Bumblebee workers from different sire groups vary in susceptibility to parasite infection

Abstract Female multiple mating with different males (polyandry) can be advantageous because the resulting genetic heterogeneity among offspring reduces the effects of parasitism. However, the underlying assumption that offspring fathered by different males vary in their susceptibility to parasites is so far only supported indirectly. Here we tested this crucial assumption using data from a study on the bumblebee Bombus terrestris L. with queens inseminated with sperm of either one or several males that originated from different sire groups (i.e. groups of brothers). We found that, under field conditions, workers from different sire groups, forming a patriline within a given colony, indeed differ in their susceptibility to the common intestinal parasite, Crithidia bombi, and do so independently of queen mating frequency.     

Male parentage and queen mating frequency in the bumblebee <Emphasis Type="Italic">Bombus ignitus</Emphasis> (Hymenoptera: bombinae)

Kin selection theory predicts conflict between queens and workers in the social insect colony with respect to male production. This conflict arises from the haplodiploid system of sex determination in Hymenoptera that creates relatedness asymmetries in which workers are more closely related to the sons of other workers than to those of the queen. In annual hymenopteran societies that are headed by a single queen, the mating frequency of the queen is the only factor that affects the colony kin structure. Therefore, we examined the mating structure of queens and the parentage of males in a monogynous bumblebee, Bombus ignitus, using DNA microsatellites. In the seven colonies that were studied, B. ignitus queens mated once, thereby leading to the prediction of conflict between the queen and workers regarding male production. In each of the five queen-right colonies, the majority of the males (95%) were produced by the colony’s queen. In contrast, workers produced approximately 47% of all the males in two queenless colonies. These results suggest that male production in B. ignitus is a conflict between queen and workers.     

Sperm reduces female longevity and increases melanization of the spermatheca in the bumblebee <Emphasis Type="Italic">Bombus terrestris</Emphasis> L.

Here we present evidence that the male mating products (sperm and gland products) reduce survival during hibernation of queens of the bumblebee B. terrestris. Most remarkably, the inseminated queens are significantly more likely to have melanized spermathecae than their virgin sisters. Although we could not detect a direct relationship between these two findings they are quite remarkable since B. terrestris is a monandrous and comparably long-lived insect where sexual conflict is unlikely to evolve. The reduced survival can probably be attributed to a general cost of maintaining the sperm, whereas the presence of melanized spermathecae in the inseminated queens may indicate a pathogen transferred during mating or genetic incompatibilities between males and queens. Received 30 December 2007; revised 27 April 2008; accepted 1 May 2008.     

Diploid male production in a rare and locally distributed bumblebee, Bombus florilegus (Hymenoptera, Apidae)

The European bumblebee B. terrestris was recently introduced in Japan for agricultural purposes and has now become naturalized. The naturalization of this exotic species may have great detrimental effects on closely related native Japanese bumblebees. The Japanese bumblebee Bombus florilegus is a rare and locally distributed species found in the Nemuro Peninsula of Hokkaido, Japan. In order to assess its population genetics, we estimated the genetic structure of B. florilegus in 16 breeding colonies (queen, workers, and males) and 20 foraging queens by analyzing microsatellite DNA markers. Of the 36 queens analyzed by genotyping and dissection, 32 had been inseminated by a male. The remaining 4 had not been inseminated at all. Of the 4 nonmated queens, one was triploid. Diploid males were found in 4 breeding colonies. Based on the microsatellite data, it appears that B. florilegus has low reproductive success. Since matched mating and nonmating within local populations are high, the extinction risk is correspondingly higher. Our results suggest that conservation of the Japanese B. florilegus is required in order to protect it from both habitat destruction and the naturalization of alien species. Received 19 July 2007; revised 13 October 2007; accepted 15 October 2007.     

No evidence that reproductive bumblebee workers reduce the production of new queens

Kin selection theory predicts potential conflict between queen and workers over male parentage in hymenopteran societies headed by one, singly mated queen, because each party is more closely related to its own male offspring. In ‘late-switching’ colonies of the bumblebee Bombus terrestris, i.e. colonies whose queens lay haploid eggs relatively late in the colony cycle, workers start to lay male eggs shortly after the queen lays the female eggs that will develop into new queens. It has been hypothesized that this occurs because workers recognize, via a signal given by the queen instructing female larvae to commence development as queens, that egg laying is now in their kin-selected interest. This hypothesis assumes that aggressive behaviour in egg-laying workers does not substantially reduce the production of new queens, which would decrease the workers' fitness payoff from producing males. We tested the hypothesis that reproductive activity inB. terrestris workers does not reduce the production of new queens. We used microsatellite genotyping to sex eggs and hence to select eight size-matched pairs of ‘late-switching’ colonies from a set of commercial colonies. From one colony of each pair we removed every egg-laying or aggressive worker observed. From the other colony, we simultaneously removed a nonegg-laying, nonaggressive worker. Removed workers were replaced with young workers from separate colonies at equal frequencies within the pair. There was no significant difference in queen productivity between colonies with reduced or normal levels of egg-laying or aggressive workers. Therefore, as predicted, reproductive B. terrestris workers did not significantly reduce the production of new queens.     

Displacement of Japanese native bumblebees by the recently introduced <Emphasis Type="Italic">Bombus terrestris</Emphasis> (L.) (Hymenoptera: Apidae)

The introduced Bombus terrestris has recently been naturalized in Japan and become dominant in some local communities. We investigated potential niche overlaps between introduced and native bumblebees in terms of morphological characteristics, seasonal flight activity, foraging and nesting habitat use, and plant species visited. There were considerable niche overlaps in flower resource use between B. terrestris and B. hypocrita sapporoensis/B. pseudobaicalensis. Bombus terrestris also potentially competes for nest sites with B. hypocrita sapporoensis. During 3-year monitoring, B. pseudobaicalensis showed no noticeable change, but B. hypocrita sapporoensis decreased while B. terrestris increased. Abundant flower resources provided by exotic plants may buffer native bumblebees from competition for food with introduced species. By contrast, the number of nest usurpers found in B. terrestris nests increased between 2003 and 2005, indicating that availability of nest sites was limiting and queens strongly competed for nest sites. Our findings suggest that competition for nest sites rather than flower resources is the major ecological mechanism for displacement of native bees. The large reduction of B. hypocrita sapporoensis queen indicates that B. terrestris may cause local extinction of native bumblebees. Control of established B. terrestris populations and prevention of further range expansion are urgently needed.     

Chemical reproductive traits of diploid Bombus terrestris males: Consequences on bumblebee conservation

The current bumblebee decline leads to inbreeding in populations that fosters a loss of allelic diversity and diploid male production. As diploid males are viable and their offspring are sterile, bumblebee populations can quickly fall in a vortex of extinction. In this article, we investigate for the first time a potential premating mechanism through a major chemical reproductive trait (male cephalic labial gland secretions) that could prevent monandrous virgin queens from mating with diploid males. We focus our study on the cephalic labial gland secretions of diploid and haploid males of Bombus terrestris (L.). Contrary to initial expectations, our results do not show any significant differentiation of cephalic labial gland secretions between diploid and haploid specimens. Queens seem therefore to be unable to avoid mating with diploid males based on their compositions of cephalic labial gland secretions. This suggests that the vortex of extinction of diploid males could not be stopped through premating avoidance based on the cephalic labial gland secretions but other mechanisms could avoid mating between diploid males and queens.     

REPRODUCTIVE CONFLICT IN BUMBLEBEES AND THE EVOLUTION OF WORKER POLICING

Worker policing (mutual repression of reproduction) in the eusocial Hymenoptera represents a leading example of how coercion can facilitate cooperation. The occurrence of worker policing in “primitively” eusocial species with low mating frequencies, which lack relatedness differences conducive to policing, suggests that separate factors may underlie the origin and maintenance of worker policing. We tested this hypothesis by investigating conflict over male parentage in the primitively eusocial, monandrous bumblebee, Bombus terrestris. Using observations, experiments, and microsatellite genotyping, we found that: (a) worker‐ but not queen‐laid male eggs are nearly all eaten (by queens, reproductive, and nonreproductive workers) soon after being laid, so accounting for low observed frequencies of larval and adult worker‐produced males; (b) queen‐ and worker‐laid male eggs have equal viabilities; (c) workers discriminate between queen‐ and worker‐laid eggs using cues on eggs and egg cells that almost certainly originate from queens. The cooccurrence in B. terrestris of these three key elements of “classical” worker policing as found in the highly eusocial, polyandrous honeybees provides novel support for the hypothesis that worker policing can originate in the absence of relatedness differences maintaining it. Worker policing in B. terrestris almost certainly arose via reproductive competition among workers, that is, as “selfish” policing.     

Colony growth and reproductive ability of feral nests of the introduced bumblebee Bombus terrestris in northern Japan

The introduced Bombus terrestris has been blamed for the decline of native bumblebee populations in Japan. To control this invasive species, it is necessary to understand its ecological traits in its newly established range. Here, we investigated the colony growth pattern and reproductive ability of feral nests of B. terrestris in northern Japan from 2003 to 2006. Nests collected at various times showed initially slow growth followed by rapid development. This pattern is consistent with findings in previous studies using laboratory-raised colonies. Our results also suggested that protandrous colonies tend to specialize in male production, whereas protogynous colonies specialize in gyne production, producing a split sex ratio in feral colonies of B. terrestris. The numerical population sex ratio was 1.40 gynes per male, calculated from the numbers of pre-emergence cocoons for gynes and males, showing a female-biased sex ratio at the population level. Mature colonies produced a mean of 376.5 cocoons and 90.2 gyne cocoons (22.1% of the total). The proportion of gynes produced by B. terrestris nests exceeded both those of conspecifics observed in other countries and those of consubgeneric native Japanese species. The propagule pressure hypothesis appears to explain the probability of establishment of this invasive species. Suitable nest sites for B. terrestris queens appeared to be in short supply, and B. terrestris may win usurpation contests against native species due to its large size, resulting in the decline of native bumblebee species.     

Effects of Male age and Size on Mating Success in the Bumblebee Bombus terrestris

In social insects, the reproductive strategies adopted by colonies emerge as a complex property of individual behaviours, but as yet we are often unable to fully explain them in evolutionary terms. In bumblebees, colonies adopt either a short-lived strategy specializing in male production, or a longer-lived strategy in which mainly new queens are produced, but this results in males emerging long before mates are available; this strategy can only easily be explained if older males are at a significant reproductive advantage. Here we examine how age and morphological characters influence mating success of male bumblebees. In two separate experiments in which single virgin males and females were confined together, we found that young males and heavy males mated more swiftly and copulated for less time compared to old males or lighter males, respectively. However, in competitive situations age proved to be unimportant and the only factors to influence mating success were the lengths of the fore and hind tibiae, with strong directional selection for long leg length. Fore and hind legs are both used in courtship, so both traits are associated with plausible mechanisms under selection. It has previously been argued that, in times of food stress, bumblebee colonies should produce males as male size is less likely to be strongly correlated with fitness than female size. Our results suggest that this may not be so, since aspects of male size directly impact on their mating success. Our results leave unexplained the emergence of males many days before new queens.     

Sexual biology of haploid and diploid males in the bumble beeBombus terrestris

Summary InB. terrestris diploid males develop normally into adults (Duchateau et al., 1994). The diploid males are similar in appearance to the haploid males, except that they are smaller. The size of the testis of diploid males, relative to the length of the radial cell, is smaller than that of haploid males. There is overlap in the frequency distribution with respect to body size and testis size. The spermatozoa of diploid males are larger than those of the haploids and the vasa deferentia contain fair less spermatozoa than those of haploid males of the same age. Countings and measurements of the spermatozoa, therefore, can give the best indication about the ploidy of the males. Diploid males are successful in mating. They mate at a younger age than haploid males and they die sooner. The number of vial offspring of diploid males, however, is very low. No queen that mated with a diploid male produced a colony, but a few queens did produce some progeny. These might have been triploid males and workers. InB. terrestris higher ploidy results in smaller individuals, whereas in several other species of the Hymenoptera it has been found to result in larger individuals.     

Kin structure and colony male reproduction in the hornet<Emphasis Type="Italic"> Vespa crabro</Emphasis> (Hymenoptera: Vespidae)

We estimated queen mating frequency, genetic relatedness among workers, and worker reproduction in Vespa crabro flavofasciata using microsatellite DNA markers. Of 20 colonies examined, 15 contained queens inseminated by a single male, 3 colonies contained queens inseminated by two males, and 2 colonies contained queens inseminated by three males. The genetic relatedness among workers was estimated to be 0.73±0.003 (mean±SE). For this high relatedness, kin selection theory predicts a potential conflict between queens and workers over male production. To verify whether males are derived from queens or workers, 260 males from 13 colonies were genotyped at four microsatellite loci. We found that all of the males were derived from the queens. This finding was further supported by the fact that only 33 of 2,990 workers dissected had developed ovaries. These workers belonged to 2 of the 20 colonies. There was no relationship between queen mating frequency and worker reproduction, and no workers produced male offspring in any of the colonies. These results suggest that male production dominated by queens in V. crabro flavofasciata is possibly due to worker policing.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.     

Evidence for hilltopping in bumblebees?

1. Male bumblebees are known to exhibit a range of mate‐location behaviours, including perching on prominent objects and darting at passing queens, patrolling of scent‐marked flight routes, and waiting outside nest entrances for virgin queens to emerge. Here we provide evidence for a fourth strategy, known as hilltopping. This behaviour is widely known from a range of invertebrates, but has not previously been described in bumblebees. 2. We studied the distribution of bumblebees along transects ascending four hills in Scotland and demonstrate that, relative to workers, males of four bumblebee species or species groups (Bombus lapidarius, B. monticola, B. pascuorum, and B. lucorum/magnus/cryptarum/terrestris) tend to congregate at or near the tops of hills. This is, to our knowledge, the first evidence for hilltopping in bumblebees and the first record of any putative mate‐locating behaviour for male B. pascuorum, a very common species in Europe. 3. We note that, in common with most previous studies of mate‐locating behaviour in bumblebees, attraction of virgin queens and mating were not observed.     

Impact of artificial photoperiodism on the colony development of the bumblebee Bombus terrestris

This study investigated the effect of the photoperiodic regimes 0 h light : 24 h dark (LD 0:24), LD 8:16, LD 16:8 and LD 24:0 at 28°C and 50% Relative Humidity (RH) on the colony development of hibernated (2.0, 2.5, 3.0, 3.5 and 4.0 months) bumblebee queens. The queens which had hibernated for 3.0 months and which were reared in a LD 8:16 photoperiod showed the highest rate of colony initiation (88.2%), colony foundation (67.6%) and progeny queen production (38.2%). The photoperiod LD 8:16 also produced the shortest period of colony initiation and colony foundation. The highest number of sexual males (171.2 ± 12.2) and queens (91.2 ± 9.9) were produced in the colonies when 3.5 and 3.0 month hibernated queens were kept in an LD 8:16 photoperiod. The results show that light regime and hibernation duration affect colony characteristics of Bombus terrestris.     

Induction of colony initiation by Japanese native bumble bees using cocoons of the exotic bumblebee Bombus terrestris

The colony initiation rates of Bombus hypocrita (a native Japanese bumblebee) and Bombus terrestris (a European species) foundresses were compared after 4 weeks of exposure to B. terrestris cocoons. The B. terrestris cocoons, when replaced weekly, were effective for inducing oviposition by foundresses of both species. There were no significant differences in the colony initiation rates of B. terrestris and B. hypocrita, either with the control treatment or with the cocoons. The cocoon method was also tested for five species and two subspecies of native Japanese bumblebees. The colony initiation rate was higher for foundresses of the subgenus Bombus s. str. than for foundresses of the subgenera Pyrobombus, Diversobombus, and Thoracobombus. When replaced weekly, the cocoons of B. terrestris are effective inducers of colony foundation in three Japanese native species, namely B. ignitus, B. hypocrita hypocrita, and B. h. sapporoensis.     

Influence of inbreeding in the early stages of artificially reared colonies of Bombus terrestris

In the present study on the bumblebee Bombus terrestris, we investigated the influence of inbreeding on queen fitness by comparing diapause survival and egg-laying success of queens mated with nestmate and non-nestmate males. We then compared the early stage of colonies with or without diploid males and analysed colony characteristics to identify a factor predictive of colony outcome. Diapause survival was no different between queens mated with nestmates and non-nestmates, but in the latter case, egg-laying success was significantly higher. Queens mated with nestmates gave rise to a percentage of diploid male colonies (52.6%) compatible with brother–sister coupling. We obtained 18.6% of colonies with diploid males even from queens mated with non-nestmates, indicating that the colonies of origin were in some way related or homozygous at the sex determination loci. There was no difference in the early growth stage between colonies with or without diploid males, except in the number of workers emerging in the first brood, which was significantly higher in the latter. Among diploid male colonies, the number of workers and the male/worker ratio in the first brood was highly variable and was not a good predictor of subsequent colony growth. Out of 49 colonies with diploid males that reached full development, only 11 had a sufficient size to assume that they could survive in the field or, in a commercial breeding, to be suitable for pollination purposes.     

Host choice in the phoretic mite Parasitellus fucorum (Mesostigmata: Parasitidae): which bumblebee caste is the best?

Host caste recognition may be important for the dispersal of phoretic mites associated with social insects. All developmental stages of the mite Parasitellus fucorum (Acari: Mesostigmata: Parasitidae) live in the nests of bumblebees (Hymenoptera: Apidae: Bombus). Dispersal occurs by specialised phoretic instars, deutonymphs, which attach to adult bumblebees. Since bumblebee colonies are annual and only young queens overwinter, deutonymphs that are able to discriminate between bumblebee castes and preferentially attach to queens should be favoured by selection. In the field, deutonymphs of P. fucorum were found to be phoretic on bumblebee workers and queens, and in behavioural experiments all castes proved to be attractive as carriers for the mites. However, they preferred queens that had hibernated as carriers when they could choose between workers and queens. In a further experiment, when given a choice, deutonymphs switched from males to young queens but never transferred from a queen to a male. These results suggest that deutonymphs preferentially attach to queens but may also use other castes for transport. Those dispersing on workers and males may try to switch to queens later. Host-switching is possible during copulation and on flowers, where bees of all castes forage. Received: 14 November 1997 / Accepted: 16 February 1998     

Bumblebee foraging rhythms under the midnight sun measured with radiofrequency identification

Background  

In the permanent daylight conditions north of the Arctic circle, there is a unique opportunity for bumblebee foragers to maximise intake, and therefore colony growth, by remaining active during the entire available 24-h period. We tested the foraging rhythms of bumblebee (Bombus terrestris and B. pascuorum) colonies in northern Finland during the summer, when the sun stays above the horizon for weeks. We used fully automatic radio-frequency identification to monitor the foraging activity of more than 1,000 workers and analysed their circadian foraging rhythms.     

Multiple mating in the ant Cataglyphis cursor: testing the sperm limitation and the diploid male load hypotheses

Multiple mating (i.e., polyandry) by queens in social Hymenoptera is expected to weaken social cohesion since it lowers within-colony relatedness, and hence, indirect fitness benefits from kin selection. Yet, there are many species where queens mate multiply. Several hypotheses have been put forward to explain the evolution and maintenance of polyandry. Here,we investigated the ‘sperm limitation’ and the ‘diploid male load’ hypotheses in the ant Cataglyphis cursor. Genetic analyses of mother-offspring combinations showed that queens mate with up to 8 males, with an effective mating frequency of 3.79. Significant paternity skew (unequal contribution of the fathers) was detected in 1 out of 5 colonies. The amount of sperm stored in the spermatheca was not correlated with the queen mating frequency, and males carry on average enough sperm in their seminal vesicles to fill one queen’s spermatheca. Analyses of the nuclear DNA-content of males also revealed that all were haploid. These results suggest that the ‘sperm limitation’ and the ‘diploid male load’ hypotheses are unlikely to account for the queen mating frequency reported in this ant. In light of our results and the life-history traits of C. cursor, we discuss alternative hypotheses to account for the adaptive significance of multiple mating by queens in this species. Received 13 August 2008; revised 19 November 2008; accepted 21 November 2008.