Reassessing the role of the honeybee (Apis mellifera) Dufour's gland in egg marking

Dufour's gland secretion may allow worker honeybees to discriminate between queen-laid and worker-laid eggs. To investigate this, we combined the chemical analysis of individually treated eggs with an egg removal bioassay. We partitioned queen Dufour's gland into hydrocarbon and ester fractions. The bioassay showed that worker-laid eggs treated with either whole gland extract, ester fraction or synthetic gland esters were removed more slowly than untreated worker-laid eggs. However, the effect only lasted up to 20 h. Worker-laid eggs treated with the hydrocarbon fraction were removed at the same rate as untreated eggs. The amount of ester which reduced the egg removal rate was far higher than that naturally found on queen-laid or worker-laid eggs, and at natural ester levels no effect was found. Our results indicate that esters or hydrocarbons probably do not function as the signal by which eggs can be discriminated.     

Non-transferable signals on ant queen eggs

How biological systems resolve internal conflicts is a major evolutionary question. Social insect workers cooperate but also pursue individual interests, such as laying male eggs. The rewards of this individual selfishness can be reduced by policing, such as by killing worker-laid eggs. However, selfish individuals may evade policing. What factors prevent individuals from being able to evade policing? In the ant Pachycondyla inversa, workers kill (police) worker-laid eggs. Because the colony keeps eggs in piles and worker-laid and queen-laid eggs are chemically distinct, worker-laid eggs might become more acceptable once placed in the egg pile by odour transfer from touching queen-laid eggs. Here, we show that such “cue scrambling” does not occur. Worker-laid eggs that were sandwiched between three queen-laid eggs for 45 min were not more acceptable in a policing bioassay than control worker-laid eggs. Chemical analyses also showed that the surface hydrocarbon profile of these eggs was unchanged. Policing, therefore, is stable against this potential cheating mechanism probably because queen-laid eggs are made chemically distinct using chemicals, that are not easily transferred by physical contact.     

Reclaiming the crown: queen to worker conflict over reproduction in <Emphasis Type="Italic">Aphaenogaster cockerelli</Emphasis>

In many social taxa, reproductively dominant individuals sometimes use aggression to secure and maintain reproductive status. In the social insects, queen aggression towards subordinate individuals or workers has been documented and is predicted to occur only in species with a small colony size and a low level of queen–worker dimorphism. We report queen aggression towards reproductive workers in the ant species Aphaenogaster cockerelli, a species with a relatively large colony size and a high level of reproductive dimorphism. Through analysis of cuticular hydrocarbon profiles, we show that queens are aggressive only to reproductively active workers. Non-reproductive workers treated with a hydrocarbon typical for reproductives are attacked by workers but not by queens, which suggests different ways of recognition. We provide possible explanations of why queen aggression is observed in this species.     

Fertility signals in the bumblebee Bombus terrestris (Hymenoptera: Apidae)

In eusocial Hymenoptera, queen control over workers is probably inseparable from the mechanism of queen recognition. In primitively eusocial bumblebees (Bombus), worker reproduction is controlled not only by the presence or absence of a dominant queen but also by other dominant workers. Furthermore, it was shown that the queen dominance is maintained by pheromonal cues. We investigated whether there is a similar odor signal released by egg-laying queens and workers that may have a function as a fertility signal. We collected cuticular surface extracts from nest-searching and breeding Bombus terrestris queens and workers that were characterized by their ovarian stages. In chemical analyses, we identified 61 compounds consisting of aldehydes, alkanes, alkenes, and fatty acid esters. Nest-searching queens and all groups of breeding females differed significantly in their odor bouquets. Furthermore, workers before the competition point (time point of colony development where workers start to develop ovaries and lay eggs) differed largely from queens and all other groups of workers. Breeding queens showed a unique bouquet of chemical compounds and certain queen-specific compounds, and the differences toward workers decrease with an increasing development of the workers' ovaries, hinting the presence of a reliable fertility signal. Among the worker groups, the smallest differences were found after the competition point. Egg-laying females contained higher total amounts of chemical compounds and of relative proportions of wax-type esters and aldehydes than nest-searching queens and workers before the competition point. Therefore, these compounds may have a function as a fertility signal present in queens and workers.     

Queens and major workers of Acanthomyrmex ferox redistribute nutrients with trophic eggs

 In Acanthomyrmex ferox, two distinct egg-types are produced: reproductive eggs that give rise to offspring and trophic eggs that serve to distribute nutrients. Queens lay both reproductive and trophic eggs, while major and minor workers lay only trophic eggs in the presence of the queen. The queen lays on average 17% of the trophic eggs in a colony, while majors and minors produce 42% and 41%, respectively. The large proportion of trophic egg production by the queen and soldiers is quite remarkable, since ant queens are expected to focus entirely on reproduction and majors, which have a defensive function in many species, to be sterile. Received: 25 April 2000 / Accepted in revised form: 16 June 2000     

Postmating changes in cuticular chemistry and visual appearance in <Emphasis Type="Italic">Ectatomma tuberculatum</Emphasis> queens (Formicidae: Ectatomminae)

In the ectatommine ant Ectatomma tuberculatum, the visual appearance of queens changes after mating and ovarian development in that their cuticle turns from shiny to matte. In this study, we have shown that this change seems to be caused by 15-fold accumulation of hydrocarbons, in particular heptacosane that covers the multiple grooves present on the cuticular surface creating a wax coat in mated fully fertile queens. Analyses of the scrapped wax revealed that it is composed largely of heptacosane. Peak-by-peak comparison of the cuticular hydrocarbon (CHC) composition of mated, virgin with developed ovaries and virgin with nondeveloped ovaries revealed significant differences between the queen groups. Although the total amount of the CHC of virgin queens with developed ovaries was not higher than virgin queens that did not have developed ovaries, the composition showed a shift toward the mated queen. While it is possible that the large accumulation of hydrocarbons may give extra physical and chemical protection to queens, we propose that the switch in the relative abundance of heptacosane and nonacosane and perhaps of other components is indicative of being a mating and fertility cue. This is the first report in social insects where external chemical changes are accompanied by changes in visual appearance.     

Chemical profiles, division of labor and social status in Pachycondyla queens (Hymenoptera: formicidae)

Queens of the neotropical ponerine ant Pachycondyla cf. 'inversa' may co-operate during colony founding. One of several co-founding queens specializes in foraging, whereas the others remain in the nest and guard the brood. Division of labor is achieved by aggressive interactions, which result in the formation of dominance hierarchies. Gas chromatography and mass spectrometry of cuticular hydrocarbons obtained from live queens by SPME revealed consistent differences between the patterns of cuticular hydrocarbons of queens with high versus low rank: only high-ranking queens showed considerable amounts of cuticular pentadecane (n-C₁₅) and heptadecene (n-C₁₇:1). These two substances presumably originate from the queens' Dufour glands.     

Costs and benefits of subordinate queens in colonies of the ant,<Emphasis Type="Italic"> Leptothorax gredleri</Emphasis>

Queens of some species of social insects form dominance hierarchies in which only the top-ranking individual reproduces. Such unequal partitioning of reproduction can be stable when subordinate queens increase their inclusive fitness indirectly by helping to rear a related dominant queen's offspring. We investigated whether subordinate queens of the ant, Leptothorax gredleri, affect the reproductive success of the dominant queen. In laboratory colonies with two queens, first eggs were laid approximately 20 days later than in colonies with a single queen, presumably due to aggressive competition among nestmate queens. Nevertheless, two-queen colonies produced on average more brood than one-queen colonies, although egg laying was completely or almost completely monopolized by one single queen. The presence of a subordinate queen therefore appears to increase the productivity of the dominant.     

A non-policing honey bee colony (Apis mellifera capensis)

In the Cape honey bee Apis mellifera capensis, workers lay female eggs without mating by thelytokous parthenogenesis. As a result, workers are as related to worker-laid eggs as they are to queen-laid eggs and therefore worker policing is expected to be lower, or even absent. This was tested by transferring worker- and queen-laid eggs into three queenright A. m. capensis discriminator colonies and monitoring their removal. Our results show that worker policing is variable in A. m. capensis and that in one colony worker-laid eggs were not removed. This is the first report of a non-policing queenright honey bee colony. DNA microsatellite and morphometric analysis suggests that the racial composition of the three discriminator colonies was different. The variation in policing rates could be explained by differences in degrees of hybridisation between A. m. capensis and A. m. scutellata, although a larger survey is needed to confirm this.     

Fertility signaling—the proximate mechanism of worker policing in a clonal ant

In eusocial insects, the ability to regulate reproduction relies on cues that signal the presence of fertile individuals. We investigated the variation of cuticular hydrocarbons (CHCs) with reproductive status in Platythyrea punctata, an ant, in which all workers are capable of producing daughters from unfertilized eggs (thelytoky). Who reproduces is determined through dominance and worker policing. New reproductives, which developed their ovaries after separation from an old reproductive for a short period of time, were attacked by nonreproductives upon reintroduction into their colony. In contrast, aggression against new reproductives with fully developed ovaries, which had been separated over a longer period, was initiated by fights between old and new reproductives. CHC profiles varied with ovarian development. New reproductives were only attacked when they expressed a CHC profile similar to old reproductives, but not when it was similar to that of nonreproductives. CHCs appear to signal the fertility of individuals and induce policing behavior towards surplus reproductive workers.     

Can Dufour��s gland compounds honestly signal fertility in the primitively eusocial wasp Ropalidia marginata?

Unlike queens of typical primitively eusocial species, Ropalidia marginata queens are docile and non-interactive, and hence cannot be using dominance to maintain their status. It appears that the queen maintains reproductive monopoly through a pheromone, of which the Dufour's gland is at least one source. Here, we reconfirm earlier results showing that queens and workers can be correctly classified on a discriminant function using the compositions of their respective Dufour's glands, and also demonstrate consistent queen-worker differences based on categories of compounds and on single compounds also in some cases. Since the queen pheromone is expected to be an honest signal of the fecundity of a queen, we investigate the correlation of Dufour's gland compounds with ovarian activation of queens. Our study shows that Dufour's gland compounds in R. marginata correlate with the state of ovarian activation of queens, suggesting that such compounds may portray the fecundity of a queen, and may indeed function as honest signals of fertility.     

Queen movement during colony emigration in the facultatively polygynous ant Pachycondyla obscuricornis

In ants, nest relocations are frequent but nevertheless perilous, especially for the reproductive caste. During emigrations, queens are exposed to predation and face the risk of becoming lost. Therefore the optimal strategy should be to move the queen(s) swiftly to a better location, while maintaining maximum worker protection at all times in the new and old nests. The timing of that event is a crucial strategic issue for the colony and may depend on queen number. In monogynous colonies, the queen is vital for colony survival, whereas in polygynous colonies a queen is less essential, if not dispensable. We tested the null hypothesis that queen movement occurs at random within the sequence of emigration events in both monogynous and polygynous colonies of the ponerine ant Pachycondyla obscuricornis. Our study, based on 16 monogynous and 16 polygynous colony emigrations, demonstrates for the first time that regardless of the number of queens per colony, the emigration serial number of a queen occurs in the middle of all emigration events and adult ant emigration events, but not during brood transport events. It therefore appears that the number of workers in both nests plays an essential role in the timing of queen movement. Our results correspond to a robust colony-level strategy since queen emigration is related neither to colony size nor to queen number. Such an optimal strategy is characteristic of ant societies working as highly integrated units and represents a new instance of group-level adaptive behaviors in social insect colonies.     

Daughters inherit colonies from mothers in the 'living-fossil' ant Nothomyrmecia macrops

Newly mated queens of monogynous (single queen) ants usually found their colonies independently, without the assistance of workers. In polygynous (multiple queen) species queens are often adopted back into their natal nest and new colonies are established by budding. We report that the Australian 'living-fossil' ant, Nothomyrmecia macrops, is exceptional in that its single queen can be replaced by one of the colony's daughters. This type of colony founding is an interesting alternative reproductive strategy in monogynous ants, which maximizes fitness under kin selection. Successive queen replacement results in a series of reproductives over time (serial polygyny), making these colonies potentially immortal. Workers raise nieces and nephews (relatedness h 0.375) the year after queen replacement. Although N. macrops is 'primitive' in many other respects, colony inheritance is likely to be a derived specialization resulting from ecological constraints on solitary founding.     

Sumo wrestling in ants: major workers fight over male production in<Emphasis Type="Italic"> Acanthomyrmex ferox</Emphasis>

In the myrmicine ant Acanthomyrmex ferox, major workers have the same number of ovarioles as queens, thrice that of minor workers, making them well suited for egg-laying. In the queen's presence, infrequent aggression allows ranking of majors but they lay only unviable trophic eggs. Major workers engage each other, but not the minors, in antennal boxing and spectacular shaking contests, a novel interaction in ants. The absence of reversals allows a clear ranking of major workers. After queen removal, aggression becomes very frequent, but previous ranking is maintained. All majors start laying reproductive eggs although they show a skew in ovary development according to ranking. The dominant major, however, actively patrols the egg-pile and cannibalises eggs laid by subordinates.     

Similar policing rates of eggs laid by virgin and mated honey-bee queens

Worker-policing is a well-documented mechanism that maintains functional worker sterility in queenright honey-bee colonies. Unknown, however, is the source of the egg-marking signal that is thought to be produced by the queen and used by policing workers to discriminate between queen- and worker-laid eggs. Here we investigate whether mating is necessary for the queen to produce the egg-marking signal. We compare the removal rate of eggs laid by virgin queens and compare this rate with that of eggs laid by mated queens. Our results show that mating does not affect the acceptability of eggs, suggesting that physiological changes linked to the act of mating do not play a role in the production of the queens egg-marking signal.     

Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

Under complementary sex determination (CSD), females of Hymenoptera arise from diploid, fertilized eggs and males from haploid, unfertilized eggs. Incidentally, fertilized eggs that inherit two identical alleles at the CSD locus will develop into diploid males. Diploid males are usually unviable or sterile. In a few species, however, they produce diploid sperm and father a triploid female progeny. Diploid males have been reported in a number of social Hymenoptera, but the occurrence of triploid females has hardly ever been documented. Here, we report the presence of triploid females, diploid males, and diploid sperm (produced by diploid males and stored in queen spermathecae) in the ant Tapinoma erraticum. Moreover, we show variations in the frequency of triploids among female castes: Triploid females are more frequent among workers than virgin queens; they are absent among mated, reproductive queens. The frequency of triploid workers also varies between populations and between nests within populations.     

Decision rules for egg recognition are related to functional roles and chemical cues in the queenless ant Dinoponera quadriceps

The capacity to distinguish colony members from strangers is a key component in social life. In social insects, this extends to the brood and involves discrimination of queen eggs. Chemical substances communicate colony affiliation for both adults and brood; thus, in theory, all colony members should be able to recognize fellow nestmates. In this study, we investigate the ability of Dinoponera quadriceps workers to discriminate nestmate and non-nestmate eggs based on cuticular hydrocarbon composition. We analyzed whether cuticular hydrocarbons present on the eggs provide cues of discrimination. The results show that egg recognition in D. quadriceps is related to both age and the functional role of workers. Brood care workers were able to distinguish nestmate from non-nestmate eggs, while callow and forager workers were unable to do so. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Scent of a queen—cuticular hydrocarbons specific for female reproductives in lower termites

In social insects, it is assumed that signals of the queen inform nestmates about her reproductive status. Thus, workers forego their own reproduction if the queen signals high fertility. In hemimetabolous termites, little is known about reproductive inhibition, but evidence exists for a royal-pair control. Workers of lower termites exhibit a high developmental flexibility and are potentially able to become reproductives, but the presence of a fertile reproductive restrains them from reaching sexual maturity. The nature of this control, however, remains unknown. Here, we report on qualitative differences in cuticular hydrocarbon profiles between queens and workers of the basal drywood termite Cryptotermes secundus. Queens were characterized by a shift to long-chained and branched hydrocarbons. Most remarkably, similar chemical patterns are regarded as fertility cues of reproductives in social Hymenoptera. This might suggest that both groups of social insects convergently evolved similar chemical signatures. The present study provides deeper insights into how termites might have socially exploited these signatures from sexual communication in their cockroach-like ancestor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aggressions and size-related fecundity of queenless workers in the ant <Emphasis Type="Italic">Cataglyphis cursor</Emphasis>

In social hymenoptera, the reproductive division of labor is often linked to differences in individual body size with the reproductive caste (the queen) being larger than the workers. Likewise, the reproductive potential may vary with size within the worker caste and could affect the evolution of worker size in social insects. Here, we tested the relationship between worker size and reproductive potential in the facultative parthenogenetic ant Cataglyphis cursor. Colonies are headed by a multiply mated queen, but workers can produce gynes (virgin queens) and workers by thelytokous parthenogenesis after the queen's death. We observed the behaviour of workers (n = 357) until the production of gynes (212 h over 3 months) in an orphaned colony (mated queen not present). The size of workers was measured, and their paternal lineage determined using six microsatellite markers, to control for an effect of patriline. Larger workers were more likely to reproduce and lay more eggs indicating that individual level selection could take place. However, paternal lineage had no effect on the reproductive potential and worker size. From the behavioural and genetic data, we also show for the first time in this species, evidence of aggressive interactions among workers and a potential for nepotism to occur in orphaned colonies, as the five gynes produced belonged to a single paternal lineage.     

Irregular brood patterns and worker reproduction in social wasps

The potential for reproductive conflict among colony members exists in all social insect societies. For example, queens and workers may be in conflict over the production of males within colonies. Kin selection theory predicts that in a colony headed by a multiply-mated queen, worker reproduction is prevented by worker policing in the form of differential oophagy. However, few studies have demonstrated that workers actually lay eggs within queenright colonies. The purpose of this study was to determine if workers laid male eggs within unmanipulated queen-right colonies of the polyandrous social wasps Vespula maculifrons and V. squamosa. We focused our analysis on an unusual brood pattern within colonies, multiple egg cells. We were primarily interested in determining if individuals reared in these irregular circumstances were queen or worker offspring. To address this question, we genotyped 318 eggs from eight V. maculifrons and two V. squamosa colonies. No worker‑reproduction was detected in any of the queenright colonies; all of the eggs found in multiple egg cells were consistent with being queen‑produced. However, the frequency of multiple egg cells differed among colonies, suggesting that queens vary in the frequency of errors they make when laying eggs within cells. Finally, we suggest that workers may not be laying eggs within queenright colonies and that worker reproduction may be controlled through mechanisms other than differential oophagy in polyandrous Vespula wasps.