humidity chamber (55% humidity, average temperature 75 °F) hatched or died after 5 ½ days. Monarch eggs in the low and high humidity chambers hatched over a period of 1 ½ days, while the eggs in the medium humidity chamber and at a lower temperature took 2 days to either hatch or die.

While eggs at high humidity exhibited the greatest hatching success at 100%, our chi-square test showed that there was not a significant difference in hatching success between the treatments (x² =2.9, df =2, p = .766).

We also observed in our experiment that A. syriaca leaves tended to desiccate and yellow after 4 days in the low humidity chamber. The milkweed leaves in the medium humidity chamber presented some signs of yellowing after 5½ days, while the leaves in the high humidity chamber remained constantly green and healthy looking throughout the experiment.

Figure 1: This graph shows that lower temperatures within our 73-83 °F range slow the rate of egg hatching in monarch butterflies.

Huntsville

Temperature and humidity measurements were monitored regularly. Humidity values in the high category ranged from 85-93% r.h., medium humidity values were 59-73% r.h. and low humidity values were 24-48% r.h. with temperature remaining relatively constant (88-98 °F) in each treatment. Figure 2 shows the average hatching success for all the trials. We found a significant difference between humidity treatments (chi-square = 10.6, df = 2, p<.001) with those in the highest humidity treatment achieving the greatest hatching success.

Figure 2: This graph shows a direct relationship between humidity and percent hatching. As relative humidity increases, percent hatching of D. plexippus eggs increases.

Minnetonka

The Minnetonka experiment found that mortality in monarch butterfly eggs is affected by humidity (Table 2). We saw a hatching success rate of 41% in the low humidity treatment (40% r.h.) and a 100% hatching success rate in the medium humidity treatment (52% r.h.). There is significant difference in egg mortality between humidity treatments (x² = 63.3, df = 1, p< .001), with higher survival in the higher humidity treatment. (Figure 3).

Table 2: This shows the results of the Minnetonka experiment. There was greater hatching success at higher humidities (52%)

Figure 3: This graph shows a direct relationship between relative humidity and hatching success. As humidity increases, so does the percent hatching.

Discussion

Chanhassen, Huntsville, Minnetonka

Three different approaches were used to determine the effect of humidity on the egg hatching success of monarch butterflies. We can conclude from all three studies that high humidity levels yield higher hatching success.

In the Chanhassen, MN experiment, where humidity chambers measured 69%, 55%, and 40% and temperatures were moderate (73 °F – 83 °F), there was not a significant difference in the egg hatching success. All three treatments demonstrated high rates of hatching success with 100% hatching at 69% humidity, 96% at 55% humidity and 92% hatching at 40% humidity. In Minnetonka, there was a hatching success rate of 100% in high humidity (59%) and temperatures ranging from 88 °F – 92 °F, whereas the egg hatching success was only 41% in low humidity (40%). The Huntsville, TX team also saw the greatest egg hatching success (88%) in the treatment with the highest humidity (85-93%). Medium humidity values were 59-73% with a hatching success of 69% and low humidity values of 24-48% showed an egg hatching success of 45%. The Huntsville experiment was carried out in temperatures ranging from 88 °F – 98 °F. In all three studies, the highest level of humidity corresponded to the highest egg hatching success.

Insect egg development, like most metabolic processes, requires water. To prevent water loss, insect eggs are covered by a layer of chorion that varies in thickness of one or more waxy layers. These layers seem to provide the egg with a constant volume of water throughout its development (Hinton, 1981). In fact, the eggs of many Hemiptera and Lepidoptera which are placed in dry, exposed conditions develop without any water uptake (Chapman, 1998). However, sufficient water inside the developing egg and high humidity levels outside seem to provide for the greatest egg hatching success.

High temperatures seem to be a factor in increasing the rate of egg mortality at both low and medium humidities. In the Chanhassen study where moderate temperatures (73 °F–83 °F) were established for all levels of humidity, the egg hatching success remained high. In Minnetonka and Huntsville where temperatures ranged from 88 °F - 92 °F and 88 °F - 98 °F respectively, there was a greater mortality rate at lower humidities (59% and 55%). In Huntsville, where temperature ranges were the highest (88 °F-98 °F), eggs in medium levels of humidity showed mortality rates averaging 31%. Higher temperatures tend to break down the protective chorion and waxy layers leading to greater egg mortality (Ferro & Chapman, 1979). The relationship between temperature and humidity is borne out in several studies of Lepidoptera including those of the Pink Bollworm and European Corn Borer, Ostrinia nubilalis.

In an article published in 1991, L. D. Godfrey studied egg hatching of the corn borer under a variety of temperatures and humidity regimes. They found that the percentage of successful hatching sharply decreased regardless of humidity at 36 °C (96.8 °F) and 39 °C (102.2 °F). Showers, et al. (1978), also studying European corn borers, concluded that moisture is dependent on temperature and has an indirect effect on mortality. Results of the Showers study showed that temperatures in excess of 30 °C (86 °F) were fatal. The mortality at the high temperatures was possibly due to evaporation and loss of water within the egg.

When conducting this experiment, all three teams (Chanhassen, Minnetonka and Huntsville) questioned the effect of the substrate on both egg hatching success and larvae survival. All groups observed that Aesclepias leaves tended to desiccate and yellow after several days in low humidity providing little, if any moisture to first instar larvae. The quality of the milkweed improved as the humidity increased, perhaps allowing for greater survival of newly hatched larvae. This observation held true with the A. curissavica plants used in Minnetonka, the A. syriaca cuttings in water tubes in Chanhassen, and the A. curissavica leaves used in the Huntsville study.

A study by Tisdale and Wagner (1990) showed that the percentage of egg hatching decreased, regardless of humidity, at temperatures exceeding 36 °C (96.8 °F). In looking at the substrate, they found that the percentage of eggs hatched on cuttings averaged 70% at 15 °C (59 °F), while on seedlings the successful hatch averaged 82% at 15 °C. The percentage of eggs hatching on cuttings sharply decreased at temperatures of 21 °C (69.8 °F) and 26 °C (78.8 °F) to 12.71% and 13% respectively. On the other hand, seedling egg hatching remained high at these elevated temperatures with hatch rates of 70% at 21 °C and 75% at 26 °C. From the Tilsdale study and our own, there appears to be a greater percentage of egg hatching in high temperatures if seedlings, rather than cuttings or leaves, are used.

The effect of substrate desiccation at low humidity levels and high temperatures as observed in our experiments remains a question for further investigation. In our observations, the plant material did not appear to offer adequate nutrition and moisture to developing larvae. Hinton (1991) points out that although there appears to be protection against water loss by the chorion and waxy layers in dry conditions, once the larva bites through the chorion, it may die from desiccation in the dry air.

Aside from further questions surrounding the relationship of temperature and humidity in egg hatching success, and the effect of substrate on egg hatching, we also had several other uncertainties for possible exploration:

• Do humidity levels less than 40% and over 70% significantly affect egg hatching success on seedlings?

• Is the percentage of successful hatch under various humidity levels the same for eggs just deposited on leaves verses those that have been on leaves for a 24-hour period?

• Does the size of the treatment chamber have an effect on egg hatch?

• Do small ranges in humidity within a treatment chamber affect the egg hatching success?

• Do eggs from different females make a difference in hatching success and development?

Literature Cited

Chapman, R. F. 1998. The insects: structure and function. Cambridge University Press.

Ferro, D. N. & R. B. Chapman. 1979. Effects of different constant humidities and temperatures on two spotted spider mite egg hatch. Environmental Entomology. 8: 701-705.

Fye, R. E. & D. E. Surber. 1971. Effects of several temperature and humidity regimens on eggs of six species of Lepidoptera pests of cotton in Arizona. Journal of Economic Entomology. Oct: 1138-1142.

Godfrey, L. D. & T. O. Holtzer. 1991. Influence of temperature and humidity on European corn borer (Lepidoptera: Pyralidae) egg hatchability. Environmental Entomology. 20(1): 8-14.

Hinton, H. E. 1981. Biology of insect eggs, Vols. 1 & 2. Pergamon Press.

Leather, S. R. & J. Hardie. 1995. Insect reproduction. CRC Press.

Morrison, W. P., B. C. Pass & C. S. Crawford. 1972. Effect of humidity on eggs of two populations of the bluegrass webworm. Environmental Entomology. 1: 218-221.

Perring, T. M., T. O. Holtzer, J. L. Toole, J. M. Norman & G. L. Meyers. 1984. Influences of temperature and humidity on pre-adult development of the Banks grass mite (Acari: Tetranychidae). Environmental Entomology. 13: 338-348.

Pyenson, L. & H. L. Sweetman. 1931. The effects of temperature and moisture on the eggs of Epilachna corrupta Mulsant (Coccinellidae, Coleoptera). Bulletin of the Brooklyn Entomological Society. 26: 221-226.

Showers, W. B., M. B. De Rozari, G. L. Reed & R. H. Shaw. 1978. Temperature-related climatic effects on survivorship of the European corn borer. Environmental Entomology. 7: 717-723.

Tisdale, R. A., & M. R. Wagner. 1990. Effects of photoperiod, temperature, and humidity on oviposition and egg development of Neodiprion fulviceps (Hymenoptera: Diprionidae) on cut branches of ponderosa pine. Environmental Entomology. 19(3): 456-458.

Acknowledgements

We would like to thank Dr. Karen Oberhauser and the Monarchs in the Classroom program at the University of Minnesota for supplying the mated females that enabled us to carry out this study, and to the National Science Foundation for supporting our research (ESI-9731429). Many thanks to Liz Goehring and David Astin for their guidance and advice throughout this project.

We would also like to acknowledge St. Hubert Catholic School for their support of our team, along with the resources they provided that made this opportunity possible. Thanks to Minnetonka East Middle School for the use of the Biotron, and finally a special thank you to our families for providing us with extra encouragement and helping us with monarch care over the summer months.