Ovapositioning Preference with Quinine Hydrate by Adult
Female Drosophila
Eric Schmit
Victoria Rottmann
Mercedez Kennedy
Department of Sciences, Loras College
1450 Alta Vista St.
Dubuque, IA 52001
Abstract: The study of ovapositioning is often done in Drosophila but they often test
whether a fly will choose the best medium compared to a lesser one, or whether they
prefer a similar medium to what they were reared in themselves compared to another of
similar qualities. The procedure performed test both of these theories against each other,
and found that the larger role is what the drosophila was reared in itself and not optimal
nutrition, however, much more research is found on what is the best nutrition selection
then how a drosophila makes this selection.
Introduction:
By rearing multiple generations of Drosophila on a mixture of standard lab quality
fly food and small levels of quinine hydrate, we had hope to raise a generation of flies that
could tolerate low levels of quinine, which is highly toxic to species such as drosophila. We
would then proceed to test this generation for ovapositioning preference, or the choice of
medium used to lay their offspring, for whether they would choose a medium with or
without the familiar quinine concentrations present to lay their eggs, with all else held
constant (Yang, Belawat). Because quinine is toxic to drosophila, the medium without any
levels of quinine would be a much higher quality medium.
To create a control variable, several generations of drosophila were reared under
the exact same conditions, except this group had no levels of quinine hydrate in their food.
This control group would be given the exact same amounts of food, water, light,
temperature and time for each generation so that other variables would not play as much
as an issue in our results.
Previous research studies has shown that the female drosophila will lay their
offspring in the best quality medium available (Sheeva), while other studies show that
female drosophila will select a medium close to what they were reared on
themselves(Yang, Belawat). With this knowledge and the must more present research that
data that female drosophila choose the better quality egg laying medium, we formed our
hypothesis that the female drosophila will choose the high quality medium without the
quinine levels, rather than the medium with the quinine levels present.
Methods:
This research was done in several stages. Our first stage was to create four different
variables or concentrations of quinine hydrate to be used to start our generations of
drosophila. We used eight glass bottles designed for rearing the drosophila, dedicating two
for each level of quinine present. One of these levels would be our control, and would only
receive 9 grams of Carolina Pre-made fly food and 30ml of deionized water each and were
appropriately labeled. The next two bottles received the same 9 grams of food, but instead
received 30 ml of .01 molar quinine hydrate. Due to the solution being a paste rather than a
liquid, 10 more milliliters of deionized water were added to allow to food medium to set.
The next two bottles were given 9 grams of fly food each, and were given 30 ml of .05 molar
quinine hydrate. The last two bottles were given 9 grams of fly food each and mixed with
30ml of .005 molar quinine hydrate.
Each bottle was allowed to set until the food formed an opaque pasty gel on the
bottom. After the food had set, groups of 40-60 drosophila melanogaster were added to
each bottle, capped with a sponge cork to allow quality air flow, and placed in an incubator
set at 20 degrees Celsius, each bottle being at the same shelf level to receive equal lighting.
The control group was the first to rear its first offspring generation. When the larva
matures in the food medium from the previous generation, it climbs up the side of the
bottle and turns a dark brown color. This means that that larva is readying to hatch and
become a young drosophila. When more than 25 of these dark larvas occur, which took the
control bottles 10 days, the bottle was emptied of all adults which were no longer needed
in this research. The control bottles remained untouched from that point for 12 hours,
allowing a new generation of drosophila to hatch. These were immediately collected in
bottles with the exact same food and water specifications, and placed back in the incubator
for five days.
To test ovapositioning, two chambers were created that consisted of large two quart
sized tubberware containers, with several small ventilation holes on the top. The
containers were flipped so that the lids became the bottom. On the lids, we placed six egg
laying mediums each.
Based on research, the best egg laying medium seemed to be an agar material made
by boiling 77.5ml water, 20 ml Welches pure grape juice (no sugars added), and 1 gram of
agar. After the substance reached a boil, we removed it from heat, and let it cool until it
reached 50 degrees Celsius. At this point, 0.5 ml glacial acid, 1.0 ml 95 percent Ethyl
Alcohol, and one drop of blue food coloring to help the eggs stand out. The solution was
poured into six empty petri dishes. To create a medium with quinine, we repeated the
above steps, but used only 67.5 ml water and 10ml of .01 molar quinine hydrate, to achieve
a desirable amount of quinine presence that would be evident to the drosophila, but not
lethal.
To test ovapositioning, 24 female drosophila were selected from each bottle and
placed in a different container, placing 4 on each medium. The flies would be subjected to
ether for a short term to knock them out, and the best of the females would be selected and
placed four on a dish. When the females awake in the breeding chamber, they are free to fly
to the medium of their choice to lay their eggs. The entire container is placed in a separate
incubator at 20 degrees Celsius for 12 hours for this process. After the 12 hour period, the
containers are removed, flies released, and the eggs are counted and recorded for each dish
under a dissection microscope.
The bottles containing .05 and .005 molar quinine levels achieved enough larvas to
hatch at the same time, seven days later than the control bottles on day 17. These bottles
were also then emptied of all adult drosophila, and sat untouched for 12 hours until the
newly hatched drosophila we removed, and placed in new bottles with the same
specifications. Due to very low levels of surviving drosophila, each quinine level group was
placed in one bottle. Now .05 and .005 levels only had one bottle. The .01bottles had yet to
achieve any larva ready to hatch, and the larva present appeared to be much smaller than
normal healthy drosophila.
After five days passed, the two bottles containing the new generation of quinine
reared drosophila were ready to be tested for ovapositioning. Again, the two containers
were set up, one for each level of quinine that the drosophila were reared one. Both
containers had the quality medium used in the control test, and the same concentration of
the quinine medium as used in the control test. The containers are treated exactly as the
control groups for 12 hours and the offspring are counted.
Results:
The presence of Quinine affected the growth and development of the drosophila.
This caused the number of offspring of each level of quinine to drop as levels increase. The
.01 group actual never succeeded at producing larva that matured enough to produce flies,
and the larva was always much smaller than the normal larva. The eggs laid by both the
.005 and .05 in the mediums were noticeably smaller then flies not reared on quinine, and
they also seemed to have laid larva rather than eggs more often than the control groups.
This effects were not found in any previous research.
The results for the ovapositioning procedure are shown per group:
Control Group (48 flies)
Quality Medium egg counts Quinine Medium egg counts
21
8
28
0
12
0
12
3
6
1
47
0
.05 Quinine Reared Group (24 flies)
Quality Medium egg counts Quinine Medium egg counts
2
6
0
3
2
14
.005 Quinine Reared Group (24 flies)
Quality Medium egg counts Quinine Medium egg counts
18
11
3
21
5
10
Without even looking at any statistical data, the two groups reared on quinine
seemed to have preferred laying their eggs in quinine-medium. When entered on statistical
software, SPSS, a Univariate Analysis of Variance proves with a significance value of .011
that there is a difference between the different groups. (Figure 1)
Figure 1: Tests of Between-Subjects Effects
Dependent Variable:egg_count
Source
Type III Sum of
Squares
df
Mean Square
F
Sig.
1402.958a
5
280.592
3.517
.022
Intercept
1793.067
1
1793.067
22.476
.000
Container
217.125
2
108.562
1.361
.282
32.267
1
32.267
.404
.533
925.792
2
462.896
5.802
.011
Error
1436.000
18
79.778
Total
5101.000
24
Corrected Total
2838.958
23
Corrected Model
Quinine_Presence
Container *
Quinine_Presence
a. R Squared = .494 (Adjusted R Squared = .354)
Figure 1. The bolded line, Container*Quinine Presence test for in-between group variance, and
shows that there is a significant difference between all three of the groups. The F-Value of 5.802
being the highest means that Groups separated by Quinine levels is the main source of variation.
Because there is a significant difference between each container, it must be broken
down to show the variance of each group from each other. This can be done by a Tukey
PostHoc test on SPSS. (Figure 2)
Figure 2: Multiple Comparisons
egg_count
Tukey HSD
(I) Container
(J) Container
Difference (I-J)
Control
.05 M reared
.005 M reared
95% Confidence Interval
Mean
Std. Error
Sig.
Lower Bound
Upper Bound
.05 M reared
7.0000
4.46592
.285
-4.3978
18.3978
.005 M reared
.1667
4.46592
.999
-11.2311
11.5644
Control
-7.0000
4.46592
.285
-18.3978
4.3978
.005 M reared
-6.8333
5.15680
.400
-19.9943
6.3277
Control
-.1667
4.46592
.999
-11.5644
11.2311
.05 M reared
6.8333
5.15680
.400
-6.3277
19.9943
Based on observed means.
The error term is Mean Square(Error) = 79.778.
Figure 2: The above table shows the significance of each group compared to other groups. None of
the groups are significantly related to one another
The best way to visually see the trend that the drosophila follow as quinine levels
increase is through a simple graph of the data of mean egg and the presence of quinine is
the egg laying medium. (Figure 3)
Discussion:
Based on our findings, we cannot reject our null hypothesis that the female
drosophila will not select the best medium available to rear their offspring on. Instead, we
could further our research by testing a new hypothesis that the female drosophila are most
likely to rear offspring on a medium that they were reared on themselves.
Possible sources of error came from not having enough flies due to the toxicity of
quinine to drosophila, and the negative effects that it had on the surviving flies. There is
much research that suggest many factors why a female drosophila may select an egg laying
medium, and it is possible that too many factors play a role to get a clear, significant choice.
Acknowledgments:
For the research, many thanks is given to Dr. Fred Schnee of Loras College for
helping to set up the lab procedure and provide necessary equipment. Dr. Schnee
continuously checked up on the research and had given much insightful information.
Thanks also go out to Melissa Herman for explaining how to properly and efficiently
proceed with the processed involved with rearing the drosophila.
Literature Cited:
Lee, K. P., Simpson, S. J., & Taylor, P. W. (2007). Lifespan and reproduction in drosophila:
New insights from nutritional geometry . National Academy of Sciences, (Sept), 109.
Sheeva, B., Madhyastha, A., & Joshi, A. (1998). Oviposition preference for novel versus normal
food resources in laboratory populations of drosophila melanogaster . Journal of
Biosciences, 23(2), 93-101.
Sheeva, B., Rajamani, M., & Joshi, A. (1999). Research communications - bimodal distribution
of oviposition preference for a novel food medium in drosophila melanogaster . Current
Science, 9, 77-80.
Vijendravarma, R. K. (2009). Effects of parental larval diet on egg size and offspring traits in
drosophila. (Master's thesis, University of Lausanne, Lausanne, Switzerland ).
Yang, C. H., Belawat, P., Hafen, E., Jan, L. Y., & Jan, Y. N. (2008). Drosophila egg-laying site
selection as a system to study simple decision-making processes.