The document summarizes research investigating how energy usage in Drosophila melanogaster larvae changes with temperature during development and early starvation. A time-lapse imaging system was used to observe embryonic development, hatching, and larval movement at different temperatures. It was found that the ratio of post-hatch survival time to embryonic development time remained consistent across temperatures, indicating energy usage scales evenly with developmental rate. Additional mutant lines involved in metabolism were tested and one named Thor showed potential differences in energy usage over temperatures that warrant further investigation. The research aims to understand the mechanisms controlling how energy is utilized as developmental and feeding rates change with temperature.

1. 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 (18˚C, 21˚C, 28˚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, are being tested
at starvation with the same range of temperatures to explain what is responsible for
controlling the rate of development forward.
Introduction
Recently, time-lapse imaging to track the progress of embryogenesis from egg lay through
hatching has established the relationship between developmental rate and temperature
(Kuntz and Eisen (2014) Plos Genetics). To better understand how energy and resources
are utilized in the face of changing developmental and feeding rates, I am investigating
how the rate of energy usage changes across a range of temperatures.
Figure 1 D. melanogaster embryos respond to temperature uniformly
A) Each point represents an event in embryogenesis. Larval growth does not scale
uniformly between warm and cold temperatures. Total of time of embryogenesis varies
over two-fold.
B) When normalized the relative timing of events scales uniformly.
Eisen and Kuntz
,PLoS Genet.2014
;10(4):e1004293
Methods
Figure 2. Embryonic development and larval survival are observed with a time-lapse assay.
I set up a time-lapse imaging assay system where developmental rates and post-hatching survival
were determined.
Time lapse camera
Tripod
Light table
96-microwell plate
Figure 3. Oxygen-permeable membrane
retains moisture over long time-scales but
prevents hypoxia. Membrane placed on top of
wells prevents evaporation and dehydration of
embryos.
Oxygen Permeable Membrane
Figure 4. Timelapse imaging captures
changes in larval movement. Up to twelve
wells (with 50µLwater and 3-5 embryos each)
in a 96-microwell plate were imaged
simultaneously.
Embryos placed in well
Figure 5. Picture of D. melanogaster embryo Figure 6. D. melanogaster 1st instar larva
Key Issues with Assay Solution
Throughput Set up imaging in 96-well plate for
simultaneous, independent observations
Focal depth Added high-contrast edges to target auto-
focus
Dehydration Filled wells with 50µL water
Evaporation Sealed wells with semi-permeable
membrane
Condensation Vented the semi-permeable membrane
Temperature inconsistency Incubation
Lighting Light table for high-constrast bright-field
imaging
Transferring embryos into wells Developed cooper wire tool
Table: Assay required maintaining quality
imaging as well as fly larval fly survival across
3-4 days
Video is a time lapse from Thor mutant
developing at 27°C (goo.gl/pdxvcf)
Results
Temperature
Figure 7. Hatching and arrest in wildtype
D. melanogaster follow similar responses to
temperature, both slowing as temperatures
decrease. Hatching is defined
as initial movement of embryos and arrest
a stoppage of movements.
Averageratio
(post-hatchsurvival
/hatching)
Figure 8. The ratio of post-hatch
survival time to embryonic
development time remains consistent
across temperatures. The time that larvae
can move scales evenly with the time it
takes embryos to develop.
Any molecular mechanism controlling the developmental rate appears to equally affect
energy usage independent of temperature.To further understand this mechanism, I analyzed
three lines with mutations in metabolic genes known to change expression with temperature.
Temperature
Averagetime(hrs)
chrb (Redd1) CG16758 (PNP) Thor (4E-BP)
Figure 9. Hatching and arrest time in mutant flies respond to temperature similarly to
wildtype. Across the three different mutations (chrb, CG16758, and Thor), embryos at
warmer temperatures developed faster than at colder temperatures,with no mutant specific
changes in developmental rate between temperatures.
Temperature
Figure 10. Ratio of hatching to
arrest may differ in Thor mutants
compared to wildtype. With the
limited trials with chrb, CG16758, and
Thor mutants, it appears that Thor
warrants further testing, as at 17°C
and 27 °C Thor is surviving
proportionally longer than wildtype
following hatching.
Time Lapse Video In wildtype Drosophila melanogaster at
17.5°C post-hatching survival lasts
27±12 hours, at 21°C survival lasts
13±5 hours, and 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.
Conclusions
The three different mutant lines that govern
metabolism, chrb, CG16758, and Thor, are still
being tested at starvation with the same range
of temperatures to further understand the
mechanism controlling energy usage. Currently,
Thor seems like the most interesting mutant to
provide unique insights to this mechanism.
Therefore, more trials are being tested on Thor.
The distribution of movement may change
across temperatures and between mutants, so
comparisons between the two will be
conducted to look for trends.
Further Work
Acknowledgements
I want like to thank my mentor and post-doc Steven Kuntz for allowing me the opportunity to
participate in research and teaching me so much. This project wouldn’t be the same without your
insight. Thank you also to the Eisen lab and to Mike Eisen for providing a fun environment to learn
and grow. Finally, thank you to Ugo Nwosu for your time and effort in helping to create this poster.
Starvation of Drosophilia melanogaster during early development reveals energy conservation across temperatures
Khalid Al-Rayess , Michael Eisen, PhD , Steven Kuntz, PhD
Molecular Cell & Biology: Cell and Developmental Emphasis
Department of Molecular & Cell Biology, University of California, Berkeley
1,2 2 2
1
2
Averageratio
(post-hatchsurvival
/hatching)
Temperature
Averagetime(hrs)