Studying Stress and Acute Toxicity Using Drosophila melanogaster
Krista Carter
Advisor: Dr. Brown
Introduction
Drosophila melanogaster has been used to study the many effects of
stress on an organism. The stress response in D. melanogaster has been
shown to increase or decrease its longevity depending on the severity of the
stress (Vermeulen and Loeschcke, 2007, 154). Resistance to certain types of
stress have been linked in D. melanogaster. For example, fruit flies resistant to
desiccation were also found to be resistant to heat stress (Bubliy and
Loeschcke, 2005, 794). However, there has been no research investigating how
stress affects the response of D. melanogaster to a toxin.
The acute toxin used in this study was the organophosphate insecticide
malathion. Insects metabolize malathion to malaoxon, which inhibits the
enzyme acetylcholinesterase and causes the neurotransmitter acetylcholine to
build up (Aker et al, 2008, 549). The continuous nerve signals caused by
acetylcholine lead to death for insects. The LC50 of D. melanogaster for a
twenty-four hour exposure to malathion has been found to be 2.12 µg/ml (Jones
et al, 2010, 1108).
The objective of this study was to compare the acute toxicity response of
fruit flies stressed by heat or cold to the response of unstressed flies. Doseresponse curves were used to determine the LC50 for stressed and unstressed
flies. A null hypothesis was used, and it was predicted that there would be no
difference in the acute toxicity response of stressed and unstressed flies.
Materials and Methods
Flies were exposed to heat or cold stress for a period of 48 hours and
then exposed to malathion for 24 hours. Unstressed flies were also exposed to
malathion for 24 hours. Mortality was recorded at the end of the malathion
exposure. Between 50 and 100 flies were used for each stress condition and
malathion concentration.
Figure 1. Stress and malathion exposure
Stress
2 days
Measure
Malathion Mortality
1 day
Flies were housed in plastic containers with a feeding tube containing a
5% glucose solution and a cotton plug. In order to expose the flies to malathion,
a variation of Miyo et al’s method was used (2001, 224). A commercially
available solution of malathion was diluted in acetone to different
concentrations. The solutions were applied to filter paper, and the acetone was
allowed to evaporate, leaving behind the malathion. The filter paper was placed
in the containers housing the flies (figure 2). Filter paper wet with acetone only
was used as a control.
Figure 2. Using filter paper
to expose flies to malathion
Results
In order to ensure that the flies were stressed, the maximum heat and
cold temperatures at which the flies could survive for 48 hours were found. The
heat stress exposure was determined to be 29° C. Flies could survive at 7° C,
but this temperature posed a problem because it restricted their movement so
that they could not reach the feeding tube. A temperature of 10° C was used for
the cold stress exposure since it was the lowest temperature at which the flies
could move enough to reach the glucose solution.
The response to different concentrations of malathion varied widely
between populations. No consistent dose-response curve for unstressed flies
could be determined, and there was a wide variability in the LC50 values for
different trials (Table 1).
Trial date
LC50 (µg/ml)
11-2-10
14.2
11-7-10
19.5
1-31-11
116.7
2-11-11
219.6
2-12-11
158.3
2-19-11
128.5
Table 1. A sample of the
LC50 values determined from
different trials
Because no consistent dose-response curve was found, a new strategy
was used. Instead of finding an average dose-response for unstressed flies,
each population of flies was tested individually to determine what
concentrations of malathion to use. Flies from each population were stressed,
and their responses were compared to the response of unstressed flies from
the same population.
Figure 3. 2-23-11
trial of testing an
individual
population. Heat
stressed flies are
not shown
because their
containers were
contaminated,
and the majority
of flies died while
being stressed.
Figure 3 shows that the unstressed flies did not display the expected
response for a dose-response curve. Malathion concentrations of 100 and 125
µg/ml caused no difference in mortality compared to the control, and only the
highest concentration tested killed a significant percentage of the population.
The cold stress flies show a trend of decreased mortality with increased
malathion concentration.
Figure 4. 3-2-11
trial of testing an
individual
population.
Figure 4 shows that the mortality of the unstressed flies did not change
significantly at any malathion concentration tested. An accurate dose-response
curve could not be constructed. For heat stressed flies, only the highest
concentration of 225 µg/ml resulted in a slightly higher mortality compared to
the control. The cold stressed flies displayed an increased rate of mortality
when exposed to malathion. However, no LC50 could be determined for the cold
stressed flies.
Figure 5. 3-2611 trial of testing
an individual
population.
The responses of the unstressed flies in figure 5 could not be used to
construct an accurate dose-response curve. The heat stressed flies not
exposed to malathion had 100% mortality, and the cold stressed flies not
exposed to malathion had 48% mortality. These high mortality rates for the
control groups suggests that the flies from this population were much more
susceptible to the stress conditions than previously studied populations.
Therefore, it cannot be determined whether the flies died from malathion toxicity
or from the effects of the stress.
Conclusions
The effects of exposure to heat and cold stress on acute malathion
toxicity for D. melanogaster have not been determined. No consistent doseresponse curve could be found for unstressed flies. When comparing
individually tested populations, there were no consistent trends in the
responses of heat and cold stressed flies compared to unstressed populations.
Several factors could have contributed to the inconsistencies seen in the
response of D. melanogaster to various malathion concentrations. The stock
populations of flies were not kept at a consistent temperature. Flies used during
winter may have been exposed to more drastic temperature changes
throughout the day compared to flies used during autumn and spring. Changes
in humidity may have also affected the flies. When testing different malathion
concentrations, the containers housing the flies were placed in a plastic storage
bin with a wet paper towel to keep the humidity fairly constant. However, the
stock populations were not kept at a consistent humidity. It is not clear how
much of an effect the age of the flies had on their response to malathion. Flies
were typically used in testing 1-2 days after eclosion, but in a few trials flies
were used up to 6 days after eclosion. However, even the responses of flies of
the same age were inconsistent.
Literature Cited
Aker W, Hu X, Wang P, Hwang H-M. 2008. Comparing the relative toxicity of malathion and
malaoxon in blue catfish Ictalurus furcatus. Environmental Toxicology 23 (4): 548-54.
Bubliy OA, Loeschcke V. 2005. Correlated responses to selection for stress resistance and
longevity in a laboratory population of Drosophila melanogaster. Journal of Evolutionary
Biology18: 789-803.
Jones RT, Bakker SE, Stone D, Shuttleworth SN, Boundy S, McCart C, Daborn PJ, ffrenchConstant RH, van den Elsen JM. 2010. Homology modelling of Drosophila cytochrome
P450 enzymes associated with insecticide resistance. Pest Management Science 66:
1106-15.
Miyo T, Takamori H, Kono Y, Ogume Y. 2001. Genetic variation and correlations among
responses to five insecticides within natural populations of Drosophila melanogaster.
Journal of Economic Entomology 94: 223-32.
Vermeulen CJ, Loeschcke V. 2007. Longevity and the stress response in Drosophila.
Experimental Gerontology 43: 153-9.
Acknowledgments
I would like to thank my advisor Dr. Brown for guiding me through this project and for giving me many
invaluable insights and unlimited support. I am also grateful to the 2010-2011 capstone class for providing
feedback about my research and presentations. This research was sponsored by the Marietta College
Biology Department.