1. 2
Acknowledgements
I
would
like
to
thank
Tokyo
Metropolitan
University
for
providing
me
with
lab
facilities
as
well
as
guidance
on
how
to
use
the
equipment
present
in
their
labs.
I
would
also
like
to
thank
Mr.
John
Fortin
for
the
supervision
and
guidance
he
provided
during
the
process
of
my
research.
And
lastly,
I
would
like
to
thank
my
parents
who
were
supportive
and
had
encouraged
me
during
this
research.
2. 3
Introduction
Drosophila
melanogaster
commonly
known
as
fruit
flies
is
well
studied
and
experimented
organism;
it
is
often
seen
around
rotten
fruits
for
instance
rotten
bananas.
Fruit
flies
have
three
major
body
components
thorax,
abdomen.
The
head
has
two
prominent
eyes,
the
heart
and
part
of
digestive
system
attached
to
the
thorax
and
the
abdomen
contains
the
remainder
of
the
digestive
system
and
reproductive
system.
The
reason
for
D.
melanogaster’s
vast
and
continued
research
is
because
it
only
has
a
generation
time
of
two
weeks
and
is
a
convenient
tool
for
genetic
research
because
their
entire
genome
has
been
sequenced.
An
adult
fly
can
lay
hundreds
of
eggs
in
one
day
and
therefore
many
generations
can
be
raised
in
relatively
short
period
of
time.
Drosophila
was
mainly
an
organism
studied
for
genetics
however
recently
researchers
and
scientists
have
started
to
look
closely
at
developmental
biology.
In
the
area
of
developmental
biology
many
areas
have
and
are
being
researched,
one
of
them
is
nutrition.
Some
studies
have
taken
place,
which
are
regarding
ideal
nutrition
for
longevity
of
flies,
testing
flies’
senses
by
using
different
types
of
nutrient
conditions
and
so
on.
Studies
have
proposed
that
the
different
varieties
of
yeast
within
various
media
were
influencing
factors
in
fruit
fly
nutrition
and
subsequent
development
(Baumberger,
1917).
Another
research
showed
that
developmental
and
fecundity
rates
varied
when
fruit
flies
were
reared
on
media
of
sugar,
tomatoes,
and
grapes
(Jaenike,
1986).
Researchers
have
found
recently
that
fruit
flies
have
pancreatic
like
cell
in
their
brain
known
as
corpora
cardiaca
(CC)
which
is
also
necessary
for
larval
glucose
homeostasis
as
well
(Brownlee,
2004).
The
resemblance
between
mammalian
structure
and
fly
will
be
helpful
to
make
connections
and
make
educated
predictions
about
subject
possibilities
for
further
studies
in
biological
areas.
The
experiment
will
examine
the
effect
of
different
levels
of
added
glucose
in
media
on
the
eclosion
rate
of
D.
melanogaster.
3. 4
The
preliminary
research
conducted
showed
that
numerous
studies
have
been
conducted
regarding
the
nutrient
media
of
fruit
flies
to
find
out
the
correlation
between
certain
nutrients,
vitamins
and
carbohydrates
and
the
fecundity
rate,
eclosion
rate
or
longevity
of
insects.
Therefore
I
formulated
an
objective
to
investigate
the
effect
of
glucose
supplementation
in
nutrient
medium
on
Drosophila
melanogaster.
The
Metropolitan
University
has
offered
to
supply
with
D.melanogaster
and
apparatus
that
will
be
required
to
conduct
this
research.
The
flies
in
the
university
are
fed
with
instant
medium
which
comes
in
powder
form
and
mixed
with
water
which
gives
it
a
spongy
quality.
The
instant
medium’s
ingredients
and
name
are
unknown
because
of
confidentiality
reasons.
The
independent
variable
in
this
experiment
will
be
glucose
concentration
since
the
ingredients
of
the
media
are
unknown
it
must
be
taken
under
consideration
that
their
may
or
may
not
be
glucose
in
the
media.
Commercially
available
dry
mixtures
of
nutrient
medium
aim
to
standardize
the
ingredients
for
rearing
the
fruit
fly
(Flagg,
2005).
Therefore
it
is
considerable
to
assume
that
there
should
be
glucose
present
in
the
media
since
all
living
organisms
need
glucose
since
it
is
essential
for
survival.
4. 5
Research
Question
Investigating
the
effect
of
glucose
supplementation
in
nutrient
medium
on
Drosophila
melanogaster
eclosion
number
Aim
The
purpose
of
the
experiment
is
to
find
out
the
ideal
level
of
glucose
required
for
D.melanogaster
to
eclose
in
the
ultimate
period
of
time.
Preliminary
research
has
shown
that
many
studies
have
been
conducted
regarding
the
nutrient
media
of
insects
to
find
out
their
ideal
nutritional
requirements,
longevity,
and
fecundity
rates.
This
study
will
show
how
glucose
will
affect
the
rate
of
flies
hatching
from
larva
the
results
of
this
experiment
may
benefit
in
further
experiments
where
there
is
need
of
controlling
the
time
when
flies
hatch
for
example
to
evade
predation
or
to
study
predation
behavior.
Hypothesis
It
is
assumed
that
D.
melanogaster
will
eclose
at
expected
time
in
10%
concentrated
glucose
media.
As
it
is
known
that
blood
glucose
level
is
maintained
by
homeostasis
in
which
case
we
can
assume
if
the
glucose
level
is
high
(and
no
insulin
to
maintain)
then
as
in
humans
diabetes
is
acquired
as
we
know
corpora
cardiaca
cells
are
similar
to
the
mammalian
pancreatic
cells
-‐
insects
may
not
be
able
to
develop
on
highly
concentrated
glucose
nutrient
medium
because
of
dehydration
that
may
occur
to
their
cells.
Apparatus
∗ Pipette
(+/-‐
0.5mL)
∗ Graduated
cylinder
(+/-‐
0.5mL)
∗ Beaker
(+/-‐
0.5mL)
∗ Electrical
scale
(+/-‐
o.oo5g)
∗ Microwave
(used
during
the
making
of
medium)
∗ 250mL
culture
vial
(quantity:
36vials
for
3
trials,
with
total
of
9
vials
for
all
the
5. 6
trials
for
all
concentrations)
∗ Incubator
with
consistant
temperature
of
25
degree
centigrade
∗ Trays
to
hold
the
culture
vials
∗ Tweezers
used
to
count
the
number
of
fruit
flies
and
to
separate
eclosed-‐dead
flies
from
the
nutrient
medium
∗ Anesthesia
(
Triethylamine)
to
immobilize
the
flies
during
data
collection
Variables
The
independent
variable
chosen
for
this
experiment
is
the
glucose
concentration.
Additional
glucose
will
be
added
to
the
nutrient
medium,
measuring
up
to
10%,
20%
and
30%.
The
result
will
be
recorded
starting
the
first
eclosion
(number
of
flies
hatched)
in
any
of
the
cultural
vials.
The
dependent
variable
in
this
experiment
is
the
number
of
flies
which
will
eclose
in
the
given
time,
until
no
more
flies
eclose.
The
controlled
variables
are
temperature,
for
which
an
incubator
set
on
constant
temperature
of
25
degree
centigrade
which
is
an
ideal
environment
for
D.melanogaster
growth
(Tatum,
1939).
Equal
amount
of
liquid
and
powder
of
the
nutrient
media
except
the
glucose
concentration
which
will
be
different
as
it
is
the
independent
variable.
Number
of
flies
added
in
each
cultural
vial,
five
male
and
females
which
will
be
added
on
the
first
day
of
the
experiment
and
taken
out
24
hours
after
they
were
put
into
all
cultural
vials
which
are
of
equal
sizes
and
shape.
To
minimize
the
error
that
may
be
introduced
and
to
control
the
experiment
the
adult
flies
which
will
be
added
in
the
cultural
vial
for
reproducing
will
be
all
from
the
same
generation.
6. 7
Method
The
investigation
includes
three
trials
of
the
experimentation
for
accuracy.
Each
trial
consisted
of
two
culture
vials
for
each
concentration
of
additional
glucose
(0%,
10%,
20%
and
30%).
The
flies
were
reared
in
250mL
culture
vials
with
foam
plugs
to
secure
the
top
opening.
The
instant
medium
was
made
using
the
following
procedure:
To
make
10%
concentrated
glucose
solution
for
the
instant
medium,
2g
of
glucose
was
measured
on
electronic
weight
scale,
water
was
added
using
a
pipette
in
the
beaker
till
it
covered
the
glucose
grains,
then
the
beaker
containing
the
glucose
and
water
were
heated
in
microwave
oven
till
it
started
to
boil.
The
dissolved
glucose
was
mixed
till
the
grains
had
melted
(this
was
essential
so
that
it
would
not
introduce
error
in
the
creation
of
instant
media
so
that
if
any
glucose
grain
remaining
left
will
not
introduce
any
error)
added
in
graduated
cylinder
and
water
was
added
till
the
solution
rose
till
20mL.
(Same
steps
were
repeated
for
20%
with
4g
of
glucose
and
30%
with
6g
of
glucose).
Five
female
and
male
adult
D.
melanogaster
were
released
in
each
culture
vial,
the
flies
were
released
according
to
the
day
trial
had
started.
(Trial
1:
On
30th
July
flies
were
released,
31st
eggs
were
laid
and
flies
were
taken
out
of
the
culture
vial.
All
this
was
conducted
at
same
time
everyday
on
2:00pm
which
was
decided
arbitrarily,
to
keep
consistency
and
avoid
any
error.
All
the
flies
were
added
in
consistent
manner
in
the
culture
vial
before
all
three
trials
and
all
nutrient
medium
was
made
at
same
time.
The
culture
vials
were
labeled
according
to
their
concentrations
and
were
kept
in
the
incubator
with
25
degree
centigrade
controlled
temperature
until
next
day.
The
adult
flies
were
taken
out
and
the
eggs
were
kept
in
the
incubator
till
expected
day
of
eclosion
which
according
to
prior
research
(from
secondary
source)
ranges
from
8
to
10
days.
Until
than
the
larva
was
left
to
feed
on
the
instant
medium,
once
the
flies
had
started
to
eclose
the
culture
vials
were
observed
daily
for
more
fly
eclosions.
7. 8
The
culture
vials
consisting
of
the
D.
melanogaster
eggs
were
observed
daily
from
the
5th
day
from
when
the
trial
had
started
and
data
was
recorded
from
the
first
fly
which
had
hatched.
When
the
flies
had
started
to
eclose,
flies
were
transferred
into
separate
culture
vials
and
were
sprayed
by
Trithylamine
(anesthesia)
until
they
were
immobilized
and
then
were
dumped
on
white
paper
if
finding
any
difficulties
to
count,
flies
were
transferred
under
the
dissecting
microscope
for
better
view.
This
way
data
collection
took
place
and
afterwards
healthy
flies
were
returned
to
the
lab
and
flies
which
have
been
dead
were
disposed
into
a
fly
morgue
(flask
containing
water
with
salt).
Exact
steps
were
repeated
for
all
three
trials
of
the
experiment.
The
D.melanogster
were
counted
until
there
were
no
further
eclosions.
Results/Findings
The
data
is
complied
using
Microsoft
Excel
spreadsheet
and
the
difference
between
each
concentration
was
compared.
As
represented
in
Figure
B1
which
clearly
suggests
that
there
is
a
peak
in
eclosion
rate
for
ideal
nutrient
medium
which
would
be
the
medium
without
any
additional
glucose
added.
The
figure
also
suggests
similar
pattern
of
eclosion
for
10%
added
glucose
however
the
number
of
flies
that
have
been
eclosed
has
decreased.
20%
added
glucose
has
similar
results
as
well.
However
the
graphical
representation
suggests
some
major
alteration
had
occurred
in
30%,
despite
the
number
of
eclosed
flies
being
fewer,
there
were
some
flies
which
did
eclose
seemingly
normal
and
healthy
however
in
delay.
In
the
30%
concentrated
glucose
culture
vial
most
larvas
had
failed
to
eclose
therefore
suggesting
that
the
additional
glucose
of
30%
had
a
detrimental
effect
on
the
flies
and
the
number
of
flies
eclosed.
8. 9
Rate
of
eclosion
when
additional
glucose
is
added
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
8
10
12
14
16
18
20
22
24
26
days
eclosionrate(averagenumberofflies)
0%
10%
20%
30%
Figure
B1
Chi
-‐squared
test
was
conducted
to
analyze
the
data
with
respect
to
D.melanogaster
selection
of
certain
glucose
concentration
(no
additional,
10%,
20%
&
30%)
and
also
to
show
any
differences
between
the
trials
of
same
concentration.
Two
sets
of
Chi-‐squared
test
were
conducted
to
test
the
Null
hypothesis,
for
1
–
there
are
no
preferences
for
specific
trial
within
the
three
trials
of
same
concentration
which
was
proven
to
be
rejected.
And
2-‐
there
are
no
preferences
for
one
specific
type
of
concentration
out
of
the
4
tested
which
was
also
proven
to
be
rejected.
The
two
tests
conclude
that
there
are
differences
within
the
each
trial
of
same
concentration
as
well
as
difference
between
each
concentration
which
greatly
contributes
to
the
research
by
showing
that
there
is
a
amount
which
his
ideal
and
popular
trend
amongst
D.melanogaster.
9. 10
Discussion
Current
research
has
show
that
D.
melanogaster
have
cells
which
sense
blood
sugar
and
secrete
insulin
as
pancreatic
beta
cells
do
in
mammals
these
cells
present
in
the
fly
are
corpora
cardiaca
cells
otherwise
known
as
CC
cells.
This
suggests
that
the
mechanism
of
homeostasis
of
fruit
flies
is
related
the
homeostatic
functions
in
humans.
The
CC
cells
are
essential
for
larval
glucose
homeostasis.
They
also
produce
adipokinetic
hormone
which
is
a
polyperptide
with
similar
functions
to
glucagon.
The
glucose
regulation
by
CC
cells
is
disabled
when
exposed
to
sulphonylureas
–
which
is
antibiotic
drug
used
in
the
management
of
diabetes
mellitus
type
2,
they
act
by
increasing
insulin
release
from
beta
cells
in
pancreas.
CC
cells
are
regulators
of
glucose
homeostasis
and
use
glucose
sensing
and
response
mechanisms
similar
to
islet
cells.
It
is
also
known
that
deficiency
of
CC
cells
does
not
result
in
growth
reduction
or
developmental
delay.
In
the
experiment
conducted
there
was
growth
reduction
and
developmental
delay
from
the
statement
above
it
is
suggested
that
CC
cells
may
or
may
not
be
in
deficiency
but
this
had
no
effect
on
the
results.The
product
of
CC
cell
is
adipokinetic
hormone
referred
as
AKH
which
is
similar
to
mammalian
glucagons
in
pancreatic
alpha
cells.
AKH
is
essential
regulator
of
haemolymph
carbohydrate
concentrations
in
D.
melanogaster.
(Kim,
Rulifson,
2004)
According
to
research
conducted
by
scientists
flies
during
starvation
had
increased
severity
of
hypoglycemia
–
pathologic
state
produced
by
a
lower
than
normal
level
of
glucose
in
blood,
in
insects
which
lacked
CC
cells.
This
also
showed
that
AKH
may
be
required
for
compensation
to
maintain
the
glucose
circulation.
(Kim,
Rulifson,
2004)
The
concentration
of
glucose
in
the
blood
is
relatively
constant.
Glucose
is
essential
for
tissue
respiration
and
to
provide
energy
to
perform
vital
activities.
The
sugar
required
immediately
for
energy
in
the
cells
is
changed
in
the
liver
to
glycogen.
The
glycogen
molecules
are
built
up
by
combining
many
glucose
molecules.
Some
of
this
insoluble
glucose
is
stored,
when
the
blood
sugar
falls
below
a
certain
level
the
glycogen
is
10. 11
changed
back
to
glucose
and
releases
it
into
the
circulation.
The
muscle
glycogen
is
not
returned
to
the
circulation
but
is
used
by
muscle
cells
as
source
of
energy
during
muscular
activity
such
as
moving
their
legs
and
wings.
Excess
glucose
not
stored
as
glycogen
is
converted
into
fat.
Generally
homeostatic
control
involves
negative
feedback
which
for
instance,
if
there
is
too
much
glucose
in
blood
(stimulus)
it
stimulates
pancreas
(beta
cells)
to
secrete
more
insulin
which
causes
the
liver
to
convert
glucose
to
glycogen
and
the
blood
glucose
level
falls
(negative
feedback).
And
when
glucose
level
raises
pancreas
(alpha
cells)
secrete
glucagons
which
causes
the
lives
to
convert
glycogen
to
glucose.
(Greenwood,
Shepherd,
Allen,
Butler
47)
Cells
are
adapted
to
keep
consistency
of
water
in
relation
to
salts
to
perform
its
functions.
Water
is
the
solvent
of
the
solutes
in
the
cell.
Each
cell
has
been
adapted
to
a
defined
quantity
of
water
in
relation
to
salts
in
it
to
perform
its
functions.
Homeostatic
mechanisms
generally
maintain
this
concentration.
High
glucose
in
the
D.
melanogaster’s
had
created
and
hypertonic
environment
(the
environment
was
more
concentrated
externally
than
cell
concentration)
which
means
the
cells
had
been
dehydrated
because
of
net
water
loss
and
high
concentration
of
glucose
in
relation
to
water
potential.
Generally
to
prevent
these
situations
cells
osmoregulate
to
keep
the
balance
but
as
the
results
show
that
in
20%
and
30%
concentrated
glucose
most
flies
has
failed
to
survive
because
of
net
water
loss.
The
results
suggest
that
the
glucose
level
in
the
medium
may
have
exceeded
the
ideal
level.
(Greenwood,
Shepherd,
Allen,
Butler
47)
In
humans
inability
to
control
blood
glucose
level
causes
diabetes,
where
the
hormones
can
not
produce
sufficient
amount
of
insulin
to
convert
glucose
into
glycogen
for
storage.
This
results’
in
high
blood
sugar
level
because
the
glucose
is
not
taken
by
body’s
cells.
11. 12
Another
possible
conclusion
of
the
results
acquired
may
be
that
the
larva
may
have
not
fed
on
the
supplementation
because
it
was
not
according
to
their
taste
and
starvation
led
to
their
delay
in
development.
According
to
one
of
the
research
where
they
had
test
the
same
independent
variable
had
results
which
show,
nymph
who
were
fed
diets
supplemented
with
glucose
consumed
less,
gained
less
weight,
developed
slower
and
had
lower
rate
of
survival
than
nymphs
who
were
fed
the
same
diet
without
added
glucose
supplementation
(Silverman
).
Sources
of
error
The
major
concerns
of
the
experiment
were
that
the
ingredients
for
the
nutrient
medium
were
unknown
due
to
confidentiality
reasons.
Therefore
it
is
possible
that
glucose
may
already
be
present
in
the
media,
the
experiment
was
conducted
keeping
in
mind
that
their
may
or
may
not
be
glucose
already
included.
It
is
not
difficult
to
see
that
there
may
already
be
glucose
added
since
the
nutrient
medium
used
in
labs
are
made
so
they
are
suited
to
the
fly
with
all
the
ideal
level
for
ingredients
used.
The
experiment
was
conducted
under
considering
that
the
(10%,
20,
30%)
were
added
glucose
and
that
the
number
0%
referred
to
no
additional
glucose
added
for
the
experiment.
Although
this
may
not
have
affected
the
results
greatly,
it
is
hard
to
conclude
regarding
the
ideal
glucose
level
for
D.melanogaster
since
it
is
unknown
whether
glucose
is
originally
present
or
not
in
the
instant
medium
used.
Looking
at
results
given
in
appendix
A,
TableA3
we
can
see
that
there
was
only
one
fly
eclosed
during
the
whole
trial
3
for
20%
concentration
which
may
suggest
that
there
may
have
been
source
of
error
during
the
experiment
for
this
particular
trial,
since
there
seemed
to
be
a
significant
difference
just
in
this
trial
we
can
assume
the
other
trials
went
fairly
accurate.
The
possible
errors
may
be
that
there
was
a
human
error
of
precision
while
making
the
instant
medium
out
of
the
instant
medium
powder.
The
proportion
of
water
in
terms
of
the
powder
may
have
been
altered
(excess
of
water
or
12. 13
less
water
causing
the
media
to
become
highly
concentrated)
which
may
have
caused
failure
in
flies
eclosing,
another
possibility
may
be
that
the
sponge
plugs
used
to
secure
the
top
of
the
cultural
vial
may
be
kept
open
which
resulted
into
the
flies
that
were
eclosed
to
escape.
Another
factor
of
slight
error
that
should
be
taken
under
consideration
is
that
the
flies
were
taken
out
of
the
incubator
every
day
after
the
first
7
days
to
observe
any
changes
or
eclosion.
This
had
exposed
the
flies
to
the
room
temperature
which
would
have
introduced
change
in
to
the
constant
temperature
of
25
degree
centigrade.
However
this
should
not
have
introduced
any
major
source
of
error.
The
instant
nutrient
medium
is
a
sticky
substance
which
may
have
also
altered
some
of
the
results.
Some
of
the
larva
or
flies
eclosed
had
got
stuck
into
the
medium,
larva
which
got
stuck
were
unable
to
hatch
and
died
and
flies
which
got
stuck
and
died
were
difficult
to
count
since
there
may
be
a
possibility
of
damaging
or
slicing
the
body
into
pieces
and
introducing
error
during
counting
even
though
the
flies
were
counted
under
the
dissecting
scope
there
is
always
possibility
of
slight
error.
13. 14
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