Behav Ecol Sociobiol (2003) 54:521–533
DOI 10.1007/s00265-003-0673-5
REVIEW ARTICLE
Boris Baer
Bumblebees as model organisms to study male sexual selection
in social insects
Received: 13 March 2003 / Revised: 1 July 2003 / Accepted: 13 July 2003 / Published online: 16 August 2003
Springer-Verlag 2003
Abstract Social-insect males are often regarded as being
merely short-lived “flying sperm containers”, which
ignores their potential influence on females and paternity
patterns as found in other animals. Consequently, socialinsect males have received only marginal attention and
sexual selection has almost never been studied in these
species. Here I present a review of the mating biology of
bumblebees (Bombus spp.), which are the best-studied
social insects to date. I follow a male’s pathway from his
birth until he successfully contributes to the next generation, and show that males have evolved adaptations and
behaviors to influence paternity patterns at various stages
of their life, which are similar to those exhibited by males
of non-social insects. By comparing the available bumblebee data with more sparse studies of male reproductive
behavior in other social Hymenoptera, I argue that such
male adaptations may indeed be widespread in social
insects. I suggest that current paradigms on sexual
selection should be challenged by using social insects as
model systems, because they offer unique features, and a
solid theoretical background in which clear predictions
can be made and appropriate experimental tests of them
can be designed.
Keywords Social Hymenoptera · Copulation · Sperm
competition · Accessory-gland compounds · Paternity
Introduction
The mating biology of social-insect males is poorly
investigated, although there is no obvious reason why
they should not have the potential influence on mating
Communicated by A. Cockburn
B. Baer ())
Department of Population Ecology,
Zoological Institute,
Universitetsparken 15, 2100 Copenhagen, Denmark
e-mail: [email protected]
Tel.: +45-35321318
Fax: +45-35321250
biology as reported for many non-social insects or
vertebrates (Birkhead and Møller 1998; Simmons 2001).
Social-insect males have been regarded as “simple and
small mating machines” (Tsuji 1996) and have received
far less attention than the highly complex eusocial
colonies produced by their mates. This lack of studies is
somewhat surprising given that social-insect males offer
several characteristics that make them interesting model
organisms to test existing sexual selection theory in
unconventional ways. First, social hymenopteran males
originate from unfertilized eggs and are therefore haploid.
This has two major consequences: (1) males produce
identical clonal sperm so that intra-ejaculatory competition for egg fertilization should be absent; (2) males are
genetically only represented in female offspring but sire
no sons. Consequently, males only transfer genes to the
female (diploid) offspring of their mates and thus prefer a
female (queen)-biased sex ratio in their offspring, whereas queens prefer an equal sex ratio among sexual
offspring (Boomsma 1996). Males therefore have no
interest in female multiple mating —not only because
they have to share fitness among each other. More
importantly, workers change their preferred sex ratio in a
polyandrous colony from a queen-biased sex ratio
towards a male-biased sex ratio. This has substantial
fitness consequences for the fathering males, because they
are not represented in male offspring. Consequently,
males should avoid mating with already-mated females.
Second, social-insect queens generally mate only
during a very short time period early in life. Consequently, males, which are often somatically short lived, have
only a limited time frame available to find females and to
copulate with them.
During recent decades, the theoretical framework of
kin selection theory for understanding social insects
(Hamilton 1964; Trivers and Hare 1976) has been
expanded to include a potential queen-male conflict over
sex ratio and paternity. A series of papers have revealed a
substantial conflict between the sexes over sperm allocation (Boomsma 1996; Boomsma and Ratnieks 1996) and
predicted that males should have evolved mating tactics
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Table 1 The major steps of male reproduction in bumblebees. The
first column (in bold) contains the headings used throughout this
paper and each identified crucial step. The second column gives
Course of action
Subjects discussed
further subtopics addressed in the text, with key references for
bumblebees (third column) and other social insects (fourth column)
Key references for bumblebees
Morphology of male sexual organs Alford (1975)
Development of
testes
Duvoisin et al. (1999)
male sexual
Duvoisin et al. (1999)
organs and sexual accessory testes
accessory glands
Duvoisin et al. (1999); Baer et al.
maturation
(2001)
external genitalia
Bolchi Serini (1993); Alford (1975)
Male maturation
Tasei et al. (1998); Duchateau and
Marien (1995)
Sperm number
Duchateau and Marien (1995)
Male multiple mating
Rseler (1973); Tasei et al. (1998)
Precopulatory
behaviors and
mating flights
Female attraction
Scent marking
Male male competition
Female choice
Copulation I:
Sperm transfer
to the female
Copulation II:
Transfer of male
accessory gland
compounds
Sperm transfer to bursa
copulatrix
Ejaculate size
Sperm viability
Mating-plug transfer
Effects of the mating plug/sign
Gland compounds vs polyandry
Other accessory-gland functions
Mate guarding
and sperm
storage
Duration of copulation
Male mate guarding
Cost of mating
Sperm morphology
Egg fertilization
and sperm
economy
Paternal effects
on colonies
Sperm length vs sperm
competition
Sperm activation and
fertilization
Sperm use
Sperm economy/depletion
Polyandry and colony
performance
Paternal inheritance
Alcock et al. (1978)
Bergman and Bergstrm (1997);
Williams (1991)
Kindl et al. (1999); O’Neill et al.
(1991)
Djegham et al. (1994); Kindl et al.
(1999); Sauter et al. (2001)
Brown et al. (2002); Duvoisin et al.
(1999)
Tasei et al. (1998)
Key references for other social insects
Hlldober and Wilson (1990)
Ball et al. (1983)
Clausen (1938)
Holldober and Bartz (1985); Ruttner
(1985)
Kerr et al. (1962)
Reichhardt and Wheeler (1996);
Danforth (1991)
Hlldobler and Bartz (1985)
Ayasse et al. (2001)
Seeley (1985); Anderson et al. (2003)
Strassmann (2001)
Robertson (1995), Ruttner (1985)
Kerr et al. (1962); Boomsma and
Ratnieks (1996)
Hunter and Birkhead (2002)
Hunter and Birkhead (2002)
Baer et al. (2000)
Monnin and Peeters (1998)
Sauter et al. (2001); Baer et al. (2001) Woyciechowski et al. (1994a, 1994b)
Baer at al. (2001)
Koeniger (1990); Baer and Boomsma
(2003)
Duvoisin et al. (1999)
Ayasse et al. (2001); Allard et al.
(2002)
Brown et al. (2002); Foster (1992)
Koeniger et al. (1979)
Duvoisin et al. (1999); Foster (1992) Yamauchi et al. (2001); Foitzik et al.
(2002)
Brown et al. (2002); Foster (1992)
Baer et al. (2003)
Jamieson et al. (1999); Lino Neto and
Dolder (2002)
Baer et al. (2003)
Lee and Wilkes (1965)
Pabalan et al. (1996)
Pabalan et al. (1996)
Rseler (1973); Paxton et al. (2001);
Estoup et al. (1995)
Rseler (1973)
Baer and Schmid-Hempel (1999a,
1999b); Schmid-Hempel (1998)
Baer and Schmid-Hempel (2003a,
2003b)
Franck et al. (2002); Haberl and Tautz
(1998)
Tschinkel (1987a, 1987b)
Schmid-Hempel (1998); Tarpy (2002)
such as queen monopolization (Tsuji 1996), sperm
clumping (Trivers and Hare 1976; Boomsma 1996) or
reduced sperm transfer as a form of male cooperation
after sperm storage (Boomsma 1996) in order to maximize reproductive success at the expense of the fitness of
their mates. Studies testing these theoretical considerations in the field are still few, but some of them have
found support for these ideas (Sundstrm and Boomsma
2000). Finally, queens typically produce several clutches
of sterile workers after colony initiation, and males
therefore invest spermatozoa into the production of sterile
helper offspring, which can be regarded as a form of
parental investment. The resulting time lag between
Robinson (1992); Page and Robinson
(1991)
colony foundation and sexual reproduction implies that
males need to supply larger numbers of viable spermatozoa to the queen’s sperm-storage organ than are used for
reproduction (Baer and Boomsma 2003). All these factors
are idiosyncratic for social Hymenoptera and suggest that
unconventional selective forces have influenced the
evolution of reproductive male traits through natural
and sexual selection.
Bumblebees are an ideal model system to study socialinsect mating behavior. In contrast to most other social
insects, several species can be bred in the laboratory
(Pomeroy and Plowright 1980; Gretenkord 1997) and
mating behavior can be observed in flight cages, green-
523
houses or sometimes even in the field (Svensson 1979;
Lloyd 1981). Additionally, the development of an artificial insemination technique for bumblebees (Baer and
Schmid-Hempel 1999a, 2000) allows manipulation of the
mating system so that aspects of sexual selection theory
can be experimentally tested. Bumblebees have also a
life-history similar to many other social insects, with
almost all of the approximately 300 known species being
primitively eusocial insects with an annual life-cycle
(Alford 1975; Prys-Jones and Corbet 1987). Queens
emerge from hibernation to found colonies. As soon as
the first workers hatch, the colony enters the eusocial
phase where workers help the queen to establish and
expand the colony. The number of workers increases
during this phase of exponential growth until colonies
switch to the production of sexual offspring. The young
queens and males leave the parental nest and mate outside
with unrelated partners, but only the inseminated queens
hibernate and start the next colony cycle in the following
year. Bumblebees produce male-biased sex ratios (Bourke
1997; Beekman and Van Stratum 1998; Duchateau et al.
2003), suggesting substantial male-male competition for
copulations, because most Bombus species appear to be
monandrous (Estoup et al. 1995; Schmid-Hempel and
Schmid-Hempel 2000). However, multiple queen mating
has been observed in Bombus hypnorum, where queens
mate up to six times (Paxton et al. 2001), as well as in B.
bifarius, B californicus, B. frigidus, B. huntii and B.
rufocinctus (Foster 1992; Crozier and Pamilo 1996).
Unfortunately, genetic data are only available for B.
hypnorum, showing that multiple copulations indeed
result in the presence of several patrilines within a colony
(Estoup et al. 1995; Paxton et al. 2001). Multiple
copulations do not necessarily always lead to multiple
paternities of workers, although they usually do in social
insects (Boomsma and Ratnieks 1996). For example,
queen multiple mating has also been inferred in B.
terrestris (Rseler 1973), but microsatellite analysis has
so far indicated that sperm from only a single male is
present in a queen’s spermatheca (Estoup et al. 1995;
Schmid-Hempel and Schmid-Hempel 2000). Even if
multiple patrilines are present in polyandrous social
insects, paternities may still be biased towards specific
males (Oldroyd et al. 1995; Sundstrom and Boomsma
2000; Fernandez-Escudero et al. 2002). These observations already indicate that sexual selection, sperm competition and cryptic female choice may all be present in
social insects and are worth being studied in more detail.
Various aspects of the mating biology of bumblebees
have been investigated in recent years and their reproductive behavior is one of the best studied among social
insects. In this review, I will follow the male’s pathway
from being born in his maternal colony until he successfully contributes his genes to the next generation,
following the steps presented in Table 1. Although I
mainly address bumblebees, it is obvious that the
dynamics of Table 1 can also be applied to other social
insects. In what follows, I typically start a section by
giving a short introduction summarizing the existing data
and conclusions for bumblebees. I then point out
promising areas of further research and compare the
information available for bumblebees with data for other
social insects and infer whether the obtained patterns and
conclusions are likely to be valid in a more general way.
The male bumblebee life-cycle
Development of male sexual organs
and sexual maturation
Similar to other insects, the sexual organs of social-insect
males have four parts that may be affected by sexual
selection (Fig. 1): (1) testes, (2) accessory testes, (3)
accessory glands, (4) external genitalia, which are often
sclerotized. The testes of social-insect males do not
produce sperm after males reach sexual maturity and
sperm number is therefore fixed in social Hymenoptera
(Ball et al. 1983; Hlldobler and Bartz 1985; Hlldobler
and Wilson 1990; Passera and Keller 1992; Heinze et al.
1998; Simmons 2001; Foitzik et al. 2002), but socialinsect males often possess enough sperm to fully inseminate at least a single female (Kerr et al. 1962; Rseler
1973; Reichardt and Wheeler 1996; Fjerdingstad and
Boomsma 1997; Tasei et al. 1998; Franck et al. 2002).The
only known exception of fixed sperm number at maturity
is the ant Cardiocondyla, where long-lived males have
testes with continuous sperm production (Heinze and
Hlldobler 1993; Heinze et al. 1998).
Fig. 1 Generalized overview of the male’s sexual organs in
bumblebees. Sperm is produced in the testes, early in the life of a
bumblebee male, but production ceases when males become older.
Mature sperm is transferred to and stored in an enlarged part of the
vas seminalis, which is known as the accessory testis. The
accessory glands produce the substances forming the bumblebee
mating plug during copulation. The outer genitalia are involved in
the attachment of the male to the female. See text for a more
detailed discussion of these morphological parts. The diagram was
drawn after Alford (1975), Duvoisin et al. (1999) and Baer and
Schmid-Hempel (2000)
524
The paired accessory testes are part of the vasa
seminales (Fig. 1) and are generally used in social insects
to store mature sperm prior to ejaculation (Ball et al.
1983; Hlldobler and Wilson 1990; Wheeler and
Krutzsch 1992; Tasei et al. 1998). Each accessory testis
is accompanied by an accessory gland that contains large
amounts of gland secretions, which are transferred to the
female during copulation. The importance of these male
accessory-gland compounds are discussed later. Finally,
the sclerotized external genitalia are complex structures,
involved in the attachment of the male to the female
during copulation and the proper transfer of sperm and
accessory-gland products. They are variable between
species and have been used for taxonomic purposes
(Clausen 1938; Hlldobler and Wilson 1990, for bumblebees see Alford 1975; Williams 1985, 1991; Bolchi
Serini 1993) but are obviously also of importance in
influencing male reproductive success.
When bumblebee males eclose within their maternal
colony, they are sexually immature and have large and
active testes (Duchateau and Marien 1995). During
maturation, sperm is transferred to the accessory testes,
which increase in size, whereas the testes become smaller
and inactive (Duchateau and Marien 1995). Bumblebee
males eclose with a fixed amount of sperm but they store
enough sperm in their accessory testes to fully inseminate
more than one female, and male bumblebees of B.
terrestris indeed mate up to eight times when given the
opportunity (Rseler 1973; Tasei et al. 1998). A mature
male stores around 500,000–600,000 sperm in the spermstorage organs (Duchateau and Marien 1995), which is
about an order of magnitude more than the 40,000–50,000
sperm found in the spermathecae of a typical inseminated
queen (Rseler 1973). Males mating more than once
transfer enough sperm to the bursa copulatrix to entirely
fill the queen’s spermatheca (Tasei et al. 1998) at least for
the first three matings, and sperm numbers found in the
spermathecae of the subsequently mated queens do not
decrease with additional male copulations. Unfortunately,
we do not know whether males copulating more than
three times still inseminate a female sufficiently, although
this seems possible. Only a small fraction (11%) of all
males observed actually copulated more than three times
in the laboratory study of Tasei et al. (1998). In the
neotropical bumblebee, B. atratus, males also mate
multiply and transfer less spermatozoa when remating
(Garofalo et al. 1986). Additionally, larger B. atratus
males have more sperm and also transfer more sperm
during copulation than smaller males.
Males start to copulate from an age of 16€7
(mean€SD) days after eclosion in B. terrestris (Duchateau
and Marien 1995), and slightly earlier in a second study of
the same species, 12€1.3 days (Tasei et al. 1998). This
post-pupal maturation time of males is longer than for
females (6.1€0.4 days) (Gretenkord 1997; Tasei et al.
1998), which indicates that colonies might face substantial costs for male maturation, especially since males
consume large amounts of pollen shortly after eclosion
(B. Baer, unpublished data). However, queens are gener-
ally assumed to be more costly to produce because they
need increased larval feeding (Ribeiro 1999; Ribeiro et al.
1999) and have a longer larval developmental time
(26 days for males vs 30 days for queens (Duchateau and
Velthuis 1988). Additionally, queens are about twice the
biomass of males (Duchateau and Velthuis 1988; Beekman and Van Stratum 1998; Duchateau et al. 2003) and
need more energy (pollen and nectar) investment after
emergence (Beekman and Van Stratum 1998). We lack an
overall body-size-corrected estimate of the reproductive
value of bumblebee males compared to queens, and need
comparative studies of reproductive investment between
species.
The external genitalia act as a strong forceps in B.
terrestris, attaching to the queen’s sting apparatus and
establishing a firm connection of the mating partners
(Williams 1985; Duvoisin et al. 1999). If mating pairs are
separated by force, the male’s sexual organs often remain
attached to the queen and are torn out of the male’s
abdomen (B. Baer, unpublished data). It seems unlikely
that queens can remove males in copula, so males most
probably control copulation duration. Male replacement
by another male thus seems unlikely and has never been
reported, although competing males have been observed
to harass a copulating pair (Duvoisin et al. 1999). The
male’s external genitalia may be important tools in
controlling not only copulation duration but also insemination success. For example, the outer genitalia form a
mating sign in some species such as stingless bees (Da
Silva et al. 1972; van Veen and Sommeijer 2000), and
may well prevent queen remating in most species. An
intensively studied case is the honeybee Apis mellifera,
where the mating sign does not prevent further copulations because following drones can remove the sign of the
previous male. The honeybee mating sign seems to have
at least two different effects. It avoids sperm leakage out
of the female (Woyciechowski et al. 1994a) and it seems
to promote multiple mating by attracting other drones
(Koeniger 1990). The latter scenario is difficult to
understand from an evolutionary viewpoint, especially
since a drone produces enough sperm to fully inseminate
a female and faces extreme male-male competition
(Seeley 1985). A possible explanation for such postmortem cooperation by males might be that males benefit
from female multiple mating in similar ways to those
suggested for females (Page 1986; Sherman et al. 1988;
Arnquist and Nilsson 2000; Jennions and Petrie 2000).
Indeed, monandrous honeybee queens have lower overall
colony performance due to decreased comb building,
honey and pollen storage and brood rearing (Oldroyd et
al. 1992; Fuchs and Schade 1994), and have reduced
parasite loads compared to polyandrous queens (Tarpy
2002, but also see Woyciechowski et al. 1994b and
Neumann and Moritz 2000).
Studies on the detailed functions of the external
genitalia are generally scarce in social insects. As these
rather anecdotal observations indicate, such studies might
be rewarding when studying sexual selection, as well as
525
the evolution of different mating systems within and
among social insects.
Precopulatory behaviors and mating flights
Precopulatory male behaviors have been described in
detail for many species, and they influence paternity in a
substantial way. The difficulty in observing mating
behavior in many social-insect species means that far
less information about these behaviors and their consequences for paternity success is available compared to
non-social insects or vertebrates.
In bumblebees, males perform a number of precopulatory sexual behaviors to find and attract females, which
have already been observed and described by Charles
Darwin (Freeman 1968). There are numerous studies
describing these behaviors for different bumblebee species (Haas 1949, 1976; Awram 1970; Schremmer 1972;
Svensson 1979; Alcock and Alcock 1983; O’Neill et al.
1991; Williams 1991; Bergman and Bergstrm 1997;
Kindl et al. 1999) or reviewing them together with similar
behaviors in other bees and wasps (Alcock et al. 1978).
Male precopulatory behaviors include searching, guarding
and patrolling of conspecific nest entrances, as well as
perching behaviors, where males wait at specific spots for
females to pass by. Several bumblebee species also scentmark objects with pheromones and patrol them later on. A
detailed study on 11 bumblebee species in Sweden
(Svensson 1979) reported substantial differences in male
precopulatory behaviors between species. Scent marks
seem important in guiding the male as he patrols his
range, but might also be involved as sex pheromones in
female attraction, species recognition and mate choice
(see Ayasse et al. 2001 for an extended review of known
sex pheromones in social insects). The chemical substances involved in scent-marking originate from the
labial gland and have been identified for several bumblebee species (O’Neill et al. 1991; Bergman and
Bergstrm 1997; Hovorka et al. 1998; Kindl et al.
1999). Bumblebees have also been found to be territorial,
aggressively excluding competing males (O’Neill et al.
1991; Kindl et al. 1999) or even fighting with them
(Williams 1991).
Male precopulatory behaviors may be very important
in influencing mating success, because female bumblebees are choosy and reject many males in the laboratory
(Djegham et al. 1994; Duvoisin et al. 1999), as well as in
the field (Kindl et al. 1999), and sometimes even sting
overly persistent males to death (Duvoisin et al. 1999).
Despite this large body of literature available for bumblebee pre-copulatory sexual behavior, we lack any
information on how these differences in male behavior
and physiology (e.g. body size, pheromone quality and
quantity) actually translate into insemination and paternity success. Further research is needed here.
Mating experiments in the laboratory have generally
neglected the above-mentioned effects of male precopulatory behavior on female mate choice because males do
not establish scent marks and patrolling behavior in flight
cages (Sauter and Brown 2001) or wooden observation
cages (Djegham et al. 1994). In the study of Sauter and
Brown (2001), queen mating status (i.e. whether she had
mated before or not), rather than male precopulatory
behaviors, determined the occurrence of copulations, but
the experimental setup might have been all too artificial to
actually see the natural dynamics of mate choice.
Precopulatory behaviors of males have also been
described in other social insects (Gries and Koeniger
1996; van Veen and Sommeijer 2000; reviewed by
Alcock et al. 1978; Hlldobler and Bartz 1985; Hlldobler and Wilson 1990; Danforth 1991) and in nonsocial hymenopterans (Alcock 1993, 2000; Van Den
Assem and Werren 1994; Willmer et al. 1994; Strohm and
Lechner 2000). Reproductive biology varies greatly
between species and results in a broad variety of male
precopulatory behaviors but, as with bumblebees, we lack
information about the evolutionary consequences of these
behaviors for reproductive success. Interesting exceptions
are species where males are sexually dimorphic and
mating flights are partially lost. This is the case in several
halictine and andrenid bees (Kukuk and Schwarz 1988;
Danforth 1991) and some ant species of Hypoponera
(Yamauchi et al 2001; Foitzik et al. 2002), Technomyrmex
(Tsuji and Yamauchi 1994) and Cardiocondyla (Heinze
and Hlldobler 1993). Although both morphs, winged
dispersing males and wingless fighter males, may coexist
in a colony of Cardiocondyla obscurior, male dimorphism is induced by environmental stress (temperature,
density), which might be of adaptive significance (Cremer
and Heinze 2003). The two male morphs differ in various
characteristics from each other (Cremer et al. 2002a).
Compared to the winged dispersing males, wingless males
stay in their maternal nest, are larger and less pigmented,
with small eyes and large sabre-shaped mandibles to fight
with each other, and have permanent spermatogenesis.
The winged males avoid aggression by their ergatoid
brothers by mimicking the chemical bouquet of virgin
queens (Cremer et al. 2002b). Sexual selection is likely to
have shaped these fundamental differences between male
morphs, and it is interesting to see how related males
(brothers) can develop into completely different morphological morphs in a common environment (colony).
Cardiocondyla ants are undoubtedly a promising study
system for further work on male sexual selection investigating the evolution and the maintenance of sexual
dimorphism, which is not only of interest for social-insect
researchers.
Copulation I: sperm transfer to the female
Sperm transfer from the male to the female is a crucial
part of the copulation process and should be especially
interesting to study in sperm-limited species such as
social insects. This is because males may transfer as few
sperm as possible for storage in a focal female to avoid
sperm depletion in future matings while simultaneously
526
ensuring that each queen gets sufficient sperm to produce
a colony. Male social insects typically do not place their
sperm directly into the female’s spermatheca, indicating
that the female may influence the sperm-storage process,
for example, through cryptic female choice (Eberhard
1996). The only known exception to this rule is A. florea,
where males transfer sperm directly to the spermatheca of
the queen (Koeniger et al. 1989). Microsatellite marker
studies in this species showed that queens mated with
more males than originally estimated by simply counting
sperm numbers within the female spermathecae (Oldroyd
et al. 1995) and comparing them with sperm numbers in
males. This suggests that males transfer either only a
sperm fraction (as predicted by Boomsma 1996) or that
females expel some sperm after mating, as in other Apis
species (reviewed in Boomsma and Ratnieks 1996).
Further work on the mating strategies of A. florea males
and females is needed.
In the bumblebees, B. terrestris, B. lucorum and B.
hypnorum, sperm is transferred to the queen’s bursa
copulatrix (Duvoisin et al. 1999) starting immediately
after the onset of copulation and lasting for only 2–5 min.
This is much shorter than the average total copulation
time of around 40 min in B. terrestris (Duvoisin et al.
1999) and around 30 min in B. hypnorum (Brown et al.
2002). A male’s endophallus places the sperm in the
lateral part of the bursa copulatrix, close to the opening of
the spermathecal duct (Duvoisin et al. 1999). As in
honeybees, males transfer more sperm to the bursa
copulatrix than will be stored in the spermatheca
(Duvoisin et al. 1999). Additionally, Rseler (1973)
found that queens store far more sperm in the spermatheca than they need to complete their colony cycle, since
the number of spermatozoa within the spermatheca did
not measurably differ between queens before and after a
colony cycle. Furthermore, females do not need a fully
inseminated spermatheca because artificially inseminated
queens with only partially filled spermathecas are also
able to complete the colony cycle (Baer and SchmidHempel 2000). If females use only a small fraction of the
spermatozoa, it remains unclear why males should deplete
their sperm stores faster by transferring more sperm than
is needed. One possible reason for increased ejaculate size
might be sperm quality rather then sperm quantity, i.e.
sperm must be successfully stored and be capable of egg
fertilization. An important factor influencing ejaculate
quality is sperm viability, i.e. the percentage of sperm that
is alive within an ejaculate. In a recent study, Hunter and
Birkhead (2002) performed pair-wise comparisons of
sperm viability between seven mono- and polyandrous
insect species. Two comparisons included social-insect
species: one between the monandrous bumblebee B.
terrestris and the polyandrous honeybee A. mellifera.
Sperm viability was generally higher in polyandrous
species than in monandrous species and was generally
high, with >95% of all sperm being alive in B. terrestris
and A. mellifera. Sperm viability seems therefore not
responsible for large ejaculation sizes in B. terrestris, at
least not for the sperm-transfer process. More information
about differences in sperm viability during the queen’s
lifetime would be very interesting for further insights into
why sperm-limited males transfer more sperm to the
female than they seem to need.
Sperm transfer has also been studied in the honeybee
A. mellifera, where copulations take only a few seconds
(Garry and Marston 1971; Koeniger et al. 1979). Male
honeybees (drones) are suicidal maters and die directly
after copulation. Unsurprisingly, they transfer all their
sperm to the female’s median and lateral oviducts, which
become large sperm reservoirs as queens remate frequently during single nuptial flights. A single male
possesses enough spermatozoa (6–12 million) to fill a
spermatheca (5.5 million sperm) (Mackensen and Roberts
1947; Kerr et al. 1962; Ruttner 1976, 1985; Franck et al.
2002), indicating that females are not polyandrous
because of sperm limitation although this has been
recently questioned (Moritz and Schlns 2002). Sperm
is transferred to the spermatheca afterwards over a time
period of about 90 h while 95% of the sperm accumulated
during the nuptial flight is expelled (Woyke 1983).
During this process, females may be able to exert
postcopulatory selection on the ejaculates (cryptic female
choice). This idea is supported by the fact that spermatozoa are transferred to the spermatheca, not only by
active migration, but also passively by muscular contractions of the spermathecal duct (Ruttner and Koeniger
1971; Gessner and Ruttner 1977). When arriving at the
spermatheca, high concentrations of Na+ and K+ ions
seem to deactivate and slow down respiration of sperm,
which might be an adaptive mechanism allowing the long
sperm survival in honeybees (Verma 1973). Interestingly,
honeybees do not have last male precedence, i.e. the last
male to copulate with a queen does not sire more
offspring than his competitors (Franck et al. 2002),
although last male precedence is widespread among nonsocial insects (Simmons 2001).
An interesting variation of male control over the
copulation process is found in the ants Hypoponera
opacior (Foitzik et al. 2002) and H. nubatama (Yamauchi
et al. 2001) where a wingless male morph mates with
females while they are still in their pupal cocoon. In H.
opacior, sperm transfer takes place at the start of the
copulation (Foitzik et al. 2002) but males remain in
copula for up to 41 h, suggesting that males guard their
mates. In H. nubatama, wingless males copulate for up to
125 min and longer when rival males are present, adding
further support to the mate-guarding hypothesis (Yamauchi et al. 2001). Further work will hopefully reveal
whether these males have complete control over the
insemination process or whether females have evolved
counter-adaptations to avoid male monopolization.
Copulation II: transfer of male
accessory-gland compounds
Accessory glands are widespread among insects (Gillott
1996, 2003; Simmons 2001) and their compounds are
527
known to influence male mating success in many different
and sophisticated ways (see Table 4.1 in Simmons 2001;
Gillott 2003). Accessory glands are also present in social
insects (Hlldobler and Wilson 1990) but our knowledge
of the biochemistry and function of their compounds is
very sparse, which is surprising given how much attention
male accessory compounds have received in non-social
insects. Accessory-gland compounds of social insects
might indeed be of special interest to study because they
might have evolved rather differently than in non-social
insects (see also Baer and Boomsma 2003). This is
because accessory-gland compounds of social insects are
not expected to become agents of chemical warfare
between the sexes. The life-long storage of sperm without
queen remating, as well as the delay in the production of
sexual offspring after sterile worker brood should prevent
the evolution of most harmful traits of accessory-gland
fluids (Baer and Boomsma 2003). Such harmful traits
have been found in many non-social insects, where they
induce excess oviposition/oogenesis or even act as toxins
and reduce female survival (Chen et al. 1988; Gillott
1996; Simmons 2001). We therefore need studies on
accessory glands of social insects, which should be easy
to conduct, given that the necessary techniques are
already established.
Male bumblebees transfer large amounts of accessorygland material into the bursa copulatrix of the queen
during copulation (Duvoisin et al. 1999). The transfer of
these products is known from B. terrestris, B. hypnorum
and B. lucorum, and takes place shortly after sperm
transfer. These substances are not necessary for a
successful insemination since artificially inseminated
females, which did not receive any gland material, were
also able to store sperm and to complete their colony
cycle (Baer and Schmid-Hempel 2000). In B. terrestris,
the transfer of gland compounds is rapid and therefore
does not explain the long copulation duration found in
this species (Duvoisin et al. 1999). The transferred
secretions form a gelatinous mass within the female’s
bursa copulatrix, known as the “mating plug”, although
recent research has shown that the main purpose of the
plug does not guarantee that the female’s reproductive
tract is sealed off. In a rare observation of a double mating
in B. terrestris, a remating male was able to push the first
plug upwards into the oviducts, and successfully placed
his sperm and plug into the bursa copulatrix (Sauter et al.
2001). Rather than physically preventing the queen from
remating, the mating plug influences female mating
behavior by reducing her willingness to remate. When
mating plugs are artificially transferred, females reject
further copulations in spite of not receiving any sperm
(Sauter et al. 2001). The mating plug of B. terrestris
contains four fatty acids (linoleic, stearic, palmitic and
oleic acids) and a small protein (cyclo-prolylproline)
(Baer et al. 2000). Only one of these substances (linoleic
acid) has a direct effect on the female by reducing her
copulation acceptance (Baer et al. 2001). That the active
substance is a common fatty acid and not a protein is
surprising, because it is proteins that are normally
involved in producing similar effects in non-social insects
(Simmons 2001; Gillott 2003). Consequently, males of B.
terrestris seem to control both copulation duration and
queen mating frequency. This might explain why females
of B. terrestris are monandrous even though they
demonstrably benefit from increased genetic heterogeneity among their worker offspring after artificial multiple
inseminations (Baer and Schmid-Hempel 1999b, 2001,
2003a). Males of B. hypnorum also transfer a mating plug
(Brown et al. 2002), but the plug disappears much faster
than in B. terrestris. Whereas the mating plug in B.
hypnorum disappears between 6 and 12 h post copulation,
it is present for up to 36 h in B. terrestris (Brown et al.
2002). The fact that B. hypnorum queens are polyandrous
is consistent with mating plugs being less efficient in B.
hypnorum than in B. terrestris, for example, because
females evolved to overcome male control via plug
compounds. If male accessory-gland secretions become
ineffective tools to control female mating frequency,
males might reduce their investment in these secretions
and increase investment in sperm production. We do not
know whether this is true for polyandrous bumblebees but
this has recently been shown in fungus-growing ants
where males of polyandrous species had smaller accessory glands and larger accessory testes compared to
monandrous species (Baer and Boomsma 2003).
Further comparative work on the biochemistry of
mating plugs is still needed to understand male accessorygland compounds in similar detail as have been obtained
for dipterans, lepidopterans or coleopterans (see Gillott
2003 for an extended review). For example, we still lack
information about the presence of larger proteins within
the bumblebee mating plug. Since many proteins are
present in the male accessory compounds of non-social
insects (Gillott 2003) and probably also in social insects
(Wheeler and Krutzsch 1992), it seems possible that the
male accessory-gland proteins of bumblebees influence
not only sperm competition, but also aspects such as
female fecundity, female behavior, ovulation/oviposition,
sperm storage and sperm protection. Further research in
this area would seem relatively easy to conduct.
Apart from B. terrestris, no information about the
chemistry of accessory-gland substances of social insects
is available so far. However, several studies do report the
transfer of accessory-gland substances from the male to
the female during copulation. Mating plugs have been
found in the ant Dinoponera quadriceps (Monnin and
Peeters 1998) and in the solitary bee Osmia rufa
(Seidelmann 1995). In honeybees, males transfer large
amounts of mucus to the female as part of the mating sign
(Ruttner 1985). Accessory-gland compounds produce
spermatophores in the ants Carebara vidua (Robertson
1995) and Diacamma sp. (Allard et al. 2002). These data
indicate that the transfer of chemical compounds may be
widespread among social insects and partly used for
similar reasons as in non-social insects (Simmons 2001).
Further data on the function and chemistry of male
accessory-gland compounds in social insects are likely to
528
provide intriguing insights into the evolution of sexual
conflicts over paternity.
Mate guarding and sperm storage
In B. terrestris, sperm are transferred from the queen’s
bursa copulatrix to the spermatheca within 30–80 min
after the onset of the copulation (Duvoisin et al. 1999).
The termination of an average copulation after 37 min
corresponds to the time when approximately half of the
spermatheca is filled with sperm. This long copulation
duration implies that males guard the queen to get at least
part of their sperm successfully transferred to the queen’s
spermatheca. Male mate guarding was also reported for B.
bifarius and B. californicus (Foster 1992). However, mate
guarding is likely to increase mating costs since copulating pairs are immobile and more vulnerable to predation.
A recent study on the multiple-mated species B. hypnorum indirectly supports the idea that copulations are
costly and that females take such costs into account when
deciding whether to remate (Brown et al. 2002). Monoand polyandrous queens both become successfully inseminated because they have fully inseminated spermathecas, but the likelihood of queen remating depends on the
duration of the first copulation. As the duration of the first
copulation decreases, the likelihood of a second copulation increases. Singly and doubly mated queens do not
differ in their overall time spent in copula but we lack
overall copulation duration measurements of males,
which might control mating duration (see above). Such
data should be easy to obtain and might indicate whether
males have different mating tactics (i.e. short vs long
copulators), and whether other factors influence their
decisions for specific copulation duration.
Another important aspect of sperm storage and sperm
use is sperm morphology. One would expect that the
limited space available in the spermatheca of a female,
together with the high sperm demands (large numbers of
spermatozoa which are viable over long periods of time),
lead to the production of numerous small sperm. However, sperm competition theory suggests that sperm could
evolve to be longer in order to swim faster, and reach
sperm-storage sites or eggs earlier. Indeed, sperm size
was found to be important in obtaining higher fertilization
success in dung flies (Otronen et al. 1997), bulb mites
(Radwan 1996) or the nematode Caenorhabditis elegans
(Lamunyon and Ward 1998), and sperm length correlated
with female multiple mating in comparative studies of
insects (Gage 1994; Morrow and Gage 2000) and
vertebrates (Gomendio and Roldan 1991; Briskie et al.
1997), but see Stockley et al. (1997), Hosken (1997) and
Gage and Freckleton (2003).
In bumblebees, sperm length has been investigated in
B. terrestris, B. lucorum and B. hypnorum (Baer et al.
2003). Sperm cells look very similar in all three species,
being elongated, with the sperm head hardly distinguishable from the tail. The multiply mated B. hypnorum has
longer sperm than the two singly mated species as
predicted by sperm competition theory but, since the
analysis was only based on three species, no general
conclusions can be drawn. More interestingly, males were
found to produce highly variable sperm lengths, which
differed between males of the same colony (brothers),
between colonies of the same species and also between
species. Sperm length is also highly variable in non-social
insects and vertebrates (Ward and Hauschteck Jungen
1993; Ward 1998; Morrow and Gage 2001a, 2001b)
although the adaptive significance of this variation is
poorly understood in these species as well.
Most of the detected variation in sperm length of
bumblebees was found within ejaculates of single males,
which is surprising given that haploid males produce
clonal sperm. The variation in (clonal) sperm length in
bumblebees is similar to that found in (non-clonal) nonsocial insects. Observations of sperm transfer in B.
terrestris indicate that this variation might have some
adaptive function (Baer et al. 2003) albeit in a rather
complex way. Longer sperm are not generally more
successful in becoming stored in the spermatheca.
Instead, both shorter and longer sperm fractions may be
preferentially stored in the spermatheca, depending on the
colony of origin of the focal male and female. Consequently, a male might be selected for increased spermlength variation to meet variable female demands. This
idea seems testable and might also add interesting new
aspects to the discussion of intraspecific sperm-length
variation in non-social insects, even though lifetime
storage of single ejaculates is possibly rare.
It is unknown whether bumblebee spermatozoa swim
actively up the spermathecal duct, whether queens
influence the sperm-storage process or whether it is a
combination of both, as suggested in honeybees (Gessner
1973; Gessner and Ruttner 1977). Again, bumblebees
seem a promising study organism to investigate the
sperm-storage process in further detail because copulations can be initiated and interrupted at any point, and
artificial insemination techniques allow the process to
take place under fully controlled conditions, so that, for
example, sperm viability can be experimentally manipulated.
Sperm morphology is variable among social insects
and this variation has mainly been used for taxonomic
purposes (Jamieson 1987; Jamieson et al. 1999; Newman
and Quicke 2000; Lino Neto and Dolder 2002). In the
wasp Dahlbominus fusciupennis, males produce five
different sperm morphs (Lee and Wilkes 1965). Some
of these morphs have a higher probability of being stored
in the spermatheca than others, indicating that the
different morphs are produced for different purposes
connected to sperm competition (Lee and Wilkes 1965).
Although details of sperm morphology are known for
various species, there are no data on the adaptive
significance of differences in sperm morphology. It is
obvious that further research on the sperm-storage process
is needed in order to understand the evolution of different
sperm morphs and the significance of sperm length for
male mating success.
529
Egg fertilization and sperm economy
The fertilization process is the last step in a male’s
pathway to contribute his genes to the next generation.
Detailed studies are lacking on the processes occurring
between the release and activation of sperm from the
spermatheca, egg penetration and fertilization. Spermathecae of social insects have accessory glands close to the
opening into the spermathecal duct (Pabalan et al. 1996;
Duvoisin et al. 1999), which are probably involved in the
de- or reactivation of sperm. Since the majority of social
insects seem to have average female-mating frequencies
close to 1 (Boomsma and Ratnieks 1996; Strassmann
2001), stored sperm often belongs to a single clonal
ejaculate so that no sperm competition is expected. This
changes in multiply mated species where the ejaculates of
several males are present within the spermatheca of a
single female. Several studies have investigated whether
sperm is randomly used in polyandrous species, or have
tried to detect possible effects of sperm competition or
cryptic female choice, but no clear-cut pattern of sperm
use was found (Keller et al. 1997; Haberl and Tautz 1998;
Fernandez-Escudero et al. 2002; Franck et al. 2002).
Obviously, more information on how biased paternities
arise and how they influence male fitness would be
beneficial in understanding sperm competition and female
influence on paternity distributions.
Social-insect queens are among the most efficient
species in terms of sperm economy. As mentioned earlier,
queens only mate during one short mating event and
afterwards keep spermatozoa alive during their entire
reproductive lives. The physiological and/or morphological pathways by which this is achieved are unknown and
need to be studied in more detail. The need for
economical sperm use might be less pronounced in
bumblebees because they do not produce large colonies,
but it becomes very important in other social-insect
species with colony sizes of several million workers, for
example, army ants and leaf-cutting ants (Wilson 1971;
Weber 1972; Tschinkel 1987b; Hlldobler and Wilson
1990). The latter case is of species interest because Atta
leaf-cutting ants are monogynous and have no queen
replacement. Nevertheless, these queens are able to
produce colonies of up to 5 million workers and are
capable of maintaining these colony sizes for up to
several decades (Weber 1972). It seems obvious that this
demand on economic sperm use is likely to have induced
special selective forces on both males and females,
especially since sperm depletion is not in the interest of
either sex. More detailed studies on egg fertilization or
sperm survival/viability over time seem practical with the
current techniques available and further insights will be
fascinating. Finally, towards the end of their lives, socialinsect queens always run the risk of sperm depletion, as
has been shown in fire ants (Tschinkel 1987a). This
should result in selection on increased sperm viability and
optimized sperm use. The large variation in colony size
and the demand for sperm among social-insect queens
offer comparative possibilities to study sperm use in more
detail.
Bumblebee queens are able to use sperm in a highly
economical way since queens still have a lot of sperm left
after a successful colony cycle (Rseler 1973). This
indicates that only a few spermatozoa are actually used
per egg, a fact that has also been reported in other social
insects, such as the fire ant Solenopsis invicta (3.5 sperm
per egg) and the honeybee (4–12 sperm per egg)
(Tschinkel 1987b; Yu and Omholt 1999).
Paternal effects on colonies
Social-insect males survive throughout the colony cycle
by their sperm stored within the spermatheca of the
queen(s) they mated with, in some cases for up to several
decades (Pamilo 1991; Schmid-Hempel 1998). Direct
paternal effects such as brood care or nuptial gifts seem to
be absent in social insects, but males can have genetic
effects on worker offspring, and thereby influence colony
performance and fitness. Queens of B. terrestris would
benefit from polyandry, because the genetic heterogeneity
among worker offspring reduces parasitism and increases
fitness (Shykoff and Schmid Hempel 1991; Liersch and
Schmid Hempel 1998; Baer and Schmid-Hempel 1999b).
Beneficial effects of polyandry were also reported in the
ant Pogonomyrmex occidentalis (Cole and Wiernasz
1999). But, as we have seen, male bumblebees seem to
effectively monopolize paternity. Since males from
different colonies differ in parasite susceptibility (Baer
and Schmid-Hempel 2003a), polyandrous queens would
increase their chances of mating with a male of superior
resistance, which would boost worker resistance, and
increases colony size and colony fitness (Baer and
Schmid-Hempel 2003b). However, in a follow-up experiment, Baer and Schmid-Hempel (2001) also showed that
multiple mating might induce a cost to the queen, because
bumblebee queens that mated only with a few males faced
a substantial decrease in fitness, so that only high queen
mating frequencies (>four matings) compensated for this
cost. Although the exact reason for this cost is still
unknown. it seems possible that this might be paternally
influenced, for example, by conflicts arising between
worker lineages of different patrilines.
Paternal effects are also present in other species
(Robinson 1992) such as honeybees, where patrilines
differ in cleaning behavior (Arathi and Spivak 2001), nest
defence (Degrandi Hoffman et al. 1998) or learning
behavior (Bhagavan et al. 1994). Honeybee queens have
been hypothesized to exploit paternal differences by being
polyandrous, and so achieving a more efficient division of
labor within their colonies (Page et al. 1995). This idea
has received empirical support since, in colonies headed
by artificially inseminated queens, patrilines were represented in different frequencies in specific behavioral
castes (Page and Robinson 1991). However, paternal
effects have not been intensively studied in social insects
and further work, especially in the other highly polyan-
530
drous species, seems necessary for a more general
understanding of the effects males have on their colonies.
Conclusions
The mating biology of social-insect males is obviously
complex. Our present knowledge indicates that males
have the opportunity to substantially manipulate final
patterns of paternity and even colony reproduction, but
the mechanisms by which this is achieved are largely
unknown. On the one hand, social-insect males have a
number of similar reproductive behaviors and traits, as do
males of non-social insects or vertebrates. However,
sperm limitation and a very short somatic life have
probably induced a suite of traits that are highly
idiosyncratic. This mixture offers the possibility of
studying aspects of reproductive conflicts between the
sexes in both conventional and unconventional settings
where, in particular, the latter could provide interesting
challenges to existing sexual selection theory. The large
variation in the mating biology among social insects, even
within a genus such as Bombus, suggests fast evolution of
these male traits, which opens up various ways of
studying the underlying evolutionary mechanisms that
have shaped them.
We already have a fairly detailed knowledge of the
mating behavior of male bumblebees, but basic data on
fundamental aspects of bumblebee reproduction are still
sparse. As a model system, bumblebees offer several
advantages (e.g. laboratory rearing, matings in the
laboratory, artificial insemination), which make them
highly suitable to be used for pioneering work in this
field. Future research will not only add further insights
into the reproductive biology of Bombus species, but will
also allow more general hypotheses for other social
insects, and possibly even non-social insects, to be
formulated.
Acknowledgements Special thanks are due to B. Baer-Imhoof, S.
Cremer, B. Hughes, and especially J. Boomsma for help and
comments on the manuscript. This work was supported by a Marie
Curie Fellowship (EU-062441) from the Swiss National Science
Foundation.
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