birkinshaw2001

Entomologia Experimentalis et Applicata 98: 345–351, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
345
Prostephanus truncatus mate choice on contact: does pheromone signalling
by males affect their mating success?
L. A. Birkinshaw1 & R. H. Smith2
1 Natural
Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK (E-mail:
[email protected]); 2 School of Biological Sciences, University of Leicester, Leicester LE1 7RH, UK
(E-mail: [email protected])
Accepted: October 10, 2000
Key words: Bostrichidae, Prostephanus truncatus, aggregation pheromone, sexual selection, female mate choice
Abstract
Prostephanus truncatus is an economically important beetle pest of stored maize and cassava in the tropics. Male
beetles signal using an aggregation pheromone that attracts both female and male beetles over large distances.
Females preferentially orientate towards the pheromone signals of particular males when given a choice. The
influence of pheromone signalling on courtship and mating success was investigated using pheromone biossays
and mating trials in both of which a female made a choice between two males. Signalling was manipulated by
exposing males to a Female Factor that inhibits pheromone production. The relative attractiveness of males to
females based on pheromone bioassays was found not to influence short-range courtship behaviour or mating
success.
Introduction
Prostephanus truncatus (Coleoptera: Bostrichidae) is
a destructive pest of stored maize and cassava in the
tropics and is commonly known as the Larger Grain
Borer or LGB (Hodges, 1996). LGB shows many of
the characteristics of wood-boring beetles and will
form complex tunnel systems with main tunnels and
blind-ending tunnels when offered a suitable host (Li,
1988). Aggregations of beetles form in response to signalling by males releasing an aggregation pheromone
that attracts both sexes over a long range (tens or 100s
of metres) (Scholz et al., 1997). Traps baited with synthetic aggregation pheromone are used to monitor both
the population dynamics and the spatial distribution of
LGB (Borgemeister et al., 1997a,b).
The hypothesis that males signal primarily to attract females (Hodges, 1994) is supported by three
observations: (1) Only the males signal (Cork et al.,
1991). (2) The sex ratio of beetles attracted by males
in the field is female-biased (Scholz et al., 1997). (3)
Males stop emitting pheromone signals when females
are present (Smith et al., 1996). Birkinshaw & Smith
(2000) investigated the role of aggregation pheromone
in sexual selection in LGB using olfactometer and
field tests, and showed that both males and females
respond consistently and preferentially to the signals
emitted by some individuals in choice tests. Their
results support the hypothesis that LGB aggregationpheromone signals are sexually selected. The males
that respond to the pheromone may be exploiting the
efforts and ability of the signaller to attract females,
and the signalling male may suffer a fitness cost of increased competition for both mates and host resources.
Males decrease the rate of production of pheromone
within 24 h of exposure to live females and they respond in a similar way to an unknown, non-volatile
substance known as ‘Female Factor’ left in grain that
has been infested by females (Smith et al., 1996).
The balance of the costs and benefits of signalling
will be determined in part by the short-range effect
of aggregation pheromone on courtship behaviour and
mating success. Adult LGB that encounter each other
in an open mating arena often push each other, and
this behaviour is especially prevalent when a female
comes into contact with a male, suggesting that push-
346
ing may be part of courtship. Females appear to be
generally reluctant to mate. Pushing behaviour may
enable beetles to assess aspects of quality, prevent copulation, or serve to stimulate further sexual behaviour
(Birkinshaw, 1998).
Males also push other males and this may be a form
of intrasexual selection, carried out either to increase
access to mates or to defend resources such as tunnel
systems. It is not known, however, whether pushing
behaviour is related to either body size or pheromone
production. Male size is often a good predictor of
mating success in arthropods (Crespi, 1989; Andersson, 1994). Small males may be more manoeuvrable
and agile than large males and this could help in obtaining mates in species that copulate in flight or in
water. Large males may perform best if there is visual
or physical assessment of ‘quality’, either within or
between sexes. Females may produce higher quality
offspring by selecting larger mates if body size and fecundity are genetically correlated (Holloway & Smith,
1987).
In the cockroach Nauphoeta cinerea, long-range
pheromones have been found to influence short-range
courtship behaviour (Moore, 1998; Moore et al.,
1995). Placing pheromone-impregnated discs of filter paper on the backs of male cockroaches elevated
their dominance status during courtship (Moore et al.,
1997). It is not known whether LGB courtship is
also influenced by aggregation pheromones. Thus the
overall question about LGB behaviour addressed here
is as follows: is a male who is more attractive than
other males at a distance more successful than those
other males in short-range interactions of courtship
and mating?
In order to address this question, we present results
from three approaches:
(1) comparison of courtship behaviour in relation
to mating success,
(2) comparison of relative attractiveness of two
males to a female in pheromone bioassays and their
performance in mating trials,
(3) manipulation of pheromone signal by exposing
one male of a pair to grain impregnated with Female
Factor.
Materials and methods
All insects used were from a laboratory culture originally established using LGB collected in Tanzania
during the 1980s and maintained on whole maize at
the Natural Resources Institute (NRI), UK. The maize
used in experiments was ‘Natco medium corn meal’
supplied by T. Cholthram and sons Ltd, Middlesex,
UK. Both insect cultures and experiments were kept
in a controlled temperature and humidity (CTH) room
at 30 ◦ C and 70% r.h. with a L12:D12 light regime.
Experimental insects were adults of mixed ages
randomly selected from sieved cultures. Individual
beetles were sexed (by examining genitalia; Birkinshaw, 1998), placed singly on medium-ground maize
in a 36 cm3 glass pot, and held for 12 days. Males were
weighed using a Cahn electrobalance 5 days after they
were sexed.
Pheromone bioassays. An olfactometer was used to
assess the preference of a single female given a choice
of signals from two males. The apparatus is described
by Birkinshaw & Smith (2000) and consists of a circular central arena (diameter 9.5 cm) in which the
walking response of a beetle (the responder) to four
plumes of air is recorded. Two of the plumes of air
were passed over control pots containing only food
(ground maize) and the other two plumes were passed
over experimental pots each containing a single male
on food. Each female therefore had a direct choice
between volatiles emitted by two different males.
Bioassays measured the number of times the head
and prothorax of the responder was placed in each tube
carrying an air plume from a pot during a 20-min period of observation; the responder was placed back in
the centre of the arena after each response (Birkinshaw
& Smith, 2000).
Mating trials. The open mating arena was a Petri
dish (9.5 cm diameter) with its floor lined with filter
paper. Two males were placed with a single female
in the arena and courtship behaviour was observed for
30 min, or until a male copulated with the female. The
identity of the male that copulated was noted.
Courtship behaviour. Three measures were recorded
for each physical contact between any two test-beetles:
(1) the identity of the beetle that approached another beetle,
(2) the identity of the beetle being approached,
(3) the identity of the pushing beetle (when a push
was observed).
The identity of the beetle that initiated an encounter was recorded as an indication of active pursuit
of mates. The strength of the push was not quantified.
347
Experiment 1. Natural variation: does long-range
attraction predict the progress and outcome of shortrange courtship? Each replicate consisted of one
female with two males. Males were chosen to differ in
weight by 0.4–0.5 mg. Three olfactometer arenas were
observed simultaneously for 20 min, and mating trials
were conducted in sequence immediately afterwards.
Initially, there were 36 replicates tested over 5 days
(data set A). One month later, 45 replicates were tested
using beetles from a different culture (data set B).
Experiment 2. Signal manipulation: does exposure of
males to Female Factor affect mating success? The
insects from the second set of 45 replicates (data set B)
were immediately used in a further experiment (data
set C). One male of each pair was randomly selected
for exposure to Female Factor, while the other male
and the female were kept separately with fresh food in
a pot containing split maize grain and a small quantity
of maize flour. The Female Factor treatment used an
equivalent amount of maize grain from cultures 8–10
weeks old; this grain was frozen for 5 days and then
allowed to equilibrate in the CTH room for 2 days.
Males remained in these media treatments for 12 days.
Males were tested in the same pairs as in data set
B. The experiment was carried out over 3 days:
– days 1 and 3 – mating trials in open arenas with
females allocated at random to day and to pairs of
males (15 replicates tested each day).
– day 2 – pheromone bioassays in order to check
the success of manipulation of pheromone signalling
by exposure to Female Factor (15 replicates tested).
Statistical analysis. The association of mating success with the outcome of the pheromone bioassay or
the relative size of the male was tested using a chisquared test with one degree of freedom. Hypotheses
about courtship behaviour in the mating trials were
tested using Wilcoxon signed-ranks tests.
Results
Does long-range attraction predict mating success?
Natural variation. Data sets A and B were used, excluding cases where there was no mating (21 cases)
or the olfactometer bioassay did not reveal preference
for either male (15 cases). Table 1a shows that the
attractiveness of the pheromone signal did not affect
the probability of being the first to mate in either data
set, although there was significant heterogeneity between data sets A and B (N = 50, contingency table
2 = 3.94, P<0.05).
χ(1)
Manipulation of pheromone signal using Female Factor. Males exposed to fresh grain were significantly
more attractive to females in olfactometer tests than
males exposed to Female Factor (mean response of
3.13 compared with 0.2 visits by a female per 20 min
trial: N = 30, z = 2.809, P<0.01). This result confirms that exposure to Female Factor caused males to
alter their emission of aggregation pheromone in this
experiment. There was, however, no significant effect
of Female Factor on mating success. The male exposed to Female Factor was the first to mate in 14/26
trials and the signalling male exposed to fresh grain
was the first to mate in 12/26 trials.
Different females were used in mating trials B and
C, yet there was strong consistency in the relative success of males in the competing pairs. Excluding the
seven cases where there was no mating in either B or in
C, the same male mated first in 19/23 cases and there
was a change in relative success in only 4/23 cases
2 = 9.78, P<0.01).
(N = 23, X(1)
Does male body size predict mating success?
Data sets A and B were used, excluding cases
where there was no mating, and were combined because there was no heterogeneity (N = 61, contin2 = 0.19, n.s.). Table 1b shows that
gency table X(1)
larger males were more likely to mate first, but the
effect was not statistically significant (overall N=61,
2 = 1.8, n.s.).
X(1)
Larger males were also visited more frequently in
bioassay tests, but not significantly so (Table 1c: data
sets A and B excluding cases where the olfactometer bioassay did not reveal preference for either male;
2 = 1.1, n.s.). Data sets A and
overall N = 66, X(1)
B could be combined because there was no hetero2
= 1.10,
geneity; N = 66, contingency table X(1)
n.s.).
Is courtship behaviour related to mating success?
Data sets A and B were used, excluding replicates
where there was no mating. The data for all physical
contacts between a male and a female were first separated into approaches involving the successful male
(first to mate) and those involving the unsuccessful
(non-mating) male, and then divided again according
to whether the female approached the male or vice
versa. The results for proportions of total encounters
involving physical contact are plotted as separate bar
charts for data sets A and B in Figure 2.
348
Table 1. The associations between males preferred by females in
pheromone-signalling bioassays, their mating success in open-arena mating
trials, and their relative sizes. Data sets A and B were tested separately using
a chi-squared test of the hypothesis of no association. They were combined
as overall figures only when a contingency-table test showed no significant
heterogeneity
(a) Female preference for signal and mating success
Data set
Preferred signaller
mated first
Non-preferred
signaller mated first
Chi-squared
statistic (1 d.f.)
A
B
15
10
8
17
1.1
0.9
2 = 3.94, P<0.05.
Test of heterogeneity: N = 50, X(1)
(b) Male size and mating success
Data set
Heavier male
mated first
Lighter male
mated first
Chi-squared
statistic (1 d.f.)
A
B
Overall
16
22
38
11
12
23
0.5
1.5
1.8
2 = 0.19, n.s.
Test of heterogeneity: N = 61, X(1)
(c) Male size and female preference for signal
Data set
Heavier male’s
signal preferred
Lighter male’s
signal preferred
Chi-squared
statistic (1 d.f.)
A
B
Overall
21
18
39
11
16
27
1.6
0.1
1.1
2 = 1.10, n.s.
Test of heterogeneity: N = 66, X(1)
Figure 1. Experimental design to show which insects contributed
to the three data sets, A, B and C. OB = Olfactometer bioassay,
MT = Mating trial.
There was no consistent or significant difference
between whether the male or the female was the first
to make contact; in data set A, females initiated more
physical encounters while the converse was true in
data set B.
The three types of push in Figure 2 were compared between males that mated and males that did not
mate. The only significant difference was for encounters involving no pushing, where males that mated
were more likely to be involved in a physical encounter involving no pushing (A: N = 27, z = −3.39,
P = 0.001; B: N = 34, z = −4.59, P<<0.001).
Out of all the physical encounters in data sets A
and B, 21% were between the two males. The percentage of encounters involving an obvious push by a
male was an order of magnitude higher for male-male
interactions (A: 30%; B: 34%) than for male-female
interactions (A: 3.5%; B: 5%). Larger males made a
higher mean number of pushes per trial in both sets
(A: larger – 1.11, smaller – 0.39; B: larger – 1.80,
349
Figure 2. Bar charts showing the proportion of physical encounters
leading to three different behaviours before mating (female pushes
male, male pushes female, or no apparent pushing). Results are
shown separately for the two sets of trials: (a) data set A (N = 27),
(b) data set B (N = 34).
smaller – 0.67 pushes per trial) but this difference was
only statistically significant in data set A (A: N = 36,
z = −2.20, p<0.05; B: N = 45, z = −1.59, p=0.11).
The males that mated successfully made significantly
more pushes than the other male in male-male interactions in set B (mated – 1.68, unmated – 0.45 pushes
per trial: N = 38, z = −2.48, p=0.01) but not in
set A (mated – 0.52, unmated – 0.56 pushes per trial:
N = 27, z = 0.07, n.s.).
Manipulating male aggregation-pheromone signalling did not result in any significant changes in
courtship behaviour (comparing set B to set C) (for
details see Birkinshaw, 1998).
Discussion
There is tremendous variation in courtship and copulation behaviour between different animal species
(reviewed by Eberhard, 1996). The results described
here need to be placed in the context of the normal
behaviour in the plant host of LGB (Birkinshaw, 1998)
and similar wood-boring beetles such as members of
the Scolytidae (Kirkendall, 1983). Birkinshaw (1998)
describes the reproductive behaviour of LGB both
within tunnel systems and in open arenas. Outside
the plant host, LGB courtship behaviour is characterised by a variable pre-copulatory period of pushing
behaviour, a period of stereotyped rubbing of the female by the male using his antennae and legs, a short
copulation, and little evidence of any post-copulatory
interaction between male and female. In contrast, behaviour within tunnel systems is characterised by a
male and a female remaining together for several days
and mating repeatedly during this time. When a male
has constructed a short lenth of tunnel (1–2 cm), he
spends most of his time positioned at the entrance
to the tunnel. Only one female cohabits with a male
within his tunnel system, unlike many polygamous
scolytid species (Kirkendall, 1983; Schlyter & Zhang,
1996; Schmitz, 1972). A typical sequence of behaviour resulting in mating is as follows: (1) the male
approaches the female in his tunnel system and appears to entice her back into a widened region of
tunnel, such as the junction of two branches or the entrance of the system, (2) the male assumes the mating
position where there is enough room for copulation,
(3) after mating, the female generally returns to the
end of a blind-ending tunnel where she lays eggs (Li,
1988), (4) the male generally returns to the tunnel
entrance. Some pushing behaviour was observed between beetles in tunnel systems and on one occasion a
female was seen to expel a male by pushing. Males appear to guard the female only within a tunnel system,
where the male will remain at the entrance in a manner very similar to many scolytid species; Kirkendall
(1983) notes of scolytids that ‘... males that stay during gallery construction remain blocking the entrance
hole, occasionally leaving their post to copulate, feed
or remove accumulation of frass’.
In the mating trials in open arenas described here,
different females showed very similar preferences for
a particular male of a pair in trials separated by 12 or
more days (data sets B and C). Females were more
likely to push males than vice versa. The males who
were most persistent in approaching females were also
most likely to secure the first mating with a female.
The suggestion by Fadamiro (1995) that males are
‘the mate-finding sex’ was not convincingly supported
here. In open arenas (and in tunnel systems), the
movement of both males and females towards each
other seemed to initiate courtship and copulation. Females appeared to exercise choice between two males
by being generally reluctant to mate and thus selecting for the most persistent males. Males also pushed
one another, and larger males on average made more
350
pushes per trial than smaller males (statistically significant in data set A). The males that were successful in
achieving the first mating also made significantly more
pushes per trial in data set B, though not in data set
A. The results for males are similar to those reported
by Hughes (1981) for Monochamus scutellatus (Cerambycidae), where larger males won contests more
often than smaller males. Pushing behaviour is also
part of male-male conflict in the Scolytidae, where it
is observed on the bark surface (Kirkendall, 1983).
Female choice in mating trials, however, could
not be predicted either from knowledge of the relative
attractiveness of pheromone signals or from relative
body size. Manipulation of pheromone signalling using Female Factor confirmed that the pheromone signal did not influence female choice during close-range
courtship. The data in Table 1 are from at least 50 mating trials in each case and it is therefore likely that the
tests are sufficiently powerful to avoid Type II Error.
All benefits of mate acquisition that result from
pheromone signalling in LGB seem therefore to accrue from manipulation of dispersal and spatial distribution of females. Beetles in the field have a high
resolution of perception of competing signals and
can distinguish consistently between two signalling
males only 0.6 m apart (Birkinshaw & Smith, 2000).
Pheromone signalling could still be a major determinant of mating success when males occupy distinct
tunnel systems, especially in non-grain hosts (Borgemeister et al., 1998). In maize seeds, however, the
tunnel systems are neither extensive nor distinct and
attraction of a female may benefit the signaller much
less than in wild hosts because of the greater likelihood
of interception of the female by another male.
It was suggested in the Introduction that males
may stop pheromone signalling when a female has
been attracted as a means of reducing competition
from other males. Another reason to stop signalling
in the presence of a female is that it may be costly
in terms of energy. Predators, however, also respond
to aggregation pheromone, e.g., Teretrius nigrescens
(Coleoptera: Histeridae) (Rees et al., 1990). Females
and their eggs could be protected from both predators and parasitoids by males guarding the entrance
of tunnel systems. There is evidence that the presence
of males increases the numbers of live offspring produced through decreased predation in the Scolytidae
(Kirkendall, 1983; Reid & Roitberg 1994). Protection
of eggs and young larvae could be the main reason
why a female stays with a single male for several
days in a tunnel system while showing no such at-
tachment in an open arena. Increased risk of offspring
predation from males that continued to signal may,
therefore, be one reason why females use cues other
than pheromone signalling to choose between mates
once a male/males have been located.
Finally, we consider whether there are any consequences for pest management. The implications of
knowledge of mating systems and sexual selection
for pest management have been reviewed by Boake
et al. (1996). In LGB, dispersing beetles are sensitive to natural variation in aggregation-pheromone
signalling. Manipulation of host selection using aggregation pheromone is therefore possible (Birkinshaw
& Smith, 2000). The results reported here, however,
show that short-range courtship behaviour is insensitive to pheromone signal. Aggregation pheromone
cannot therefore be used as a courtship disruptant in
this species.
Acknowledgements
This publication is an output from a research project
funded by the United Kingdom Department for International Development (DFID) for the benefit of
developing countries. The views expressed are not
necessarily those of DFID. R6201 ‘Crop Post Harvest
Research Programme’.
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