Two experimental models, one for testing the attraction behaviour (Petri-dish ... behaviour, Blattella germanica, Galleria mellonella, Locust migratoria ...
Vol.30
No.1/2
Japanese Journal
Experimental
Models
for Testing
Steinernema
Anwar
Attraction glaseri
L. BILGRAMI1,2, Eizo
December,
of Nematology
and Preferential
to Several
KONDO1 and
Behaviour
2000
of
Insects
Toyoshi
YOSHIGA1
Two experimental models, one for testing the attraction behaviour (Petri-dish scoring system) and the other for analyzing preferential behaviour (preference test chambers) of infective juveniles of Steinernema glaseri are described and proposed. Petri-dish scoring system was based on the probability of nematode distribution in the presence and absence of the insects. It was given weightage, depending upon the number of nematodes reaching to the insects. The weighted responses were given ranks and scores to make quantitative analysis of the attraction behaviour of nematodes. The preference test chambers were designed to test preferential behaviour of the nematodes for the larvae of four insect species: Galleria mellonella, Spodoptera litura, Blattella germanica and Locust migratoria. The attraction rates of S. glaseri increased with the time and reached to maximum (67.6%) 6 hr after inoculation of the nematodes. S. glaseri responded to homogenate of G. mellonella larvae in a dose dependent manner. Maximum values for the absolute and relative preferential indices showed that the nematodes preferred G. mellonella most among the four insects examined. Jpn. J. Nematol. 30 (1/2): 35-46 (2000). Key words: attractants, behaviour, Blattella germanica, Galleria mellonella, Locust migratoria, Spodoptera litura.
The attraction aspect of study.
behaviour
of nematodes
is a fascinated
It is all the more so, since nematodes
under physio-chemical influences medium similar to their natural
but most difficult
are tiny and microscopic
and challenged organisms
living
of the soil environment. No doubt, experimental models using habitat has an advantage as it allows nematodes to exhibit
normal behaviour
but accomplishment
to the microhabitat
and microscopic
of such a technique is rather difficult, if not impossible, due size of the nematodes.
Good results have been obtained using
soil and sand columns or by pot experiments (5, 8), but many important behavioural milestones may go unrecorded that would otherwise provide significant information about sensing behaviour of the nematodes.
The other option to study behaviour
is the two-dimensional
agar or agarose as the medium with conditions maintained as possible.
For such observations,
a hypothesis
as close to the natural
explaining behaviour
of nematodes
bioassay
using
habitat
(3, 4)
under natural
conditions may be erected depending upon the factual data. To be able to define chemical cues and stimuli involved in the migration of nematodes it is required to develop simpler and uniform systems based on quantitative
and numerical
estimations
of nematode
responses
to chemical cues
by excluding most of the possible external factors that may cause coincidental redistribution of nematodes. Numerous bioassays have been designed to suit particular nematodes viz., predatory 1 Department of Applied 2 JSPS Visiting Fellow:
Biological Sciences , Faculty of Agriculture, to whom correspondence may be addressed
―35―
Saga University, Saga 840-8502, Japan. at email: bilgramil956@hotmail .com
第30巻
日本線 虫学会 誌
第1/2号
nematodes
(3), plant parasitic
living nematodes
nematodes
(10) but neither
2000年12月
(2, 4), entomopathogenic
was based
on the system
nematodes
which could
(7), and free-
assess
nematode's
response quantitatively except the two studies (2, 9). Therefore, a system is needed where the nematode responses to chemical cues may be assessed quantitatively by giving weighted ranks and scores to their responses depending upon patterns of their distribution. Keeping in view the lack of standard and uniform ordinal system to test the attraction preferential behaviour of Steinernema glaseri to insect emitted models were designed and proposed in this paper. MATERIALS Infective SDS
3rd
Biotech
stage
K.
preferential
K.,
buffered
at
28•}1•Žbefore 3rd
germanica, the
0.01%
being
instars
of
and
the
laboratory.
experiments
Petri-dish
The
last
cutworm, Locust
experiments out
at
solution. of
surface-washed
Japan
and They
the
Spodoptera migratoria
Biotopia(R)) for
models.
phosphate
instar
name:
Ibaraki,
experimental
were
two experimental
METHODS
(Product
Center,
streptomycin
grasshopper,
carried
S. glaseri
potassium
common
all
of
proposed
mM
tested.
the
For were
the (20
with
(IJs) Technology
using saline
surface-sterilized
the
Tsukuba
behaviour
phosphate
juveniles
AND
attractants,
and
mM
were
larvae
in
from
attraction
were
extracted
chloride, saline
Galleria
German
of
cultures
insects
from and into
pH
7.2)
and
for
one
day
mellonella,
cockroach,
the
the
obtained
their
fresh
moth,
the
obtained
sodium
left
wax
litura,
testing
Nematodes
150
greater
were
and Blattella
maintained were
used.
in All
28•}1•Ž.
scoring system: The test arena for the proposed
Petri-dish
scoring system was an
8.5 cm diam. Petri-dish (Fig. 1). The Petri-dish was divided into three zones viz., the inner, middle, and outer by drawing in two concentric circles of 1.5 and 3.5 cm in diam. on the bottom surface
of the Petri-dish.
transitional,
The inner circle was designated
as the incubation,
the middle as the
and the outer as the free zone. A 2mm thick layer of 1% water agar was made in
Fig. 1.
Petri-dish scoring system designed to test attraction of Steinernema glaseri to insect emitted kairomones. ―36―
response
Vol. 30 No. 1/2
the Petri-dish
Japanese Journal
and allowed to cool.
December,
of Nematology
A plastic straw pipe measuring
2000
1.5 cm in length and 10mm
and 8 mm in the inner upper and bottom diam., respectively was taken. One of its end was glued with a piece of 200 mesh nylon sieve. Larvae of the insect to be tested were released into this pipe. To prevent the escape of the larvae, the open end of the pipe was sealed with a piece of cello-tape. The straw pipe containing
insect larvae was then placed vertically
in the middle of the incubation
zone so as the end that was sealed with the nylon sieve remained
inside the agar.
The whole set
was then left for 12 hr to allow insect attractants to diffuse and develop minimum perceptible attraction gradient in the medium. After incubation 10 IJs of S. glaseri were released at various points of the periphery of the transitional zone that is referred here as the inoculation circle (Fig. 1). The distribution of nematodes was examined in the three zones of the test arena 1, 2, 3, 4, 5, and 6 hr after inoculation. combinations
The nematode's
distribution
(Table 1). Each of the 66 distribution
in the three zones corresponds
patterns
to 66
were given a rank depending upon
the number of nematodes reaching to the incubation zone (Table 1) . The area of each zone was calculated and weighted factors (Wf) were obtained by dividing the area of the free zone with each of the three zones with the help of the following. Weighted The
area
factor
and weighted
Scores their
were
attractants
factors
obtained
corresponding
thus obtained
Area
(Wf)=
of the free zone Area of a zone
for each
by summing
weighted
zone are given
up the products
factors.
The scores
were used to calculate
attraction
The
response
maximum
incubation
tabulated
zone
Where:
Niz,
(Table
NTZ,
score
1) .
NFZ
respectively free
zones,
The
presence
100% obtaining
The
referred
of to
The
as
is
321
scores
the
mean
28•{1•Ž.
The
control
Responses
Table
of
insect
6
hr
WfTZ,
of
their
the
were attraction of
when
of nematodes
the insect
emitted
•~ 100 all
obtained
nematodes
WfFZ
are
in the
the
as
nematodes
were
present
in
the
follows.
present
the
in the
weighted
incubation,
factors
responses 20
for
towards
transitional
the
and
incubation,
were same.
distribution
free
transitional
scores,
last
ranks
―37―
in
mean
of
mean
all
i.e.,
"321"
the
66 and
was
or
show
of
taken were
were
for
absence
was
response
experiments
thus
considered
presence
replicates
attraction The
by
using instar
nematodes
1.
of
and
used
to
conducted
at
insects.
emitted tested
Two of
of
without
ranking
score
nematodes
attractants.
attractants
attractants
corresponding
run
high
the
The
values
insect
were
of
times.
The to
show
Therefore,
replicated response.
remained
zone
1).
attraction
experiments
The
incubation
(Table
nematodes
S. glaseri
zone. and
i.e.,
were
of
attractants
emitting
conditions
incubation
number
nematodes
values
responses
All
all
experiments
the
towards
the
Wfiz,
insect
represent
and
patterns
towards
(NFZ•~WfFZ)
are
and
relative
insects.
the
distribution
of nematodes
in each zone with
respectively.
response
above.
for different
response
Observed score Maximum tabulated score
(%)=
Score=(Niz•~Wfiz)+(NTZ•~WfTZ)
and
2. of nematodes
as follows.
Attraction
zones,
in Table
of number
larvae in
and
the
insects:
Petri-dish
each attraction
of zone
The
responses
scoring
system
G. was
mellonella
were
recorded
responses
of as
incubated
after were
S. glaseri described in
1, 2, 3, 4, 5,
obtained
from
第30巻
第1/2号
Table
日本線 虫学 会誌
1. Sixty six possible combinations of nematode distribution of the Petri-dish scoring system with their corresponding attraction responses.
―38―
2000年12月
in the three zones ranks, scores and
Vol.30 No.1/2
Japanese Journal
Table
Responses
of
attractants
S. by
described
above.
The
the
number
of
same
The
number
of
Responses was
and
of
source
mellonella
used
was
recorded
at
as
3, 6, 9, and
bottom
of
was to
same
as
was shown
used
to
in
each
Fig.
on to
2 was
determine
left
the
the
to
each 24
of
canal
obtained
the to
observation
To
of
the
10
ml
the
distribution
of
stock
concentration
with
G.
prepare in
was
of
nematodes
20 replicates
each
on
made
for
each
in
such as
holes
with
canal
fixed
four
as
that
preference
test
touch
one
(test
chambers).
chamber.
Thus, This
the
the
One
canal
Petri-dish
Similarly,
of
and
Petri-dishes
should
The
such
length,
chamber.
chambers.
indices
make
in five
they
adhesive.
firmly.
inoculation
preferential
the
inoculation
Petri-dishes
the
of
2cm
of in diam.
To
of
one
a way
an
5.5cm
chambers. each
of
the
the
to
test
wall
behaviour
measuring
pieces,
the
to
of the
relative
preferential
four
four
let
named
and
The
this
fluids)
experiment,
each
each
four
referred
connecting
and
absolute
of
100%.
the
conducted
and
into
made
were
of hr
to of
determining
canals, cut
were
was
for
time
for
homogenized
ml
Petri-dishes,
four was
holes
walls
each
for
plastic
cm-d
The
nematodes.
(body
and
prior
zone. was
of
tested.
water.
cm-d)
0.5
80
and
were
system
the
as
mellonella
inoculation
were
1
G.
scoring
60,
Just
incubation
chambers
Petri-dish
outside
made
attached
(0.5
40,
and
experiment
chamber,
tube
then
the
distilled
Test
holes,
from
was
then
transparent
This
diameter
dish
The
inoculation glass
was
of
five
positions.
canals
middle
with
of
used.
of
homogenate
mellonella
towards system
migratoria
excepting
20,
G.
concentrations
run
Petri-dish.
attached
four
the
viz.,
it was
larvae
the
insects,
nematodes scoring
L.
after
above,
of
until
instar
Petri-dish
live
larvae
incubation.
12•‹ clock
the
then the
in
6 hr
described and
of
Petri-dish
germanica,
homogenate:
as
4•Ž
last
B.
recorded
intact
above
the
the
litura,
concentrations
at
the
was
Four
was
instar
stored
the
an
long
canal.
five
using of
S.
insect
of
chambers:
of
8 cm
named
after
consisted
consisted
zone
last
to pipe
test were
chambers
at
and
of
conditions
10
Control
Preference
was
tested
straw
6 hr
concentration.
nematodes
the
diluted
the
each
Responses
tested
individuals
larvae
Instead
filtered,
in
intact
towards
with
was
was
introduced
in glaseri
(100%)
water,
and instar
insects:
were
2000
of test arena.
different
insects
live 3rd
attraction.
larvae
homogenate
It
S.
homogenate
distilled
two the
2. Measurements
towards
different
nematodes of
experiment
stock
glaseri
emitted
December,
of Nematology
glued
hole
of
One
Petri-
the
assembly
model
nematodes
the
was
for
then
different
insects. Absolute attraction relation
to
glaseri
for
described the
test
preferential response the
total
the
number
the larvae
of
thick
including
other
three of
G.
S.
glaseri:
that
emitted
A 4 mm
chambers"A"
last
of
nematodes
insect
above.
connecting instar
indices of
test mellonella
The
occurred
nematodes
the
were of
1%
canal
chambers and
obtained
water
agar
"B","C", same
―39―
it and
number
insect.
absolute using
was to
"D"
the
the
It
was
defined
was
determined
preferential
preferential
made
were of
index
single
The
connecting
the
preferential the
inoculated.
attractants layer
absolute
towards
in
the
inoculation sealed 3rd
test
filter
of
chamber
inoculation
larvae
the in
indices
and
chamber. with
instar
as
as one
The paper. of
S.
S.
of
canal Fifteen
litura,
B.
第30巻
日本線 虫学 会誌
第1/2号
Fig. 2.
germanica,
and
L.
chambers"A", to
prevent
the
the
insects
chamber.
after
6 hr
nematodes run
the
that
without
in
In
experiment
the
insects
its
host
was
then
for
test
nematodes in
corresponding
test
12
hr
them
in
closed
by
at
nematodes
were canal
each
species
of
were
chamber.
The
to in
the
allow test in
the
recorded insect
along
control
lids
the
was
counted
test
the
released
and
canal,
the
28+1•Ž
gradient
chamber for
the
then
attraction 50
in the
present
releasing were
left
chambers
of
by
chambers
perceptible
test
index
and
L.
were four to
After of
if
the
present
that
was
with
the
experiments
were
lids in four
and
incubation,
50 in
indices
each of
the
of
was
perceptible
were
for
each
counted
species along
―40―
then
released
chamber
of with
G. B,
the
D,
the
canal insect
were
the
nematodes
S.
same.
litura,
at
28+1•Ž
gradient
All assembly to
in
the
chamber.
recorded obtained that
B.
respectively. whole
inoculation was
described the
The 12 hr
was
S. glaseri
as
mellonella,
the
It
of
remained
C, and
for
as
others.
chamber
insects.
left
defined
of
attraction in
and
test
•~ 100
indices
temperature
A,
escape
chambers
observation
were
chambers
the
presence
and
minimum
nematodes
nematodes canal
test
develop
the
together
test
prevent
in
was
preferential
preferential and
tested
index
insect
relative
layer
in the
to
an
using
agar
were
released
the
insects
attractants
for The
of
by the insect inoculated
preferential
insects. obtained
insects
were by
Relative
occurred of
were
four
closed
in
glaseri:
species
migratoria
nematodes
preferential
S.
concentration
different emit
of
attractants
then
Number of nematodes attracted Total number of nematodes
(%)=
other
and all
distribution
nematodes,
nematodes
if
nematodes
to
thickness
chambers.
relative
index
of
emitted
The
containing
in
index
relation
insect
above.
chambers
present
response
determined
germanica,
the
of
the
test
assembly
from
nematodes
preferential
preferential
this
insects
as and
minimum
preferential
The
were
whole develop
distribution
absolute
preferential
Relative
the
the
separately
inoculation
of I:
insects.
Absolute
for
The and
The
follows.
the
insects.
removing
and as
tested
Both
attractants
chamber.
obtained
were
a time. of
emit
After
inoculation
at
escape
to
Preference test chambers designed to test preference Steinernema glaseri. A, B, C, and D: test chambers; inoculation chamber.
migratoria
one
2000年12月
after as were
allow test The
6 hr
follows. present
and The in
Vol.30
No.1/2
Japanese Journal
its corresponding
test chamber.
Relative preferential Nematodes
the
index
attracted
While inoculation Statistical
of
the
preferential
med
before
failed
to
rank
as
mean •}
and
test
standard
chamber the
among
and
four
at
among from
and
the
SCHEFFE
nematodes
variances departures
sum
test for
t-test.
of
equalize
significant
the
differences
responses
to
MANN-WHITNEY reported
overall
attraction
in index
by the insect/ by all the insects its
insects,
×100
canal.
the
nematodes
present
in
considered.
(ANOVA)
analysis correct
not
The
variance
nematodes
preferential
were
analysis:
analysis
of
relative
chamber
Number of nematodes attracted Total number of nematodes attracted
(%)=
=number
determining
way
December.2000
of Nematology
means test
p
