Characterization of Glutamate receptors at the Drosophila Neuromuscular Junction DEVAL BHATT AND ROBIN L. COOPER, DEPARTMENT OF BIOLOGY, UNIVERSITY OF KENTUCKY, LEXINGTON, KY 40506-0225

1

Educational material integrating math and biology
for middle school, high school, and introductory
college sciences classes Population dynamics and
physiology of Drosophila.
SAMUEL POTTER1, PHILIP H. CROWLEY1, REBECCA M.
KRALL2, SUSAN MAYO3, DIANE JOHNSON3, KIM
ZEIDLER-WATTERS2,3, RICHARD COOPER1 and ROBIN L.
COOPER1.

Module 3 Pupa and adult behaviors in response
to diet as a larvae .
1Dept. of Biol., Univ. of KY. 2Dept. of STEM,
Univ. of KY and 3P-12 Math and Science Outreach
Unit of PIMSER.

Module 2 Food choice of adults and effects on
larvae
Before starting the modules a pretest will given
to examine the knowledge base of the students on
population dynamics and thoughts on the effect of
nutrition and amount of resources on the
population survival.
INTRODUCTION
The driving principle is to develop educational
material which integrates math, biology and
environmental factors for middle and high school
sciences classes as well as introductory college
courses. Here we outline an approach to modeling
the life cycle and its implications for
population growth in laboratory Drosophila
populations. The goal is to develop a viable
example of methodology that can be implemented in
high schools and middle schools to introduce
modeling that links with empirical studies in a
way that is accessible to students. The approach
is intended to encourage conceptual thinking and
open a range of student-driven explorations at
the intersection of biology and
mathematics. Activity without understanding
opposes what we know about effective science
teaching (Windschitl, Thompson, Braaten,
2008a,b). In addition, the lack of cross
disciplinary training in K-20 grades
compartmentalizes learning and hampers
educational integration. This is even evident
within the sciences such as with math and
biology. We are developing modules which will
be cross-disciplinary and can be used for middle
school through college level courses. We are
developing modules for measuring fecundity with
various numbers of females and males, various
environmental factors such as temperature and
types of food. With the data collected we will
develop a means for teachers and students to use
mathematical models to explain the collected
observations. The mathematical models can be used
to predict the impact of environmental
disturbances which could influence fecundity (egg
laying). Then experiments can be performed to
test if the predictions in the mathematical model
hold or if adjustments in the model need to be
performed. The module begins with students
observing Drosophila from nymphs through the
adult stage. In time we plan to have students use
the parameters and data collected from their
studies and use Simulink (The MathWorks, Natick,
MA) to simulate the population dynamics over
time. In this study we also examined how diet
can effect larval development and behavior as
well as adult behaviors. The food choices were
lemon, banana, and peaches as well as corn meal
and sugar water. Adult flies did not show any
real difference for food type to eat but tended
to lay more eggs on the lemon however, all the
larvae died in lemon due to mold/fungal growth
more common on lemon than the other food choices.
Larvae only fed sugar water delayed pupation
for up to 14 days as compared to 4 days with a
healthy diet. The nutrient deprived larvae were
smaller in body length for the same instar
stage. Behavioral assays in these larvae were
carried out as a measure of health.
Module 1 Rate of population growth depending on
of females and elclosin rate
An example of 2 sets of conditions, repeated 3
times. 1 female 1 male vs 3 females 1 male
The aquarium and the fruits under them. Peach,
lemon, and banana . Ever 5 minutes count the
number of adults on the various food s. Repeat
trails at various time intervals. Graph data
(left)
1 female 1 male
3 females 1 male
Mark each pupa as they form. Take out adults at
a particular day before pups elcose. Then count
the rate of eclosion by releasing the flies each
day.
These graphs are showing the number of flies that
landed on each fruit. The left panel is the raw
data of the total number of flies for the 5, 10,
20 and 50 min time points for the five different
trials. The right side panel is the converted
data set when transferred to percent of total
number of flies.
Time to pupation and then the time for the pupa
to become an adult with larvae raised on 2, 4 or
6 days on sugar water before being placed in corn
meal.
1 female 1 male
Righting response after being vortexed. This type
of test is used in adult Drosophila to examine if
the brain and sensory neurons are functioning
well. The results did not show any difference in
the adults from the larvae fed sugar water 2, 4,
or 6 days. All the flies quickly got up after
being vortexed. They were all able to do this in
less than 10 seconds.
Another example of 2 sets of conditions,
repeated 3 times. 1 female 1 male vs 3 females
1 male These large vials one can keep the
adults which form and count continuously the
number of pupa which form until it become too
crowded.
Number of eggs
3 females 1 male
METHODS
Determining pupation rates Adults at one of two
ratios (one male to one female or one male to
three females) were placed into food containing
vials. They were allowed to lay eggs and were
monitored daily. Once larvae began pupating, they
were circled and numbered on the exterior of the
vial. Each pupa was then documented with the time
at which it was first seen. This continued as
pupa continued to form. Determining eclosion
rates Using the same vials used for calculation
pupation rates, they were monitored for the
emergence of new adults. When the first fly
eclosed in a vial, it was documented, and all
adult flies were removed. The vials were then
monitored daily for newly eclosed flies. Adults
were continuously removed, and records were kept
as to how many flies were removed in each
session. This continued until no new flies
eclosed in a given vial. Closed system adult
counts Three large containers were setup with
ample water and a different amounts of food. A
male and female fruit fly were placed in each.
They were set aside and allowed to grow
indefinitely. The population continued to thrive
until the food ran out. Once all of the flies had
died, the containers were opened and adults
counted. Food choices and effects in quality of
food Adults laid eggs on apple juice containing
agar. The eggs were taken off and placed in the
various environments containing sugar or a corn
meal diet at different developmental times. The
developmental growth was examined every 12 hours.
The shapes of the mouth hooks were used to
determine the larvae stages. To conduct this part
of experiments, mouth hooks had to be removed
from the larvae and placed on microscope slides
to be viewed with a compound microscope at 100x.
The adults were tested for various behavioral
assays based on sensory function. The 3rd instar
larvae were tested for behaviors.   Egg Lying In
this experiment around 100 adults were placed in
a glass cage, adult taste preference and egg
laying were examined together in this assay. 3
Petri dishes containing lemon, peach and banana
as food were placed inside the cage. These foods
were also used to test survival of the larvae.
After the adults have had 4 hours on these dishes
they were removed and how many eggs were laid on
each dish were counted. This showed egg laying
preference. Then every day larva developed and
formation of pupa in the different foods were
examined.   Body Wall Movements (BWM) for
larvae Early 3rd instar larvae were used for
behavioral assays after they fed on sugar water
for 2, 4 and 6 days. Feeding and locomotory
behavior were performed as described in
Neckameyer (1996) and Li et al., (2001). In
brief, single animals were placed on a 2 agar
surface and the number of body wall contractions
counted for 1 min. The 1st instar larvae were
placed in sugar water and examined after the set
number of days.   Mouth Hook Movements (MHM) for
larvae Larvae were placed in a 2 yeast solution
on an agar plate after they fed on sugar water
for 2, 4 and 6 days. In this condition,
Drosophila larvae immediately fed, causing a
pattern of repetitive mouth hook movements. The
number of full mouth hook contractions in 1
minute was counted (Li et al., 2001). The
results of these behaviors are plotted as body
wall contractions or mouth hook movements per
minute. Behaviors for the newly elcosed adults
A plastic tube on a wooden stick with a small
flashlight on top of the tube was used. The light
was placed on the top of the tube to examine the
locomotive ability of the flies to go to the top
of the tube. The time it took flies to reach the
12 and 24 cm marks was measured. A righting
response of the adults was also tested after
they were shaken for 20 seconds. This was done by
placing the flies in a plastic vial and vortexed
and then quickly examined . Animals that had 2,
4, or 6 days in sugar water as a larvae and then
placed in cornmeal for pupation were the
experimental conditions for this set of
experiments.
The adult test with light responses with larvae
fed sugar water for various days.
CONCLUSIONS
The number of eggs on each dish for the five
trails. Since there was not an equal number of
eggs in each trial, calculate a percent of eggs
on each dish related to counting the number of
eggs on the banana as 100.
1. Modules examining population dynamics can be
successfully implemented in biology classes by
altering several factors. Sex ratios in the
parental generation and food availability
measurably affects how to population grows. 2.
Students can use mathematical modeling, along
with an understanding of the fruit fly life
cycle, to describe how the population changes.
This can be accomplished by using a flow diagram
or with the aid of computer modeling
software. 3. Lemon was the fruit that fruit
flies liked the most to lay their eggs, but it
turned out that lemon was the worst fruit for the
larvae to grow on. 4. Sugar water alone was one
of the foods that increased their life span by
nearly 2 weeks. Behavioral tests showed no huge
changes in larvae fed only sugar water. Only
mouth hook movements and body wall movements
showed some minor decreases then the ones fed
normal food. In the future it would be
interesting to test the effects of different
types of amino acids on larval development and
behavior.
TABLE 1   Survival Peach Lemon Banana  
Egg laying Many pupa Larvae died Many
pupa adults adults   1st
instar 28 pupa All larvae died 30 pupa
25 adults (fungus) 28 adults
 
TABLE 2 These are showing the survival rate of
the different diets.     Sugar water Corn
meal Peach Banana Form Pupa 15 days (3/60)
5 days 5 days 5 days (14 day larvae
but sick) Adults All died as larvae
Day 10 15 18 23 The 3 pupa died Day
11 78 81 77 Day 12
98 98 98
The three fruits, cornmeal, and sugar water diets
all in their containers.
Closed system 1 male 1 female but each container
with different amounts of food. Let the chamber
go until populations die out and then count total
number of adults.
References
Now one can start to express the population
dynamics in a flow chart. And even graph the data
gathered and construct mathematical
expressions. Students collaborate on developing
a flow diagram expressing these ideas. This
should start simple (e.g. the four boxes linked
horizontally in the diagram below) and then take
on more realistic detail. The result should be
something approximating the figure on the left.
Aceves-Piña EO, Booker R, Duerr JS, Livingstone
MS, Quinn WG, Smith RF, Sziber PP, Tempel BL,
Tully TP (1983) Learning and memory in
Drosophila, studied with mutants. Cold Spring
Harb Symp Quant Biol 48 Pt 2 831840. Alpatov,
W. Growth and Variation of the Larbae of
Drosophila Melanogaster. J. Exp. Zoology,
Vol.52(No.3) Hendel T, Michels B, Neuser K,
Schipanski A, Kaun K, Sokolowski MB, Marohn F,
Michel R, Heisenberg M, Gerber B (2005) The
carrot, not the stick appetitive rather than
aversive gustatory stimuli support associative
olfactory learning in individually assayed
Drosophila larvae. J Comp Physiol A.
191265279. Kaznowski, E.C. Schneiderman, H.A.,
and Bryant, J.P. (1985) Cuticle secretion during
larval growth in drosophila melanogaster. J.
Insect Physiology. Vol 31(No.10), 801-813
(1985) Li, H., Harrison, D., Jones, G., Jones,
D., and Cooper, R.L. (2001) Alterations in
development, behavior, and physiology in
Drosophila larva that have reduced ecdysone
production. Journal of Neurophysiology
8598-104. Watts, Thomas., Woods, Arthur.,
Hargand, Sarah., Elser, James., Markow, Therese
Biological stoichiometry of growth in Drosophila
melanogaster. J. Insect Physiology, 52 (2006),
187-193 White LA, Ringo JM, Dowse HB (1992)
Effects of deuterium oxide and temperature on
heart rate in Drosophila melanogaster. J Comp
Physiol B 162278283 Websites Larva Food
http//flystocks.boi.indiana.edu/german-food.htm
These graphs show the body wall and the mouth
hook movement results with the different days of
larvae eating sugar water.