Factors affecting engorgement behavior in the salt marsh horse fly, Tabanus nigrovittatus Macquart (Diptera: Tabanidae)


Downer, K.E.; Nachman, R.J.; Stoffolano, J.G.

Journal of Insect Behavior 20(4): 403-412

2007


Female Tabanus nigrovittatus were field collected and used in laboratory experimentation to explore physiological and behavioral factors that affect engorgement. Previous studies showed sulfakinins act as feeding satiety factors in insects. This study demonstrates that sulfakinins have differing effects on engorgement that is dependent on the feeding technique used in the laboratory. The satiety effect of sulfakinin on flies engorging using the blood-soaked Kimwipep feeding technique is not in agreement with previous experiments using an alternate feeding technique with artificial membranes. This study is the first to demonstrate that the temperature of blood is a significant factor for engorgement in this species. Also, the percentage of flies engorging is significantly different depending on the feeding technique used.

J
Insect
Behav
(2007)
20:403-412
DOI
10.1007/s10905
-007-9086-z
Factors
Affecting
Engorgement
Behavior
in
the
Salt
Marsh
Horse
Fly,
Tabanus
nigrovittatus
Macquart
(Diptera:
Tabanidae)
K.
E.
Downer
R.
J.
Nachman
J.
G.
Stoffolano
Jr
Published
online:
19
June
2007
©
Springer
Science
+
Business
Media,
LLC
2007
Abstract
Female
Tabanus
nigrovittatus
were
fi
eld
collected
and
used
in
laboratory
experimentation
to
explore
physiological
and
behavioral
factors
that
affect
engorge-
ment.
Previous
studies
showed
sulfakinins
act
as
feeding
satiety
factors
in
insects.
This
study
demonstrates
that
sulfakinins
have
differing
effects
on
engorgement
that
is
dependent
on
the
feeding
technique
used
in
the
laboratory.
The
satiety
effect
of
sulfakinin
on
fl
ies
engorging
using
the
blood-soaked
Kimwipe®
feeding
technique
is
not
in
agreement
with
previous
experiments
using
an
alternate
feeding
technique
with
artificial
membranes.
This
study
is
the
first
to
demonstrate
that
the
temperature
of
blood
is
a
significant
factor
for
engorgement
in
this
species.
Also,
the
percentage
of
fl
ies
engorging
is
significantly
different
depending
on
the
feeding
technique
used.
Keywords
Sulfakinins
temperature
engorgement
feeding
techniques
horse
fl
ies
Introduction
Tabanus
nigrovittatus,
commonly
referred
to
as
the
greenhead
horse
fl
y,
inhabits
the
salt
marshes
along
the
Atlantic
coast
of
Massachusetts.
This
fl
y
is
only
present
on
the
K.
E.
Downer
J.
G.
Stoffolano
Jr
Department
of
Plant,
Soil
and
Insect
Sciences, Division
of
Entomology,
University
of
Massachusetts,
Amherst,
MA
01003,
USA
R.
J.
Nachman
Area
Wide
Pest
Management
Research,
Southern
Plains
Agricultural
Research
Center,
USDA,
College Station,
TX
77845,
USA
K.
E.
Downer
(E)
USDA
-APHIS,
1398
W
Truck
Rd.,
Otis
ANGB,
MA
02545,
USA
e-mail:
Kelley.E.Downer@aphis.usda.gov
Springer
404
J
Insect
Behav
(2007)
20:403-412
marsh
for
3-4
weeks,
but
can
be
collected
in
extremely
high
numbers.
While
research
on
its
biology
has
come
a
long
way
over
the
years
(see
Stoffolano
1979;
Magnarelli
and
Stoffolano
1980;
Friend
and
Stoffolano
1983;
Stoffolano
1983;
Graham
and
Stoffolano
1983;
Downer
and
Stoffolano
2006),
a
great
deal
of
information
remains
unknown
about
what
controls
blood
feeding.
Understanding
the
feeding
behavior
of
insects
continues
to
be
one
of
the
most
vital
aspects
of
entomology,
especially
when
trying
to
control
hematophagous
insects.
Perisulfakinin
and
Engorgement
Very
little
is
known
about
short-term
feeding
satiety
regulation
in
insects
(see
Bernays
and
Simpson
1982),
and
even
less
is
known
about
long-term
satiety
regulation.
Recent
research
with
the
neuropeptide
sulfakinin
has
demonstrated
short-term
feeding
inhibition
in
three
non-hematophagous
insects
(Downer
et
al.
2007a;
Maestro
et
al.
2001;
Wei
et
al.
2000).
Previous
research
in
our
laboratory
investigating
the
role
of
sulfakinin
in
the
regulation
of
blood
feeding
satiety
in
T
nigrovittatus
showed
a
similar
inhibiting
effect
of
45-60%
when
injected
with
1
nmol
of
perisulfakinin
(PSK)
and
blood
-fed
using
artificial
membranes
(Downer
et
al.
2007b).
The
artificial
membranes
require
the
deployment
of
the
piercing
mouthparts
prior
to
engorgement.
PSK
at
the
1
nmol
dose
inhibited
individuals
from
engorging
a
blood
meal
and
raised
the
question
of
whether
PSK
was
affecting
the
probing
mechanism
or
the
ingestion
process
as
a
whole
(Downer
et
al.
2007b).
The
following
experiments
were
conducted
in
order
to
determine
whether
sulfakinin
has
the
same
effect
on
engorgement
when
exposed
to
blood
through
an
alternate
laboratory
feeding
technique.
The
blood-soaked
Kimwipes®
provide
an
alternative
method
of
feeding
that
may
not
require
the
probing
mechanism
and
piercing
mouthparts
to
be
employed
prior
to
feeding.
Temperature
and
Engorgement
Thermal
stimuli
are
well
known
to
be
important
factors
in
the
successful
engorgement
of
a
blood
meal
in
tsetse
fl
ies,
mosquitoes,
and
Rhodnius
spp.
(Dethier
1954,
1957;
Friend
and
Smith
1977).
However,
to
our
knowledge,
the
effect
of
blood
temperature
on
engorgement
has
not
previously
been
shown
for
this
species
or
other
Tabanidae.
Thus,
experiments
were
conducted
with
blood-soaked
Kimwipes®
in
order
to
elucidate
the
effect
of
thermal
stimuli
on
engorgement.
Parafilm®
versus
Kimwipe®
Feeding
Techniques
Stoffolano
(1979)
found
that
the
best
method
for
feeding
T
nigrovittatus
was
by
providing
grouped
females
with
blood-soaked
Kimwipes®.
He
found
that
Kimwipe®-
soaked,
group
-fed
females
ingested
significantly
more
blood
(51.4
ill)
than
did
Parafilm®,
group
-fed
females
(40
pi).
However,
the
author
did
not
look
at
the
percentage
of
engorged
fl
ies
per
trial.
In
fact,
an
overwhelming
majority
of
the
papers
on
hematophagous
arthropods
and
their
feeding
habits
do
not
report
the
percentage
of
engorgement.
Reporting
percentages
is
important
for
understanding
feeding
success
rates
of
blood
feeders.
1
Springer
J
Insect
Behav
(2007)
20:403-412
405
Materials
and
Methods
Collecting
and
Maintaining
Flies
Female
host
-seeking
Tabanus
nigrovittatus
were
fi
eld
collected
from
box
traps
on
the
salt
marshes
of
Essex
Co.,
Massachusetts,
during
July
2005.
Flies
were
laboratory
maintained
as
previously
described
by
Downer
and
Stoffolano
(2006).
Prior
to
experimentation,
all
fl
ies
were
deprived
of
granulated
sucrose
for
16-20
h.
All
fl
ies
used
in
experimentation
were
tested
the
day
after
being
collected
in
the
field.
The
exact
chronological
ages
of
all
fl
ies
used
are
unknown.
The
first
collection
date
for
the
2005
experiments
was
4
July
2005
and
field
collections
were
made
every
other
day
consecutively
throughout
the
season.
Perisulfakinin
and
Engorgement
Prior
to
experimentation,
fl
ies
of
approximately
the
same
size
were
cold
immobilized
in
the
fr
eezer.
Sham
-injected
and
treatment
fl
ies
were
injected
with
1µl
of
solution
in
the
second
to
last
intersegmental
membrane
on
the
right
ventral
side
of
the
abdomen.
All
fl
ies
were
injected
using
a
30
-gauge
needle
attached
to
a
25-µ1
glass
gastight
Hamilton
#1750
syringe
(Hamilton
Co.,
Reno,
Nevada).
Sham
-injected
fl
ies
were
injected
with
Phormia
saline
(Chen
and
Friedman
1975)
and
treatment
fl
ies
were
injected
with
perisulfakinin
(Bachem,
PA,
USA)
dissolved
in
Phormia
saline.
The
sulfakinin
was
prepared
in
a
stock
solution
of
80%
acetonitrile
and
20%
water,
made
up
to
0.01%
trifluoroacetic
acid.
Treatment
fl
ies
were
injected
with
1-nmol
and
10-nmol
doses
of
perisulfakinin.
Absolute
control
fl
ies
(used
in
the
experiments
described below)
were
cold
immobilized
and
set
on
ice
for
the
same
duration
as
the
sham
-injected
and
treatment
fl
ies.
After
an
individual
fl
y
was
injected
it
was
placed
back
on
ice
until
the
entire
experimental
group
(n=20)
was
completed.
An
experimental
group
consisted
of
the
control,
sham
-control,
and
treatment.
The
entire
injection
process
for
each
group
took
less
than
10
min.
Each
experimental
group
of
fl
ies
was
placed
in
a
23-cm
3
metal
-screened
cage
and
the
feeding
assay
was
started.
There
was
zero
mortality
with
all
injections.
All
fl
ies
recovered
fr
om
injections
and
resumed
normal
behavior
in
less
than
5
min.
For
the
feeding
assay,
citrated
beef
blood
was
warmed
on
a
hot
plate
to
37°C
and
stirred
with
a
magnetic
stirrer.
Kimwipes®
were
placed
on
top
of
each
23-cm
3
cage
and
the
warmed
blood
was
pipetted
on
to
the
Kimwipes®
until
they
had
been
completely
soaked.
A
lamp
with
a
60-W
bulb
was
positioned
over
it
to
provide
adequate
light
and
keep
the
blood
warmed
at
approximately
37°C.
The
fl
ies
were
then
allowed
to
feed
ad
libitum
for
one
hour.
After
the
feeding
assays
were
completed,
fl
ies
were
killed
in
the
freezer.
Once
dead,
they
were
submerged
in
70%
ethanol
and
each
one
was
held
up
to
a
light
bulb
to
check
for
the
presence
of
a
blood
meal.
The
abdomen
of
the
fl
ies
appeared
red
if
a
blood
meal
was
taken
and
appeared
yellow
if
lacking
one.
Any
questionable
individuals
were
dissected
to
check
for
the
presence
of
blood
in
the
midgut.
A
total
of
587
fl
ies
were
used
during
experimentation
with
ten
replicates
performed.
All
statistical
comparisons
were
performed
using
ANOVA
to
compare
the
percentage
of
engorgement
by
treatment
and
post
-hoc
comparison
using
the
Tukey—Kramer
HSD
Springer
406
J
Insect
Behav
(2007)
20:403-412
test
(JMP,
SAS
Institute
Inc.
2005).
The
percentage
of
difference
between
the
sham
-
injected
group
and
the
treatment
group
was
calculated
by:
(%
engorged
treatment—
%
engorged
sham)/%
engorged
sham
*
100.
Temperature
and
Engorgement
The
experiments
testing
the
effect
of
blood
temperature
were
run
simultaneously
with
the
perisulfakinin
experiments.
The
procedures
remained
the
same,
but
there
were
no
injections
made
and
'cold
blood'
was
kept
at
room
temperature
(no
heating
involved,
approximately
21-23°C)
for
the
feeding
assay.
In
addition,
a
lamp
was
not
positioned
directly
over
the
Kimwipes®
in
the
'cold
blood'
experimental
group,
but
the
cage
was
placed
next
to
the
warmed,
blood-soaked
Kimwipe®
cage
in
order
to
still
have
access
to
adequate
light.
Ten
replicates
were
performed
with
648
fl
ies
used
in
these
experiments.
An
ANOVA
was
used
to
compare
the
percentage
of
engorgement
by
treatment
and
Tukey—Kramer
HSD
test
in
post
-hoc
comparison
(JMP,
SAS
Institute
Inc.
2005).
Comparison
of
Feeding
Techniques
We
compared
the
percentage
of
fl
ies
that
engorged
on
an
artificial
membrane
to
the
percentage
of
fl
ies
engorged
on
blood-soaked
Kimwipes®.
We
also
compared
the
two
methods
in
alternate
and
successive
feeding
assays.
A
group
of
fl
ies
(n=20)
were
allowed
to
blood
feed
ad
libitum
for
1
h
using
a
Parafilm®
membrane,
by
the
method
previously
described
by
Downer
and
Stoffolano
(2006).
After
the
time
trial
was
finished,
flies
were
cold
immobilized
and
the
number
of
fl
ies
that
had
taken
a
blood
meal
was
counted.
Those
fl
ies
that
blood
fed
were
discarded
and
the
flies
that
had
not
taken
a
blood
meal
were
put
back
into
another
metal
-screened
cage
and
allowed
to
feed
ad
libitum
for
30
min,
using
the
Kimwipe®
feeding technique.
At
the
end
of
the
feeding
assay
the
fl
ies
were
killed
and
then
recounted
to
determine
how
many
had
engorged
during
the
subsequent
feeding
method.
The
opposite
feeding
assay
(i.e.,
Kimwipe-fed
fi
rst
and
then
Parafilm®-fed)
was
also
tested
to
see
if
the
order
of
the
feeding
technique
had
any
effect.
A
total
of
429
flies
were
used
and
ten
replicates
performed
for
each
feeding
technique.
A
t
test
was
used
to
compare
the
mean
percentage
(+
SEM)
of
flies
that
had
engorged
in
the
first
part
of
the
feeding
trial
to
those
that
had
fed
in
the
second
part
of
the
feeding
trial
(JMP,
SAS
Institute
Inc.
2005).
However,
the
data
is
presented
in
the
form
of
the
number
of
flies
that
fed
out
of
the
total
fl
ies
exposed
to
blood
using
the
two
different
techniques
in
order
to
amplify
the
differences.
Results
Perisulfakinin
and
Engorgement
Perisulfakinin
(PSK)
had
no
statistically
significant
effect
on
engorgement
by
T
nigrovittatus
when
fed
using
the
warmed,
blood-soaked
Kimwipe®
technique
(P=
0.06,
F2
,
27=3.1).
The
percentage
of
sham
-injected
flies
engorged
was
58.7%,
while
the
percentage
engorged
for
the
10
nmol
and
1
nmol
treatments
was
72.6%
and
70.5%,
respectively
(Fig.
1).
While
not
statistically
significant,
10
nmol
PSK
stimulated
1
Springer
J
Insect
Behav
(2007)
20:403-412
407
The
Effect
of
PSK
on
Engorgement
by
T.
nigrovittatus
(Warmed
Kimwipe
Technique)
90
80
-
7,
70-
Lb
60
-
o
50
-
c
40
-
W
30
-
'a
l?.
20
-
10-
0
58.7
I
Sham
72.7
70.5
I
10nmol
1nmol
n=
587
flies;
reps.=
10
Fig.
1
The
effect
of
perisulfakinin
(PSK)
on
engorgement
using
warmed,
blood-soaked
Kimwipes®
by
T
nigrovittatus
(July
2005).
Engorgement
was
increased
by
24%
and
20%
when
injected
with
10
nmol
and
1
nmol
of
PSK
(respectively),
compared
to
the
sham
-injected
group.
However,
the
effect
of
PSK
on
engorgement
using
this
feeding
technique
was
not
significant
(P=0.06,
F
2
,
27
=3.1).
60
fl
ies
were
used
per
replicate
with
ten
replicates
used
throughout
experimentation.
Small
bars
represent
SEM.
engorgement
by
24%
and
1-nmol
stimulated
engorgement
by
20%,
compared
to
the
sham
-injected
experimental
group.
Temperature
and
Engorgement
The
percentage
of
T
nigrovittatus
engorging
when
offered
warmed
blood-soaked
Kimwipes®
was
82.1%,
while
only
12%
of
the
flies
offered
room
temperature
blood-
soaked
Kimwipes®
engorged.
Only
19.2%
of
T.
nigrovittatus
engorged
when
fed
using
the
Parafilm®
membrane,
compared
to
the
82.1%
offered
blood-soaked
Kimwipes®.
The
effect
of
feeding
technique
and
the
temperature
of
blood
significantly
effects
engorgement
(P<0.0001,
F2
,
27=91.9)
(Fig.
2).
According
to
a
Tukey—Kramer
HSD
test,
the
Parafilm®
control
and
the
Kimwipes®
soaked
in
room
temperature
blood
are
not
significantly
different.
Percentage
Engorged
100
90
80
70
60
50
40
30
20
10
0
Effect
of
Feeding
Technique
and
Blood
Temperature
on
Engorgement
by
T.
nigrovittatus
A
B
Parafilm
(Warm
Blood)
Kimwipe
(Warm
Blood)
Kimwipe
(Cold
Blood)
Fig.
2
The
effect
of
blood
temperature
and
feeding
technique
on
the
percentage
of
engorged
female
T
nigrovittatus.
The
percentage
of
fl
ies
engorged
when
offered
warmed,
blood-soaked
Kimwipes®
was
82.1%.
Flies
offered
room
temperature
(`cold
blood'),
blood-soaked
Kimwipes®
engorged
less
(12%),
while
19.2%
of
the
flies
offered
warmed
blood
through
artificial
membranes
(Parafilm®)
engorged.
The
temperature
of
the
blood
meal
and
the
feeding
technique
has
a
significant
effect
on
engorgement
(P<
0.0001,
F
2
,
27
=91.9).
Sixty-five
fl
ies
were
used
per
replicate
with
ten
replicates
performed
throughout
experimentation.
Bars
with
different
letters
denote
significant
difference.
Small
bars
represent
SEM.
Springer
408
J
Insect
Behav
(2007)
20:403-412
Comparison
of
Two
Feeding
Techniques
There
is
no
significant
difference
in
the
mean
percentage
of
females
engorged
when
exposed
to
Parafilm®
membranes
first
(15.8+7.5)
or
second
(32.0+7.5)
in
the
two
separate
assays
(P=0.14,
F
1
,
20
=2.3).
Nor
was
there
was
a
statistically
significant
difference
in
the
mean
percentage
of
females
engorged
when
Kimwipe®-exposed
first
(74.7+5.5)
compared
to
fl
ies
Kimwipe®-exposed
second
(59.5+5.5)
in
the
two
assays
(P<0.07,
F
1
,
18
=3.9).
Thus,
the
order
of
the
feeding
technique
made
no
difference
in
the
percentage
of
females
engorging.
When
fl
ies
were
exposed
first
to
the
membranes
and
second
to
the
Kimwipes®,
the
mean
percentage
of
fl
ies
that
fed
using
the
Parafilm®
(15.8+4.9)
was
significantly
different
from
the
mean
percentage
of
fl
ies
that
fed
using
Kimwipes®
(59.5+4.9)
(P<
0.0001,
F
1
,
18
=39.1).
In
other
words,
of
the
total
number
of
fl
ies
(211)
exposed
to
blood
using
a
Parafilm®
membrane
first,
only
34
fl
ies
engorged.
Of
the
remaining
174
fl
ies,
those
that
did
not
engorge
by
probing
the
Parafilm®
membrane,
101
fl
ies
did
successfully
engorge
blood
using
the
warmed,
blood-soaked
Kimwipes®
(Fig.
3).
For
the
assay
where
the
fl
ies
were
exposed
fi
rst
to
warmed,
blood-soaked
Kimwipes®
and
second
to
the
membranes,
the
mean
percentage
of
fl
ies
that
fed
using
Engorgement
Response
to
Two
Different
Techiniques
of
Blood
Feeding
Number
of
Flies
Engorged
240
210
-
180
-
150
-
120
-
90
-
60
-
30
-
0
1st
meal-
parafilm
2nd
meal-
Kimwipe
1st
meal-
2nd
Kimwipe
meal-
parafilm
Total
Flies
Used
211
174
218
163
#
of
Flies
Engorged
34
101
52
Feeding
Technique
20
fl
ies
per
replicate;
reps
=
10
22
Total
Flies
Used
#
of
Flies
Engorged
Fig.
3
The
engorgement
response
of
female
T
nigrovittatus
during
alternate
and
successive
feeding
assays.
The
data
was
analyzed
using
t
-test
to
compare
the
mean
percentage
of
fl
ies
that
fed
when
exposed
to
parafilm
fi
rst
and
blood-soaked
Kimwipes
second.
There
was
a
significant
difference
in
mean
percentage
SEM)
of
fl
ies
that
engorged
using
the
Parafilm®
assay
fi
rst
(15.8±4.9)
and
the
Kimwipe®
assay
second
(59.5±4.9)
(P<0.0001;
F
1
,
18
=39.1).
There
were
20
fl
ies
used
per
experimental
group
(i.e.,
Parafilm®
and
Kimwipes®)
and
ten
replicates
performed.
A
total
of
218
fl
ies
used
in
experimentation
in
the
fi
rst
assay.
Important
to
note
is
that
the
data
is
graphed
as
the
number
of
fl
ies
that
engorged
out
of
the
total
fl
ies
exposed
to
blood
using
each
feeding
technique
in
order
to
amplify
the
differences
between
the
techniques.
The
alternate
design
was
also
tested,
where
fl
ies
were
exposed
to
blood-soaked
Kimwipes®
fi
rst
and
then
to
Parafilm®
membranes
second.
There
was
a
significant
difference
in
mean
percentage
SEM)
of
fl
ies
that
engorged
when
exposed
to
blood-soaked
Kimwipes®
fi
rst
(74.7±7.8)
and
Parafilm®
second
(32.0±7.8)
(P<0.0012;
F
1
,
20=14.9).
There
were
20
fl
ies
used
per
experimental
group
and
ten
replicates
performed.
A
total
of
211
fl
ies
were
used
in
experimentation
during
the
assay.
1
Springer
J
Insect
Behav
(2007)
20:403-412
409
the
Kimwipes®
(74.7+7.8)
was
significantly
different
from
the
mean
percentage
of
fl
ies
that
fed
using
the
membranes
(32.0+7.8)
(P
<0.0012,
F
1
,
20
=14.9).
Therefore,
of
the
218
fl
ies
allowed
to
feed
on
warmed,
blood-soaked
Kimwipes®
fi
rst,
163
fl
ies
successfully
engorged.
Out
of
the
remaining
52
fl
ies
that
failed
to
engorge
a
blood
meal,
22
flies
did
successfully
engorge
blood
through
the
Parafilm®
membrane
(Fig.
3)
(Note:
Some
fl
ies
were
lost
during
the
transferring
phase,
accounting
for
the
difference
in
numbers
between
feeding
assays).
Discussion
Perisulfakinin
and
Engorgement
Previous
research
(Downer
et
al.
2007b)
examining
the
effect
of
PSK
on
engorgement
through
a
parafilm
membrane
in
T
nigrovittatus
showed
that
10
nmol
of
PSK
stimulated
engorgement
by
45-70%,
while
1
nmol
inhibited
engorgement
by
45-60%
relative
to
the
sham
-injected
group
(2005-2006
data).
This
study
demonstrates
the
same
trend,
although
not
statistically
significant,
of
increased
engorgement
by
the
10-nmol
dose
of
PSK.
However,
the
percentage
of
increased
engorgement
is
lower
when
fed
using
blood-soaked
Kimwipes®
(24%)
compared
to
using
Parafilm®
membranes
(45%)
from
previous
experiments
(Downer
et
al.
2007b).
Interestingly,
the
1-nmol
dose
of
PSK
does
not
demonstrate
the
same
inhibition
as
previously
observed
in
the
Parafilm®
membrane
study
(e.g.,
increased
engorgement
of
20%
when
fed
using
blood-soaked
Kimwipes®
compared
to
inhibition
of
engorgement
by
45%
when
fed
using
Parafilm®
membranes).
These
experiments
were
designed
to
determine
what
effect
sulfakinin
has
on
engorgement
in
hematophagous
insects
and
to
determine
if
the
type
of
laboratory
feeding
technique
plays
any
role
in
the
expression
of
the
drug
effect.
Parafilm®
feeding
requires
lacerating
and
probing
the
membrane,
whereas
Kimwipe®
feeding
does
not
require
the
initiation
of
the
piercing
mouthparts
prior
to
feeding.
The
difference
between
the
contrasting
effects
at
the
1-nmol
dose
for
the
Parafilm®
feeding
technique
(inhibition
of
engorgement
by
45%,
Downer
et
al.
2007b)
and
the
Kimwipe®
feeding
technique
(stimulation
of
engorgement
by
20%)
suggests
that
PSK
may
be
influencing
the
probing
mechanism
more
since
the
Parafilm®
technique
requires
the
fl
ies
to
puncture
the
membrane
in
order
to
blood
feed.
Other
research
found
that
sulfakinins,
at
all
doses,
inhibited
feeding
in
non-
hematophagous
insects
(Black
blowfly,
Phormia
regina:
Downer
et
al.
2007a;
German
cockroach,
Blattodea
germanica:
Maestro
et
al.
2001;
Desert
locust,
Schistocerca
gregaria:
Wei
et
al.
2000).
The
endogenous
amounts
of
sulfakinins
that
naturally
occur
in
the
tabanid,
as
other
insects,
are
unknown
at
this
time,
thus
adding
to
the
difficulty
of
determining
whether
the
observed
effects
are
a
physiological
or
a
pharmacological
effect.
It
is
possible
that
the
lack
of
statistical
significance
is
due
to
experimental
testing
being
conducted
on
a
wild
population
of
fl
ies
and
not
ones
raised
in
a
laboratory.
The
chronological
and
physiological
ages
of
the
fl
ies
were
unknown
when
testing
and
those
factors
could
influence
the
fl
y's
response
to
PSK.
If
sulfakinin
was
affecting short-term
satiety,
it
would
be
expected
that
meal
size
would
be
regulated
in
blood
feeding
(as
was
shown
in
the
other
non-hematophagous
insects).
The
role
of
stretch
receptors
in
regulating
the
blood
meal
size
(i.e.,
short-term
Springer
410
J
Insect
Behav
(2007)
20:403-412
feeding
satiety)
in
hematophagous
insects
have
been
well
documented
(Gwadz
1969;
Friend
and
Smith
1977).
Further,
horse
fl
ies
only
require
one
blood
meal
between
each
gonadotrophic
cycle.
The
implication
of
sulfakinin
(specifically
1
nmol
PSK)
inhibiting
probing
suggests
that
it
may
act
as
a
long-term
satiety
regulation
mechanism,
terminating
probing
and
theoretically
blood
feeding
until
after
oogenesis.
It
may
be
that
sulfakinins
act
additively
with
the
stretch
receptors
to
produce
short-
term
satiety
and/or
act
on
long-term
blood
-feeding
regulation
in
this
species.
Temperature
and
Engorgement
Most
studies
examining
engorgement
in
hematophagous
insects
do
not
report
the
percentage
of
insects
that
engorge,
but
rather
report
the
amount
of
blood
engorged.
In
the
few
studies
that
do
report
the
percentages
of
hematophagous
insects
that
successfully
engorge
(either
through
artificial
membranes
or
live
hosts),
those
percentages
are
relatively
low
(30%
by
Aedes
spp.
on
live
hosts:
Mather
and
DeFoliart
1984;
27.3%
by
T
nigrovittatus
fed
by
artificial
membrane
in
the
field:
Thompson
and
Krauter
1978).
Important
to
note
is
that
the
percentage
of
engorgement
by
T
nigrovittatus
using
the
Parafilm®
membrane
feeding
technique
(19.2%)
is
in
agreement
with
the
few
studies
that
have
reported
percentages
of
engorgement.
Reporting
the
percentage
of
engorgement
is
beneficial
for
those
studies
looking
at
feeding
success
rates
and
consequences
of
blood
engorgement.
There
is
a
62.9%
difference
in
engorgement
between
the
two
feeding
techniques
(19.2%
for
Parafilm®
membrane
and
83.1%
for
Kimwipes®).
It
is
interesting
how
much
of
a
significant
difference
there
is
in
the
engorgement
response
between
the
two
different
feeding
techniques,
especially
when
the
method
thought
to
be
employed
normally
in
the
wild
(probing
through
a
membrane)
by
the
fl
y
is
so
much
lower
than
the
uncharacteristic
way
(open
pools
of
blood)
of
encountering
a
blood
meal.
It
is
highly
unlikely
that
the
reason
for
increased
engorgement
on
Kimwipes®
is
because
of
tarsal
stimulation
from
contacting
the
blood.
Stoffolano
et
al.
(1990)
tested
the
chemosensilla
from
the
tarsi,
tibia,
terminal
end
of
the
antenna,
labrum
and
the
labral
groove
to
various
substances.
The
authors
found
that
all
of
the
chemosensilla
responded
to
salts
and
sugars;
however,
the
only
chemosensilla
that
responded
to
sera
and
plasma
were
those
at
the
tip
of
the
labrum
and
lining
the
labral
groove.
Chirov
and
Alekseyev
(1970)
were
also
able
to
show
that
tabanids
would
feed
from
free
liquids
(blood,
water,
and
sugar
water)
when
a
pipette
was
inserted
over
the
mouthparts
with
the
labium
moved
aside.
Friend
and
Stoffolano
(1984)
suggest,
"...Mouthpart
deployment
may
not
play
as
significant
a
role
in
establishing
the
blood
-feeding
mode
in
pool
feeders
as
it
does
in
vessel
feeders."
However,
it
is
unlikely
that
the
fl
ies
would
often
encounter
open
pools
of
blood
in
the
wild.
We
propose
that
the
fl
ies
are
thermally
stimulated
by
the
warmed
blood
on
the
Kimwipes®
and
once
the
chemosensilla
on
the
labrum
and
labral
groove
contact
the
blood
they
are
stimulated
to
engorge.
The
fl
ies
exposed
to
Kimwipes®
soaked
in
cold
blood
lacked
the
thermal
stimulation
and
failed
to
engorge.
The
Parafilm®
feeding
assays
are
probably
not
as
successful
in
eliciting
engorgement
because
of
the
lack
of
other
natural
stimuli,
which
are
missing
in
the
laboratory.
Thermal
stimuli
have
proven
to
be
an
important
factor
in
probing
by
other
hema-
tophagous
insects
(Dethier
1954;
Friend
and
Smith
1977)
and
a
pair
of
thermoreceptor
sensilla
on
the
antennae
have
been
identified
in
the
mosquito,
Aedes
agypti
(Davis
1
Springer
J
Insect
Behav
(2007)
20:403-412
411
and
Sokolove
1975).
However,
this
is
the
first
study
to
examine
the
effect
of
blood
temperature
on
the
engorgement
response
in
Tabanidae,
especially
using
the
Kimwipe®
feeding
technique.
T
nigrovittatus
engorged
significantly
more
on
warmed
blood
than
on
blood
that
was
kept
at
room
temperature.
Comparison
of
Two
Feeding
Techniques
Stoffolano
(1979)
showed
that
there
are
two
types
of
techniques
that
can
be
used
to
feed
T
nigrovittatus
in
the
laboratory.
The
alternate
and
successive
feeding
assays
were
designed
in
order
to
have
a
better
understanding
of
why
there
were
low
percentages
of
fl
ies
engorging
using
the
Parafilm®
feeding
technique.
We
wanted
to
determine
if
the
fl
ies
were
not
engorging
because
they
were
not
in
the
blood
-feeding
mode
or
if
the
failure
to
engorge
was
because
of
missing
stimuli.
In
both
techniques,
Parafilm®
membrane
and
Kimwipes®,
there
were
always
some
flies
that
failed
to
engorge.
When
we
took
the
flies
that
failed
to
feed
through
Parafilm®
membranes
and
presented
them
with
blood-soaked
Kimwipes®,
we
always
observed
a
higher
percentage
of
engorgement
than
in
the
original
Parafilm®
assay.
We
observed
the
same
pattern
when
the
Kimwipe®
assay
was
performed
first
in
that
more
flies
engorged
by
the
Kimwipe®
technique.
While
there
was
no
statistical
difference
in
the
percentage
of
engorgement
when
examining
the
order
of
exposure
using
Parafilm®
membranes,
engorgement
was
higher
for
those
flies
previously
exposed
to
blood-soaked
Kimwipes®.
This
may
be
due
to
the
central
excitatory
state
being
elevated
after
having
more
contact
with
open
pools
of
blood.
It
is
interesting
that
100%
engorgement
is
never
observed,
especially
because
all
of
the
fl
ies
should
be
in
the
blood
-feeding
mode
since
they
were
engaging
in
host
-
seeking
behavior
(i.e.,
collected
from
the
field
traps).
This
study
demonstrates
that
the
lack
of
successful
engorgement
when
fl
ies
are
exposed
to
blood
using
an
artificial
membrane
is
not
because
the
flies
are
not
in
the
blood
feeding
mode.
Rather,
there
are
probably
missing
stimuli
since
the
fl
ies
that
failed
to
engorge
using
membranes
fed
more
readily
when
exposed
to
blood-soaked
Kimwipes.
Our
results
suggest
that
there
are
cues
or
stimuli
missing
from
the
Parafilm®
membrane
feeding
technique
essential
to
stimulate
the
fl
ies
to
successfully
probe
and
ingest
a
blood
meal
in
the
laboratory.
Acknowledgements
The
authors
wish
to
acknowledge
Jack
Card,
Esteban
Cuebas-Incle,
and
Walter
Montgomery
of
the
Northeast
Massachusetts
Mosquito
Control
and
Wetlands
Management
District
for
the
use
of
box
traps,
their
expertise,
and
their
dedication
to
the
salt
marsh
hematophagous
Diptera.
We
gratefully
acknowledge
Rick
Adams
of
Adam's
Farm
(Athol,
MA)
for
supplying
fresh
bovine
blood.
This
research
was
supported
by
the
Massachusetts
Agricultural
Experiment
Station
(Hatch
817
to
J.
G.
S.)
and
is
published
as
contribution
No.
3394
from
the
Massachusetts
Agricultural
Experiment
Station.
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SJ
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