The cephalic labial gland secretions of two socially parasitic bumblebees Bombus hyperboreus (Alpinobombus) and Bombus inexspectatus (Thoracobombus) question their inquiline strategy

Social parasitic Hymenopterans have evolved morphological, chemical, and behavioral adaptations to overcome the sophisticated recognition and defense systems of their social host to invade host nests and exploit their worker force. In bumblebees, social parasitism appeared in at least 3 subgenera independently: in the subgenus Psithyrus consisting entirely of parasitic species, in the subgenus Alpinobombus with Bombus hyperboreus, and in the subgenus Thoracobombus with B. inexspectatus. Cuckoo bumblebee males utilize species‐specific cephalic labial gland secretions for mating purposes that can impact their inquiline strategy. We performed cephalic labial gland secretions in B. hyperboreus, B. inexspectatus and their hosts. Males of both parasitic species exhibited high species specific levels of cephalic gland secretions, including different main compounds. Our results showed no chemical mimicry in the cephalic gland secretions between inquilines and their host and we did not identify the repellent compounds already known in other cuckoo bumblebees.     

Atypical secretions of the male cephalic labial glands in bumblebees: the case of Bombus (Rhodobombus) mesomelas Gerstaecker (Hymenoptera, Apidae)

In bumblebees, the male secretion of the cephalic labial gland is species-specific. It is highly involved in the nuptial behavior, acting as a sexual attracting pheromone. Therefore, it is also used to accurately identify the species. In contrast to this common scheme, the secretions of Bombus mesomelas are found to be strongly reduced and do not include the most volatile compounds that are present in the secretions of all the other studied bumblebee species. These secretions correspond to cuticular hydrocarbons that can be found in all bumblebee species. This was also the case for another bumblebee species from the same Rhodobombus subgenus: Bombus pomorum. This atypical composition of the male cephalic labial gland secretions seems to indicate that, at least for these two species of Rhodobombus, these secretions are not used to attract virgin females from a long distance, as it is the case for all the other bumblebee species studied.     

Diploid male production in a leaf‐cutting ant

1. In haplodiploid social insects where males are haploid and females are diploid, inbreeding depression is expressed as the production of diploid males when homozygosity at the sex‐determining locus results in the production of diploid individuals with a male phenotype. Diploid males are often assumed to have reduced fitness compared with their haploid brothers. 2. While studying the reproductive biology of a leaf‐cutting ant, Atta sexdens, in Gamboa, Republic of Panama, we detected the presence of a larger male morph. Using microsatellite markers we were able to confirm that the large male morph was diploid in 87% of cases. 3. We infer that the Gamboa population of A. sexdens experiences inbreeding depression because diploid males were found in three out of five mature colonies. However, their frequencies were relatively low because queens were multiply mated and our estimates suggest that many diploid male larvae may not survive to adulthood. 4. We measured two traits potentially linked to male reproductive success: sperm length and sperm number, and showed that diploid males produced fewer but longer sperm. These results provide indirect evidence that diploid male reproductive success would be reduced compared with haploid males if they were able to copulate. 5. We conclude that diploid male production is likely to affect the fitness of A. sexdens queens with a matched mating, as these males are produced at the cost of workers and, if the colony survives to reach mature size, also gynes.     

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.     

Reduced cephalic labial glands in the male bumblebees of the subgenus Rhodobombus Dalla Torre (Hymenoptera: Apidae: Bombus Latreille)

Until now, all males of bumblebees are known to attract conspecific females by marking places with a chemical signal secreted by their cephalic labial gland. The specific combination of patrolling flight and scentmarking is of outmost importance to bumblebees as it is their main species specific recognition system. We report here the lack of that recognition system in species of the subgenus Rhodobombus by comparing the morphology and histology of the cephalic labial glands of Bombus (Rhodobombus) mesomelas with those of a well known species, B. (Bombus) terrestris (L.) The cephalic labial glands are much smaller in B. mesomelas than in other bumblebees species and most likely non-functional. This morphology is also observed in B. pomorum and B. brodmanni which also belong to the subgenus Rhodobombus. Our morphological observations are consistent with the chemical analyses of the secretions in B. mesomelas and B. pomorum which are very limited and of most unusual composition for a bumblebee. In addition, whereas other species are thought to use their barbae mandibularis to spread their secretion onto the substrate, these structures are absent in all Rhodobombus. All these observations would mean that the males of Rhodobombus do not attract females from far away by the use of their cephalic labial gland secretions.     

Exocrine gland secretions of virgin queens of five bumblebee species (Hymenoptera: Apidae, Bombini)

Secretions of three different glands (mandibular gland, labial gland, and Dufour's gland) of virgin queens of five bumblebee species (Bombus lucorum, B. lapidarius, B. hypnorum, B. pascuorum, and B. terrestris) were analysed. Around 200 compounds were identified in the secretions. The compositions of the secretions of labial and mandibular glands were species-specific. Dufour's gland of all species produced mainly hydrocarbons, both saturated and unsaturated, the proportions of which differed quantitatively between the species studied.     

First Chemical Analysis and Characterization of the Male Species‐Specific Cephalic Labial‐Gland Secretions of South American Bumblebees

The evolution of signals and reproductive traits involved in the pre‐mating recognition has been in focus of abundant research in several model species, such as bumblebees (genus Bombus). However, the most‐studied bumblebee reproductive trait, the male cephalic labial gland secretions (CLGS), remains unknown among bumblebee species from South America. In this study, the CLGS of five South American bumblebees of the subgenera Thoracobombus (Bombus excellens and B. atratus) and Cullumanobombus (B. rubicundus, B. hortulanus, and B. melaleucus) were investigated, by comparing the chemical compositions of their secretions to those of closely related European species. The results showed an obvious interspecific differentiation in both subgenera. The interspecific differentiation among the species of the Thoracobombus subgenus involved different compounds present at high contents (main compounds), while those of the Cullumanobombus subgenus shared the same main components. This suggests that among the species of the Cullumanobombus subgenus, the differentiation in minor components could lead to species discrimination.     

Sex, bugs and Haldane's rule: the nematode genus Pristionchus in the United States

The haplodiploid sex determining mechanism in Hymenoptera (males are haploid, females are diploid) has played an important role in the evolution of this insect order. In Hymenoptera sex is usually determined by a single locus, heterozygotes are female and hemizygotes are male. Under inbreeding, homozygous diploid and sterile males occur which form a genetic burden for a population. We review life history and genetical traits that may overcome the disadvantages of single locus complementary sex determination (sl-CSD). Behavioural adaptations to avoid matings between relatives include active dispersal from natal patches and mating preferences for non-relatives. In non-social species, temporal and spatial segregation of male and female offspring reduces the burden of sl-CSD. In social species, diploid males are produced at the expense of workers and female reproductives. In some social species, diploid males and diploid male producing queens are killed by workers. Diploid male production may have played a role in the evolution or maintenance of polygyny (multiple queens) and polyandry (multiple mating). Some forms of thelytoky (parthenogenetic female production) increase homozygosity and are therefore incompatible with sl-CSD. We discuss a number of hypothetical adaptations to sl-CSD which should be considered in future studies of this insect order.     

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.     

Male labial gland secretions and mitochondrial DNA markers support species status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae)

Summary. Spring queens of Bombus cryptarum and B. magnus from 2 localities in Brandenburg/Germany and Scotland/United Kingdom respectively were determined by morphological characteristics. The lateral border of the collare at the border of the pronotallobus or at the episternum proved to be an especially useful character. Artificial colonies were reared from safely determined spring queens and the cephalic part of the labial glands of males from these colonies were investigated by GC/MS. The investigation identified approximately 50 compounds, as a mixture of straight chain fatty acid derivatives (alcohols, esters and hydrocarbons). The labial secretions of B. cryptarum and B. magnus are significantly different. Mitochondrial cytochrome oxidase 1 (CO1) of two queens from each locality and species were sequenced. Each species from the different localities formed a cluster. Sequence divergence between B. cryptarum and B. magnus was about 30 base substitutions and approximately 0.04 in Tamura-Nei genetic distance. Bombus cryptarum and B. magnus were closer to each other than to B. lucorum and made the sister group in the topology of the tree. Both the CO1 sequences and the labial gland secretions of males of B. cryptarum from Brandenburg and of males from artificial colonies reared from safely determined spring queens from Scotland are identical. B. cryptarum has thus, for the first time, been identified as part of the British bumble bee fauna. The differences of both the labial gland secretions, used as species recognition signals, and the genetic differences established by sequencing CO1 confirm the morphological findings that B. cryptarum and B. magnus are distinct taxa which should be treated as distinct species.Received 25 March 2004; revised 13 June 2004; accepted 15 June 2004.     

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.     

Effect of a Founder Event on Variation in the Genetic Sex-Determining System of the Fire Ant Solenopsis Invicta

Effects of a recent founder event on genetic diversity in wild populations of the fire ant Solenopsis invicta were studied, with particular attention given to the genetic sex-determining system. Diploid males are far more common relative to haploid males in introduced populations than in native populations of fire ants, and queens that produce diploid males account for a significantly larger proportion of the mated queens in introduced than in native populations. Differences between native and introduced populations in attributes of the mating systems (i.e., queen mating frequency or level of inbreeding) can be excluded as factors contributing to these different levels of diploid male production. Thus, we conclude that diploid males have increased in frequency in introduced populations because of a loss of allelic diversity at the sex-determining locus (loci). This loss of sex alleles has generated a substantial increase in the estimated segregational genetic load associated with production of sterile diploid males in introduced populations over the load in native populations. The loss of allelic diversity in the sex-determining system in introduced S. invicta is paralleled by a loss of electrophoretically detectable rare alleles at protein-encoding loci. Such concordance between these different types of markers is predicted because each of the many sex alleles present in the native populations is expected to be rare. Estimates of expected heterozygosity (H(exp)) based on 76 electrophoretic loci do not differ significantly between the native and introduced fire ant populations, illustrating the lack of sensitivity of this measure for detecting many types of bottlenecks.     

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.     

Analysis of the labial gland secretions of the male bumblebee Bombus griseocollis (Hymenoptera: Apidae)

The labial gland secretions from males of the bumblebee Bombus (Separatobombus) griseocollis De Geer, a bumblebee exhibiting perching behaviour, were analysed by gas chromatography/mass spectrometry (GC/MS) in the electron impact and positive ion chemical ionization mode. The major compound of the complex mixture of alkenols, acetates, hydrocarbons, wax type esters and steroids is tetradecyl acetate, considerable amounts of hexadecyl, geranyllinaloyl, geranylgeranyl, docosyl, tetracosenyl and hexacosenyl acetate were also found. 1,3-Tetradecanediol diacetate, detected as a minor component, has not yet been identified in male bumblebee labial gland secretions. Besides small amounts of primary alcohols (tetradecanol and hexadecanol) the tertiary alcohol geranyllinalool (3,7,11,15-tetramethyl-hexadeca-1,6,10,14-tetraene-3-ol) was also present. The primary alcohols were also present as esters of butanoic, dodecanoic, tetradecanoic, and hexadecanoic acid. Besides the usual mixture of un- and mono-unsaturated straight chain hydrocarbons, the labial gland contains the isoprenoid hydrocarbons beta-springene [(6E, 10E)-7,11,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene] and two isomers of a-springene [(3Z,6E,10E)- and (3E,6E,10E)-3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene]. The close relationship in chemical composition in male bumblebees with perching and flight pass behaviour is discussed.     

Male reproductive investment and queen mating-frequency in fungus-growing ants

Sperm number and male accessory gland compounds are often importantdeterminants of male mating success but have been little studiedin social insects. This is because mating in social insectsis often difficult to manipulate experimentally, and first evidencefor an explicit influence of accessory gland secretions on malemating success in social insects was obtained only recently.Here we perform a comparative analysis of male sexual organsacross 11 species of attine fungus-growing ants, representingboth genera with single- and multiple-queen mating. We foundthat the general morphology of the male sexual organs was verysimilar across all species, but the relative sizes of the accessoryglands and the sperm-containing accessory testes vary significantlyacross species. Small testes and large accessory glands characterizespecies with singly mated queens, whereas the opposite is foundin species with multiply mated queens. However, in the socialparasite Acromyrmex insinuator, in which queens have secondarilyreverted to single mating, males have accessory gland characteristicsreminiscent of the lower attine ants, but without having significantlyreduced their investment in sperm production. We hypothesizethat the main function of accessory gland compounds in attineants is to monopolize male paternity in similar ways as knownfrom other social insects. This would imply that the evolutionof polyandry in the terminal clade of the fungus-growing ants(the leafcutter ants) has resulted in selection for decreasedinvestment by males in accessory gland secretions and increasedinvestment in sperm number, in response to sperm competitionfor sperm storage.     

Impacts of inbreeding on bumblebee colony fitness under field conditions

Background  

Inbreeding and the loss of genetic diversity are known to be significant threats to small, isolated populations. Hymenoptera represent a special case regarding the impact of inbreeding. Haplodiploidy may permit purging of deleterious recessive alleles in haploid males, meaning inbreeding depression is reduced relative to diploid species. In contrast, the impact of inbreeding may be exacerbated in Hymenopteran species that have a single-locus complementary sex determination system, due to the production of sterile or inviable diploid males. We investigated the costs of brother-sister mating in the bumblebee Bombus terrestris. We compared inbred colonies that produced diploid males and inbred colonies that did not produce diploid males with outbred colonies. Mating, hibernation and colony founding took place in the laboratory. Once colonies had produced 15 offspring they were placed in the field and left to forage under natural conditions.     

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.     

Similar Performance of Diploid and Haploid Males in an Ant Species without Inbreeding Avoidance

Under haplodiploidy, a characteristic trait of all Hymenoptera, females develop from fertilised eggs, and males from unfertilised ones. Males are therefore typically haploid. Yet, inbreeding can lead to the production of diploid males that often fail in development, are sterile or are of lower fertility. In most Hymenoptera, inbreeding is avoided by dispersal flights of one or both sexes, leading to low diploid male loads. We investigated causes for the production of diploid males and their performance in a highly inbred social Hymenopteran species. In the ant Hypoponera opacior, inbreeding occurs between wingless sexuals, which mate within the mother nest, whereas winged sexuals outbreed during mating flights earlier in the season. Wingless males mate with queen pupae and guard their mating partners. We found that they mated randomly with respect to relatedness, indicating that males do not avoid mating with close kin. These frequent sib‐matings lead to the production of diploid males, which are able to sire sterile triploid offspring. We compared mating activity and lifespan of haploid and diploid wingless males. As sexual selection acts on the time of emergence and body size in this species, we also investigated these traits. Diploid males resembled haploid ones in all investigated traits. Hence, albeit diploid males cannot produce fertile offspring, they keep up with haploid males in their lifetime mating success. Moreover, by fathering viable triploid workers, they contribute to the colonies' work force. In conclusion, the lack of inbreeding avoidance led to frequent sib‐matings of wingless sexuals, which in turn resulted in the regular production of diploid males. However, in contrast to many other Hymenopteran species, diploid males exhibit normal sexual behaviour and sire viable, albeit sterile daughters.     

Control of male production in the swarm‐founding wasp,Polybioides tabidus

In social insect colonies, male production may involve conflicts over the sex ratio, worker vs. queen reproduction, and each queen's contribution to the males when there are multiple queens. We examined male production in the swarm‐founding, multiple‐queened wasp, Polybioides tabidus, for which previous work suggested worker control of the sex ratios. We found that queens produced the males in accord with the collective worker preference. We also found that diploid males were produced, but only in association with haploid males. Simulations show they should have been produced in other colonies as well and their absence indicates that they were killed in some of these other colonies. The pattern of their removal indicates that P. tabidus cannot distinguish diploid from haploid males, and that haploid males would have been removed from these colonies too. This provides evidence that the workers are able to manipulate male production when collective preferences dictate.     

Methods for species delimitation in bumblebees (Hymenoptera,Apidae, Bombus): towards an integrative approach

Delimitation of closely related species is often hindered by the lack of discrete diagnostic morphological characters. This is exemplified in bumblebees (genus Bombus). There have been many attempts to clarify bumblebee taxonomy by using alternative features to discrete morphological characters such as wing shape, DNA, or eco‐chemical traits. Nevertheless each approach has its own limitations. Recent studies have used a multisource approach to gather different lines of speciation evidence in order to draw a strongly supported taxonomic hypothesis in bumblebees. Yet, the resulting taxonomic status is not independent of selected evidence and of consensus methodology (i.e. unanimous procedure, majority, different weighting of evidence). In this article, we compare taxonomic conclusions for a group of taxonomically doubtful species (the Bombus lapidarius‐group) obtained from the four commonly used lines of evidence for species delimitation in bumblebees (geometric morphometric of wing shape, genetic differentiation assessment, sequence‐based species delimitation methods and differentiation of cephalic labial gland secretions). We ultimately aim to assess the usefulness of these lines of evidence as components of an integrative decision framework to delimit bumblebee species. Our results show that analyses based on wing shape do not delineate any obvious cluster. In contrast, nuclear/mitochondrial, sequence‐based species delimitation methods, and analyses based on cephalic labial gland secretions are congruent with each other. This allows setting up an integrative decision framework to establish strongly supported species and subspecies status within bumblebees.