The Development and Coloration of Monarch Larvae at Controlled Temperatures

Elizabeth Larkin 1998-99

 

Abstract: Monarch larvae are raised in labs in controlled temperatures to regulate the speed of their growth. The different temperatures also affect their development and coloration. Larvae were raised at three different temperatures: warm 22°C to 32°C, control 17°C to 27°C, and cold 6°C to 16°C. The larvae were closely monitored and measured for the duration of their growth and using a presence/absence method, their percent color was measured. The metabolic rates of the larvae increased in relation to the temperature surrounding them, thus the larvae increased in length, mass, and instar at a faster speed at a heightened temperature and at a decreased speed at lessened temperatures. The temperature has a direct influence on the percent of black color and light color present on larvae. The average black color was 65% in the cold treatment, 29% in the warm treatment, and 49.5% in the control treatment. The data has been graphed with a line of best fit. The line of best fit has a mean square error of 6.82x10-35, or too small to be significant.

 

Figure 1. Monarch larval stages

 

Introduction

The purpose of this project was to study the effects of temperature on the coloration of the Danaus plexippus (monarch butterfly) larvae and the development (i.e.: mass, length, speed of growth, and mortality at different stages) from egg stage through emergence as adults. Although very little work has been done which studies the coloration of monarchs in relation to the temperature, previous work regarding development in different climates has shown that cold climates slow the metabolic rate of larvae and adult monarch. The first hypothesis for this project was that the coloration of larvae in a cold climate would be darker than that of larvae in a warm climate since, dark colors absorb light and warmth to maintain warmer body temperature and in a warm climate, excess warmth from light is unnecessary, so the coloration would be lighter than that of larvae in a cold climate. The second hypothesis for this study was that the development of the larvae would be slowed in a cold climate and would be sped up in a warm climate. The mortality rates would be higher in both the cold and warm climate due to extreme temperatures. To keep a stable perspective, there was a control climate.

Background

Monarchs, as well as all other moths and butterflies, go through complete metamorphosis. The life cycle of the monarch, as seen in Figure 1, is divided into four distinct stages: egg, larva, pupa, and adult. The first stage of development is the egg stage, which lasts for four days under ideal conditions. Once the monarch hatches from its egg, it enters into the larva stage. The larva period is divided into five different stages called instars. An instar level shows how many times a larva has molted and can be distinguished by comparing the size of the head capsule to tentacle length.

 

Figure 2 - A fourth level instar molting to a fifth level instar



Since a larva cannot grow excessively with the protection of its cuticle, it must molt frequently as it develops. This can be seen in Figure 2. After molting for the last time and reaching the fifth instar level, a larva creates a silk pad then pupates to enter into the pupal/chrysalis stage of development. A monarch remains in chrysalis for up to two weeks under ideal conditions. During its tenure in a chrysalis, a larva’s inner organs and outer appearance rapidly change into those of an adult butterfly (Kuda and Oberhauser).

 

The coloration of monarch larvae varies in unique patterns of black, white, and yellow. As a larva develops, its color becomes more vivid and defined (Kuda and Oberhauser). Most larval coloration is one-half black, one quarter yellow, and one quarter white. However, different variations have been known to exist. For example, a coloration pattern consisting of black and white has been observed (Oberhauser). Those particular larvae with that color pattern are called “zebra” larvae (Solensky).

 

No research has yet been done that quantifies the color differences between larvae in warm temperatures and larvae in cold temperatures. However, color differences between climate larvae have been noted. Research performed by Dr. David James in New South Whales, Australia, proved that the larvae adapt to their environment by changing their colors and thus manipulating their body temperature. Although he did not quantify the color differences, Dr. James gave reason for their existence (James).

 

It was observed at the beginning of this project that, when moving larvae from a cold climate (used to slow metabolism and development in the monarch lab), that coloration was significantly darker than that of the larvae that remained in the room temperature (Solensky and Prysby). From this, the question was raised as to why those larvae were darker and whether larvae in warm temperatures would have lighter coloration. This developed into the question proposed by this study.

 

Procedure

Fifty eggs were placed into each of three treatment environments (as seen in Figure 3): a warm treatment that fluctuated between 22°C and 32°C during night and day respectively, a cold treatment that fluctuated from 6°C to 16°C, and a control treatment that fluctuated between 17°C and 27°C. The ten degrees of fluctuation mimicked a twenty-four hour day where the lighting in the treatments were programmed to be on during the day and off during the night hours. “Day” lasted from 5:00 A.M. until 9:00 P.M.

 

Figure 3 - A larvae cage in a cooler

 

The development of monarchs was monitored from the egg stage to the full adult. The eggs were monitored from the time they hatched until the point at which they emerged from the chrysalis. Ten larvae were randomly selected from each treatment environment (warm 22-32°C, cold 6-16°C, and control 17-27°C), their instar was determined [as according to A Field Guide to Monarch Caterpillars (Danaus plexippus)] and recorded. Then, as seen in Figures 4 and 5, each larvawas  measured with digital calipers (length from the head capsule to the anterior end not including the tentacles) and then was weighed on an electronic balance.

 

Figure 4. A larvae having its length measured

 

Figure 5. Massing a larvae

 

The masses and lengths of the ten larvae were averaged and recorded. All larvae present in each treatment were counted to determine the number that died. Once all the larvae in a treatment had reached the fifth instar level, the color of the ten random larvae was quantified, using the following unique presence absence method, as seen in Figures 6-9. The larva was placed on a post-it note and allowed to straighten. A small ruler was placed beside the larva, and the number of millimeters that contain black stripes, the number of millimeters that contain yellow stripes, and the number of millimeters that contain white stripes were counted and recorded. This was repeated for all ten larvae.

 

Figure 6. This figure shows how the color measurements took place. Black was recorded as 10 mm, yellow was recorded as 9 mm, and white was recorded as 6 mm.

 

Figure 7. A larvae on a post-it note ready to have its color measured

 

 

Figure 8. The color measurements being taken

 

 

As the butterflies emerged, their treatment, gender, personal identification number, and date of emergence (D.O.E.) were recorded.

 

Figure 9. An adult monarch having its wings measured

 

After all information was recorded, adults were released into the wild. Finally, mortality rates, color ratios, and development rates in the different treatments were analyzed and studied.

Results

As seen in Figure 10, the average of black color on the larvae was 65% in the cold treatment (6°C to 16°C). As seen in Figure 11, the average of black color on the larvae was 29% in the warm treatment (22°C to 32°C). As seen in Figure 12, the control treatment (17°C to 27°C) showed an average color of 49.5% black and 50.5% light (white and yellow). The significant difference between the three treatments is seen on Figure 13, which shows the difference in the percents of color. The range of yellow coloration was 32.9% (in the warm treatment) to 19.5% (in the cold treatment). The range of white coloration was 38.2% (in the warm treatment) to 15.5% (in the cold treatment). The percent of black coloration ranged from 65% (in the cold treatment) to 28.8% (in the cold treatment).

 

Figure 10

 

Figure 11

 

Figure 12

 

Figure 13

 

As seen on Figure 14, the population declined in all of the treatments, but the cold and warm treatments showed a more severe drop in population, and the control treatment had a less extreme decline in larval population. Figure 15 and 16 show that the average masses and lengths of the larvae increased gradually and showed a slight decrease in mass and length near the end of the measuring period. The period in which measurements could be taken lasted from the time at which the larvae are large enough to handle (about second instar level) to the time at which they pupate (directly following the fifth instar level). There was a 423.2 mg drop in average mass and a 4.85 mm drop in average length in the cold treatment. There was a 0.70 mm drop in average length and no drop in average mass in the control treatment. The larvae in the warm treatment had no drop in average mass or average length. As seen in Figure 17, the average instars increased in the warm and control treatments, but in the cold treatment, the second instars were too small to handle, so the measurements were taken at the beginning of the fourth instar level and lasted until pupation.

 

 

Figure 14

 

Figure 15

 

Figure 16

 

 

 

Figure 17

 

As seen in Table 1, the larvae in the warm treatment consumed 12 milkweed plants, the larvae in the cold treatment consumed 17 milkweed plants, and the larvae in the control treatment consumed 16 milkweed plants. As seen in Table 2, the average mass of the adult monarch butterflies was 417 mg in the warm treatment, 571 mg in the cold treatment, and 477 mg in the control treatment.

 

 

 

As seen in Figure 18, the three data points fall on the line of best fit with the equation f(x) = A + Bx + Cx2. When A = 45.3, B = 3.72, and C = -0.160, the mean square error in 6.82x10-35, which means that the average deviation from the line of best fit is insignificant

 

 

Figure 18

 

Conclusion

 

The larvae in the warm treatment developed at a greater rate with increased temperature, because increased temperature increases metabolic rate. This increased rate of development from the egg stage to the adult stage was the sole result of temperature and not of the amount of milkweed consumed, because the larvae in the warm treatment consumed the same amount of milkweed per day as the control group. Because the larvae in the warm treatment ate the same amount as the control group per day but developed significantly faster, they emerged as smaller butterflies than the larvae in the control group. As a result of the high temperature, the percent of dark color present on the larvae in the warm treatment was significantly lower than the percent of dark color on the larvae in the control treatment. This indicates that difference in coloration is an adaptation that helps larvae maintain an appropriate body temperature. The high temperature also increased death rate due to high humidity, the brown death (a bacteria found in lab situations that grows inside a larva and eventually kills it), and difficulties molting or pupating, all of which were seen in the experiment.

 

The larvae in the cold treatment developed at a slow rate with decreased temperature, because lowered temperature slows metabolic rate. Again, decreased rate of development was the sole result of temperature and not of the amount of milkweed consumed, because the larvae in the cold treatment consumed more milkweed than the larvae in the control group. Because the larvae in the cold group ate more than the control group per day but developed more slowly, they emerged as larger butterflies than the larvae in the control group. As a result of the low temperature, the percent of dark color present on the larvae in the cold treatment was higher than the percent of dark color on the larvae in the control treatment. Again, this strongly indicates that dark color is an adaptation to enable the larvae to absorb radiation from the light source to maintain an appropriate body temperature. The lower temperature also increased death rate. Another issue was the original temperature of the climate. The temperature was initially set from 6°C to 16°C, however, the eggs did not hatch at that range. After day nineteen, it was decided that the best course of action was to remove the eggs from the cold and see if the eggs could hatch at all. Refusing to completely give up on the eggs, the temperature in the climate was increased to 20°C during the day. The three-degree difference was enough for the larvae to hatch. One problem that followed the temperature change was that the larvae hatched at different times. When the majority of larvae were large enough to handle, some of the larvae had already reached the fifth-instar level. As a result of the variance, the instar levels, lengths, and masses were greatly different. This explains why the average lengths, masses, and instars were so small.

 

All results at the control temperature fell directly between the cold and warm treatments with the exception of population. The population for the control treatment was larger than the normal population of the cold and warm treatments, because there was little death, given ideal conditions of humidity, food, and pupating/molting not present at normal temperatures. The average masses, lengths, and instars increased in a similar way as the warm treatment, but remained a median between the cold and warm treatments. The dark color on the larvae in the control treatment comprised about fifty percent of the larvae’s coloration, a fact that also places it about halfway between the cold and warm treatment. It also proves that at normal temperatures, larvae will most likely not develop extremely dark or extremely light colors.

 

The percent dark color had an indirect relationship to the temperature. The data collected in this experiment were almost an exact fit to the line of best fit. The mean square error is so insignificant that it may as well not exist.

 

To further proceed in this study, there are questions that could be address. First how the humidity affected the growth of the larvae, because the humidity was a factor that was not addressed, how the abundance of food affected the growth, how the two different types of milkweed affected the growth, if there was any chance of the larvae hatching at the original temperature in the cold climate, how cold would it have to be before the larvae cannot hatch, how warm would it have to be before the larvae cannot hatch, what an adequate control temperature under ideal conditions would be, would the results have been different if the temperature was not fluctuating during the day, if the sixteen hour day was an appropriate length for the temperatures, if the constant light during the day was too strong for the larvae, and how the absence of shade affected the larvae.

 

Bibliography


Donnelley, Elizabeth. “Journey North.” Annenberg & CPB Math and Science Project August 1998. Learner. Online. Netscape. 25 Sept. 1998.
Address: http://www.learner.org/jnorth/

James,David. “Thermoregulation in Danaus Plexippus (L.) (Lepidoptera Nymphalidae): Two Cool Climate Adaption.” Diss. Macquarie University, 1986.

Kuda, Kristen and Oberhouser, Karen. A Field Guide to Monarch Caterpillars. St. Paul: National Science Foundation, 1997.

Oberhauser, Karen. Monarchs in the Classroom. St. Paul: National Science Foundation, 1997.

Oberhauser, Karen. Personal interview. a series of interviews beginning 6 June 1998 and ending 25 Sept. 1998.

Prysby, Michelle. Personal interview. a series of interviews beginning 6 June 1998 and ending 25 Sept. 1998.

Prysby, Michelle. “Impact of Natural Enemies on the Survival and Foraging of the Lepidopteran Herbivore, Danaun Plexippus.” Diss. University of Minnesota, 1998.

Solensky, Michelle. Personal interview. a series of interviews beginning 6 June 1998 and ending 25 Sept. 1998.