The conundrum of the yellow crazy ant (Anoplolepis gracilipes) reproductive mode: no evidence for dependent lineage genetic caste determination

Asexual reproduction and hybridisation are often found among highly invasive plants and marine invertebrates. Recently, it has been suggested that clonality may enhance the success of invasive ants. In contrast, obligate hybridisation (dependent lineage genetic caste determination or DL GCD in ants) may decrease the chances of population persistence if one lineage is less prevalent than the other (asymmetry in lineage ratio). Genetic data available for the invasive yellow crazy ant (Anoplolepis gracilipes) suggest that it has an unconventional mode of reproduction that may involve asexual reproduction by workers or queens, or a form of genetic caste determination. Here, we investigated whether A. gracilipes reproduction involved DL GCD. The potential for worker reproduction was also assessed. We used microsatellite markers to assess the population structure of A. gracilipes workers, males, queens and sperm in queen spermathecae, from field collections in Arnhem Land. We found that a single queen lineage is present in Arnhem Land. The presence of a single lineage of queens discounts the possibility of DL GCD. Population structure separated queens and workers into different lineages, suggesting that these castes are determined genetically in A. gracilipes, or the mode of reproduction differs between workers and queens. Evidence for worker reproduction was weak. We conclude that the reproductive mode of A. gracilipes does not involve DL GCD. The resolution of the reproductive mode of A. gracilipes is complicated by a high prevalence of diploid males. The determination of the A. gracilipes reproductive mode remains a fascinating research question, and its resolution will improve our understanding of the contribution of the reproductive system to invasion success.     

Potential and realized reproduction by different worker castes in queen-less and queen-right colonies of <Emphasis Type="Italic">Pogonomyrmex badius</Emphasis>

Workers of the Florida harvester ant (Pogonomyrmex badius), the only North American Pogonomyrmex with a polymorphic worker caste, produce males when colonies are orphaned. In this study,we assessed the reproductive potential of workers of each caste group, minors and majors, in the presence and absence of the queen, and tested whether males produced in natural queen-right colonies are derived from workers. Worker size was positively correlated with ovariole number such that major workers had approximately double the number of ovarioles as minor workers. The number of vitellogenic oocytes, a measure of reproductive potential, was greater in major compared to minor workers and increased in both worker castes when queens were removed. Major workers have greater reproductive potential than minors although they represent a minority within the colony (~5% of workers are majors). Worker produced eggs were visible in colonies 28 – 35 days after queen removal. This time lag, from queen removal to egg production, is similar to other ants and bees. Though workers are capable of producing viable eggs, we found no evidence that they do so in queen-right colonies, suggesting that worker reproduction is controlled via some social mechanism (self restraint, policing, or inhibition). This result supports predictions of kin selection theory – that due to multiple mating by the queen workers are more related to queen-produced males than most worker-produced males and should thus favor reproduction by the queen and inhibit reproduction by other workers. Received 25 January 2007; revised 1 May 2007; accepted 21 May 2007.     

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.     

Male parentage in dependent-lineage populations of the harvester ant Pogonomyrmex barbatus

We investigated the extent to which workers reproduce in a dependent-lineage population of the monogynous harvester ant Pogonomyrmex barbatus. Dependent-lineage populations contain two interbreeding, yet genetically distinct mitochondrial lineages, each associated with specific alleles at nuclear loci. Workers develop from matings between lineages, and queens develop from matings within lineages, so queens must mate with males of both lineages to produce daughter queens and workers. Males develop from unfertilized eggs and are haploid. Worker production of males could lead to male-mediated gene flow between the lineages if worker-produced males were reproductively capable. This could result in the loss of the dependent-lineage system, because its persistence depends on the maintenance of allelic differences between the lineages. To investigate the extent of worker reproduction in P. barbatus, we genotyped 19-20 males and workers from seven colonies, at seven microsatellite loci, and 1239 additional males at two microsatellite loci. Our methods were powerful enough to detect worker reproduction if workers produced more than 0.39% of males in the population. We detected no worker-produced males; all males appeared to be produced by queens. Thus, worker reproduction is sufficiently infrequent to have little impact on the dependent-lineage system. These results are consistent with predictions based on inclusive fitness theory because the effective queen mating frequency calculated from worker genotypes was 4.26, which is sufficiently high for workers to police those that attempt to reproduce.     

Male production by workers in the polygynous ant Prolasius advenus

The ability of workers to produce male individuals is reported here for the first time in a species of the formicine ant genus Prolasius. We show that Prolasius advenus workers possess ovaries and demonstrate that they are able to produce adult males in queenless colonies. We also experimentally tested the influence of queen volatiles on the level of worker reproduction. Workers produced fewer eggs in treatments where they could perceive odors from queens. Some volatile compounds emitted by queens may thus have a signaling or inhibitory effect on worker reproduction. This effect of queen presence did not entirely stop worker reproduction, however, as adult males still emerged under these conditions. Worker-produced males were absent only in treatments with the physical presence of queens. Dissections of workers collected from queenright nests in the field revealed signs of egg-laying activity in more than half of individuals. Together, these results suggest that in nature P. advenus workers produce males at least in orphaned colonies or in situations where the physical presence of queens is limited.     

Mating structure and male production in Vespa analis and Vespa simillima (Hymenoptera: Vespidae)

We estimated queen mating frequency, genetic relatedness between workers and worker reproduction in the hornets Vespa analis and Vespa simillima using microsatellite DNA genotyping. The 20 V. analis colonies studied each contained a queen inseminated by a single male. Of the 15 V. simillima colonies studied, nine had a queen inseminated by a single male, four had a queen inseminated by two males, and two had a queen inseminated by three males. The estimated effective number of matings was 1.33 ± 0.74 (mean ± SD), with 75–85% of the offspring of the six multiply mated queens sired by single males. The values for genetic relatedness between the workers of V. analis and V. simillima were 0.739 ± 0.004 and 0.698 ± 0.013 (mean ± SD), respectively. We conclude that V. analis and V. simillima colonies are genetically monogynous and monandrous. When high relatedness between the workers occurs within colonies, kin selection theory predicts a potential conflict between queens and workers over male production. To determine whether males were derived from queens or workers, males from V. analis and V. simillima colonies were genotyped at four microsatellite loci and the level of ovary activation in workers was determined. None of the 787 V. analis workers and only 15 of 3520 V. simillima workers had developed ovaries. Furthermore, the genotyping identified no worker‐produced males in any colony. The presence of reproductive workers correlated positively with the number of workers within the colony. These results suggest that eusocial colonies with an annual life cycle tend to break down socially when they become large and are close to dying.     

The potential for worker reproduction in the ant <Emphasis Type="Italic">Aphaenogaster cockerelli</Emphasis> and its absence in the field

A reproductive division of labor between subordinates and established reproductives is a hallmark of eusociality. In most groups, however, workers retain some reproductive capabilities. Across insect societies, measures of successful worker reproduction in the presence of a queen, with few exceptions, indicate that worker reproduction is kept at very low levels. There are, however, certain colony-level characteristics that may influence the degree to which worker reproduction is promoted, such as queen number, queen mating frequency, and physical presence of a queen in species with multiple nesting sites (polydomy). In this study, the level of worker reproduction in field colonies of the ant species Aphaenogaster cockerelli was measured. A. cockerelli is a monogynous and polydomous species, so worker reproduction across nesting sites was investigated. None of the 297 males sampled provided any evidence of worker reproduction. Worker reproduction would have been detectable if it was present at or above a level of 1.5 % of the total males per colony. An effective mating frequency for queens of this species was found to be 1.03. Although A. cockerelli colonies have many colony-level factors potentially promoting worker reproduction (workers with active, trophic egg-producing ovaries, a single singly-mated queen, workers who are physically separated from the queen), it is evident that worker reproduction is highly regulated. Synthesizing the extensive amount of policing and fertility signaling data previously reported on this species, A. cockerelli is presented as case study for how worker reproduction is repressed and cooperation is maintained in insect societies.     

A quantitative study of worker reproduction in queenright colonies of the Cape honey bee, Apis mellifera capensis

Reproduction by workers is rare in honey bee colonies that have an active queen. By not producing their own offspring and preventing other workers from producing theirs, workers are thought to increase their inclusive fitness due to their higher average relatedness towards queen-produced male offspring compared with worker-produced male offspring. But there is one exception. Workers of the Cape honey bee, Apis mellifera capensis, are able to produce diploid female offspring via thelytokous parthenogenesis and thus produce clones of themselves. As a result, worker reproduction and tolerance towards worker-produced offspring is expected to be more permissive than in arrhenotokous (sub)species where worker offspring are male. Here we quantify the extent to which A. m. capensis workers contribute to reproduction in queenright colonies using microsatellite analyses of pre-emergent brood. We show that workers produced 10.5% of workers and 0.48% of drones. Most of the workers' contribution towards the production of new workers coincided with the colonies producing new queens during reproductive swarming.     

Colony strength and queen replacement in Melipona marginata (Apidae: Meliponini).

Physogastric queens of Melipona marginata were removed from their colonies in order to verify the acceptance of a new queen by workers. Colony strength was evaluated according to queen oviposition rate and comb diameters. Replacement was observed seven times. Its occurrence and speed related positively to colony strength, independently of queen's age. In weak colonies, queen replacement was observed only once, following colony population increase that occurred after introduction of combs from another colony. Worker oviposition after queen removal was observed three times: in a strong colony with virgin queens and males, and in two of the weak colonies. In the first two or three days of new queen oviposition, during which most of the eggs were eaten by the queen, worker oviposition preceded almost all provisioning and oviposition processes (POPs). After this period, worker oviposition decreased until it reached around 25% of the POPs. Daily oviposition rate of young queens decreased or was even interrupted by hatching of their first brood.     

Clustered male production by workers in the stingless bee Melipona subnitida Ducke (Apidae, Meliponinae)

Summary: In stingless bees brood cells are sequentially filled with liquid larval food (mass-provisioning), upon which the queen lays an egg. Thereafter the cell is closed by a worker. This study showed that during these processes workers of Melipona subnitida regularly laid eggs that served as food for the queen. Occasionally cells were oviposited in and immediately closed by a worker. These cells always rendered males. Some of these reproductive workers were seen to lay a trophic egg as well. Cells which were exclusively oviposited in by the physogastric queen gave rise to workers and queens only. In one colony it could be verified that three workers alone, which differed in age by one day, laid 15 male-producing eggs within a period of two successive weeks. Among them the number of ovipositions was positively related to the order in which workers eclosed - the oldest worker laying most eggs - and inversely related to the number of times they closed cells oviposited in exclusively by the queen. Apparently the physogastric queen was not able to stop certain workers from reproducing. We therefore conclude that some workers in M. subnitida temporarily dominated their queens in egg-laying.     

Variation in patriline reproductive success during queen production in orphaned colonies of the thelytokous ant Cataglyphis cursor

In genetically diverse insect societies (polygynous or polyandrous queens), the production of new queens can set the ground for competition among lineages. This competition can be very intense when workers can reproduce using thelytoky as worker lineages that manage to produce new queens gain a huge benefit. Selection at the individual level might then lead to the evolution of cheating genotypes, i.e. genotypes that reproduce more than their fair share. We studied the variation in reproductive success among worker patrilines in the thelytokous and highly polyandrous ant Cataglyphis cursor. Workers produce new queens by thelytoky in orphaned colonies. The reproductive success of each patriline was assessed in 13 orphaned colonies using genetic analysis of 433 workers and 326 worker-produced queens. Our results show that patrilines contributed unequally to queen production in half of the colonies, and the success of patrilines was function of their frequencies in workers. However, over all colonies, we observed a significant difference in the distribution of patrilines between workers and worker-produced queens, and this difference was significant in three of 13 colonies. In addition, six colonies contained a low percentage of foreign workers (drifters), and in one colony, they produced a disproportionably high number of queens. Hence, we found some evidence for the occurrence of rare cheating genotypes. Nevertheless, cheating appears to be less pronounced than in the Cape Honey bee, a species with a similar reproductive system. We argue that worker reproduction by parthenogenesis might not be common in natural populations of C. cursor.     

QUEEN SIGNALING IN SOCIAL WASPS

Social Hymenoptera are characterized by a reproductive division of labor, whereby queens perform most of the reproduction and workers help to raise her offspring. A long‐lasting debate is whether queens maintain this reproductive dominance by manipulating their daughter workers into remaining sterile (queen control), or if instead queens honestly signal their fertility and workers reproduce according to their own evolutionary incentives (queen signaling). Here, we test these competing hypotheses using data from Vespine wasps. We show that in natural colonies of the Saxon wasp, Dolichovespula saxonica, queens emit reliable chemical cues of their true fertility and that these putative queen signals decrease as the colony develops and worker reproduction increases. Moreover, these putative pheromones of D. saxonica show significant conservation with those of Vespula vulgaris and other Vespinae, thereby arguing against fast evolution of signals as a result of a queen–worker arms race ensuing from queen control. Lastly, levels of worker reproduction in these species correspond well with their average colony kin structures, as predicted by the queen signaling hypothesis but not the queen control hypothesis. Altogether, this correlative yet comprehensive analysis provides compelling evidence that honest signaling explains levels of reproductive division of labor in social wasps.     

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.     

Worker reproduction and related behavior in orphan colonies of a Japanese paper wasp,Polistes jadwigae (Hymenoptera,Vespidae)

Worker reproduction and related behavior in 2 orphan colonies ofPolistes jadwigae, one of which had been maintained by a queen and 5 artificially introduced alien workers, were observed. After the queen loss, a dominance hierarchy was established among workers, and several workers laid eggs without physical interference from other workers. Only males emerged from worker-laid eggs, however, a few new queens were produced from queen-laid eggs. Investment sex ratio of queenright (=normal) colonies (0.27) fell between the 2 theoretical values, corresponding to worker control (0.20 or 0.18) and queen control (0.48 or 0.46) of the sex ratio, both being calculated by considering the excess of males produced in the orphan colonies.     

Mating system and population structure in the desert ant Cataglyphis livida

We investigated population genetic structure, mating system, worker reproduction and thelytokous parthenogenesis in the desert ant Cataglyphis livida. Pedigree analyses at polymorphic microsatellite loci show that colonies are headed by a single queen, and that queens are mated with two to eight males. No inbreeding was found in the population sampled. Colonies are genetically differentiated and exhibit no isolation-by-distance pattern, consistent with independent foundation of new colonies. Workers do reproduce and lay haploid (arrhenotokous) eggs in queenless colonies; conversely, we found no evidence of worker reproduction in queenright nests. In contrast with C. cursor, where new queens are produced by thelytokous parthenogenesis, female sexuals and workers of C. livida arise from classical sexual reproduction. We discuss the parallels and contrasts between the mating system and population structure in C. livida and the other Cataglyphis species studied so far.     

Queen mating frequency and maternity of males in the stingless bee Trigona carbonaria Smith

Summary. Aspects of the sociobiology of the native Australian stingless bee Trigona carbonaria (Hymenoptera, Meliponini) were investigated using highly variable microsatellite markers. The queen mating frequency was estimated by examining genotypes of samples of workers from five colonies across three to five microsatellite loci. In each case, results were consistent with the workers being progeny of a queen mated with a single male. Microsatellite analysis of haploid males from two of the colonies suggested they predominantly arose from queen-laid eggs. As workers are more related to sons-of-workers than to sons-of-queens in monandrous colonies, this is somewhat surprising, and suggests that there may be colony-level costs associated with worker reproduction, or that queens are able to regulate worker reproduction via the ritualized cell provisioning and oviposition process. Males from another T. carbonaria colony were found to be diploid.     

Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming

Kin selection theory predicts that honeybee (Apis mellifera) workers should largely refrain from producing their own offspring, as the workers collectively have higher inclusive fitness if they rear the sons of their mother, the queen. Studies that have quantified levels of ovary activation and reproduction among workers have largely supported this prediction. We sampled pre‐emergent male pupae and adult workers from seven colonies at regular intervals throughout the reproductive part of the season. We show that the overall contribution of workers to male (drone) production is 4.2%, nearly 40 times higher than is generally reported, and is highest during reproductive swarming, when an average of 6.2% of the males genotyped are worker‐produced. Similarly, workers in our samples were 100 times more likely to have active ovaries than previously assumed. Worker reproduction is seasonally influenced and peaks when colonies are rearing new queens. Not all worker subfamilies contribute equally to reproduction. Instead, certain subfamilies are massively over‐represented in drone brood. By laying eggs within the period in which many colonies produce virgin queens, these rare worker subfamilies increase their direct fitness via their well‐timed sons.     

Influence of queen weight and colony origin on worker response in Solenopsis geminata

Abstract. The influence of weight and colony origin of the queen of Solenopsis geminata (F.) (Hymenoptera: Formicidae) on worker attraction is studied under laboratory conditions. In the first experiment, worker response to individual queens of different weight from the same colony is evaluated. Heavier queens are more attractive than smaller queens to their own workers. In subsequent experiments, the colony origin effect is investigated and worker response to a pair of queens of the same weight from the same or different colonies is compared. When queens are from the same colony, workers do not show a significant preference between queens. However, when queens are from a different colony, workers are significantly more attracted to their own queen than to the foreign queen. Finally, the response of workers to queens of different weight from the same or different colonies is investigated. In both cases, workers are significantly more attracted to a heavier queen than a lighter queen, even if the lighter queen is their own queen. A putative pheromonal component (E)‐6‐(1‐pentenyl)‐2H‐2‐pyranone, is not positively correlated with queen weight.     

Experimentally induced variation in the physical reproductive potential and mating success in honey bee queens

In honeybee colonies, reproduction is monopolized by the queen while her daughter workers are facultatively sterile. Caste determination is a consequence of environmental conditions during development, during which female larvae may become either queens or workers depending on their larval diet. This bipotency introduces significant variation in the reproductive potential of queen bees, with queens raised from young worker larvae exhibiting high reproductive potential and queens raised from older worker larvae exhibiting lower reproductive potential. We verify that low-quality queens are indeed produced from older worker larvae, as measured morphometrically (e.g., body size) and by stored sperm counts. We also show, for the first time, that low-quality queens mate with significantly fewer males, which significantly influences the resultant intracolony genetic diversity of the worker force of their future colonies. These results demonstrate a reproductive continuum of honeybee queens and provide insights into the reproductive constraints of social insects.     

Conflict over male parentage in stingless bees

Summary Stingless bees usually have one, singly-mated queen. This can lead to a genetic conflict of interest between the queen and the non-laying workers over who should produce the males. In many stingless bee species workers have developed ovaries and can produce male-destined eggs. In this study we compile the available data on who produces the males in stingless bees. Worker reproduction is common but less frequent than expected from predictions built on relatedness-based preferences of non-laying workers. We tested whether the pattern in worker reproduction can be explained best by queen control, by an arms race between workers and their queen, by the costs of losing workers to reproductive competition, or by phylogenetic constraints. The data are consistent with the view that there is ongoing conflict over male production that is resolved differently depending on the specific dynamics of costs and benefits of worker reproduction. There was also a role for phylogeny; Melipona workers often reproduced while Plebeia and Australian stingless bee workers seldom or never did. The high worker reproduction in Melipona may reflect low costs, because many of the replaced queen-laid eggs would become excess queens.Received 17 April 2003; revised 2 September 2003; accepted 5 September 2003.