Growth and food utilization parameters of germ-free house crickets, Acheta domesticus


Kaufman, M.G.; Klug, M.J.; Merritt, R.W.

Journal of Insect Physiology 35(12): 957-967

1989


Conventional and germ-free larvae of Acheta domesticus are reared under three diet conditions varying in nutritional quality in order to assess possible roles of the gut microbial community in these insects. Although growth rates, adult size, and maturation times were nearly identical for the two groups across all diet treatments, conventional larvae digested and converted food into biomass more efficiently than germ-free larvae in all cases. Enhanced food utilization efficiency in conventional animals was related to the scavenging of soluble carbohydrates by gut microorganisms. Comparisons of enzymatic activity in both groups also indicated that the gut bacteria increased the level of several carbohydrase classes in conventional larvae and were the sole source of a -galactosidase activity. The microorganisms may also have aided in conservation of nitrogenous compounds, but this was influenced by age and diet. Microbial metabolism of uric acid could not consistently be related to overall food utilization or nitrogen economy, and did not appear to be an important means of conserving nitrogen.

J.
Insect
Physic!.
Vol.
35,
No.
12,
pp.
957-967,
1989
0022-1910/89
$3.00
+
0.00
Printed
in
Great
Britain.
All
rights
reserved
Copyright
©
1989
Pergamon
Press
plc
GROWTH
AND
FOOD
UTILIZATION
PARAMETERS
OF
GERM
-FREE
HOUSE
CRICKETS,
AC'HE1A
Dumt6'1lc;uS
MICHAEL
G.
YAUFMAN,1*
MICHAEL
J.
KTTtnl
and
I2
TrIT
A.Pn
W.
MPRFITT
2
'
Michigan
State
University,
W.
K.
Kellogg
Biological
Station,
Hickory
Corners,
MI
49060
and
2
Michigan
State
University,
Department
of
Entomology,
East
Lansing,
MI
48824
U.S.A.
(Received
30
March
1989;
revised
27
June
1989)
Abstract
—Conventional
and
germ
-free
larvae
of
Acheta
domesticus
are
reared
under
three
diet
conditions
varying
in
nutritional
quality
in
order
to
assess
possible
roles
of
the
gut
microbial
community
in
these
insects.
Although
growth
rates,
adult
size,
and
maturation
times
were
nearly
identical
for
the
two
groups
across
all
diet
treatments,
conventional
larvae
digested
and
converted
food
into
biomass
more
efficiently
than
germ
-free
larvae
in
all
cases.
Enhanced
food
utilization
efficiency
in
conventional
animals
was
related
to
the
scavenging
of
soluble
carbohydrates
by
gut
microorganisms.
Comparisons
of
enzymatic
activity
in
both
groups
also
indicated
that
the
gut
bacteria
increased
the
level
of
several
carbohydrase
classes
in
conventional
larvae
and
were
the
sole
source
of
a
-galactosidase
activity.
The
microorganisms
may
also
have
aided
in
conservation
of
nitrogenous
compounds,
but
this
was
influenced
by
age
and
diet.
Microbial
metabolism
of
uric
acid
could
not
consistently
be
related
to
overall
food
utilization
or
nitrogen
economy,
and
did
not
appear
to
be
an
important
means
of
conserving
nitrogen.
Key
Word
Index:
A.
domesticus,
Gryllidae,
germ
-free
rearing,
digestibility,
food
utilization,
carbohydrates,
nitrogen
excretion,
uric
acid
excretion
INTRODUCTION
The
roles
of
microorganisms
found
in
the
digestive
tracts
of
insects
are,
with
a
few
exceptions,
poorly
understood.
Non
-transient
populations
of
gut
microorganisms
in
insects
have
usually
been
correlated
with
a
particular
diet
which
is
refractory
and/or
deficient
in
specific
nutrients
(Buchner,
1965;
Breznak,
1982).
However,
several
groups
of
omnivo-
rous
insects
harbour
extensive
communities
of
gut
microorganisms;
most
notably
cockroaches
(e.g.
Periplaneta
americana)
and
crickets
(e.g.
Gryllus
spp,
Achetv
,
domesticus)
and
MAricoVeI7,
1984;
Martoja,
1966;
Ulrich
et
al.,
1981).
Possible
nutri-
tional
interactions
between
insect
and
microoganisms
are
less
obvious
in
these
cases
because
of
the
variable
quality
of
consumed
food
items.
Many
types
of
mutualistic
nutritional
interactions
can
be
postulated
for
gut
symbionts
and
omnivorous
insects,
but
equally
pertinent
questions
centre
around
how
the
interactions
may
vary
with
age
and
diet
conditions.
Crickets
(Gryllidae,
Ciryllotalpidae)
are
excellent
examples
of
omnivorous
insects
with
associated
gut
microorganisms;
a
large,
permanent
bacterial
community
is
characteristic
of
their
anterior
hindgut
segments
(Martoja,
1966;
Ulrich
et
al.,
1981;
Nation,
1983).The
association
of
gut
microorganisms
with
fi
eld
crickets
(Gryllus
bimaculatus)
is
apparently
consistent
through
developmental
stages,
but
nonobligatory
to
the
insect
(Martoja,
1966).
Crickets
are
also
likely
to
encounter
and
consume
a
variety
of
plant
and
animal
material
during
the
course
of
their
development
crpnnic,
1
Q521
;
Rate
1969;
Mathenv,
1981;
Gangwere,
1961).
It
has
also
been
shown
that
*To
whom
all
correspondence
should
be
addressed.
considerable
digestion
and
transport
of
nutritionally
valuable
compounds
occur
in
the
gryilid
anterior
hindgut
(Thomas
and
Nation,
1984a,
b),
but
the
contribution
of
the
bacterial
community
to
these
processes
is
not
yet
understood.
In
omnivorous
cockroaches,
hindgut
microorgan-
isms
are
thought
to
contribute
to
carbon
and
nitrogen
metabolism
in
the
insect
through
the
degradation
of
cellulose
(Bignell,
1977,
1981)
and
uric
acid
(Cochran,
1985).
nigestinn
of
refractory
enrhnn
PnliflpnlIndc
such
as
cellulose
has
been
reported
to
occur
in
some
cricket
species
(Martoja,
1966;
McFarlane
and
Distler,
1982),
'a
nd
a
high
pci
1..,cnia
g
c
Of
i,acterial
isolates
from
the
house
cricket
hindgut
were
uricolytic
(Ulrich
et
al.,
1981).
These
fi
ndings
suggested
that
the
bacterial
community
in
the
gryilid
hindgut
might
play
a
role
in
carbon
and
nitrogen
economy.
The
following
report
details
our
initial
investiga-
tions
into
the
nutritional
interactions
between
Acheta
domesticus
(L.)
(Orthoptera:
Gryllidae)
and
the
bacterial
community
inhabiting
its
anterior
hindgut.
The
objective
was
to
determine
the
overall
effects
of
the
bacterial
community's
presence
on
food
utiliza-
tion
by,
and
development
of,
A.
domesticus
larvae
reared
under
dietary
conditions
varying
in
carbohy-
drate
and
nitrnirn
rnntent
Cnnriirren
tl
y
we
sniigh
t
to
develop
,germ
-free
rearing
techniques
for
future
investigations
of
this
symbiosis.
MATERIALS
AND
METHODS
Insects
A.
domesticus
individuals
were
obtained
from
stock
cultures
maintained
in
our
laboratory
for
several
I.P.
35112-0
957
958
MICHAEL
G.
KAUFMAN
et
al.
years.
Originally,
crickets
were
obtained
from
a
local
commercial
operation
(Top
Hat
Cricket
Farm,
Kalamazoo,
Mich.).
The
stock
cultures
were
kept
at
30°C,
60%
r.h.
and
on
a
12
h
light
-12
h
dark
cycle.
Crickets
were
fed
a
commerical
diet
(described
below).
Germ
free
techniques
The
terminology
employed
here
to
describe
animals
reared
with
and
without
microorganisms
is
discussed
by
Coates
and
Gustafsson
(1984).
"Germ
-
free"
(
=
axenic)
will
refer
to
conditions
of
no
detectable
bacteria,
fungi,
or
protozoa,
while
"conventional"
will
apply
to
insects
with
a
"normal",
undefined
compliment
of
microorganisms.
Germ
-free
cricket
hatchlings
were
obtained
in
the
following
manner.
Stock
-culture
females
were
allowed
to
oviposit
into
a
dish
of
moist,
autoclaved
sand
for
a
24
h
period.
Eggs
incubated
in
the
oviposition
dish
for
5-7
days
were
separated
from
the
sand
by
washing
with
distilled
water.
Broken
and
discoloured
eggs
were
discarded,
and
the
remainder
rinsed
several
times
with
sterile
distilled
water_
The
eggs
were
then
immersed
in
a
0.3%
solution
of
benzalkonium
chloride
and
gently
agitated
for
"
min
Fr
,
11
,
swing
several
rinses
with
sterile
AistilleA
water,
eggs
were
transferred
individually
to
brain
-
heart
infusion
(Difco
Co.,
Detroit,
Mich.)
plates
using
aseptic
techniques.
After
24
h
incubation
at
30°C,
eggs
showing
no
sign
of
contamination
were
aseptically
transferred
to
fresh
brain
-heart
infusion
plates
and
incubated
for
an
additional
24
it
at
30°C.
Groups
of
20-25
eggs
which
showed
no
evidence
of
microbial
growth
on
the
plates
were
transferred
to
sterile
plastic
Petri
plates
(100
x
80
x
25
mm)
containing
moistened
fi
lter
paper
and
incubated
at
30°C
until
hatching.
All
brain
-heart
infusion
plates
used
were
also
incubated
for
an
additional
4-7
days
to
assay
for
slower
growing
contaminants.
In
practice,
the
majority
of
contamination
manifested
itself
in
the
fi
rst
24
h
and
subsequent
prolonged
incubations
rarely
indicated
new
growth.
Subsamples
(2-3)
of
young
larvae
(5-7
days
after
hatch)
from
each
group
were
assayed
for
contaminants
by
crushing
them
with
forceps
to
expose
and
open
the
digestive
tract,
and
then
inoculating
media
with
the
entire
carcass.
Several
media
were
initially
employed
as
screens;
including
brain
-heart
infusion
agar
and
broth,
blood
agar,
peptone
-yeast
extract
-glucose
plates,
nutrient
agar,
and
2%
malt
extract
broth.
Preliminary
work
indicated
that
brain
-heart
infusion
plates
and
broth
were
equal
to
or
better
than
other
aerobic
media
in
detection
efficacy.
Anaerobic
brain
-
heart
infusion
or
peptone
-yeast
extract
glucose
broth
(Holdeman
et
al.,
1977)
were
also
used
after
transfer-
ring
subsets
of
young
larvae
to
an
anaerobic
glove
bag
(Coy
Manufacturers,
Ann
Arbor,
Mich.).
Though
contaminant
growth
was
rarely
seen
in
malt
extract
broth,
the
media
was
used
as
a
screen
for
yeasts
in
all
initial
and
fi
nal
tests.
All
manipulations
of
surface
-sterilized
eggs
and
subsequent
transfers
of
germ
-free
crickets
were
done
at
a
laminar
-flow
work
station.
Plates
and
growth
chambers
were
kept
in
microisolator
units
(Lab
Products
Inc.,
Haywood,
NJ.)
which
provided
an
effective
seccnidary
barrier
during
incubations
in
environmental
chambers.
Groups
of
hatchlings
deemed
germ
-free
with
the
above
tests
were
subdivided
into
smaller
groups
as
they
matured
by
transferring
the
insects
to
fresh
Petri
plates.
Sterile
food
and
water
were
aseptically
placed
in
the
100
x
80
x
25
mm
plastic!
Petri
plates
("grctwth
chambers")
prior
to
the
introduction
of
insects.
All
food
was
steam
-sterilized
(121°C)
for
30
min
and
dried.
Water
was
'-f-oduced
in
3
dram
vials
with
cotton
rolls
as
wicks.
The
vials,
placed
horizontally
in
the
growth
chambers,
were
equipped
with
stainless
steel
wire
collars
to
reduce
roiling
movements.
Crickets
were
transferred
to
new
growth
chambers
at
approximately
weekly
intervals.
Transferring
the
insects
was
facilitated
by
chilling
them
(5°C,
approx.
30
min)
prior
to
handling.
Routine
assay
for
contam-
inants
was
accomplished
by
plating
faecal
material
and
moist
cotton
swabbings
of
used
chambers
on
aerobic
brain
-heart
infusion.
Critical
assays
for
contamination
performed
at
the
end
of
an
experiment
included:
(1)
aerobic
and
anaerobic
incubation
of
faecal
material
and
swabs
of
each
chamber;
(2)
phase
microscopic
examination
of
faecal
material;
(3)
placing
whole,
crushed
insects
in
aerobic
and
anaero-
bic
broth;
and
(4)
examination
of
the
anterior-
hirtArrrif timn !lc C.1).1
,
11 01,11,1
,
110 vz4+1
.1
or...11A1,er
5,11 11111.111.1
6.3,
V
,11.11 llllll
LL
1,3
VV
1111
J
5
electron
micrscopy
(Ulrich et
al.,
1981).
Efficacy
of
aerobic
brain
-heart
infusion
plates
for
routine
containment
checks
was
confirmed
by
fail
-
are
to
detect
microorganisms
with
any
of
the
above
methods
when
the
plates
showed
no
sign
of
growth.
Successive
generations
of
germ
-free
A.
domesticus
were
obtained
by
tranferring
mated
females
to
larger
chambers
(Pyrex
Corning
No.
3250
glass
storage
jars)
containing
oviposition
dishes.
Diets
A
"natural"
diet
for
crickets
and
other
opportunis-
tic
omnivores
is
unknown
and
variable
enough
to
be
essentially
undefinable
A
commercially
available
"cricket
feed"
(Country
Mark
Inc.,
Columbus,
Ohio)
was
used
as
a
basic
diet
for
the
maintenance
of
stock
cultures
and
as
an
"optimal"
reference
diet
(Diet
1)
for
growth
studies
since
it
was,
in
effect,
the
"natural"
diet
for
A.
domesticus
populations
used
in
the
study.
It
also
contained
-_•__r_____
of
unrefined
plant
and
animal
material
that
would
be
similar
to
the
general
classes
of
material
consumed
by
most
gryllid
species.
The
commercial
diet
consisted
largely
of
a
mixture
of
wheat
middlings
and
soy
bean
meal,
with
animal
protein
and
fat,
vitamins,
and
minerals
added
as
supplements.
The
diet
was
similar
to
that
employed
by
Woodring
et
al.
(1977)
in
studies
of
growth
and
food
utilization
by
A.
domesticus.
Though
ill-defined
crickets
feed
supported
good
growth
of
A.
domesti-
cus,
contained
no
antibiotics,
and
remained
palatable
to
the
insects
after
autoclaving.
Diets
2
and
3
consisted
of
50:50
(wt
:
wt)
dilutions
of
Diet
1
with
alfalfa
hay
and
cellulose
(ball
-milled
fi
lter
paper),
respeotively.
Viet
increased
the
level
a
plant
structural
carbohydrates
in
the
diet
while
only
marginally
decreasing
total
nitrogen
levels.
Diet
3
increased
structural
carbohydrates
and.
halved
nitrogen
content
of
ingested
material.
Diets
2
and
3
also
presumably
diluted
fat
and
vitamin
pools.
Basic
component
percentages
for
ea
-
ch
diet
are
presented
with
the
results
(Table
2).
All
diets
were
ball
milled
Germ
-free
A.
domesticus
959
for
48
h,
sieved
(No.
40
mesh),
and
stored
under
desiccation
prior
to
autoclaving.
Growth
and
food
utilization
experiments
Ten
groups
consisting
of
three
larval
crickets
of
each
type
(conventional
and
germfree)
were
reared
from
hatching
to
10
days
beyond
the
adult
moult
on
each
of
the
three
diets.
Germ
-free
animals
were
third
generation
under
axenic
conditions
and
conventional
larvae
were
taken
from
a
parallel
stock
culture
originating
from
the
same
batch
of
eggs.
Conven-
tional
animals
were
reared
in
chambers
identical
to
those
described
for
germ
-free
larvae
except
that
the
Petri
dish
lids
were
perforated.
The
food
utilization
parameters
Approximate
digestibility,
efficiency
of
conversion
of
ingested
matter
to
biomass,
efficiency
of
conver-
sion
of
digested
matter
to
biomass,
and
relative
consumption
rates
were
calculated
following
Waldbauer
(1968)
and
were
determined
during
four
consecutive
periods
encompassing
the
most
linear
portion
of
the
larval
growth
curve.
Faecal
material
weight
was
corrected
for
uric
acid
content
in
estimates
of
digestibility
(Waldbauer,
1968).
The
growth
periods
were
arbitrary
divisions,
but
most
larvae
had
reached
the
last
instar
by
the
start
of
period
No.
4
on
all
diets.
Larvae
were
weighed
after
each
period
and
transferred
to
fresh
chambers.
Remaining
food
and
faecal
material
was
dried
(50°C,
72
h),
sorted,
and
weighed.
Faecal
material
was
then
ground
and
stored
under
desiccation
for
later
chemical
analysis.
Larval
fresh
weights
were
converted
to
dry
mass
with
linear
regression
equations
derived
from
wet
-to
-dry
measurements
of
larvae
taken
from
parallel
cultures
of
germ
-free
and
conventional
insects.
Larvae
were
separated
prior
to
the
fi
nal
moult
and
were
weighed
5
days
after
reach-
ing
the
adult
stage.
Adult
virgin
females
were
also
weighed
and
sacrificed
10,
days
after
the
fi
nal
moult
in
order
to
count
eggs
present
in
the
body
cavity
and
thereby
estimate
fecundity.
Chemical
analyses
Uric
acid
and
nitrogen
content
of
faecal
material
was
determined
for
each
replicate
group
of
crickets
at
each
growth
period.
For
uric
acid
determination,
duplicate
10
mg
subsamples
of
material
were
extracted
c....
30
min
at
6,0°C.,
in
a.
Li
2
CO
3
/1-,oric
acid
buffer
(Cox
et
al.,
1976),
centrifuged,
and
the
super-
natant
analyzed
with
a
modified
version
of
an
HPLC
method
devised
by
Marquardt
et
al.
(1983).
A
Regis
ODS
C18
column
(25
x
4.6
mm)
coupled
with
u.v.
detection
at
305
nm
were
used
for
separation
and
quantitation
of
the
compound.
The
mobile
phase
was
50
mM
Na
PO,
acidified
to
pH
2
with
phosphoric
acid.
The
fl
ow
rate
was
1.0/ml/min
at
40°C.
Total
nitrogen
content
was
measured
for
triplicate
subsamples
of
each
faecal
sample
using
a
Carlo
Erba
Elemental
Analyzer
(Model
1102).
Nitrogen
content
was
also
determined
for
six
subsamples
of
each
autoclaved
diet
using
the
above
methods.
Total
lipid,
soluble
carbohydrate,
insoluble
carbohydrate,
protein
and
ash
content
were
estimated
for
food
and
faecal
material
collected
during
growth
period
4.
Food
material
analyses
were
done
on
six
subsamples
of
each
autoclaved
diet,
while
analysis
of
faecal
material
was
done
on
material
from
individual
larvae
replicates.
Total
lipids
were
estimated
by
the
methods
of
Bligh
and
Dyer
(1959).
Soluble
carbohy-
drates
were
determined
as
those
sugars
released
after
extraction
with
0.1
N
H
2
SO
4
and
quantified
with
the
phenol
-sulphuric
method
(Dubois
et
al.,
1956).
Woodring
et
al.
(1979)
reported
that
virtually
all
carbohydrate
available
to
the
normal
A.
domesticus
digestive
system
was
released
upon
extraction
with
0.1
N
H
2
SO
4
and
the
soluble
carbohydrate
referred
to
here
is
equivalent
to
their
"digestible"
carbohy-
drate.
Total
residual
(
=
insoluble)
carbohydrate
was
estimated
by
successive
extraction
of
the
material
with
hot
water
(100°C,
30
min)
and
hot
1.0
N
H
2
SO
4
(100°C,
30
min),
followed
by
chloroform/methanol
(25°C,
30
min)
to
remove
li
pids
and
serve
as
a
fi
nal
wash.
The
ash
-free
dry
weight
of
the
material
remaining
after
these
extractions
was
taken
to
be
a
relative
estimate
of
refractory
structural
carbohy-
drate
content.
Protein
content
was
estimated
from
total
nitrogen
values
(
x
6.25)
after
adjustment
for
uric
acid,
and
per
cent
ash
was
determined
by
standard
methods.
Approximate
digestibility
values
for
each
component
were
calculated
as
described
above
for
total
dry
weight
(Waldbauer,
1968).
Enzyme
comparison
Comparisons
of
germ
free
and
conventional
enzyme
activity
were
made
using
API
ZYM
system
(API
Analytical
Products,
Sherwood
Medical,
Plain-
view,
N.Y.).
Digestive
tracts
were
excised
from
eight
insects
of
each
type
and
subdivided
into
two
regions.
The
midgut
region
included
gastric
caeca
and
the
ventriculus,
and
the
hindgut
segment
consisted
of
the
anterior
hindgut
segment
located
immediately
posterior
to
the
ventriculus.
Gut
segments
from
each
category
were
pooled
and
homogenized
in
2
ml
of
deionized
water
with
a
tissue
grinder.
Eight
each
of
germ
-free
and
conventional
adult
A.
domesticus
midgut
sections
were
pooled
for
the
analyses,
and
eight
germ
-free
and
four
conventional
hindgut
segments
were
used.
Fewer
conventional
hindgut
segments
were
used
in
order
to
maintain
roughly
equivalent
weights
of
homogenized
material:
conven-
tional
anterior
hindgut
segments
are
approximately
twice
as
large
on
a
fresh
weight
basis
as
germ
-free
Y
unpublished
data).
A
subsample
of
each
homogenate
was
inactivated
by
heating
at
95°C
for
15
min
and
used
as
a
blank.
Triplicate
subsamples
of
the
homogenate
were
added
to
substrate
containers
as
per
API
instructions,
and
incubated
for
4
h
at
30°C.
Semi
-quantitative
measures
of
enzyme,
activity
levels
were
then
determined
by
comparisons
of
colour
development
in
active
vs
blank
homogenate
treatments
for
each
gut
segment
category.
We
refer
to
activity
levels
measured
in
this
assay
as
"relative
activity"
since
reaction
products
and
rates
of
formation
were
not
quantified.
Activity
levels,
as
defined
here,
can
be
used
to
compare
particular
enzyme
classes,
but
are
not
intended
to
allow
comparison
of
the
activities
of
different
enzymes.
960
300
200
5
g
100
u
..
DIET
ONE
-
period
1
0
5
10
15
20
25
30
35
40
AGE
(days)
MICHAEL
G.
KAUFMAN
et
al.
DIET
TWO
-n-
conventional
germfree
.
period
1
. 1 1
1
1
10
15
20
25
30
35
AGE
(days)
40
DIET
THREE
period
1
20
25
30
35
40
45
50
AGE
(days)
Fig.
1.
Growth
of
germ
-free
and
conventional
A.
domesticus
larvae
on
three
diets.
Bars
are
SE,
n
=
25-30.
Statistics
Parameters
were
compared
as
their
log
transformed
values
with
standard
ANOVA
and
regression
techniques
(Sokal
and
Rohlf,
1969).
Experiments
were
initially
analysed
in
a
3
-way
factorial
design.
When
significant
interaction
was
found,
further
analyses
were
performed
with
separate
ANOVA
of
parameters
within
each
diet
or
growth
period.
Unless
otherwise
indicated,
P
<0.05
were
considered
significant.
RESULTS
Growth
parameters
Growth
rates
(Fig.
1),
size
at
maturity,
maturation
time,
and
fecundity
estimates
(Table
1)
were,
in
general,
very
similar
for
germ
-free
and
conventional
insects.
Although
conventional
animals
tended
to
be
slightly
larger
than
germ
-free
counterparts
on
diets
1
and
2,
and
slightly
smaller
on
diet
3,
the
only
statistically
significant
difference
in
any
of
the
growth
parameters
was
found
between
fecundity
estimates
for
crickets
reared
on
diet
3;
conventional
crickets
produced
fewer
eggs
during
the
fi
rst
10
days
of
maturity.
Food
utilization
Differences
between
germ
-free
and
conventional
animals
were
manifested
in
the
insects'
abilities
to
digest
food.
Conventional
crickets
digested
a
greater
percentage
of
each
diet
(AD,
Fig.
2)
and
converted
food
into
biomass
more
efficiently
(ECI,
Fig.
2).
Overall,
crickets
with
hindgut
microorganisms
digested
34-50%
more
of
ingestia
than
germ
-free
crickets
and
gained
21-39%
more
biomass
per
unit
of
food
ingested
(average
of
combined
data
over
the
four
time
periods).
Germ
-free
crickets
tended
to
convert
digested
food
into
biomass
more
efficiently
(ECD,
Fig.
2).
Though
conventional
animals
were
more
efficient
in
converting
food
to
biomass,
growth
rates
were
similar
to
those
in
germ
-free
animals
because
conventional
crickets
consumed
significantly
less
(RCR,
Fig.
2)
across
all
diets
and
time
periods.
Food
utilization
parameters
changed
similarly
with
diet
treatments
for
both
germ
-free
and
conventional
crickets.
Food
utilization
efficiencies
decreased
from
diets
1-3,
while
consumption
rates
generally
increased
as
diet
quality
decreased.
Changes
in
food
utilization
parameters
across
diets
were
the
same
for
germ
-free
and
conventional
animals;
i.e.
differences
between
germ
-free
and
conventional
animals
in
these
parameters
remained
nearly
constant
as
diet
quality
was
decreased.
Total
nitrogen
and
uric
acid
excretion
Nitrogen
economy
(N
excreted,
Fig.
3)
roughly
paralleled
overall
food
utilization
parameters.
Conventional
larvae,
in
general,
consumed
less
total
nitrogen
and
excreted
a
smaller
proportion
of
ingested
nitrogen.
However,
older
instars
of
conven-
tional
larvae
reared
on
diet
3
excreted
the
same
proportion
of
ingested
nitrogen
as
germ
-free
larae.
Additionally,
while
germ
-free
larvae
tended
to
excrete
less
nitrogen
as
they
matured,
conventional
larvae
showed
the
opposite
trend.
Although
germfree
animals
excreted
more
nitrogen
in
the
form
of
uric
acid
over
all
than
conventional
Table
1.
Adult
size,
maturation
time,
mortality,
and
fecundity
values
for
germ
-free
and
conventional
A.
domesticus
on
three
diets
Diet
1
Diet
2
Diet
3
Male
wt
Conventional
369.9
±
8.9
(14)
283.4
+
7.9
(15)
215.0
+
6.8
(11)
(mg)
Germ
-free
327.5
+
8.5
(15)
274.6
+
7.7
(16)
250.6
+
5.4
(14)
Female
wt
Conventional
432.6
+
11.7
(16)
370.4
+
10.1
(15)
257.2
±
7.3
(14)
(mg)
Germ
-free
408.3
+
7.3
(12)
339.6
+
8.5
(14)
254.2
+
8.9
(11)
Maturation
Conventional
38
±
0.4
(30)
39
±
0.6
(30)
51
±
1.3
(25)
time
(days)
Germ
-free
39
+
0.6
(27)
39
±
0.4
(30)
51
+0.6
(25)
No.
eggs
per
Conventional
444
±
13.9
(16)
331
+
10.3
(15)
137
+
14.9
(14)
female
Germ
-free
441
±
13.1
(12)
324
+
11.8
(14)
*207
+
9.6
(11)
Total
%
Conventional
0 0
16.7
mortality
Germ
-free
0
0
16.7
Adult
weights
were
taken
5
days
after
the
fi
nal
moult.
Values
are
mean
±
SE
(n).
*Significant
difference
at
P
=
0.05.
Germ
-free
A.
domesticus
961
70
60
50
40
3
4
C
0
20
60
C
a
40
C.)
30
LU
C.)
10
RCR
(mg/mg/day)
50
20
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
DIET
ONE
ECD
ECI
1
2
3
4
GROWTH
PERIOD
DIET
TWO
conventional
—0—
germfree
U
ECD
ECI
1
2
3
4
GROWTH
PERIOD
DIET
THREE
ECD
ECI
1
2
3
4
GROWTH
PERIOD
Fig.
2.
Approximate
digestibility
(AD),
food
utilization
efficiencies
(ECD,
ECT),
and
relative
consumption
rates
(RCR)
for
germ
-free
and
conventional
A.
domesticus
larvae
on
three
diets.
Periods
are
growth
intervals
illustrated
in
Fig.
1.
Bars
are
SE,
n
=
6-10.
*significant
differences
at
P
=
0.05.
animals,
both
groups
excreted
similar
amounts
of
uric
acid
as
a
function
of
ingested
nitrogen
(Fig.
3).
Early
instar
conventional
larvae
reared
on
diets
1
and
2,
however,
excreted
a
smaller
proportion
of
ingested
nitrogen
as
uric
acid
than
germ
-free
counterparts,
while
older
conventional
instars
reared
on
the
first
two
diets
excreted
the
same
or
greater
proportion
of
ingested
nitrogen
as
uric
acid.
The
presence
of
microorganisms
did
not
significantly
affect
the
proportion
of
consumed
nitrogen
excreted
as
uric
acid
by
larvae
reared
on
the
low
nitrogen
diet.
Differences
(or
similarities)
in
overall
nitrogen
economy
between
germ
-free
and
conventional
larvae
were
independent
of
uric
acid
excretion
rates;
particularly
in
the
case
of
larvae
reared
on
diet
3.
Utilization
of
crude
diet
components
Conventional
larvae
were
more
efficient
at
removing
water-soluble
and
acid
-soluble
carbohy-
drates
from
food
material.
Faecal
material
from
germfree
animals
contained
a
high
proportion
of
soluble
carbohydrate,
while
material
from
conven-
tional
crickets
was
correspondingly
higher
in
residual
(insoluble)
carbohydrates
(Table
2)
on
all
diets.
Differences
in
calculated
digestibility
of
the
acid
soluble
pool
were
significant
between
groups,
while
digestibility
of
the
remaining
insoluble
carbohydrate
was
virtually
identical
in
germ
-free
and
conventional
animals.
Conventional
animals
also
digested
protein
(nitrogen)
more
efficiently
than
germ
-free
animals
on
diets
1
and
2.
Calculated
digestibilities
of
other
crude
dietary
components
did
not
differ
between
the
two
types
of
larvae.
Enzyme
activity
Enzyme
activity
levels
of
gut
sections
from
germ
-
free
and
conventional
A.
domesticus
indicated
that
no
major
differences
existed
between
the
two
larval
types
in
midgut
preparations
(Fig.
4).
Additionally,
midgut
preparations
in
general
showed
higher
activity
levels
than
hindgut
preparations.
Conventional
hindgut
homogenates,
however,
showed
higher
activity
levels
toward
most
carbohydrates
than
did
germ
-free
preparations.
Activity
against
oc-galactoside
linkages
was
evident
only
in
conventional
larvae
digestive
tracts
and
was
associated
primarily
with
the
hindgut.
DISCUSSION
Growth
and
food
utilization
efficiency
Based
solely
on
growth
performance,
the
presence
of
microorganisms
in
the
digestive
tract
of
A.
domes-
ticus
appears
to
be
inconsequential,
or
even
detrimen-
tal
to
reproductive
allocation
when
diet
conditions
962
MICHAEL
G.
KAUFMAN
et
al.
70
CI
60
¢c
8
50
LUZ
°
40
20
25
ILI
10
Q
Z
5
0
DIET
ONE
*
*
*
DIET
TWO
*
_u--
conventional
-u--
germfree
1
2
3
4
1
2
3
4
GROWTH
PERIOD
GROWTH
PERIOD
DIET
THREE
1
2
3
4
GROWTH
PERIOD
Fig.
3.
Total
nitrogen
(N)
and
uric
acid
(UA)
excreted
by
germ
-free
and
conventional
A.
domesticus
larvae
on
three
diets.
Periods
are
growth
intervals
illustrated
in
Fig.
1.
Bars
are
SE,
n
=
6-10.
*significant
differences
at
P
=
0.05.
Table
2.
Composition
of
crude
nutrient
fractions
in
diets
and
faecal
material,
and
approximate
digestibility
estimates
for
each
fraction
in
germ
-free
and
conventional
A.
domesticus
during
growth
period
4
Diet
Component
Food
%
dry
wt
Conventional.
Germ
-free
Faeces
%
dry
wt
AD
Faeces
%
dry
wt
AD
1
Residual
carbohydrate
26.1
±
0.8
39.0
±
0.9
45
26.0
±
1.2
47
Soluble
carbohydrate*
47.1
±
0.9
20.2
±
0.3
85
39.0
±
1.0
58
Lipid
3.8
+
0.3
1.5
+
0.1
85
1.4
+
0.2
81
Protein*
18.1
+
0.6
9.6
+
0.6
80
11.3
+
0.4
66
Ash
9.0
+
0.2
13.8
+
0.7
43
9.8
+
0.4
42
Uric
acid
3.4
+
0.2
1.9
+
0.4
2
Residual
carbohydrate
37.8
±
1.0
45.9
±
0.6
33
36.4
±
1.0
33
Soluble
carbohydrate*
32.6
±
0.8
12.9
±
0.3
78
21.6
±
0.8
54
Lipid
3.1
+
0.1
2.4
+
0.1
57
1.7
+
0.1
62
Protein*
16.9
+
0.6
11.6
+
0.3
63
11.5
+
0.2
52
Ash
10.3
+
0.2
13.7
+
1.2
26
10.6
+
0.9
28
Uric
acid
1.7
±
0.07
1.3
±
0.02
-
3
Residual
carbohydrate
57.4
+
0.7
72.0
+
1.0
12
64.
±
1.3
11
Soluble
carbohydrate*
22.3
±
0.6
10.3
±
0.3
69
14.0
±
0.6
50
Lipid
1.9
+
0.2
1.3
+
0.1
52
1.1
+
0.1
54
Protein
8.8
+
0.7
5.5
+
0.2
56
4.9
+
0.1
55
Ash
5.7
+
0.1
5.8
+
0.4
17
5.0
+
0.2
18
Uric
acid
0.7
+
0.1
0.6
+
0.02
Values
are
mean
±
SE,
n
=
6.
*Significant
differences
(P
=
0.05)
in
AD
(approximate
digestibility)
between
germ
-free
and
conventional
larvae
for
that
diet
component.
Germ
-free
A.
domesticus
963
6
RELATIVE
ACTIVITY
3
2
1
6
4
3
2
1
0
HINE/GUT
C
0
C.) C.)
U
7
esterase
LiJ
LU
t
U
arninopeptidase
(7
)
›-
cc
CHYMOTRYPSIN
protease
at-GALACTOSIDASE
p-GALACTOSIDASE
p-GLUCURONIDASE
ac-GLUCOSIDASE
Ui
W
Li/
ILI
C/)
N
CO
co
< <
p
0
CT
)
(7)
0
0
U
.
2
c7
o
<
N-ACETLYGL
carbohydrase
Fig.
4.
Activity
levels
of
major
enzyme
classes
in
the
digestive
tract
of
germ
-free
and
conventional
A.
domesticus
as
measured
with
the
API-zyme
system.
are
poor.
Upon
closer
examination,
however,
the
gut
symbionts
enhanced
the
extraction
of
soluble
compounds
from
the
food
bolus;
reducing
the
amount
of
ingested
material
necessary
for
optimal
growth
across
a
range
of
nutrient
concentrations.
The
microbial
contribution
to
overall
food
utilization
efficiency
was
generally
consistent
during
larval
development
and
over
the
dietary
conditions
examined.
However,
the
results
indicated
that
nitrogen
metabolism
interactions
between
gryllids
and
their
hindgut
bacterial
community
are
strongly
influenced
by
age
and
diet.
Comparisons
of
germ
-free
and
conventional
versions
of
insects
in
other
nutritional
studies
have
usually
focused
on
differential
survival,
growth,
or
fecundity
in
the
presence
or
absence
of
symbionts.
Relatively
few
insects
with
large
and
permanent
gut
microorganism
populations
have
been
examined
with
a
germ
-free
approach,
and
none
have
quantified
food
utilization
efficiency
parameters.
Charnley
et
al.
(1985)
found
little
distinction
in
growth
and
develop-
ment
between
germ
-free
and
conventional
locusts
free
of
gregarine
parasites.
Bracke
et
al.
(1978)
reported
that
cockroaches
developed
poorly
when
a
large
portion
of
the
normal
gut
bacterial
community
was
eliminated
with
antibiotics.
Although
not
quantified,
Martoja
(1966)
notes
that
G.
bimaculatus
seemed
to
develop
normally
when
reared
without
gut
bacteria.
Studies
of
vertebrate
animals
are
much
more
extensive
(reviews
in
Savage,
1984;
Gordon
and
Pesti,
1971;
Luckey,
1969),
and
indicate
that
germ
-free
animals
generally
show
better
growth
characteristics
than
conventional
individuals
if
the
diet
is
"complete".
In
contrast,
our
results
suggest
that
microorganisms
in
house
crickets
do
not
present
an
obvious
cost
to
the
insect's
growth
and
development
on
a
relatively
high
quality
diet.
The
mechanisms
of
increased
food
utilization
efficiency
by
conventional
crickets
are
presently
unknown,
but
appear
to
be
linked
to
the
utilization
of
soluble
carbohydrates.
This
diet
fraction
was
the
only
crude
component
that
was
digested
more
efficiently
by
conventional
animals
on
all
diet
treat-
ments.
Efficiency
of
food
utilization
in
conventional
larvae
exceeded
that
of
germ
-free
larvae
even
when
overall
nitrogen
digestion
and
excretion
patterns
from
the
two
larval
types
were
identical
in
late
growth
periods
of
the
diet
3
treatment.
This
would
also
imply
that
the
mechanism
of
enhanced
efficiency
is
primar-
ily
channelled
through
carbon
compounds.
The
latter
964
MICHAEL
G.
KAUFMAN
et
al.
stages
of
larval
growth
in
house
crickets
is
character-
ized
principally
by
the
accumulation
of
lipid
(Lipitz
and
McFarlane,
1971;
Woodring
et
al.,
1979)
and
weight
gain
would
be
largely
independent
of
dietary
nitrogen.
We
did
not
partition
biomass
gain
into
carbohydrate,
lipid,
and
protein
during
this
study,
and
therefore
cannot
be
certain
of
hbw
dietary
fractions
were
actually
utilized
by
the
larvae
for
growth.
However,
carbohydrates
serve
as
lipid
precursors
and
their
conservation
by
the
activities
of
the
hindgut
microorganisms
would
be
consistent
with
growth
and
food
utilization
patterns
we
observed
in
conventional
cricket
larvae.
Enzyme
activity
Enzyme
activity
in
the
hindgut
of
crickets
is
thought
to
result
largely
from
enzymes
secreted
in
the
midgut
(Teo
and
Woodring,
1985;
Thomas
and
Nation,
1984b)
and
activity
in
the
hindguts
of
both
germ
-free
and
conventional
larvae
generally
reflected
midgut
activity
(Fig.
4).
However,
activity
levels
in
conventional
hindguts
also
indicated
that
cricket
gut
microorganisms
enhanced
the
ability
of
the
insect
to
attack
carbohydrates,
and
to
some
extent
peptides.
It
is
noteworthy
that
twice
as
many
germ
-free
hindguts
were
used
in
the
assay
as
conventional
hindguts
(see
Materials
and
Methods).
Thus,
on
a
per
cricket
basis,
the
differences
between
germ
-free
and
conventional
hindgut
enzyme
activity
would
be
more
pronounced
than
illustrated.
The
results
are
important
from
two
standpoints;
gut
microbes
apparently
(1)
added
to
the
diversity
of
hydrolytic
activity
in
the
cricket
digestive
tract,
and
(2)
duplicated
the
insect's
own
enzymatic
activities;
providing
an
additional
attack
on
compounds
that
escape
midgut
digestive/absorptive
processes.
The
fi
ndings
also
indicate
that
gut
microorganisms
in
crickets
do
not
substantially
inactivate
host
enzymes.
In
some
vertebrate
systems,
for
example,
host
enzyme
activity
levels
drop
in
the
hindgut
of
conventional
animals;
presumably
because
gut
microorganisms
use
the
enzymes
as
protein
sources
for
growth
(Yoshida
et
al.,
1968;
Coates,
1984).
Charnley
et
al.
(1985)
showed,
however,
that
activity
of
a
-glucosidase
was
increased
in
the
locust
hindgut
in
the
presence
of
gut
microorganisms.
They
suggested
that
the
fi
nding
reflected
an
enzyme
of
microbial
origin
and
not
an
enhancement
of
the
activity
of
host
enzyme
present
Since
the
results
presented
here
only
illustrate
activity
levels
of
broad
classifications
of
enzyme
activity
in
a
semi
-quantitative
manner,
it
cannot
be
determined
how
much
of
the
extra
activity
found
in
conventional
hindguts
resulted
from
a
duplication
of
the
insect's
degradative
capabilities.
It
is
likely
that
the
bacteria
provide
different
isozymes
within
a
broad
substrate
category,
and
that
this
allows
more
efficient
attack
of
carbohydrate
classes.
It
is
also
possible
that
physiochemical
conditions
in
the
germ
-free
hindgut
were
sufficiently
altered
in
the
absence
of
microorgan-
isms
to
reduce
activity
levels
of
enzymes
entering
the
region.
Midgut
pH
and
anterior
hindgut
pH
are
similar
in
conventional
A.
domesticus
(Teo
and
Woodring,
1985);
implying
that
insect
enzymes
would
operate
with
similar
efficiency
throughout
most
of
the
digestive
tract
in
this
species.
Hindgut
environmental
condition
differences
in
germ
-free
and
conventional
crickets
have
not
been
compared
as
yet;
however,
pH
and
osmolarity
in
rat
caeca
are
considerably
altered
by
germ
-free
conditions
(Gordon
and
Bruckner,
1984;
Gordon,
1968).
Lower
activity
levels
of
enzyme
classes
in
germ
-free
cricket
hindguts
may
reflect
a
similar
change.
The
fi
nding
here
that
microorganisms
are
responsi-
ble
for
a
-galactosidase
activity
confirms
the
suspicion
of
Teo
and
Woodring
(1985)
that
the
raffinase
activity
found
in
the
anterior
hindgut
of
A.
domesti-
cus
was
produced
by
microorganisms.
Raffinase
is
a
subclass
of
the
a
-galactosidases
assayed
for
in
this
study.
The
presence
of
raffinase/a
-galactosidase
ac-
tivity
probably
represents
only
one
example
of
micro-
bial
metabolism
found
in
conventional
crickets
that
increases
the
insect's
overall
metabolic
capabilities.
Digestion
of
cellulose
is
often
postulated
as
a
primary
role
of
gut
symbionts
in
insect
systems.
Our
results
do
not
support
this
concept
for
the
cricket/microorganism
sybiosis.
Calculated
digestibil-
ity
for
the
residual
carbohydrate
fractions
did
not
differ
with
the
presence
and
absence
of
gut
microor-
ganisms
(Table
2),
and
growth
of
conventional
crickets
is
not
enhanced
as
this
fraction
increases
as
a
dietary
constituent.
Additionally,
approximate
digestabilities
for
both
types
of
crickets
are
almost
exactly
halved
as
the
basic
diet
is
diluted
by
50%
with
cellulose
(Fig.
2).
Although
/3
-glucosidase
activity
was
enhanced
in
the
anterior
hindgut
of
conventional
larvae,
germ
-free
larvae
were
also
capable
of
breaking
the
linkage
of
the
polymer.
Recent
work
in
our
laboratory
indicates
that
both
conventional
and
germ
-free
A.
domesticus
are
incapable
of
digesting
purified
cellulose,
but
that
both
types
degrade
soluble
forms
of
the
compound
(e.g.
carboxymethylcellulose)
(Kaufman,
1988;
Kaufman,
unpublished),
Assays
with
different
forms
of
cellulose
may
explain
why
McFarlane
and
Distler
(1982)
reported
extensive
digestion
of
the
compound
by
"normal"
house
crickets,
while
Teo
and
Woodring
(1985)
and
Ulrich
et
al.
(1981)
found
no
evidence
of
cellulase
activity
in
the
insect's
digestive
tract
or
isolates
from
gut,
respectively.
Whether
or
not
gut
microorganisms
in
crickets
act
chiefly
on
common
substrates
or
on
those
that
are
normally
unavailable
to
host
enzymes,
it
is
clear
that
the
bacteria
process
remnants
of
midgut
digestive
processes
and
make
a
portion
of
the
energy
available
to
the
insect.
The
fact
that
faecal
material
from
germ
-free
insects
contains
a
substantial
portion
of
soluble
carbohydrate
indicates
that
the
house
cricket
digestive
system
is
relatively
inefficient
to
begin
with,
and
that
microorganisms
are
not
competing
with
the
animal
under
normal
conditions.
The
soluble
carbo-
hydrate
fraction
assayed
in
this
study
could
include
a
wide
range
of
compounds
including
free
sugar
monomers,
hemicelluloses,
gums,
and
fragments
thereof.
A
further
partitioning
of
extracted
carbohy-
drates
will
be
necessary
to
address
the
question.
It
also
remains
to
be
seen
how
microbial
trapping
of
"leftover"
energy
in
the
carbohydrates
that
reach
the
hindgut
is
transferred
to
the
insect.
Anaerobic
metabolism
of
carbohydrates
by
microorganisms
has
been
shown
to
provide
energy
and
biosynthetic
precursors
to
host
animals
in
several
well
-studied
gut
systems;
including
reminants
and
termites
(reviews
in
Germ
-free
A.
domesticus
965
Hungate,
1975;
McBee,
1977;
Breznak,
1982).
It
is
likely
that
this
activity
accounts
for
at
least
a
portion
of
the
carbon
economy
in
conventional
cricket
larvae
(Kaufman,
1988).
Consumption
rates
Crickets
in
this
study
were
apparently
unable
to
totally
compensate
for
nutrient
dilution
with
increased
consumption
as
has
been
demonstrated
for
locusts
(McGinnis
and
Kastins,
1967)
and
cockroaches
(Gordon,
1972).
In
general,
food
was
consumed
at
a
higher
rate
by
young
cricket
larvae
and
individuals
reared
on
diet
3:
Patterns
of
consumption
appeared
to
be
different
in
larvae
reared
on
diet
3,
but
the
high
relative
consumption
rates
of
larvae
during
growth
period
4
on
diets
1
and
2
(RCR,
Fig.
2)
is
most
likely
an
artifact
of
the
growth
period
duration.
The
fourth
growth
period
of
diets
one
and
two
treatments
was
truncated
at
3
days
because
food
was
being
depleted
in
some
germ
-free
chambers.
At
this
time,
most
larvae
were
in
the
middle
of
the
last
instar
and
consumption
rates
decline
rapidly
to
near
zero
after
that
point
(Woodring
et
al.,
1979).
Therefore,
if
the
period
had
been
extended
to
7
days
(length
of
period
4
in
diet
3
treatment),
little
additional
food
would
have
been
consumed,
but
the
calculated
relative
consumption
rate
(mg/mg/day)
would
have
dropped
to
approximately
half
of
the
rate
illustrated
in
Fig.
2;
effectively
making
the
patterns
of
consumption
with
age
similar
across
the
diet
treat-
ments.
It
is
difficult
to
reconcile
the
facts
that
crickets
with
microorganisms
used
food
more
efficiently,
but
failed
to
translate
the
benefit
into
better
growth.
The
reduc-
tion
in
consumption
of
food
by
conventional
animals,
which
accounts
for
the
discrepancy,
may
be
related
to
a
change
in
physiology
due
to
microbial
activity
or
the
physical
presence
of
the
bacterial
community:
e.g.
the
mass
of
microorganisms
in
the
anterior
hindgut
acts
on
stretch
receptors
that
signal
a
slowing
of
feeding
because
the
region
is
perceived
to
be
"full".
There
is
evidence
to
suggest
that
food
movement
and
feeding
behaviour
can
be
partially
controlled
by
such
feedback
sensors
in
various
regions
of
insect
digestive
tracts,
including
the
hindgut
(Simpson,
1981;
Bernays,
1985).
Gut
retention
times
were
not
dis-
cernibly
different
between
germfree
and
conventional
house
crickets
(Kaufman,
1988),
but
the
regulation
of
feeding
behaviour
is
certainly
more
complex
than
merely
controlling
the
passage
of
ingestia.
Internal
physiological
status
(e.g.
haemolymph
titres
of
sugars
and
amino
acids)
may
play
a
role
in
determining
how
frequently
and
how
much
an
insect
consumes,
and
osmolarity
of
the
haemolymph
has
been
shown
to
affect
feeding
behaviour
of
locusts
(Bernays,
1985).
It
is
possible
that
microbial
activity
in
the
cricket
anterior
hindgut
interacts
at
that
level
by
releasing
metabolites
and
products
of
digestion
to
the
haemolymph,
which
subsequently
act
to
inhibit
ingestion.
Total
nitrogen
and
uric
acid
metabolism
The
role
gut
microorganisms
play
in
overall
nitrogen
metabolism
in
animals
is
apparently
complex
(Salter,
1984;
Prins,
1977),
and
nitrogenous
excretion
patterns
of
cricket
larvae
do
not
clarify
the
issue.
The
observed
effects
of
microorganisms
on
excretion
of
total
nitrogen
and
uric
acid
were
obscured
by
interaction
between
diet
and
age
of
the
larvae.
A
recycling
of
nitrogen
via
microbial
metabolism
of
uric
acid
was
suggested
by
excretion
rates
of
young
instars
reared
on
diets
1
and
2,
however,
older
larvae
and
those
reared
on
the
low
nitrogen
diet
showed
little
evidence
that
nitrogen
conservation
is
enhanced
by
gut
microorganism
metabolism
of
uric
acid.
In
fact,
since
egg
production
is
a
highly
nitrogen
-dependent
process
(Chen,
1985),
the
reduced
number
of
eggs
found
in
conventional
females
reared
on
diet
three
indicates
that
gut
microorganisms
competed
with
the
insect
for
nitrogen
sources.
The
results
presented
seem
to
constitute
a
paradox:
gut
symbionts
in
house
crickets
aid
in
nitrogen
conservation
only
when
nitrogen
is
abundant.
A
scavenging/recycling
of
nitrogen
as
it
becomes
scarce
in
the
diet
is
often
postulated
as
a
role
for
gut
microorganisms
(Salter,
1984;
Cochran,
1985),
but
the
evidence
from
the
cricket
system
suggests
other-
wise.
Our
study
did
not
specifically
control
for
nitro-
gen;
diet
3
was
also
dilute
in
available
carbohydrates
and
other
nutrients.
The
hindgut
bacterial
community
may
have
switched
to
alternative
substrates
(e.g.
nitrogenous
compounds)
as
carbohy-
drates
become
scarce.
Under
conditions
of
low
available
nitrogen
(diet
3),
germ
-free
larvae
tended
to
excrete
a
smaller
portion
of
dietary
nitrogen
as
they
matured
while
conventional
larvae
excreted
a
larger
portion.
This
suggests
that
activities
of
the
bacterial
community
may
have
interfered
with
the
insect's
own
nitrogen
reclamation
mechanisms.
It
is
likely
that
nitrogen
dynamics
in
the
cricket
hindgut
are
highly
dependent
upon
the
population
dynamics
and
fl
uctu-
ating
metabolic
activities
of
the
bacterial
community;
both
of
which
may
be
influenced
by
diet
and
status
of
the
host
insect.
The
results
point
out
the
facultative
nature
of
the
cricket/microorganism
association
and
the
need
for
additional,
more
-detailed
studies
in
this
area.
The
fact
that
conventional
larvae
excreted
less
uric
acid
overall
than
did
germ
-free
larvae
without
necessarily
showing
an
enhanced
ability
to
conserve
nitrogen
suggests
that
gut
microorganisms
are
degrading
uric
acid,
but
that
the
metabolites
are
not
readily
available
to
the
insect.
Gut
bacteria
have
been
shown
to
recycle
uric
acid
in
termites
(Potrikus
and
Breznak,
1981)
and
have
been
implicated
in
perform-
ing
a
similar
function
in
cockroaches
(Cochran,
1985)
and,
birds
(Mortensen,
1984;
Salter,•
1984).
Termites
and
cockroaches
are
known
to
store
uric
acid
internally
for
mobilization
under
conditions
of
nitrogen
stress
(Mullins
and
Cochran,
1975a,
b).
Such
a
system
of
"storage
excretion"
(Cochran,
1985)
is
not
evident
in
house
crickets
(Cochran,
1976;
Ulrich
et
al.,
1981;
Nation
and
Patton,
1961),
however,
and
strategies
for
nitrogen
conservation
are
apparently
different
or
less
important
in
this
insect.
Nitrogen
excretion
results
must
be
viewed
with
some
caution.
Ammonia
nitrogen
was
not
measured
during
the
course
of
this
study
and
may
be
a
consid-
erable
component
of
nitrogen
excretion
in
some
terrestrial
insects
(Cochran,
1985).
Crickets
have
been
shown
to
be
primarily
uricotelic
(Nation
and
Patton,
966
MICHAEL
G.
KAUFMAN
et
al.
1961;
Cochran,
1976);
however,
the
concern
is
partic-
ularly
relevant
here
since
ammonia
is
a
product
of
gut
microorganism
metabolism.
From
our
preliminary
investigations
of
this
question,
we
have
found
that
ammonia
is
produced
and
excreted
to
a
greater
extent
by
conventional
larvae
compared
to
germ
-free
versions,
but
that
ammonia
nitrogen
would
account
for
less
than
10%
of
total
excreted
nitrogen,
using
our
most
conservative
estimates
(Kaufman,
unpublished
data).
The
excreted
nitrogen
values,
(Fig.
3)
for
conventional
animals
are
therefore
slightly
underesti-
mated,
but not
enough
to
affect
overall
patterns
and
differences
with
germ
-free
larvae.
Extrapolation
to
other
insects
The
applicability
of
results
presented
here
to
nutritional
symbioses
in
other
species
of
gryllids
is
open
to
question.
A.
domesticus
is
cosmopolitan
in
its
native
Europe
and
Asia
(Bate,
1969,
1971),
but
it
is
an
introduced
species
in
the
U.S.A.;
reared
commer-
cially
in
large
concentrated
populations
(cricket
"farms")
or
maintained
in
laboratories
as
a
tool
for
insect
physiologists.
It
remains
to
be
seen
if
labora-
tory
populations
of
crickets
contain
a
"normal"
bacterial
community
and
some
previous
work
would
suggest
that
rearing
conditions
and
diet
do
affect
gut
microorganism
community
composition
in
other
insects
(Brooks,
1963;
Charpentier
et
al.,
1978;
Hunt
and
Charnley,
1981).
However,
researchers
studying
vertebrate
systems
have
emphasized
the
stability
in
gut
microorganism
community
composition
over
a
range
of
conditions
(Savage,
1977;
Lee,
1985).
Studies
of
bacterial
fermentation
metabolites
in
several
gryllid
species
(Kaufman,
1988)
indicated
that
the
bacterial
community
in
crickets
is
functionally
similar
between
laboratory
and
fi
eld
-collected
individuals.
Furthermore,
because
fermentation
metabolites
of
microorganisms
in
the
anterior
hindgut
of
A.
domesticus
were
nearly
identical
to
those
seen
in
native
populations
of
fi
eld
crickets
(Gryllus
spp),
it
appears
that
members
of
the
Gryllidae
as
a
group
are
likely
to
possess
a
functionally
similar
microbial
community
(Kaufman,
1988).
Although
growth
performance
of
house
crickets
was
not
enhanced
by
the
presence
of
microorganisms
during
this
laboratory
study,
a
general
increase
in
food
utilization
efficiency
across
a
range
of
diets
might
be
of
substantial
benefit
to
crickets
in
natural
environments.
Bacteria
in
the
hindgut
augmented
the
ability
of
the
insect
to
digest
a
portion
of
consumed
carbohydrates
and
provided
enzymatic
activity
against
at
least
one
polymer
that
would
not
be
available
to
the
insect's
endemic
enzyme
system.
Hindgut
bacteria
in
gryllids
may
provide
a
small
but
important
buffer
as
dietary
carbon
fl
uctuates
in
quality
and
quantity.
Gryllids
are
omnivores
which
feed
sporadically;
food
resources
are
likely
to
be
limited
and
variable.
An
ability
to
more
efficiently
extract
nutrients
from
an
unpredictable
resource
would
certainly
be
advantageous
to
these
insects
and
other
omnivorous
groups.
We
are
currently
investi-
gating
the
cricket
bacterial
community's
metabolic
characteristics
and
its
response
to
dietary
change.
Acknowledgements
—We
gratefully
acknowledge
the
technical
assistance
of
C.
Fullmer
and
S.
Huggett,
and
the
typing
skills
of
A.
Gillespie.
This
work
was
supported
by
NSF
grant
No.
BSR-8706480
and
NIH
grant
BMRG
No.
2-S07
RR007049-15.
This
paper
is
contribution
No.
668,
W.K.
Kellogg
Biological
Station.
REFERENCES
Bate
J.
(1969)
On
some
aspects
of
the
biology
of
Acheta
domesticus
(Insecta,
Orthoptera,
Gryllidae)
with
refer-
ence
to
the
infestation
of
refuse
tips.
Pedobiology
9,
300-322.
Bate
J.
(1971)
Life
history
of
Acheta
domesticus
(Insecta,
Orthoptera,
Gryllidae).
Pedobiology
11,
159-172.
Bernays
E.
A.
(1985)
Regulation
of
feeding
behaviour.
In
Comprehensive
Insect
Physiology
Biochemistry
and
Pharmacology
(Ed.
by
Kerkut
G.
A.
and
Gilbert
L.
I.),
pp.
1-32.
Pergamon
Press,
Oxford.
Bignell
D.
E.
(1977)
An
experimental
study
of
cellulose
and
hemicellulose
degradation
in
the
alimentary
canal
of
the
American
cockroach.
Can.
J.
Zool.
55,
579-589.
Bignell
D.
E.
(1981)
Nutrition
and
digestion.
In
The
American
Cockroach
(Ed.
by
Bell
W.
J.
and
Adiyedi
K.
G.),
pp.
57-86.
Chapman
&
Hall,
London.
Bligh
E.
G.
and
Dyer
W.
J.
(1959)
A
rapid
method
of
total
lipid
extraction
and
purification.
J.
Biochem.
Physiol.
37,
911-917.
Bracke
J.
E.,
Cruden
D.
L.
and
Markovetz
A.
J.
(1978)
Effect
of
metronidazole
on
the
intestinal
microflora
of
the
American
cockroach,
Periplaneta
americana
(L.)
Antimicrob.
Agent.
Chemoth.
13,
115-120.
Breznak
J.
A.
(1982)
Intestinal
microbiota
of
termites
and
other
xylophagous
insects.
Ann.
Rev.
Microbiol.
36,
323-343.
Brooks
M.
A.
(1963)
Microorganisms
of
healthy
insects.
In
Insect
Pathology
(Ed.
by
Steinhaus
E.
A.),
Vol.
VI,
pp.
215-250.
Academic
Press,
New
York.
Buchner
P.
(1965)
Endosymbiosis
of
Animals
with
Plant
Microorganisms,
909
pp.
Interscience,
New
York.
Charnley
A.
K.,
Hunt
J.
and
Dillon
R.
J.
(1985)
The
germ
-free
culture
of
desert
locusts,
Schistocerca
gregaria.
J.
Insect
Physiol.
31,
477-485.
Charpentier
R.,
Charpentier
B.
and
Zethner
0.
(1978)
The
bacterial
flora
of
the
midgut
of
two
Danish
populations
of
healthy
fi
fth
instar
larvae
of
the
turnip
oth,
Scotia
segetum.
J.
Invert.
Pathol.
32,
59-63.
Chen
P.
S.
(1985)
Amino
acid
and
protein
metabolism.
In
Comprehensive
Insect
Physiology
Biochemistry
and
Pharmacology
(Ed.
by
Kerkut
G.
A.
and
Gilbert
L.
I.),
Vol.
4,
pp.
177-218.
Pergamon
Press,
Oxford.
Coates
M.
E.
(1984)
Vitamins.
In
The
Germ
-Free
Animal
in
Biomedical
Research
(Ed.
by
Coates
M.
E.
and
Gustafsson
B.
E.),
pp.
269-274.
Laboratory
Animals
Ltd,
Oxford.
Coates
M.
E.
and
Gustafsson
B.
E.
(Eds)
(1984)
The
Germ
-Free
Animal
in
Biomedical
Research.
442
pp.
Laboratory
Animals
Ltd,
Oxford.
Cochran
D.
G.
(1976)
Excretia
analysis
on
additional
cockroach
species
and
the
house
cricket.
Comp.
Biochem.
Physiol.
53A,
79-81.
Cochran
D.
G.
(1985)
Nitrogenous
excretion.
In
Comprehensive
Insect
Physiology
Biochemistry
and
Pharmacology
(Ed.
by
Kerkut
G.
A.
and
Gilbert
L.
I.),
Vol.
4,
pp.
467-506.
Pergamon
Press,
Oxford.
Cox
G.
B.,
Loscombe
C.
R.
and
Upfield
J.
A.
(1976)
The
determination
of
uric
acid
in
animal
feeding
stuffs
using
high-performance
li
quid
chromatography.
Analyst
101,
381-385.
Cruden
D.
and
Markovetz
A.
J.
(1984)
Microbial
aspects
of
the
cockroach
hindgut.
Archs
Microbiol.
138,
131-139.
Dubois
M.,
Giles
K.
A.,
Hamilton
J.
K.,
Rebers
P.
A.
and
Smith
F.
(1965)
Colorimetric
method
for
determination
of
sugars
and
related
substances.
Analyt.
Chem.
28,
350-356.
Germ
-free
A.
domesticus
967
Gangwere
S.
K.
(1961).A
monograph
on
food
selection
in
Orthoptera.
Trans
Am.
Ent.
Soc.
87,
67-230.
Gordon
H.
A.
(1968)
Is
the
germfree
animal
normal?
A
review
of
its
anomolies
in
young
and
old
age.
In
The
Germ
-free
animal
in
Research
(Ed.
by
Coates
M.
E.,
Gordon
H.
A.
and
Wostmann
B.
S.),
pp.
127-150.
Academic
Press,
New
York.
Gordon
H.
T.
(1972)
Interpretations
of
insect
quantitative
nutrition.
In
Insect
and
Mite
Nutrition
(Ed.
by
Rodriguez
J.
G.),
pp.
73-105.
North
Holland,
London.
Gordon
H.
A.
and
Bruckner
G.
(1984)
Anomolous
lower
bowel
function
and
related
phenomena
in
germfree
animals.
In
The
Germ
-Free
Animal
in
Biomedical
Research
(Ed.
by
Coates
M.
E.
and
Gustafsson
B.
E.),
pp.
193-214.
Laboratory
1
,
;--
1
-
Ltd
0xford.
Gordon
H.
A.
and
Pesti
L.
(1971)
The
gnotobiotic
animal
as
a
tool
in
the
study
of
host
microbial
relationships.
Racterfol
Rev
35(4)
390-479.
Holdeman
L.
V.,
Cato
E.
P.
and
Moore
W.
E.
C.
(Eds)
(1977)
Anaerobe
Laboratory
Manual.
Virginia
Polytechnic
Institute
Anaerobe
Laboratory,
VPI,
Va.
Hungate
R.
E.
(1975)
The
rumen
microbial
ecosystem.
Ann.
Rev.
Ecol.
Syst.
6,
39-66.
Hunt
J.
and
Charnley
A.
K.
(1981)
Abundance
and
distribu-
tion
of
the
gut
fl
ora
of
the
desert
locust,
Schistocerca
gregaria.
J.
Invert.
Pathol.
38,
378-385.
Kaufman
M.
(1988)
The
role
of
anaerobic
bacterial
metabolism
in
the
nutrition
of
crickets
(Orthoptera:
Gryllidae).
Ph.D.
thesis,
Michigan
State
University,
Mich.
Lee
A.
(1985)
Neglected
niches:
the
microbial
ecology
()idle
gastrointestinal
tract.
In
Advances
in
Microbial
Ecology
(Ed.
by
Marshall
K.
C.),
Vol.
8,
pp.
115-162.
Lipitz
E.
Y.
and
McFarlane
J.
E.
(1971)
Analysis
of
lipid
during
the
life
cycle
of
the
house
cricket,
Acheta
domesti-
cus.
Insect
Biochem.
1,
446-460.
Luckey
T.
D.
(1969)
Effects
of
microbes
on
germfree
animals.
Adv.
appl.
Microbiol.
7,
169-223.
McBee
R.
H.
(1977)
Fermentation
in
the
hindgut,
In
Microbial
Fcology
tyr
the
Gut
(Rd.
by
Clarke
R.
T.
I
and
Bauchop
T.),
pp.
185-222.
Academic
Press,
London.
McFarlane
J.
E.
and
Distler
M.
H.
W.
(1982)
The
effect
of
rutin
on
growth,
fecundity,
and
food
utilization
in
Acheta
domesticus
(L.).
J.
Insect.
Physiol.
28,
85-88.
McGinnis
A.
J.
and
Kasting
R.
(1967)
Dietary
cellulose:
effect
on
food
consumption
and
growth
of
a
grasshopper.
Can.
J.
Zool.
45,
365-367.
Marquandt
R.
R.,
Ward
A.
T.
and
Campbell
L.
D.
(1983)
A
rapid
high-performance
liquid
chromatographic
method
for
the
quantitation
of
uric
acid
in
excreta
and
tissue
samples.
Poult.
Sci.
62,
2099-2105.
Martoja
R.
(1966)
Sur
quelques
aspects
de
la
biologie
des
orthoptires
in
relation
avec
la
presence
de
concentrations
microaiennes
(bacterial
intestinals,
rickettsies).
Ann.
Ent.
Fr.
2,
753-940.
Mathew,'
F,.
.h
.
(1981)
Contrasting
feeding
habits of
pest
mole
cricket
species.
J.
Econ.
Ent.
74,
444-445.
Mortensen
A.
(1984)
Importance
of
microbial
nitrogen
metal+nliem
in
the
reel,
of
hirriq
In
current
P
,
rspPcam
,
v
in
Microbial
Ecology
(Ed.
by
Klug
M.
J.
and
Reddy
C.
A.),
pp.
273-278,
Proc.
Third
Int.
Symp.
Micro.
Ecol.,
Am.
Soc.
Micro.,
Washington,
D.C.
Mullins
D.
E.
and
Cochran
D.
G.
(1975a)
Nitrogen
metabolism
in
the
American
cockroach
—I.
An
examina-
tion
of
positive
nitrogen
balance
with
respect
to
uric
acid
stores.
Comp.
Biochem.
Physiol.
50A,
489-500.
Mullins
D.
E.
and
Cochran
D.
G.
(1975b)
Nitrogen
metabolism
in
the
American
cockroach
—II.
An
examina-
tion
of
negative
nitrogen
balance
with
respect
to
mobiliza-
tion
of
uric
acid
stores.
Comp.
Biochem.
Physiol.
50A,
501-510.
Nation
J.
L.
(1983)
Specialization
in
the
alimentary
canal
of
some
mole
crickets
(Orthoptera:
Uryllotalpidae).
Int.
J.
Insect
Morph.
Embryo!.
12,
201-210.
Nation
J.
L.
and
Patton
R.
L.
(1961)
A
study
of
nitrogen
excretion
in
insects.
.1.
Insect
Physiol.
6,
299-308.
Potrikus
C.
and
Breznak
J.
(1981)
Gut
bacteria
recycle
uric
acid
nitrogen
in
termites:
a
strategy
for
nutrient
conserva-
tion
Proc
natn
Acad
Sc:
78
4601-4'605.
Prins
R.
A.
(1977)
Biochemical
activities
of
gut
microorgan-
isms.
In
Microbial
Ecology
of
the
Gut
(Ed.
by
Clarke
R.
T
J
and
Ranehnp
T)
pp.
71-1R4.
Academic
Preqc,
London.
Salter
D.
N.
(1984)
Nitrogen
metabolism.
In
The
Germ
-Free
Animal
in
Biomedical
Research
(Ed.
by
Coates
M.
E.
and
Gustafsson
B.
E.),
pp.
235-263.
Laboratory
Animals
Ltd,
Oxford.
Savage
D.
C.
(1977)
Microbial
ecology
of
the
gastrointesti-
nal
tract.
Ann.
Rev.
Microbiol.
31,
107-133.
Savage
D.
C.
(1984)
Present
view
of
the
normal
flora.
In
The
Germ
-Free
Animal
in
Biomedical
Research
(Ed.
by
Coates
M.
E.
and
Gustafsson
B.
E.),
pp.
119-140.
Laboratory
Animals
Ltd,
Oxford.
Simpson
J.
(1981)
The
role
of
volumetric
feedback
from
the
hindgut
in
the
regulation
of
meal
size
in
fi
fth
instar
Locusta
migratoria
nymphs.
Physiol.
Ent.
12,
451-467.
Sokal
R. R.
and
Rohlf
F.
J.
(1969)
Biometry,
Freeman
&
Co.,
San
Francisco.
Tennis
P.
(1981)
Survivorship,
spatial
pattern,
and
habitat
structure
of
fi
eld
crickets
(Orthoptera:
Gryllidae)
in
two
old
fi
elds.
Environ.
Ent.
12,
110-116.
Teo
L.
H.
and
Woodring
J.
P.
(1985)
Digestive
enzymes
in
the
house
cricket
Acheta
domesticus
with
special
refer-
ence
to
amylase.
Comp.
Rinrhem.
Physiol
R2A,
R71
—R77
Thomas
K.
K.
and
Nation
J.
L.
(1984a)
Absorption
of
glucose,
glycine
and
palmitic
acid
by
isolated
midgut
and
hindgut
from
crickets.
Comp.
Biochem.
Physiol.
79A,
289-295.
Thomas
K.
K.
and
Nation
J.
L.
(1984b)
Protease,
amylase
and
li
pase
activities
in
the
midgut
and
hindgut
of
the
cricket,
Gryllus
rubens
and
mole
cricket,
Scapteriscus
acletus.
Comp.
Biochem.
Physiol.
79A,
297-304.
Ulrich
R.
G.,
Buthala
D.
A.
and
Klung
M.
J.
(1981)
Microbiota
associated
with
the
gastrointestinal
tract
of
the
common
house
cricket,
Acheta
domesticus.
Appl.
Environ.
Microbiol.
41,
246-254.
Waldbauer
G.
P.
(1968)
The
consumption
and
utilization
of
food
by
insects.
Adv.
Insect
Physiol.
5,
229-287.
Woodring
J.
P.,
Clifford
C.
W.
and
Beckman
B.
R.
(1979)
Food
utilization
and
metabolic
efficiency
in
larval
arid
adult
house
crickets.
J.
Insect
Physiol.
25,
903-912.
Woodring
J.
P.,
Roe
R.
M.
and
Clifford
C.
W.
(1977)
Rela
tinn
of
feeding,
grnwth,
and
metahnlism
to
age
in
the
larval
female
house
cricket.
J.
Insect
Physiol.
23,
207-212.
Yoshida
Y.,
Pleasants
J.
R.,
Reddy
B.
S.
and
Wostmann
B.
S.
(1968)
Efficiency
of
digestion
in
germ
-free
and
conventional
rabbits.
Br.
J.
Nutr.
22,
723-737.