1. The Effects of Different Temperatures on Energy Levels in Wooly Bear
Caterpillars Pyrrharctia isabella
Megan Mignogna and Stacy Cameron
Department of Biology, University of Mount Union
INTRODUCTION
• Animals need energy to perform work and maintain their molecular and structural integrity. How
an animal uses energy resources may determine survival and reproduction success, and thus
overall fitness.
• Rapid climate change may have a detrimental impact on many animal species, especially
ectotherms. The global mean surface temperature is predicted to increase by 1.4 to 5.8°C from
1990 to 2100 [6]. Increasing temperatures may cause winters to be warmer, spring to arrive
sooner and summers to last longer. The impact of global warming is uncertain. Earlier springs
may mean ectotherms, such as insect larva may progress into the pupal stage sooner. Longer
summers may allow the overwintering larva to eat more and thus store more energy reserves
prior to winter dormancy. Since many ectotherms use debris and snow as a source of insulation
to survive, increased temperatures along with reduced snowfall can lead to decreasing
overwintering survival. Changes in winter temperatures and the amount of time spent in colder
and warmer temperatures can affect the overwintering energy expended and may impact the
populations of insects in the spring months [2].
• The primary energy storage molecule for insects is triglycerides. Triglycerides are efficient energy
storing molecules, as more energy can be stored per unit weight of fat than in glycogen [5]. Most
insects possess fat bodies composed of cells called adipocytes. The function is to store and
release energy in response to energy demands of the insect [1]. The amount of energy reserves
the insect has may affect the rate of growth, timing of metamorphosis and reproduction. During
larval stages, accumulation of enough energy reserves is important for surviving the dormant
stage of metamorphosis and possibly fueling mating activity of the non-feeding adult stage.
• We used the Isabella Tiger moth larva (Pyrrharctia Isabella) to investigate how the varying
overwintering temperatures would affect their energy reserves. Triglyceride levels were
examined from caterpillar maintained in temperature treatments of 2°C, 5°C, and 8°C. Because
body temperature is strongly correlated with an ectotherms metabolic rate, we hypothesized that
the triglyceride levels would be lower with increasing temperatures.
MATERIALS AND METHODS
• Animal Maintenance
Pyrrharctia isabella caterpillars were collected in November 2013 from Columbiana County, OH.
They were maintained in a 21.5 cm X 34 cm X 19 cm, clear container that contained leaf litter and
holes in the lid. Paper towels were placed within the enclosure and damped once a week to
maintain moisture. The caterpillars were maintained in a 12:12 LD photoperiod.
• Treatments
In November, caterpillars were weighed to obtain their initial masses. A subsample (n=12) were
flash frozen in liquid nitrogen to determine the starting concentrations of triglycerides and glycerol.
The remainder of animals were divided into three temperature treatments: 2 °C, 5 °C, and 8 °C (n=24
per treatment). Animals were maintained at these temperatures for 120 days in the dark (0:24 h).
• Energy Reserve Assays
At the end of the 120 days, we measured the total triglyceride and free glycerol of individuals from
each treatment group. Twelve randomly selected animals from each treatment group were flash
frozen in liquid nitrogen. Animals were weighed to acquire wet masses and then placed at 65 °C for
7 days to determine the dry masses. Dried caterpillars were stored at -70 °C before performing
triglyceride and free glycerol analysis. Caterpillars were homogenized in 10mL of 0.05% Tween 20,
then centrifuged (7 min.). A 20X dilution of the supernatant with 0.05% Tween 20 was frozen at -
20°C until the triglyceride concentrations were measured (Triglyceride Colorimetric Assay Kit
#10010303; Cayman Chemical Company, USA), and a 100X dilution of the supernatant with 0.05%
Tween 20 was frozen at -20 °C until the glycerol concentrations were measured (Sigma-Aldrich,
catalog # MAK117, Canada). The triglyceride and glycerol in the supernatant were then assayed in a
triplicate using spectrophotometric assays. The Triglyceride Colorimetric Assay Kit obtained the total
amount of glycerol from each animal. The Sigma-Aldrich Glycerol Assay Kit obtained the amount of
free glycerol from each animal. The amount of free glycerol was subtracted from the total glycerol
amount obtained from the Triglyceride Colorimetric Assay Kit to determine the amount of
triglycerides in each animal. An analysis was then conducted to compare the different temperature
treatment groups.
• Data Analysis/Statistics
SPSS (IMB SPSS Version 21) software was used to calculate the statistics. A T-test was used to test
between the starting group compared to each of the temperature treatments. ANOVA was used to
make comparisons across the three treatment groups.
RESULTS AND DISCUSSION
• Ectotherms constitute the vast majority of terrestrial biodiversity and are
especially likely to be vulnerable to climate change because their basic physiology
functions such as locomotion, growth and reproduction which are strongly
influenced by environmental temperature [3].
• We examined the effects of temperature on the energy reserves (i.e. triglycerides
and glycerol) in the overwintering ectotherm, Pyrrharctia isabella larvae.
• Average dry masses, were significantly different between the starting group and
each of the three treatment groups. However, there was no significant difference
across the three treatment groups, suggesting temperatures between 2°C and 5°C
does not significantly alter energy use during this period (Figure 1, Table 1).
• Free glycerol concentrations did not significantly differ between the starting group
and each of the three treatment groups but were significantly different across the
treatments (Figure 3, Table 3). Hahn et. al, (2007) reported that as the temperature
decreases during diapause, the glycogen storage is converted to glycerol. At the
end of the overwintering period, unused free glycerol may be recycled into other
metabolic substances, such as glycogen or triglycerides [5]. This would suggest
glycerol values would start low, increase during the overwintering period, and
decline as temperatures begin to increase at the end of the overwintering period.
The opposite would occur with glycogen values, where the glycogen is converted
to glycerol as temperatures decrease and will rise as the temperatures increase at
the end of the overwintering period.
• We observed a significant increase in triglycerides with increasing temperature. If
metabolic rate does not significantly change over the range of the treatment
temperatures, then possibly the unused glycerol may have been recycled into
metabolic substances, such as triglycerides [5]. Accumulation of triglycerides may
be beneficial in fueling metamorphosis and mating during the post-diapause stage,
especially since adults do not feed.
• A future direction for this study would be to examine the metabolic rates of the
larvae over a wider range of temperatures in addition to other storage molecules,
such as glycogen and protein. This would allow use to examine a wider profile of
the energetics of overwintering larvae over a greater range of environmental
temperatures.
REFERENCES
1. Arrese, Estela L. "Insect Fat Body: Energy, Metabolism, and Regulation." Annu Rev Entomol 55
(2010): 207-25.
2. Bale, JS., Hayward, SAL. “Inset overwintering in a changing climate.” The Journal of Experimental
Biology 213 (2010): 980-994.
3. Deutsch, Curtis A. "Impacts of Climate Warming on Terrestrial Ectotherms across Latitude."
Proceedings of the National Academy of Sciences 105.18 (2008): n. pag.
4. Ed Meyertholen. "Lipids." Austin Community College. SCILINKS from NSTA, n.d. Web. 18 Apr. 2014.
5. Hahn, Daniel A., and David L. Danlinger. "Meeting the Energetic Demands of Insect Diapause:
Nutrient Storage and Utilizations." ScienceDirect 53 (2007): 760-33.
6. Karuppaiah, V., and G.K. Ujayanad. "Impact of Climate Change on Population Dynamics of Insect
Pests." World Journal of Agricultural Sciences 8.3 (2012): 240-46. IDOSI Publications.
ACKNOWLEDGEMENTS
We thank Dr. Spiro Mavroidis for all his help and guidance during this project. We also
would like to thank Tom Wise for his assistance with this project and the Biology
Department for supplies and instrumentation needed to conduct the research.