Transcript Document

THE EFFECTS OF CO2 AND HYPOXIA ON THE PHYSICAL BEHAVIOR AND HEART RATE IN DROSOPHILA LARVAE
NICOLAS H. BADRE AND ROBIN L. COOPER
DEPARTMENT OF BIOLOGY, UNIVERSITY OF KENTUCKY, LEXINGTON, KY 40506-0225
RESULTS
INTRODUCTION AND BACKGROUND
Time (in sec)
# of Body wall movements in the first 2 min
We repeated the experiment with N2 to make sure that the results were
specific to CO2. We also had a control, recording the natural bwm and HB of
the larvae, without the injection of any gas.
Here is a model of the experiment. CO2 and N2 are represented on the left
and the control on the right.
400
Carbon Dioxide
Air
Nitrogen
300
# of Body wall movements in the last 2 min
4. Under a high concentration of CO2, the larvae cease movement in less
than minute.
0
2
Air
N2
120
Body wall movements
100
80
60
40
20
100
The results of the HB test show difference between the CO2, and Air
and N2. However, under a high concentration of CO2, the larvae
cease cardiac activity.. The average time for the movement ending
under high concentration of CO2 is 57 sec. Even under a high
concentration of N2 or with air, the heart beat does not stop as with
CO2.
80
60
40
20
0
CO2
Air
N2
CO2
Carbon Dioxide
Air
Nitrogen
Air
N2
The results of those bwm tests show difference in all three
conditions. The normal (air) average number of body wall
movements for the first two minutes is 76 bwm. However, with
the N2, the results were twice smaller with an average of 35.4
bwm for the first two minutes. Furthermore, with CO2, the
results were very small with an average of 10 bwm which is 7
times smaller. The normal (air) average number of body wall
movements for the last two minutes is 98.2 bwm. However, with
the N2, the results were five times smaller with an average of
19.4 bwm for the first two minutes. Furthermore, with CO2, the
larvae stopped moving so the average for the last two minutes is
0 bwm.
The trend is that, under a high concentration of N2, the
larvae have a decreased locomotion which seems to increase
with time. For CO2, the result seem comparable to N2, except
that the effects are stronger as the results are smaller for the
first two minutes and inexistent for the last two.
Time at which body wall movements stop
700
600
500
Carbon Dioxide
Air
Nitrogen
400
300
200
100
0
CO2
Air
N2
The results of this bwm test show difference for the CO2.
Under a high concentration of N2, the larvae does not react by
stopping its bwm. However, under a high concentration of
CO2, the larvae cease movement in less than minute. The
average time for the movement ending under high
concentration of CO2 is 40 sec.
3. Under a high concentration of N2, the larvae does not react by stopping
its BWM.
200
CO
Time (in sec)
Part II – The reaction of the larvae with the CO2
In our effort to identify particular characteristics of the larval response to
carbon dioxide, we designed several terms to quantify those responses.
Shell position designates larvae which are in a curved position. Elongated
position designates larvae which are flaccid and which look longer than
usual. Contracted position designated larvae which had returned to their
normal shape after being in elongated position. Those response were
tested by placing the larvae under anesthesia for approximately 5 minutes
and recording the different behaviors of the larvae during the first minutes
and the minutes following the end of the CO2 injection. This test has for
objective to understand and detail the reaction of the larvae to CO2.
2. Under a high concentration of CO2, the larvae have a even stronger
decrease in locomotion which leads to a stopping of movements.
500
100
METHODOLOGY
Part I – Body wall movements (bwm) & Heart Beats (HB)
We injected CO2 in the sealed container for a period of 10 minutes, after
which the container was opened. We recorded the bwm for the first and last
two minutes. If at any time bwm or the HB stopped, the time would be
recorded. If the HB stopped, the time when the HB started again once the
container was open, would be recorded. The objective of this test is to
quantify the difference between CO2 and hypoxia in the larvae using
common features of the animal.
1. Under a high concentration of N2, the larvae have a decreased
locomotion which seems to increase with time.
700
600
0
We tested Canton S, the common ‘wild-type’ laboratory strain of Drosophila
melanogaster. This experiment focused on larvae at the beginning of the
“wandering” phase of the third instar. Many of the techniques used in this
experiment have already used in Cooper and Neckameyer (1999). Each
larva was in a sealed agar plate with carbon dioxide injected into the
container.
Time at which Heart Beat stops
Body Wall Movements
Body wall movements
Carbon dioxide is commonly used as an anesthesia for adult
Drosophila melanogaster, however, the mechanism of its actions is
unknown. This is important, as it might lead to the discovery of
new types of insecticides that would be innocuous to plants and
plant eaters. Since mosquitoes have been shown to have sensory
structures that detect carbon dioxide, we postulated that
Drosophila must also contain similar types of receptors because
they share the same kind of environment. Laval insects have never
been examined for carbon dioxide sensory neurons. Previous
experiment supposed that carbon dioxide affected larvae the same
way than humans: a increase in body fluid acidity causing different
behaviors such as anesthesia (Sillans and Biston, 1979). Those
experiments also showed that carbon dioxide had different effects
to hypoxia as a high concentration of carbon dioxide and oxygen
could also cause anesthesia (Sillians et al., 1969). However, this
current research has an objective to find sensory neurons on the
larvae capable of detecting the CO2.
CONCLUSION
Heart Beats
Another figure determined by this experiment is the time needed for
the larvae to recover its cardiac activity once the CO2 injection is
stopped and that the container is opened. The average for the
recovery time of the larvae is: 59.6 sec. (This figure is only available
for the CO2 as the larvae would only stop their cardiac activity under
high concentrations of CO2).
5. Under a high concentration of N2 or with air, the larvae do not have any
kind of cardiac arrest.
6. Under a high concentration of CO2, the larvae cease cardiac activity.
7. The larvae needs a recovery time close to a minute to regain cardiac
activity after a being in an environment high in CO2.
8. CO2 provokes to the larvae behaviors, which are different to the one
caused by hypoxia and, which are similar to the one of anesthesia.
9. In 67 % of the CO2 exposure, the larvae will go in shell position before
stopping its cardiac activity.
10. In 100 % of the CO2 exposure, the larvae will have go in elongated
position once the cardiac activity is stopped.
11. 4.63 minutes is the time needed on average by a larvae to regain its
natural shape once it has been exposed to CO2.
12. 8.8 minutes is the time needed on average by a larvae to regain its
natural mobility once it has been exposed to CO2.
Physical Behavior
Current Research Objectives
shell position
elongated position
contracted position
Determining where the CO2 receptors are located on the larvae
by inhibiting certain part of the body at detecting the presence of
CO2.
Understanding which parts of the larvae are still
active/responding while the animal is under anesthesia.
The physical behavior test showed that in 67 % of time, the larvae
would go in shell position before stopping its cardiac activity. During
the other cases, the larvae would directly go in elongated position.
In all of the trials (100%), the larvae would go in elongated position
once the HB was stopped. When the injection of CO2 was stopped,
the larvae would have many different behaviors which did appears
in the same order every time. However, slight head turns and mouth
hooks movements were very common behaviors in the first three to
four minutes. It took on average 4.63 minutes for the larvae to gain
back their common size: contracted position. Once the larvae is in
contracted position, it usually does a couple of non-motional bwm.
Before starting its natural motion, the larvae often turns its head left
and right and then starts its motion. The average time for the larvae
to come back to a normal behavior after five minutes of exposition to
CO2 is 8.8 minutes.
Finding out how the larvae knows when to stop its anesthesia
mode to return to a normal activity.
Explaining the benefit of having such quick responses to CO2.
Recording the activity of the sensory neurons by
electrophysiology.
REFERENCES
Biston J, Sillans D (1979) Studies on the anesthetic mechanism of carbon
dioxide by using Bombyx mori larvae. Biochimie 61, nº2:153-156
Sillans D, Esteve J, Legay JM (1969) C.R. Acad. Sci., 269: 1209-1212
Cooper RL, Neckameyer WS (1999) Dopaminergic neuromodulation
of motor neuron activity and neuromuscular function in
Drosophila melanogaster. Comp Biochem Physiol [B] 122:199-210.