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Chemical defence in ladybird beetles (Coccinellidae). I. Distribution of coccinelline and individual variation in defence in 7-spot ladybirds (Coccinella septempunctata)

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Abstract

7-spot ladybirds secrete alkaloid (coccinelline)-rich fluid (reflex blood) from leg joints as a defence mechanism against predators. A technique is described that enables the collection and accurate quantification of reflex blood produced, and the amount of coccinelline therein. Coccinelline was found distributed throughout the body, although concentrated in the reflex blood. Reflex blood was collected from a large set of beetles at several time points. Significant variation was found among beetles in the amount of reflex blood produced (for males and for females corrected for body weight) and the coccinelline concentration of the reflex blood. The results are discussed in relation to automimicry and the maintenance of variation through energy trade-offs. The relationships between tendency to aggregate, ability to reflex bleed and the possession of aposematic coloration are also considered.
Chemoecology
2
(1991)
7-14
Chemical
defence
in
ladybird
beetles
(Coccinellidae).
I.
Distribution
of
coccinelline
and
individual variation
in
defence
in
7-spot
ladybirds
(Coccinella
septempunctata}
Graham
J.
Holloway,
Peter
W. de
Jong,
Paul
M.
Brakefield,
and
Helene
de Vos
Section
of
Evolutionary Biology, Department
of
Population
Biology, University
of
Leiden, Schelpenkade 14a,
NL-2313
ZT
Leiden,
The
Netherlands
Received
November
20,
1990
/
Revision
accepted
February
21,
1991
Summary
7-spot ladybirds secrete alkaloid (coccinel-
line)-rich
fluid
(reflex blood) from
leg
joints
as a
defence
mechanism
against
predators.
A
technique
is
described
that
enables
the
collection
and
accurate quantification
of
reflex
blood produced,
and the
amount
of
coccinelline therein.
Coccinelline
was
found distributed throughout
the
body,
al-
though concentrated
in the
reflex
blood.
Reflex
blood
was
collected
from
a
large
set of
beetles
at
several time points.
Significant
variation
was
found among beetles
in the
amount
of
reflex
blood produced (for males
and for
females cor-
rected
for
body weight)
and the
coccinelline concentration
of
the
reflex
blood.
The
results
are
discussed
in
relation
to au-
tomimicry
and the
maintenance
of
variation through energy
trade-offs.
The
relationships between tendency
to
aggregate,
ability
to
reflex
bleed
and the
possession
of
aposematic
colo-
ration
are
also
considered.
Key
words
chemical defence, mimicry,
reflex
bleeding,
variation, alkaloid, coccinelline, Coleoptera, Coccinellidae,
Coccinella
septempunctata
Introduction
Mimicry
provides
one of the
most obvious
and
clear-cut examples
of a
consequence
of
natural selection
(Fisher
1930). Mimetic systems have been studied
at a
number
of
different
levels, including
the
genetics
and
evolution
of
aposematic (warning) coloration (Turner 1977, 1984; Shep-
pard
et al.
1985)
and the
factors causing distastefulness
(e.g.
Brower
et al.
1967; Brower 1984; Rothschild 1985; Malcolm
&
Brower
1989). Variation
in
chemical defence
as a
consequence
of
among
individual
differences
in
physiology, rather than
variation
in the
host plant,
has
been less studied (but
see de
Jong
et al.
1991), perhaps
due to the
difficulty
commonly
ex-
perienced
in
accurately
quantifying
toxic components.
The
conditions required
for the
evolution
of
bright
and
obvious warning coloration
are far
from clear (see
Guilford
(1988)
for
consideration
of
problems), although
once
established, normalizing selection
is
expected
to
remove
ge-
netic variation
and
maintain
a
uniform
colour
pattern
throughout
the
population (Turner 1984). Indeed,
in
single
(panmictic)
field
populations
of
species, there
is
usually very
little
variation
in
warning coloration,
at
least
for
Mullerian
mimics.
This
is
true
for the
7-spot ladybird beetle, Coccinella
septempunctata
L.,
although some variation
within
the
apose-
matic colour scheme,
e.g.
variable spot size,
is
present
(Dobz-
hansky
&
Sivertzev-Dobzhansky 1927; Hodek 1973). Further-
more, ladybirds, including
C.
septempunctata,
are
considered
to
form mimetic assemblages (Brakefield 1985a).
©
Georg
Thieme Verlag Stuttgart
New
York
Guilford
(1988) argued that aposematic
colo-
ration
was
most
likely
to
evolve
in a
species that
was
already
distasteful.
The
7-spot ladybird
is
distasteful
and
toxic
to
some bird predators (Marples
et al.
1989)
and the
alkaloid,
coccinelline (Tursch
et al.
1971,
1975, 1976; Meuller
et al.
1984), which
is
synthesised
by the
beetles themselves (Pasteels
et al.
1973),
is the
major toxic component
of
their chemical
defence
(Marples 1990). Indeed, many ladybird species syn-
thesise alkaloids internally
as
defensive substances (Pasteels
et
al.
1973; Ayer
&
Browne 1977). Defence
fluid
is
exuded
by an
active ladybird
as
soon
as it is
attacked
by a
predator;
a be-
haviour
called reflex bleeding (Cuenot 1896; Hollande 1911;
Frazer
&
Rothschild 1960;
Happ
&
Eisner 1961), although
7-
spot ladybird defence
fluid
does
not
contain
the
blood
cells
found
in the
haemolymph (Kay
et al.
1969).
In
well
defended species, such
as the
7-spot
la-
dybird (Marples
et al.
1989),
the
evolution
of
warning mecha-
nisms
is
expected. This
is
achieved through bright aposematic
coloration
and the
emission
of
volatile
repellent
compounds,
such
as
pyrazines (Rothschild
1961;
Guilford
et al.
1987).
The
basic warning
coloration,
at
least,
is
invariable
within
popula-
tion. However,
the
possession
of
signals advertising
toxicity
and
distastefulness
may
alter
the
selective
influences
on the
production
of
defence
fluid
in
ladybirds. Internal synthesis
of
defensive
molecules
may be
metabolically more costly than
se-
questration,
but the
production
of
copious quantities
of
fluid
to
transport
these chemicals must surely
be
energetically
cost-
ly.
As a
result there
may be
selection
to
reduce
the
amount
of
energy
allocated
to
defence
and to
relocate resources
to
other
functions,
for
example oviposition
(Williams
1966;
Sibly
&
Calow
1986; Smith
et al.
1987; Holloway
et al.
1990a,
b).
8
Chemoecology
2
(1991)
Holloway
et al.
Thus,
if all
conspecifics
are
defended
and
warningly coloured,
it
may pay an
individual
to
become
a
Batesian mimic
of
con-
specific
(or
nonconspecific) models, i.e. automimicry (Brower
era/.
1967, 1970; Pough 1973; Gibson 1974). Guilford (1988)
acknowledged
the
possibility
of
this type
of
'cheating'
in re-
cognizably
well
defended species, such
as
7-spot ladybirds.
The
evolution
of a
mixed evolutionary stable strategy (ESS)
(Maynard Smith 1982) that balances
the
cost
of
unpalatability
against increased chance
of
prédation becomes
a
possibility.
The
development
of
automimicry becomes
still
more
likely
in
an
aposematically colored species that aggregates. Here,
if a
naive
predator discovers
an
aggregation,
the
chance
of
being
sampled
is
clearly much less than
if a
single animal
is
found
(providing
the
predator finds
the
defended prey distasteful).
7-spot ladybirds form such aggregations, particularly during
winter
hibernation (Hemptinne 1988;
Majerus
&
Kearns
1989)
and
summer aestivation (pers.
obs.).
It
seems plausible that although
the
apose-
matic
coloration
is
invariable
in
7-spot
ladybirds, there
may
be
substantial variation
in the
amount
or
toxicity
of
defence
fluid
produced.
The
purpose
of the
present study, therefore,
was two
fold:
1.
to
establish
how the
7-spot
ladybird defends itself, i.e.
is
the
defensive alkaloid distributed throughout
the
body
or
is
energy
channelled
into
producing
only
the
toxic
secre-
tion?
Whether ladybirds remain
toxic
during
the
winter
months
and are
still
able
to
reflex
bleed
may be in-
fluential
in
determining
the
need
to
aggregate
in
groups
at
cer-
tain
times
of the
year.
Furthermore,
we
also
wished
to
test:
2.
whether among beetle variation exists
in the
amount
of
fluid
produced
and the
concentration
of
alkaloid therein
and,
if so,
whether
the
findings
are
consistent with
the
exis-
tence
of a
mixed
ESS
suggesting
the
development
of au-
tomimicry?
Materials
and
Methods
Insects
Adult
7-spot ladybirds were collected from
ivy
(Hedera
helix)
in
Leiden (The Netherlands)
as
they
emerged from winter hibernation
in
February, 1990.
210 un-
sexed insects were distributed
at
random
across
four
groups.
Three
of the
groups
(A to C)
contained
20
beetles
whilst
the
fourth
group
(D)
contained
the
remaining 150. Each ladybird
was
maintained separately
in
a-
5 cm
diameter plastic petri-
dish
and
kept
in a
constant climate cabinet
(20°C,
80%
rela-
tive
humidity,
18:6 light: dark).
In
addition, each animal
in
group
D was
sexed using ventral surface abdominal character-
istics.
The
ladybirds were provided with
a
clean petri dish
and
fresh
pea
aphids
(Acyrthosiphon
pisum)
every
day
derived
from
infested laboratory bean plants,
so
that
an
ample food
supply
was
always available. After
a
period
of
time, many
of
the
female ladybirds
began
to lay
batches
of
eggs.
The
num-
bers
of
eggs laid were counted.
Extraction
and
collection
of
reflex
blood
A
length
of
flexible silicone rubber tubing
(2.3
mm
diameter)
was
attached
to a
tap-operated vacuum
pump.
The
suction created
by
touching
the
tube down
onto
the
elytra
was
sufficient
to
pick
up an
individual ladybird.
This rarely induced reflex bleeding;
the use of
forceps
or
fin-
gers almost always did.
The
beetle could then
be
held ventral
side
upwards
and
stimulated
to
produce
reflex
blood
by
touching
the
exposed thorax
or
legs with
a 10
|xl
capacity
ca-
pillary
tube. When
a
drop
of
fluid
was
produced
(usually
in-
stantly)
the
capillary tube
was
turned through 180°
and the
fluid
taken
up in the
opposite
end of the
tube.
The
beetle
could then
be
stimulated
to
produce
more
reflex
blood
by us-
ing
the dry end of the
capillary tube, which again
was
col-
lected
in the
opposite
end of the
tube
and so on
until
secretion
ceased.
Occasionally
a
second
tube
was
required
to
complete
collection.
The
amount
of the
fluid
produced
was
calcu-
lated
by
weighing
the
capillary tube before
and
after
bleeding
on a
Metier
AE 160 top pan
balance
(to
±0.01
mg).
The
tube
plus
fluid
was
quickly
transferred
to an
Eppendorf
vial
con-
taining
0.2 ml
methanol.
It was
then shaken vigorously
for a
few
seconds
to
disperse
the
solvent along
the
capillary tube.
The
capillary tubes were left overnight
in the
methanol
in
sealed Eppendorfs.
A
gentle stream
of air was
then used
to
blow
any
methanol from
the
tube into
the
Eppendorf.
The
solvent
was
allowed
to
evaporate
off at
room
temperature.
All
ladybirds were weighed after being reflex
bled
to the
nearest
0.1
mg.
Groups
A, B and C
ladybirds were
examined
on
days
1, 3 and 5,
respectively,
to
determine
the
rate
at
which
reflex
blood
could build
up
following hiberna-
tion
and
also
to
establish
an
appropriate length
of
time
to
leave
the
beetles
in
group
D
between bleedings.
Group
D
lady-
birds
were used
to
examine variation
among
beetles.
These
la-
dybirds
were bled
and
weighed
on
days
8, 15 and 22, so
that
for
each beetle there were three values
of
fluid
weight, body
weight
and
amount
of
coccinelline. After
the
final
bleeding
and
weighing
on day 22, all
beetles were frozen
at
30°C.
Alkaloid
analysis
Following
evaporation
of the
methanol,
the
Eppendorfs were kept
at
4°C.
The
amount
of
alkaloid
in
each
extract
was
measured using
a
Packard
433 Gas
Chromato-
graph (GC). Prior
to
injection
each
extract
was
redissolved
in
(exactly)
0.2 ml
methanol.
0.2
u.1
of
each resulting solution
was
injected into
the GC.
Material
was
allowed
to
pass
through
the
column
(25 m
long, 0.53
mm
inner diameter
CP-
Sil
8) for
10
min at a
temperature
of
185°C
using
H2
(7.18
ml/min)
as
carrier gas.
The
coccinelline appeared
on the
trace
in
less