Larval Color Project
Elizabeth Larkin
Breck High School
Minneapolis, MN
Abstract | Introduction | Background
| Procedure | Results |
Conclusions | Bibliography
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.
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 (ie:
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 extream 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 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, and a second level instar can be seen in Figure 1. 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. 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).
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Figure 1. A second instar.
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Figure 2. A fourth instar molting to a fifth instar.
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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.
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Figure 4. Measuring the length of a larva.
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Figure 5. Weighing a larva.
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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 larva was weighed on an
electronic balance and measured with digital calipers (length from the head capsule
to the anterior end not including the tentacles). 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, 7, 8,
9, and 10. 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 would be
recorded as 10 mm, yellow would be recorded as 9 mm, and white would be recorded
as 6 mm.
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Figure 7. A larva on a Post-it note, ready to have its color measured.
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Figure 8. The color measurements being taken
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Figure 9. A finished Post-it note
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Figure 10. An adult monarch having its wings measured
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As the butterflies emerged, their treatment, gender, personal identification number,
and date of emergence (D.O.E.) were recorded. 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 12, the average of black color on the larvae
was 65% in the cold treatment (6°C to 16°C). As seen in Figure 13,
the average of black color on the larvae was 29% in the warm treatment (22°C
to 32°C). As seen in Figure 14, 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
15, 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).
As seen in Figure 16, 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. Figures
17 and 18 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 19, 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.
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 20, 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.
Conclusions
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 which 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, 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.
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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, Danaus Plexippus." Diss. University of Minnesota,
1998.
Solensky, Michelle. Personal interview. a series of interviews beginning 6 June
1998 and ending 25 Sept. 1998.