Journal of Chemical Ecology, Vol. 26, No. 8, 2000
CHEMISTRY OF A MATING PLUG IN BUMBLEBEES
BORIS BAER,1,* ROLAND MAILE,2 PAUL SCHMID-HEMPEL,1
E. DAVID MORGAN,2 and GRAEME R. JONES2
1ETH
Zurich, Experimental Ecology, ETH-Zentrum NW
CH-8092 Zurich, Switzerland
2Keele University, Chemistry Department
Keele, Staffordshire, ST5 5BG, England
(Received November 10, 1999; accepted April 4, 2000)
Abstract—In the bumblebee B. terrestris males transfer a mating plug into
the queen’s sexual tract shortly after sperm transfer. The plug is a sticky,
opaque secretion of the male accessory gland. In order to clarify the meaning
of the mating plug, we collected the plug substance directly from the male’s
accessory gland and identified the chemical substances present with gas
chromatography. The main compounds found in the mating plug were four
fatty acids (palmitic, linoleic, oleic, and stearic acids) and a cyclic peptide
(cycloprolylproline). Mixing the four fatty acids resulted in a similar sticky,
opaque mass as found in natural plugs, indicating that cycloprolylproline
is not necessary for the physical attributes of the plug. The function of
the fatty acids may therefore be to build up a physical barrier, optimizing
sperm placement before the spermathecal duct or preventing sperm backflow.
Cycloprolylproline, on the other hand, may influence female mating behavior
so as to reduce her receptivity. In fact, peptides are known to reduce female
receptivity in other insects. This would explain why queens of B. terrestris are
only singly mated, although multiple mating is beneficial during the colony
cycle with respect to parasitism and fitness.
Key Words—Mating plug, Bombus, cycloprolylproline, palmitic acid, linoleic
acid, oleic acid, stearic acid.
INTRODUCTION
Male–male competition over access to females has led to the evolution of various adaptations to maximize male mating success and paternity among offspring.
* To whom correspondence should be addressed. e-mail: baer@eco.umnw.ethz.ch
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For example, during mating males may transfer not only sperm, but also chemical substances into the female’s sexual tract, which increase the likelihood of
paternity (male mating success) (Price et al., 1999; Gonzalez et al., 1999; Soller
et al., 1997). Such chemical compounds are produced in the male accessory
gland of insects (Gillott, 1996). They are known to induce behavioral and
physiological changes in mated females (Wolfner, 1997; Yi Shu and Gillott,
1999). In some insects compounds from the male accessory gland form a mating plug or part of a mating sign, which are reported to have different functions;
such plugs or signs inhibit further matings (Polak et al., 1998; Dickinson and
Rutowski, 1989; Monnin and Peeters, 1998), prevent sperm from flowing out
of the female’s sexual tract (Woyciechowski et al., 1994), ensure the paternity
of the last male (Lachmann, 1998), retain sperm near the opening of the sperm
storage (Polak et al., 1998), or serve as a biochemical signal influencing femaleto-male behavior (Monnin and Peeters, 1998). Mating success in social insects
is especially interesting. In particular, whether one or several males succeed in
mating with a single queen affects the average relatedness among workers in
the colony. This leads to variation in a number of key parameters of sociality, such as the level of cooperation (Keller and Reeve, 1994) or sex allocation
to offspring (Boomsma and Grafen, 1990). For the social Hymenoptera (bees,
ants, and wasps) information on female (queen) mating behavior is available,
but male reproductive skews, i.e., sperm bias, are poorly investigated including the function and biology of the male accessory gland. Given the observed
variation in female mating frequency and sperm bias (Boomsma and Ratnieks,
1996), more information is needed about the function and the biochemistry of
these compounds in social insects. This could help in understanding recently
discussed topics such as the observed differences in queen mating frequencies
among females and among taxa.
Queens of B. terrestris appear to be singly mated in Central Europe
(Schmid-Hempel and Schmid-Hempel, 2000). They also often refuse to remate
in flight cages (personal observation), although queens would benefit from multiple mating by higher fitness and lower parasite loads (Baer and Schmid-Hempel,
1999). Therefore, queens do not take advantage of the benefits of multiple mating. Male bumblebees may control queen mating frequency, for example, by
transferring chemical compounds that influence the queen’s mating behavior.
This idea is supported by the observation that bumblebee males transfer a mating
plug into queens shortly after sperm transfer. The plug is a gelatinous secretion
of the male accessory gland and forms a characteristic, sticky mass in the queen’s
bursa copulatrix. The plug remains in place for several days and could potentially act as a sperm barrier (Duvoisin et al., 1999). Here we have identified the
chemical compounds of the plug.
MATING PLUG CHEMISTRY OF BUMBLEBEES
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METHODS AND MATERIALS
Colonies of B. terrestris were reared in the laboratory from queens caught
in spring 1998 around Zürich, Switzerland. The colonies were kept in climate
chambers in red light at 268 C and 60% humidity and were fed ad libitum with
pollen and sugar water. To avoid measuring chemical compounds of the female’s
sexual tract rather than those from the male, we extracted the secretion that forms
the plug directly from the male accessory gland instead of dissecting freshly
mated queens. The males were removed from the colonies shortly after eclosion,
kept in brother groups in plastic boxes (14 × 18 × 10 cm) and were fed ad libitum
with pollen and sugar water. Males of B. terrestris normally mate at an age of 16
± 7 days (Duchateau and Marien, 1995). For practical reasons, we used males
of an approximate age of 10 days for our experiments.
Identification of Plug Compounds by Gas Chromatography. To collect the
plug secretion, males were killed with CO2 . The abdomen was opened and
the accessory gland as well as the ejaculatory duct and the endophallus were
removed and washed in insect Ringer. With this procedure, the accessory male
gland secretion (i.e., the plug) starts to leak out of the endophallus and forms a
typical white, opaque mass that can be picked up with a fine insect needle and
transferred into a glass capillary (1.6 mm diam.). The capillary was sealed by
melting both ends and was used directly for gas chromatography. To verify the
presence and concentration of plug compounds, we later synthesized a plug using
commercially available chemicals in the same concentrations as found in the natural plug and repeated the chromatography. The analyses were carried out with a
Hewlett Packard 5890 gas chromatograph directly coupled to a 5970B selective
detector (quadrupole mass spectrometer, 70 eV electron impact ionization). The
system was controlled and data accumulated on a Hewlett Packard series 300
computer with HP 5970C Chemstation. Mass spectra were scanned from m/ z 35
to m/ z 550. Scan time was about 2.4 sec. Chromatography was performed on
an immobilized polydimethylsiloxane phase in a fused silica column (12 m ×
0.2 mm, 0.33-mm film thickness) (SGE, Milton Keynes, UK). Helium was used
as carrier gas at 1 ml/ min. The glass capillary containing the plug material was
crushed inside the injector port. The oven was programmed from 308 C (3 min)
at 88 C/ min to 2508 C. The split valve was closed before crushing the sample
and reopened 30 sec later. Identification of compounds was confirmed by comparison of their mass spectra and retention times with those of standards and by
comparison with MS databases.
RESULTS
The chromatograms of a single plug and the synthetic mixture of the compounds are shown in Figure 1a, 1b. Chromatograms of five natural plugs were
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FIG. 1. (a) Reconstructed total-ion chromatogram (TIC) of a GC-MS analysis from injection of the mating plug content of one Bombus terrestris drone by solid-sampling technique. 1: Cycloprolylproline; 2: palmitic acid, 3: linoleic acid, 4: oleic acid, 5: stearic
acid. (b) Reconstructed chromatogram of a GC-MS analysis from injection of a synthetic plug of commercially available chemicals in similar concentrations as found in a
natural mating plug of Bombus terrestris.
practically identical. The identity of the compounds is given in Table 1. The
main compounds detected in the five plugs were linoleic acid, followed by oleic,
palmitic, and stearic acids. A cyclic dipeptide, cycloprolylproline was found in
smaller amounts (Table 1). Mixing the four commercially available fatty acids
(linoleic, oleic, palmitic, and stearic acids) at the same concentrations as found
in natural plugs gave the same kind of sticky, opaque mass. Cycloprolylproline
is obviously not necessary for the physical attributes of the plug. The synthetic
plug containing the five substances detected produced a chromatogram similar
to that of a natural plug.
DISCUSSION
Analyses of the chemical compounds in male accessory gland secretions
of male bumblebees showed relatively few main components. The fatty acids
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MATING PLUG CHEMISTRY OF BUMBLEBEES
TABLE 1. COMPOSITION OF MATING PLUGS REMOVED FROM MALES OF Bombus
terrestries, TOGETHER WITH STANDARD DEVIATIONS (N c 5 Drones)
1
2
3
4
5
Compound
Retention time
(min)
Cycloprolylproline
Palmitic acid
Linoleic acid
Oleic acid
Stearic acid
24.95
25.36
27.75
27.88
28.09
Mol. mass and
(diagnostic ions)
196
256
280
282
284
(70,
(43,
(67,
(55,
(43,
98,
73,
41,
69,
73,
110)
60)
55, 81)
60, 83)
60)
Mean proportion
(ng/ plug ± SD)
230
980
5400
1700
440
±
±
±
±
±
40
300
1000
750
290
detected (Table 1) are common biological substances and are known to be used in
other social insects for communication purposes. For example, oleic and linoleic
acid levels increase in dead ants and induce necrophoretic behavior in different
ant species (Akino and Yamaoka, 1996). Other social insects such as wasps use
these two fatty acids as predator (ant) repellents (Dani et al., 1996) where they
also form a gelatinous mass. In general, fatty acids are common but minor components of insect cuticular lipids (Lockey, 1988). The ubiquitous presence of
fatty acids in tissue means that caution must be used when ascribing a function
to them. However, in this case, the fatty acids are found as a massive quantity.
This is the first report of the combination of palmitic-, linoleic-, oleic, and stearic
acids to be involved in insect reproduction. Since the fatty acids detected here
have the potential to be a messenger, it would be very interesting to see whether
males or queens use them in the organization of mating behavior, for example,
to manipulate the readiness for copulation.
Duvoisin et al. (1999) found that the plug persists in the queen’s sexual
tract for several days indicating that it is not, or only slowly, absorbed by the
queen. Additionally Duvoisin et al. (1999) demonstrated that the plug can act
as a sperm barrier. Therefore, the main function of the fatty acids in the plug in
the case of B. terrestris may be a physical barrier. Additionally, the plug may
aid in the placement of sperm near the entrance to the queen’s spermatheca or
aid in preventing sperm backflow. Both effects could help maximize a male’s
paternity.
Cycloprolylproline has never been reported before in social insects. As our
analysis showed, this compound is not necessary for the physical properties of
the plug. Many cyclic peptides, such as cycloleucylphenylalanine in the venom
of the ant Pachycondyla (Cruz Lopez and Morgan, 1997) have very bitter tastes
to humans (Matoba and Rata, 1972). However, cycloprolylproline is reported to
have a flat taste (Ishibashi et al., 1988). While caution is necessary in extrapolating from human to insect senses, cycloprolylproline may have a role in sensory
detection. Peptides are known from other insects to reduce receptivity in females
(Chen et al., 1988), and cycloprolylproline may have the potential to act as a
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pheromone influencing the mating behavior of queens, and in particular reducing
her receptivity. This should substantially increase a male’s reproductive success
at the expense of competitors since bumblebee males do not seem able to remove
another male’s sperm or plug from the queen’s spermatheca.
Acknowledgments—We thank B. Imhoof for comments on the manuscript. This work was supported through an European Network for Training and Mobility in Research (TMR) no. ERBFMRXCT960072 “Social Evolution” of the Universities of Aarhus, Firenze, Keele, Sheffield, Uppsala,
Wurzburg, and ETH Zürich.
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