1 Effect of Daylight Hours and Exercise on Drosophila melanogaster Activity Florence Judge-­‐Clayden Contents
Effect of Daylight Hours and Exercise on Drosophila melanogaster Activity ......................................... 1 Abstract .............................................................................................................................................. 2 Introduction ....................................................................................................................................... 2 Methodology ...................................................................................................................................... 4 The Effect of Exercise on Drosophila Activity ............................................................................. 4 The Effect of Light Treatments on Drosophila Activity ................................................................ 8 Results and discussion ..................................................................................................................... 10 The Effect of Exercise on Drosophila Activity ........................................................................... 10 The Effect of Light Treatments on Drosophila Activity .............................................................. 24 Evaluation ........................................................................................................................................ 28 Appendix ......................................................................................................................................... 30 Bibliography ..................................................................................................................................... 31 Acknowledgements ......................................................................................................................... 32 Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 2 Abstract
The aim of these two studies is to investigate the effect of exercise at different times of the day on
level of activity and sleep in Drosophila melanogaster (common fruit fly) with the hope of finding
a method to reduce interrupted sleep in humans. Flies were exercised in the morning, afternoon
and evening in full space or confined space by tapping them down in a measuring cylinder. The
effect of different light treatments on their activity and sleep levels were also investigated. The
flies were placed in light treatments of normal daylight hours, reversed daylight hours,
inconsistent daylight hours or twenty-four hour light.
Restrained space in the flies that were tapped down was found to increase the number of sleep
bouts compared to their counterparts who had access to the full cylinder (p<0.005) . Exercise
taken in the afternoon or evening was shown to induce less-interrupted night-time sleep (2000 to
0800) as they had significantly less sleep bouts and very similar activity as the morning flies. In
the second study, when in darkness (0800 to 2000) ‘Opposite’ had generally less sleep bouts and
more activity periods than ‘Normal’, suggesting that ‘Opposite’ hours led to more interrupted
sleep. There were no significant differences between treatments for total sleep bouts and total
number of active periods.
Florence Judge-Clayden 25/9/2015 11:45
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Introduction
A recent study discovered that night-shift work leads to a shortened lifespan of humans, impaired
cognitive performance and longer term health issues such as Diabetes Type II and Coronary Heart
Disease (Gu, et al. 2015). It follows that other impacts of light should be investigated on direct
effect on sleep length, quality and daytime activity . Many studies focus on specific effects of
sleep, for example melatonin and Cortisol levels (Koller M, et al. 1994). Drosophila have been
proven to have circadian rhythms (Hendricks, et al. 2000), so different light treatments were used
to analyse the effect of exposure to light/dark pattern that mimic the pattern many night shift
workers are exposed to.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 3 Drosophila are a useful study species because their daylight hours can easily be changed, they
enable a larger sample size to be taken and other lifestyle elements can be controlled more easily.
Using the Drosophila Activity Monitor (DAM) their exact activity levels can be measured and
sleep quality can be worked out from it. Their physiology is also very similar to humans (Udai
Bhan Pandey and Charles D. Nichols 2011) and their very short breeding cycle fitted in well with
the length of time for this experiment.
In a separate experiment, flies were exercised at different times of the day and the focus was
looking at the effect on activity and sleep levels throughout the day, as well as on total activity
and sleep levels, as there is a possibility that different exercise regimes could lead to a treatment
of poor sleep quality in humans without using medication.
(Piazza, et al. June 2009) used a piece of apparatus to exercise the flies, called the Power Tower;
this idea was adapted for this experiment. Although many previous studies have been completed
on the time of day of exercise in humans (Baxter and Reilly 1983), and (Benloucif, et al. 2004)
which showed that morning or evening exercise improves neuropsychological performance,
many of these studies did not look at the subsequent change in sleep patterns in humans or in flies,
and if they did, the subject size was very small (Horne and Porter February 1976). Other papers
looked more at types and number of days of exercise, rather than the time at which the exercise
was undertaken.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 Florence Judge-Clayden 23/9/2015 11:27
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4 Methodology
The Effect of Exercise on Drosophila Activity
Drosophila have negative geo-taxis (move away from gravitational pull); when they are tapped to
the bottom of a container, they naturally climb up the sides in order to get to the top. Therefore
this natural behaviour was used to exercise the flies by putting them in a tall measuring cylinder
and tapping it down. A control group was not subjected to the tapping treatment to detect any
difference caused by the tapping.
Three groups of flies were set up and they were exercised for either two, four or eleven days (this
will be referred to as the Length of Exercise).
Within each of these lengths of exercise, the flies were split into eight treatments. They were
exercised at different times of the day and either had a stopper at the top or the bottom of the
cylinder. The change in location of the stopper was in order to determine the effect of exercise and
non-exercise. When the stopper was at the top, the full length of the cylinder was available to run
up (25 centimetres) so the flies would be exercised more (Figure 1). When the stopper was at the
bottom, the length was limited to three centimetres, therefore only allowing limited movement and
little exercise.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 5 Figure 1 : The measuring cylinder on the left has the stopper pushed down (small space for
flies) whilst the measuring cylinder on the right has the stopper at the top (full space for
flies)Height: 25 cm Inner Bore: 2.8cm
The eight treatments are as follows:
1. Morning exercise at 11:30am with stopper at the top
2. Morning exercise at 11:30am with the stopper pushed down
3. Afternoon exercise at 1:30pm with stopper at the top
4. Afternoon exercise at 1:30pm with stopper pushed down
5. Afternoon non-exercise at 1:30pm with stopper at the top. This was used as a control to see the
effect that the tapping had on the flies.
6. Afternoon non-exercise at 1:30pm with stopper pushed down. This was used as a control to see
the effect that the tapping had on the flies.
7. Evening exercise at 3:30pm with stopper at the top
8. Evening exercise at 3:30pm with stopper pushed down
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 6 When flies had an exercise session, the group of flies was transferred from their food vials into a
measuring cylinder using a funnel. The stopper was placed in the required position. The
measuring cylinder was then tapped down by hand onto a table twice within two seconds every
thirty seconds, which dislodged the flies so they fell to the bottom. The flies then climbed up to
the stopper and mostly stayed near the stopper until being tapped down again.
This cycle
happened continuously for thirty minutes.
In order to ensure that the flies had very similar tapping treatments, the same individual tapped
down the cylinders. As there were always two cylinders being tapped down at the same time e.g.
Treatments 1 and 2, the cylinders were swapped between hands after seven and a half minutes,
after fifteen minutes, then after twenty-two and a half minutes in order to compensate for the
potential that one hand tapped down stronger than the other.
After the thirty minutes of exercise were over, the group of flies was then transferred back into
their food vial using a funnel and the cotton wool ball was replaced to prevent them escaping, but
still allowed air to enter.
After the allotted number of days of exercise, the flies were transferred from their vials into small
glass tubes. One fly was put in one glass tube which had a lid on one end with agar based medium
in it to provide moisture and cotton wool in the other end. The tubes were then put the glass tubes
half way in to the Drosophila Activity Monitor (DAM) as used in previous experiments (Piazza, et
al. June 2009). When the fly moved from one end of the glass tube to the other end, it broke the
infra-red beam which was emitted from the DAM and cut across the middle of the tube (Figure 2).
The number of times the beam was broken was recorded and counted up every thirty seconds. The
flies remained in the DAM for seventy-two hours.
The flies that were exercised for two and four days had a sample size which ranged from between
seventeen to twenty flies each. The flies that were exercised over eleven days had a sample size
which was on average six to seven flies.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 7 All flies that were used were aged between two and five days old when exercise commenced and
only male flies were used. All Drosophila were of the Canton-S Strain. The room that all they
flies were kept in remained 27°C throughout the experiment and remained lit 24 hours of the day.
All flies were kept in the same-sized vials when they were not being exercised and the food agar
placed in the bottom of them was the same recipe for all flies. The vials all contained the same
number of flies at the start, but some flies died or escaped during the experiment. When the flies
were exercised, they were transferred into the same-sized and same-branded measuring cylinders.
The stoppers that were placed in the top or bottom of the measuring cylinder were all the same
sponge material.
Tapping was the chosen method of exercising the flies as a method of rotating the flies did not
work. This was because there was not any equipment available that could rotate the flies at a slow
enough speed, so they just remained in the middle of the tube they were put in. This therefore did
not exercise them.
There were not many safety precautions, apart from the careful handling of the glass tubes when
fitting the lids to the tubes so that they did not break. Also when separating the female flies from
the males, Carbon Dioxide gas was used to temporarily knock-out the flies so that they did not fly
off. It was important that the carbon dioxide pump did not remain on too long for the health of the
flies and the scientists in the laboratory.
Figure
2
:
The
(DAM)
with
glass
Drosophila Activity Monitor
Florence Judge-Clayden 23/9/2015 11:29
through the holes,
tubes
placed
halfway
which an infra-red beam cut
across.
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... [1]
Florence Judge-Clayden 23/9/2015 11:29
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8 The Effect of Light Treatments on Drosophila Activity
Flies were kept in the same room at twenty-seven degrees Celsius and were given one of four
different light treatments, by placing them in identical, light proof cardboard boxes with LED
lamps in. These boxes were all kept at the same height in the room, therefore they were all the
same distance away from the heater in the ceiling. The boxes measured 31cm x 23cm x 22cm and
were all the same shade of brown inside. Duct tape was placed over any gaps in the box structure
to prevent light entering. A slit one centimetre wide and 3.4 centimetres deep was cut into the 31
cm side half way along. A slit of similar size and position, but 0.8cm wide was cut into the lid of
the box. This allowed the neck and head of the LED lamp to be put into the box, but to have the
base standing outside of the box. (Figure 3)All the lamp heads were the same distance away from
the bottom of the box and from all of the sides of the box. The LED lamps were plugged into
timers, which could turn the lights on or off automatically according to the set programme.
Figure 3 : The four cardboard boxes with LED Lamps inserted through slits in the side.
Thirty flies were subjected to each treatment of light, but approximately nineteen to twenty-four
flies were recorded for each treatment of light due to some flies dying or escaping during
transferal. Ten flies were put in each vial, which were the same size and with the same recipe of
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 9 food in the bottom of each vial. Three vials were then placed in each of the four boxes which all
had a different light treatment.
The four treatments are as follows:
1. Normal Daylight hours – light was switched on at 08:00 and switched off at 20:00.
2. Reversed Daylight hours – light was switched on at 20:00 and switched off at 08:00
3. Rotating Daylight hours – light was switched on for six hours, then off for six hours, then on for
twelve hours, then off for six hours, then on for six hours, then off for twelve hours.
4. Twenty-four hour light
The flies were kept in their boxes for seven days, to adjust to their new light treatments. After this,
the flies were transferred from their vials to individual glass tubes. One glass tube contained one
fly, along with food agar in one end to prevent starvation and in the other end, there was cotton
wool to prevent escape.
All the flies that were used were female and were aged between two and three days old when they
were put into their boxes. All of the flies had previously been kept in twenty-four hour light
before being placed in the box. The lamps that were used were all the same brand of LED lamps
so they gave off the same heat, however, the increase in temperature due to the LED lamps was
only two degrees Celsius. All flies were subjected to the same number of hours of light and dark
throughout the experiment, apart from the box which had twenty-four hour light. We then put the
glass tubes half way in to the Drosophila Activity Monitor (DAM). When a fly moved from one
end of the glass tube to the other end, it broke the infra-red beam which was emitted from the
DAM and cut across the middle of the tube (Figure 2). The number of times the beam was broken
was recorded and counted up every thirty seconds. Flies remained in the DAM for forty-eight
hours.
The only safety precaution that was to be taken was handling the glass tubes carefully when fitting
the lids so that the glass did not break.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 10 Results and discussion
The Effect of Exercise on Drosophila Activity
Flies were tapped down or not tapped down at three times of the day, with or without a stopper and
for different numbers of days. The eight different treatments are as follows:
Legend
1. Morning exercise at 11:30am with stopper at the top = AM TAP FULL SPACE
2. Morning exercise at 11:30am with the stopper pushed down = AM TAP SMALL SPACE
3. Afternoon exercise at 1:30pm with stopper at the top =AFT TAP FULL SPACE
4. Afternoon exercise at 1:30pm with stopper pushed down = AFT TAP SMALL SPACE
5. Afternoon non-exercise at 1:30pm with stopper at the top. This was used as a control to
see the effect that the tapping had on the flies. = AFT NO TAP FULL SPACE
6. Afternoon non-exercise at 1:30pm with stopper pushed down. This was used as a control
to see the effect that the tapping had on the flies. = AFT NO TAP SMALL SPACE
7. Evening exercise at 3:30pm with stopper at the top = EVE TAP FULL SPACE
8. Evening exercise at 3:30pm with stopper pushed down = EVE TAP SMALL SPACE
Treatment numbers within the following paragraphs will refer to these treatments above.
The number of thirty second periods where the fly was active was counted up – this will be called
‘Number of Active Periods’.
One ‘Sleep Bout’ was classed as inactivity for a duration of five or more minutes.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 11 This is set as the standard of the sleep in many studies, but it is important to consider that this may
not be fully representative of a fly’s sleep. This is because a large number of bouts could mean
that the fly could have a longer length of sleep, or that it is having the same length of sleep, but
just more interrupted sleep. A small number of bouts could mean that the fly is sleeping less, or
that the fly is having the same amount of sleep but it is less interrupted, or is actually consistently
inactive for less than five minutes at a time, therefore not qualifying for the sleep status. Total
duration of sleep could have been used to avoid this problem, but that is not fully representative as
25
20
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10
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5
Active 30s periods per hour
30
the length of inactivity following a fly’s death would have been counted as sleep.
0
5
10
15
20
Hours from midnight
Figure 4 : Number of Active Periods is shown through twenty-four - Colour indicates
treatment and error bars show one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 12 0.8
0.6
0.4
0.2
1
2
3
4
5
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7
8
0.0
Sleep bouts started within hour
1.0
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Hours from midnight
Figure 5: Number of Sleep Bouts started in an hour is shown through twenty-four hours Colour indicates treatment and error bars show one standard error
In terms of the treatments, when active periods and sleep bouts are compared hourly, the
following can be noticed:
The effect of space for exercise
It is very interesting to note that there is a significant difference between those flies that were
tapped at the same time but had different space limitations i.e. exercised and not exercised. This
was the case for flies exercised at all times of the day; morning, afternoon and evening. I.e. 1-2
(Morning ex vs no ex), 3-4 (Afternoon ex vs no ex) and 7-8 (Eve ex vs no ex). P<0.0005
Within the first five hours, Treatment 1 and 2 are very similar in activity levels, with their activity
peaking at 2 hours at 23 periods. At 6 hours, Treatment 2’s activity rises to 20, whilst 1 drops
down to 17. Both of their activity then decreases at roughly the same rate, but 2 remains more
active. Their lowest activity levels are recorded at 18 hours, with 1 at 9 periods and 2 at 11
periods. Both of their activity peaks at 20 hours at 23 periods. Both their activity then decreases
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 13 until 23 hours, when it starts to rise again. Treatment 2 has marginally more activity within this
time.
Treatment 2 has a much greater number of sleep bouts within the first twelve hours than
Treatment 1. After this, number of sleep bouts falls dramatically to 0.4 for Treatment 2 and then
rises very steeply to 0.8 at 21 hours, whilst sleep bout number remains fairly constant for
Treatment 1 at around 0.6. (Figures 6 and 7)
Therefore, it can be concluded that, because activity levels are very similar from 20 hrs to 6 hrs,
but the number of sleep bouts for 2 is higher during this time period except at 22 hrs, 2 has more
interrupted sleep than 1 during the night-time. Morning exercise gives less-interrupted sleep than
non-exercise in the morning.
Figure 6, 7 : (Left) Number of thirty second Active periods is shown through twenty-four
hours. (Right) Number of sleep bouts started within the hour through twenty-four hours.
Treatment 1 (AM TAP FULL SPACE) is shown in black, Treatment 2 (AM TAP SMALL SPACE) is
shown in red. - Error bars show one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 14 Comparing Treatments 3 and 4, 4 has a lower activity than 3 within the first 5 hours. However
Treatment 4 activity rises at 7 hours slightly, but then declines, meaning that 4 has a higher
activity level between hours 5 to 9. During this time, Treatment 3 continues to decline. From 10
hours to 20, both gradients are positive with the exception of a dip from 15 to 18 hrs, with 3
having more active periods than 4. As the graph peaks at 20 hours, Treatment 4 has more activity
than 3 for two hours until their activity remains the same as each other from 22 hours to 24 hours,
when both of their activity starts to increase again. (Figures 8 and 9).
It is therefore hard to conclude exactly that treatment 3 has more interrupted sleep than treatment
4, but from 2200 to 0600, Treatment 3 has a higher number of activity bouts and a lower number
of sleep bouts, suggesting that it is too active to register for sleep. In the afternoon, exercise is
shown to induce more interrupted sleep than not exercising, perhaps because it is interrupting the
flies’ natural sleep time at midday.
Figure 8, 9 : (Left) Number of thirty second Active periods is shown through twenty-four
hours. (Right) Number of sleep bouts started within the hour through twenty-four hours.
Treatment 3 (AFT TAP FULL SPACE) is shown in green, Treatment 4 (AFT TAP SMALL SPACE) is
shown in blue - Error bars show one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 15 In the first 5 hours, Treatment 8 has a higher number of active sessions than Treatment 7. Activity
peaks from both at 2 hours. Then after 5 hours, as both gradients become negative, 7 is more
active than 8. Another peak occurs at 13 – 14 hours and then until 18 hours activity plateaus. Only
at 20 hours does treatment 8 have a higher activity level, then both drop and return to around the
same level as each other. Activity then starts increasing at 22 hours to 24 hours. (Figure 10 and
11)
It can be concluded that from 0000 to 0500 hours Treatment 7 has less-interrupted sleep because it
has a smaller activity bout number and sleep bout number. Evening exercise gives less interrupted
sleep than non-exercise in the evening.
Figure 10, 11 : (Left) Number of thirty second Active periods is shown through twenty-four
hours. (Right) Number of sleep bouts started within the hour through twenty-four hours.
Treatment 7 (EVE TAP FULL SPACE) is shown in orange, Treatment 8 (EVE TAP SMALL SPACE) is
shown in grey - Error bars show one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 16 Effect of Exercise at different Times of the day:
There is a clear difference between those flies tapped at different times of the day. There were
significant differences in hourly sleep in the tapped exercised flies between the morning and
afternoon, as well as morning and evening. The same pattern was observed in the flies that were
tapped and restrained too. It is important to point out that there is no significant difference
between exercising flies in the afternoon and the evening. There are significant differences in
sleep bout numbers between the following treatments: 1-3 (morning ex vs afternoon ex), 1-7
(morning ex vs eve ex), but nothing for afternoon to evening,
2-4 (morning non exercise vs aft
non ex), 2-8 (morning non ex vs eve non ex), but nothing for afternoon to evening. P<0.05
Figure 12 and 13 : Shows activity and sleep levels comparing 1 (AM TAP FULL SPACE) – 3
(AFT TAP FULL SPACE). (Treatment 1 in black, Treatment 3 in Green) - Error bars show
one standard error
Treatment Three has substantially less activity bouts than 1 in the morning from 5 hours from
midnight to 10 hrs. 3 then has substantially more activity bouts than 1 from 10 hrs until 20 hrs,
when activity levels are very similar. 3 has less sleep bouts than 1 throughout the day. Therefore,
we can say that 3 is more active during the latter half of the day until 20hrs, when 3 and 5 have the
same activity levels from 20hrs to 5 hrs. However, because 3 has less sleep bouts, we can say that
3 has less interrupted sleep during the night than 1. (Figure 12 and 13)
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 17 Figure 14 and 15 : Activity levels and sleep levels 1 (AM TAP FULL SPACE)– 7 (EVE TAP
FULL SPACE) (Treatment 1 in black, Treatment 7 in yellow) - Error bars show one
standard error
Treatment 7 has substantially more activity levels from 5 hrs to 21 hrs than 1. From 21 hrs to 5 hrs
activity levels are very similar. 7 has substantially less sleep bouts than 1 throughout the whole
day, apart from at 3 hrs when 1 and 7 have the same number of sleep bouts. Therefore, it can be
said that 7 is more active than 1 from 5hrs to 21 hrs, so 7’s sleep is more interrupted during the
day. However, from 21 hrs to 5 hrs, when activity levels are similar, 7 has a smaller number of
sleep bouts so 7 has less interrupted sleep during the night. (Figure 14 and 15)
Figure 16 and 17 : Activity levels and sleep levels 2 (AM TAP SMALL SPACE shown in red)
– 4 (AFT TAP SMALL SPACE shown in blue) - Error bars show one standard error
2 has a greater level of activity than 4 from 23 hrs to 12 hrs, apart from at 6hrs when their activity
level is very close. From 12 hrs to 23 hrs, 4 has greater level of activity. From 0 hrs to 15 hrs, 2
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 18 has a greater number of sleep bouts, apart from at 4-5hrs when 4 just goes about 2. From 15 hrs –
17 hrs, sleep bout number is very similar. From 17hrs to 20 hrs 4 has a greater number of sleep
bouts than 2. From 20 hrs to 0 hrs, sleep bout number varies a lot per hour for both of them, but in
opposite directions. It can be said that because 2 has a higher level of activity and a higher sleep
bout number, 2 has more interrupted sleep and 4 has less interrupted sleep from 0 hrs to 13 hrs.
(Figure 16 and 17)
Figure 18 and 19 : Activity levels and sleep levels 2 (AM TAP SMALL SPACE shown in red)– 8 (EVE TAP SMALL SPACE shown in grey) - Error bars show one standard error From 0 to 2 hrs, activity is very similar. 8 has a greater level of activity than 2 from 2hrs to 6hrs.
From 6hrs to 12 hrs, 2 is more active than 8. Activity is then similar from 12 hrs to 22 hrs except
at 20 hrs, 8 is more active than 2. From 22 hrs to 0 hrs, 2 is more active. 2 has more sleep bouts
from 20 hrs to 17 hrs, except at 4-5hrs, when 8 has more sleep bouts. From 17 hrs to 20 hrs, 8 has
more sleep bouts. It can be said from 22 hrs to 2 hrs, 2 has more interrupted sleep because 2 has a
greater number of sleep bouts during the night but activity is very similar. However, it can also be
said that between 2 and 6 hrs, 8 has more interrupted sleep, because 8 is more active between 2-6
hours and at 4-5 hrs also has a greater number of sleep bouts. It can be understood that inactivity
was for too short a time to be counted as sleep between 2-4 hrs, as less sleep is recorded but more
activity during that period. (Figure 18 and 19)
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 19 Effect of Tapping and Not Tapping:
Also we can see a clear difference (p < 0.001) in those tapped and not tapped unrestrained tube in
the afternoon – 3-5 (afternoon ex vs afternoon rest full space), but there is no significant
difference between Treatment 4 (tapped stoppered) and Treatment 6 (untapped stoppered).
Therefore we have to consider that the difference seen is not because of the force of the tapping
causing damage to the flies, but either the effect of falling continuously from a great height or the
fact that with the tapping, the flies have to run up the tube more often.
Figure 20, 21 : (Left) Number of thirty second Active periods is shown through twenty-four
hours. (Right) Number of sleep bouts started within the hour through twenty-four hours.
Treatment 3 (AFT TAP FULL SPACE) is shown in green, Treatment 5 (AFT NO TAP FULL SPACE) is
shown in light blue - Error bars show one standard error
Because Treatment 5 (untapped and unrestrained) is always above Treatment 3 (tapped and
unrestrained) on (Figure 21) and has a greater total bouts of sleep, but the fact that 3 and 5 have
little difference in total bouts of activity, it suggests that 3, who has less sleep bouts, has lessinterrupted sleep. Therefore, I conclude that Treatment 3 ie tapping exercise induces less
interrupted sleep.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 20 Effect of Different Days:
While the flies were in the DAMs, their sleep levels across the four days differed significantly,
apart from the difference in activity between Day 1 and Day 3. 1-2, 1-4, 2-3, 2-4, 3-4.
This is to be expected, as we can see that across the whole time that the flies were in the DAMs,
the number of active periods decreased as time went on. (Figures 22 and 23)
The first twenty-four hours exhibit extremely high and low numbers of active periods. It is
unknown exactly what has caused this, but it can be speculated that acclimatisation to the new
environment of the narrow glass tubes could be the cause. The most likely reason that activity
80
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Active 30s periods per hour
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decreases over time in the DAM is because the flies had no food.
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Hours since midnight of day 1
Figure 22, 23 : Number of Active Periods are shown through ninety-­‐six hours with and without error bars (Left and Right respectively). Colour indicates treatment and error bars show one
standard error Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 21 Total Number of Active Periods and Sleep bouts
The total number of active periods and sleep bouts were not significantly different between
treatments. However, there were significant differences between the Length of Exercises (how
many days the group of flies had been exercised for). P<0.0001
When looking at total number of sleep bouts, and total number of active bouts (Figures 24 and
25), although differences can be seen, they were not significant. It is interesting to note though
that within the flies that were tapped down (all treatments except Treatment 5 and 6), those flies
with limited space to exercise (Treatments 2, 4 and 8) consistently had more sleep bouts than their
counterparts who had the full length of the tube to run up and down. However, between the
treatments that were not tapped down, Treatment 5 had a fractionally greater number of sleep
1000
900
800
600
700
Total 30s periods active
1100
1200
bouts than Treatment 6, maybe because the flies ran up the tube more.
All days exercised pooled
1
2
3
4
5
6
7
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Treatment
Figure 24 : Total number of Active Periods for each treatment - Error bars show one
standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 22 50
40
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30
Total bouts of sleep
60
All days exercised pooled
1
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Treatment
Figure 25 : Total number of bouts of sleep for each treatment - Error bars show one
standard error
Effect of Length of Exercise
It is interesting to note that that there is a significant difference between exercised flies for two
days and four days, as well as between two days and eleven days. It can therefore be concluded
that a larger difference in sleep and activity is obtained between exercise over a shorter period and
longer period, but that extra exercise does not have as great an effect.
It is also very interesting to note that whilst the flies exercised for two days had the largest number
of total sleep bouts (Figure 26), they also had the largest number of activity bouts (Figure 27).
Therefore, it can therefore be concluded that exercise for two days led to more interrupted sleep,
whilst four and eleven days of exercise led to less interrupted sleep as they had less sleep bouts
and less active bouts. However an alternative explanation could be that the flies that were
exercised for two days slept more, and had more active bouts during the non-sleep time compared
to the four and eleven days exercised flies. Total sleep duration cannot be used because some flies
will have died before others, therefore, inactivity due to death would contribute to total sleep
duration so is not representative. It must also be noted with importance that exercising the for
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 23 different lengths of time mean that they were not the same age when they went into the DAM, as
40
20
Total sleep bouts
60
80
all flies started to be exercise when they were two to five days old.
0
All treatments pooled
2
4
11
Days exercised
Figure 26 : Total bouts of sleep for each Length of Exercise- Error bars show one standard
1400
1200
1000
800
600
Total 30s periods active
1600
error
All treatments pooled
2
4
11
Days exercised
Figure 27 :Total Number of Active periods for each Length of Exercise - Error bars show
one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 24 The Effect of Light Treatments on Drosophila Activity
In terms of activity bouts (Active), there was a significant difference depending on the treatment,
hour of the day and which day it was. In terms of sleep bouts, there was a significant difference
depending on the treatment and on which day it was.
Legend 1- Normal Daylight Hours (0800 to 2000)
2- Reversed Daylight Hours (2000 to 0800)
3- Rotating Daylight Hours
4- 24 Hour Light
Numbers may be used to indicate the above
treatments in the following paragraphs.
There were significant differences (p<0.005) between the hourly activities of the four different light
treatments, apart from between ‘reversed daylight’ and ‘rotating daylight’. ‘Reversed daylight’ and
‘rotating daylight’ were more active than ‘normal’ and ‘24’ from 0000 to 1800. From 1800 to 2100
‘normal’ showed a massive increase to be more active than ‘reversed’ , ‘rotating’ and ‘24’.
However, there were significant differences between ‘reversed daylight’ and ‘rotating daylight’ with
regard to hourly sleep bout number, with their sleep pattern being completely opposite to each other
between the hours of 0500 and 1500, and 2000 and 0200, with ‘reversed’ having much less sleep
bouts. It must be noted that between different hours of the day, treatments ranked differently in
activity and sleep bout number, so there is no significant difference to be seen in Total Sleep Bouts
and Total Active bouts.
The activity levels of ‘Reversed’ and ‘Normal’ were very different from each other when comparing
them by hour. When comparing the hourly activities when it was dark or light for both of them,
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 25 their patterns were not the same. When in darkness (0800 to 2000) ‘Opposite’ had generally less
sleep bouts and more activity periods than when ‘Normal’ was in darkness (2000 to 0800) apart
from at 2000, when the activity bout number of ‘Normal’ increases to 22. Overall this suggests that
‘Opposite’ hours lead to more interrupted sleep.
It is hard to compare ‘Rotating Daylight’ to ‘Normal’ as there are no consistent hours that should be
considered as ‘sleeping hours’ in the ‘Rotating Daylight’ flies. The flies that had ‘24 hour light’
share this problem too. However, ‘Rotating Daylight’ and ’24 Hour Daylight’ do not share the same
pattern of activity or sleep even though their treatments share this characteristic.
Although the difference is not significant between treatments when looking at Total Activity bouts
and Total Sleep bouts (Figures 30 and 31), it is interesting to see that the means of activity bouts
is highest in treatment 2 – ‘reversed daylight’, followed marginally by treatment 3 – ‘rotating
daylight’, followed by treatment 1 – ‘normal daylight’, then by treatment 4 – ‘24 hour light’.
The opposite is found in sleep bouts, suggesting that those who have more sleep bouts have less
active bouts, suggesting that they either sleep for longer in total so that they have less time to be
active, or that their sleep is very interrupted and when they are active, they are not active for long.
In the opposite way, those with less sleep bouts and more active bouts could sleep for a lesser time
and be active more of the time, or they sleep for a longer time and have more activity when
awake. However, the latter is less likely as there is not unlimited time in the day.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 26 Figure 28 : Active periods shown through twenty-four hours – Colour indicates treatment
and error bars show one standard error
Figure 29: Sleep bouts shown through twenty-four hours – Colour indicates treatment and
error bars show one standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 27 Figure 30: Total number of sleep bouts for each treatment - Error bars show one standard
error
Figure 31: Total number of Active periods for each treatment - Error bars show one
standard error
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 28 Evaluation
In the first study, it can be shown that the time of exercise is important. Exercise taken in the
afternoon or evening induces less-interrupted night-time sleep, as those flies had significantly less
sleep bouts and very similar activity to the morning flies.
Exercising and not exercising (tapped down in full tube or restrained space) also had an effect,
however this effect was different depending on the time of the day. In the morning, exercise was
shown to induce less-interrupted sleep than no exercise in the morning. In the afternoon, no
exercise was shown to induce less- interrupted sleep than not exercising, perhaps because it was
not interrupting the flies’ natural sleep time at midday as much as exercising at this time of day. In
the evening, exercise induced less interrupted sleep than no exercise in the evening.
Therefore it can be concluded overall that Evening exercise is the best time to exercise in order to
get less-interrupted sleep as flies exercised at this time has less-interrupted sleep compared to flies
exercised at other times of the day and to flies not exercised (tapped in limited space) in the
evening.
It is recognised that the tapping method of the exercise could lead to damage as was seen in (Piazza,
et al. June 2009), however, it was identified in this study that it is not the force of the tapping itself
that may lead to damage (no significant difference was seen between Treatment 4 and 6). However,
a significant difference was seen between 3 and 5, with Treatment 3 (tapped with full tube in
afternoon) had less-interrupted sleep. Whether this is because of damage is unknown. Therefore, in
a future study, it would be useful to see whether the results of this study are shown when flies are
exercised by rotation.
In the second study, it can be said that ‘Opposite’ hours induce more-interrupted night-time sleep
because flies have less sleep bouts and more activity periods than ‘Normal’ when both are compared
in darkness. This needs to be considered by organisations that employ people on night-shifts as
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 29 these workers are potentially having more interrupted sleep which could lead to decline in health,
although different studies come to different conclusions.
A further experiment that could be completed would be one where the flies are kept in normal
daylight hours (08:00 to 20:00) to see whether that sets a stronger circadian rhythm in the flies, so
that they respond better to the exercise at different times of day. Another one would be to compare
exercise sessions taking place at more times of the day which start earlier and finish later than in
this experiment e.g. early morning, late morning, lunch time, afternoon and early evening. It would
also be very interesting to compare the same length of exercise, but either completed all at once, or
split up into two or three smaller sessions throughout the day.
If a larger study were to take place, I think an automated tapping machine would work better, as
the flies could be exercised for a longer period of time and the tapping technique would be exactly
the same for all the exercise sessions too.
Another experiment that could be developed from this one would be to compare the lengths of
exercise where the flies are all the same age by the time they are put into the DAM, to see whether
the results in this experiment are due to the age differences or exercise length differences.
In determining the effect of light on Drosophila a further experiment would be to observe the
difference in length of lifespan to see whether different Daylight Hours are a stressing factor or
not. Another further experiment would be to use different coloured lights to see which colours
they respond to better.
This experiment could be improved by using virgin flies that are all the same day old, as this
would allow only the independent variable to be investigated. A tapping machine could have been
used to control the strength of the taps.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 30 Appendix
It may be of interest to look at Figures (24 and 25) which show the means for Effect of Daylight
hours.
Figure 32: Mean number of Sleep Bouts for Effect of Daylight Hours.
Figure 33: Mean number of Active Periods for Effect of Daylight Hours.
Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 31 Bibliography Baekeland, Frederick, and Richard Lasky. "Exercise and Sleep Patterns in College Athletes." Perceptual and Motor Skills, 1966: Volume 23, Issue , pp. 1203-­‐1207. Baxter, C., and T. Reilly. "Influence of time of day on all-­‐out swimming." Br J Sports Med, 1983: 17:122-­‐127. BBC. "Night shifts 'significantly shorten lifespan'." Today Programme 27/07/15. 2015. Benloucif, Susan, et al. "Morning or Evening Activity Improves Neuropsychological Performance and." SLEEP, 2004: 27(8):1542-­‐51. Bernard, T., M. Giacomoni, O. Gavarry, M. Seymat, and G. Falgairette. "Time-­‐of-­‐day effects in maximal anaerobic leg exercise ." European Journal of Applied Physiology and Occupational Physiology , December 1997: Volume 77, Issue 1-­‐2, pp 133-­‐138. Davis, JC, and PJ O'Connor. "Psychobiologic responses to exercise at different times of day." Medicine and Science in Sports and Exercise, 1992: 24(6):714-­‐719. Gu, Fangyi, et al. "Total and Cause-­‐Specific Mortality of U.S. Nurses Working Rotating Night Shifts." American Journal of Preventive Medicine, 2015: 241-­‐252. Hendricks, Joan C. "Invited Review: Sleeping flies don't lie: the use of Drosophila melanogaster to study sleep and circadian rhythms." Journal of Applied Physiology , 2003: Vol. 94 no. 4, 1660-­‐
1672 . Hendricks, Joan C, et al. "Rest in Drosophila Is a Sleep-­‐like State." Neuron 25, no. 1 (January 2000): 129–138. Horne, J.A, and J.M. Porter. "Time of day effects with standardized exercise upon subsequent sleep." Electroencephalography and Clinical Neurophysiology, February 1976: 178–184. Koller M, Harma M, Laitinen JT, Kundi M, and Piegler B. "Different patterns of light exposure in relation to melatonin and Cortisol rhythms and sleep of night workers." Journal of Pineal Research, 1994: 127-­‐135 Volume 16, Issue 3. Kubitz, Karla A., Daniel M. Landers, Steven J. Petruzzello, and Myungwoo Han. "The Effects of Acute and Chronic Exercise on Sleep." Sports Medicine 21, no. 4 (April 1996): 227-­‐291. MI, Härmä, Ilmarinen J, Knauth P, Rutenfranz J, and Hänninen O. "Physical training intervention in female shift workers: I. The effects of intervention on fitness, fatigue, sleep, and psychosomatic symptoms." Ergonomics, 1988: 31(1):39-­‐50. Moatt, Joshua P., Calvin Dytham, and Michael D.F. Thom. "Sperm production responds to perceived sperm competition risk in male Drosophila melanogaster." Physiology & Behavior, May 2014: 111-­‐114. Piazza, Nicole, Babina Gosangi, Shawn Devilla, Robert Arking, and Robert Wessells. "Exercise-­‐Training in Young Drosophila melanogaster Reduces Age-­‐Related Decline in Mobility and Cardiac Performance." PLoS ONE , June 2009. Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015 32 Reilly, T., and S. Marshall. "Circadian rhythms in power output on a swim bench." J. Swim. Res , 1991: 11-­‐13. Reilly, Thomas, and George A. Brooks. "Selective Persistence of Circadian Rhythms in Physiological Responses to Exercise." Chronobiology International, 1990: Vol. 7 No.1 p59-­‐67. Reilly, Thomas, G. Robinson, and D. S. Minors. "Some circulatory responses to exercise at different times of day." Medicine & Science in Sports & Exercise, Oct 1984: Vol 16(5), 477-­‐482. Thayer, Robert E. "Problem perception, optimism, and related states as a function of time of day (diurnal rhythm) and moderate exercise: Two arousal systems in interaction ." Motivation and Emotion , March 1987: Volume 11, Issue 1, pp 19-­‐36 . Tworoger, Shelley S., et al. "Effects of a Yearlong Moderate-­‐Intensity Exercise and a Stretching Intervention." SLEEP, 2003: 26(7):830-­‐6. Udai Bhan Pandey , and Charles D. Nichols. "Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery." Pharmacological Reviews, 2011: 411-­‐436. Vuori, I., H. Urponen, J. Hasan, and M. Partinen. "Epidemiology of exercise effects on sleep." Acta Physiologica Scandinavica. Supplementum, 1988: 574:3-­‐7. Youngstedt, Shawn D. "Effects of Exercise on Sleep." Clin Sports Med, 2005: 355– 365. Acknowledgements
I would like to thank Prof Calvin Dytham, Antje Kuhrs and Emily Rose Churchill profusely for being my supervisors throughout the duration of this project and for giving up their time to help me and teach me the techniques needed. I would also like to thank the University of York for letting me come and do this summer project. Finally I would like to thank the Nuffield Foundation for funding my placement and to Dr David Ward who organised the regional placements. This project has been very worthwhile because I have experienced what it would be like to be a researcher. Furthermore, I have learnt a lot about preparation, time management and organisation. I definitely would like to pursue research as part of my future career. Florence Judge-­‐Clayden – Nuffield Summer Research Report -­‐ August 2015