1. Starvation of Drosophilia melanogaster during early development reveals energy
conservation across temperatures
Khalid Al-Rayess, Michael Eisen, Steven Kuntz
University of California, Berkeley
Molecular and Cell Biology Honors Thesis
Abstract
Cold-blooded animals develop faster at warmer temperatures than cold temperatures, but
not all processes change evenly with temperature. Developmental processes, energy
usage, and feeding behavior are all affected by temperature, but separating these
processes has remained elusive. Though the total time of embryogenesis varies over two-
fold, the relative timing of events in embryogenesis scales uniformly. However, larval
growth does not scale uniformly between warmer temperatures and colder temperatures.
Therefore, how energy is utilized as developmental and feeding rates change with
temperature is unknown. To understand this mechanism better, I set up a time-lapse
imaging assay system where developmental rates and post-hatching survival were
determined. I observed the effect of starvation in Drosophila melanogaster through the
first instar following incubation and hatching at different temperatures (17˚C, 21˚C, and
27˚C) for 3-4 days. On average, post-hatch survival lasted 83% of embryonic
development, with no significant changes between temperatures detected. I found that
energy usage likely scales evenly across temperatures during embryogenesis and early
development. Therefore, any molecular mechanism driving development forward equally
affects energy usage independent of temperature. To further understand this mechanism,
different mutant lines that govern metabolism, chrb, CG16758, and Thor, were tested at
starvation with the same range of temperatures to explain whether one of these mutants
play a role in controlling the rate of development forward. Surprisingly, the mutant that
had most interesting results was Thor. Thor survived proportionally longer than any of
the mutants, thus indicating that that it could be an important regulator in maintaining the
uniform timing of events in D. melanogaster development despite stress induced.
Introduction
We understand that temperature impacts development at warm and cold temperatures.
However, it imposes a challenge when comparing development among species as it was
unclear whether species should be compared at different temperatures or at the same
temperature in order to maintain the same developmental rate (Kuntz and Eisen (2014)
PLOS Genetics). The work of Steven Kuntz and Michael Eisen addressed this challenge.
They used time-lapse imaging to track the progress of embryogenesis from egg lay
through hatching using 7 different temperatures between 17.5°C and 32.5°C. Using
different annotations to track changes in the embryo. They found that although certain

2. milestones in development vary over two-fold, the timing of these certain events is
constant across temperatures. (Kuntz and Eisen (2014) PLOS Genetics). Thus, this
allowed developmental biologists to compare species by developing them at different
temperatures. However, not much is known about how energy and resources are utilized
in the face of changing developmental temperatures. I tried to understand this by
investigating whether energy usage is conserved across temperatures like development
rate.
Methods
I developed a time-lapse assay where developmental rates and post-hatching survival
were determined. This time-lapse assay is a lot different than the one utilized by Kuntz
and Eisen. The time-lapse assay they utilized was more particular and precise as it looked
at specific milestones in embryogenesis. Therefore, they were only able to image a small
number of embryos. The time-lapse assay I created used a 96-well plate that imaged
about twelve wells at once. 3-5 embryos were placed in each of these wells. Due to the
number of embryos imaged at once and the fact that fly larvae lasted 3-4 days, it was hard
to establish good imaging quality until certain issues were resolved.
I. Imaging Assay
In my assay, I used a time-lapse camera (JVC HD:GZ-HM690) attached to a tripod. This
camera was able to zoom in to the region on the 96-mircowell plate where the embryos
were placed. However, it was difficult to a capture a quality image because of increased
depth of focus. This was resolved by adding high-contrast edges to target auto-focus on
the region of interest. To avoid dehydration of the embryos, the wells were filled with
50μL water. To avoid evaporation of water the wells were sealed with an oxygen semi-
permeable membrane. This membrane was able to retain moisture over long-time scales
but prevent hypoxia. The membrane was also vented so that no condensation appeared on
the membrane so that embryos could still be tracked. Furthermore, the entire 96-mirco
well plate was placed on a light table, allowing for high-contrast bright field imaging. In
order to transfer the embryos to the mircowell plate I developed a cooper wire tool that
was able to maintain the integrity of the embryo. Finally, these embryos developed at
different temperatures (17°C, 21°C, and 27°C) but sometimes the temperatures were
inconsistent in different areas that the embryos were grown. Therefore, to avoid
inconsistency these embryos were incubated in an incubator that had adjusted
temperatures.
II. Embryo observations
I observed the effect of starvation in the Drosophila melanogaster through the first
instar.The D. melanogaster were first placed in the well as considered embryos.
However, after incubation for 3-4 days the D.Melanogaster develops into a larva through
the 1st instar. I was able to observe this by manually annotating hatching and arrest in the
D. melanogaster time-lapse video. Hatching is defined as initial movement of embryos
and arrest is defined as full stoppage of these movements (usually the larva).
Results and Discussion
1. Wildtype D.melanogaster

3. After multiple trials of the wildtype D. Melanogaster (OreR), I tried to characterize
average hatching and arrest times in different temperatures:17°C, 21°C, and 27°C (Figure
1). What I found is that at 17 °C hatching takes a long time to initiate. The average
hatching time for 17 °C is approximately 32 hrs. The average arrest time for 17 °C is
approximately 60 hrs. For 21 °C the average hatching time is approximately 17 hrs and
average arrest time is approximately 31 hours. For 27 °C the average hatching time is
approximately 15 hours and average arrest time is approximately 27 hours. These results
indicate that hatching and arrest in wildtype D.melanogaster follow similar responses to
temperature both slowing as temperatures decrease.
Figure 1. Hatching and arrest in wildtype D.melanogaster
Second, I characterized the ratio of post-hatching survival/ hatching in order to
understand whether this ratio is consistent across temperatures (Figure 2). The average
ratio I found among all temperatures is approximately 0.8 with no significant difference
found between temperatures. Thus, the ratio of post-hatch survival time to embryonic
development time remains consistent across temperatures. Indicating that the time it takes
the larvae to move scales evenly with the time it takes embryos to develop.
Figure 2. The ratio of post-hatch survival time to embryonic developmental time

4. 2. Mutants
Any molecular mechanism controlling the developmental rate appears to equally affect
energy usage independent of temperature. To further understand this mechanism, I
analyzed three with mutations in metabolic genes known to change expression with
temperature. These three mutations tested are chrb (Redd 1), CG16758 (PNP), and Thor
(4E-BP). Furthermore, these genes are involved in the input and output of the Tor
pathway (which is important in cell growth and survival). Similar to the wildtype
D.melangoster I set to characterize hatching and arrest times in each mutant strain across
different temperatures (Figure 3). I found that the average hatching times in each strain
chrb, CG16758, and Thor were approximately 32-34 hours at 17°C and average arrest
times for chrb, CG16758, and Thor were 68 hours, 77 hours, and 80 hours respectively at
17°C. The average hatching times in chrb, CG16758, and Thor were approximately 20
hours at 21°C and average arrest times in chrb, CG16758, and Thor were 40 hours at
21°C. The average hatching times in chrb, CG16758, and Thor at 27°C were
approximately 17 hours and the average arrest times in chrb, CG16758, and Thor were
approximately 32-35 hours. Therefore, the hatching and arrest time in mutant flies
respond to temperature similarly to wildtype. This means that across the three different
mutations embryos at warmer temperatures developed faster than at colder temperatures
with no mutant specific changes in developmental rate between species.
Figure 3. Hatching and arrest time in mutant flies
Finally, I set to characterize the ratio of post-hatch survival/ hatching in these mutant
strains (Figure 4). Specifically, among all temperatures in wildtype D.melangoster the
average ratio is approximately 0.8 and similarly at 21°C and 27°C chrb and CG16758 had
ratios of approximately 1. Thus, indicating that at temperatures of 21°C and 27°C chrb
and CG16758 survived just as long as wildtype. Although, chrb and CG16758 had ratios
of approximately 1.5 at 17°C and survived longer than wildtype at this temperature, this
result wasn’t as interesting and significant as the Thor responses. At 21°C Thor had a
ratio of approximately 1.7 compared to approximately 0.8 for wildtype. And at 27°C Thor
had a ratio of approximately 1.5 compared to 0.8 for wildtype. With the limited trials
with chrb, CG16758, and Thor mutants, it appears Thor at 21°C and 27°C is surviving
proportionally longer than wild type following hatching. This is important because it
seems as if Thor plays a role as an important regulator in controlling developmental rate
forward.

5. Figure 4. Ratio of hatching to arrest in mutants compared to wildtype
Conclusions/Further work
After comprehensive tests in wildtype Drosophilia melanogaster at 17.5°C post-hatching
survival lasts 27 ±12 hours, at 21°C survival lasts 13 ± 5 hours, and at 27. 5°C lasts 9 ± 4
hours. On average, post-hatch survival lasted 83% of embryonic development, with no
significant changes between temperatures detected. Thus, energy usage is likely
conserved across temperatures during embryogenesis and early development. Out of the
three different mutant lines tested chrb, CG16758, and Thor, Thor is the most interesting
mutant that provides insights to the mechanism that controls developmental rate. This is
important in developmental biology because it can offer an understanding of how a
species can still develop at a uniform same rate regardless of different stresses induced in
it’s environment. Therefore, a species can be studied at different conditions with the
understanding that energy usage and development rate won’t be affected. In order to
characterize whether Thor is important, more precise tests of the mutant need to be taken.
A time-lapse assay that closely annotates events in embryogenesis through instar 1 will
further confirm whether Thor is regulating developmental rate.
Acknowledgements
I would like to thank my mentor and post-doc Steven Kuntz for allowing me the
opportunity to participate in research and teaching me so much over the last couple of
years.You have inspired me to be a better scientist and continue to be involved in
research. Thank you also to the Eisen lab and Michael for providing a fun environment to
learn and grow.
References
1.Kuntz SG, Eisen MB (2014). Drosophila embryogenesis scales uniformly across
temperature in developmentally diverse species. PLoS Genet 10(4): e1004293. doi:
10.1371/journal.pgen.1004293.