Characterization of cuticular hydrocarbons of diploid and haploid males, workers and queens of the stingless bee Melipona quadrifasciata

Males, queens and workers of stingless bees show differences in external morphology, behaviour and roles within a colony. In addition, each individual has a cuticular chemical signature responsible for mutual communication that is essential for maintaining the integrity of the colony. In this paper we characterize the cuticular hydrocarbon composition of newly emerged diploid and haploid males, workers and virgin queens of Melipona quadrifasciata by gas chromatography-mass spectrometry (GC/MS) analysis. This is the first time that the cuticular profile of diploid males in a species of stingless bee has been characterized. We found differences in the cuticular hydrocarbon composition among males, workers and virgin queens, recording both qualitative and quantitative differences among individuals of different phenotypes. However, no compound was found exclusively in diploid males. The cuticular chemical profiles of haploid and diploid males were very similar to those of workers. Moreover, the cuticular lipids of males and workers were significantly different from those of queens. Tricosane, pentacosene-2 and 7-methyl-heptacosane were the compounds responsible for this significant separation. This result correlates with the behavioural and morphological differences among these phenotypes.     

Rank integration in dominance hierarchies of host colonies by the paper wasp social parasite Polistes sulcifer (Hymenoptera, Vespidae)

In multiple-foundress nests of the wasp Polistes dominulus, dominance hierarchies are established among foundresses, and only the dominant (=alpha) individual lays eggs. The alpha female can be distinguished from subordinate females and workers on the basis of the proportions of some hydrocarbons present on the cuticle, suggesting that chemical signaling of her reproductive status could occur. P. dominulus is also the host species of the obligate social parasite Polistes sulcifer. After aggressively usurping host colonies and behaviorally replacing the host alpha female, parasites are characterized by a change in the proportions of their cuticular hydrocarbons to match that of the host cuticular profile at both species and colony levels. In the current study, we demonstrate that P. sulcifer queens also modify their cuticular hydrocarbon proportions after usurpation to match that of the host alpha female. Parasite females, therefore, acquire the dominant rank in host colonies both reproductively and chemically by mimicking the typical alpha profile of the host. Parasite females were not able to fully inhibit ovary development in host foundresses, and 10 days after usurpation, parasites, alpha and beta foundresses show similar chemical profiles and ovarian development.     

Workers and queens of the European hornetVespa crabro L. have colony-specific cuticular hydrocarbon profiles (Hymenoptera: Vespidae)

Summary Cuticular hydrocarbons were extracted from sixty individual workers from six colonies ofVespa crabro L. and analyzed by combined gas-chromatography/mass spectrometry. Discriminant analysis of the cuticular hydrocarbon profiles of workers and queens showed that the wasps could be grouped by colony and by caste. Stepwise discriminant analysis selected the components which were weighted most heavily in these analyses. Different combinations of cuticular hydrocarbons were important in grouping workers by colony, queens and workers by colony, and workers and queens by caste.     

Castes and asynchronous colony cycle in Polybia bistriata (Fabricius) (Hymenoptera: Vespidae)

Neotropical swarm-founding wasps, the Epiponini, are an outstanding group of social insects whose societies are polygynic and complex nest builders. Caste dimorphism in these wasps ranges from incipient to highly distinct. Morphometric analyses of nine body parts, ovarian status, relative age and development of the 5th gastral sternite gland (Richards' gland) of Polybia bistriata Fabricius were undertaken in order to estimate caste differentiation in nine colonies. Morphological and physiological data were used in multivariate analyses in order to evaluate the level of discrimination between inseminated and non-inseminated females. Clear physiological differences were found: queens had highly developed ovaries and they were inseminated, and workers had totally undeveloped ovaries or they had few developed oocytes (only in two colonies), but in both cases insemination was not detected. ANOVA and discriminant function analysis detected slight, but significant differences between castes. In relation to colony cycle, colonies were considered to be in the following stages: one in pre-emergence, four in worker production and four in sexuals production. Richards' gland analyses indicated that in small colonies (<100 females) queens had a less developed gland than in medium (100-200 females), and large colonies (>200 females). Taking the whole data, it was possible to conclude that caste differences were slight, but more evident in some phases of the colony cycle, a phenomenon previously described for other epiponines.     

Comparative study of the cuticular hydrocarbon composition of Melipona bicolor Lepeletier, 1836 (Hymenoptera, Meliponini) workers and queens

In social insects, cuticular hydrocarbons are involved in species, kin, caste and nestmate recognition. Gas chromatography and mass spectrometry were used to compare the cuticular hydrocarbon composition of workers, males and queens of Melipona bicolor. The cuticular hydrocarbon composition of this species was found to consist mainly of C23, C25:1, C25, C27:1, C27, C29:1 and C29, which are already present in imagoes that have not yet abandoned the brood cell. This composition varied quantitatively and qualitatively between and within the castes and sexes. The newly emerged workers and young queens (virgins) had similar cuticular hydrocarbon profiles, which were different from those of the males. When the females start executing their tasks in the colony, the cuticular hydrocarbon profile differences appear. The workers have less variety, while the queens conserve or increase the number of cuticular hydrocarbon compounds. The queens have more abdominal tegumentary glands than the workers, which apparently are the source of the new cuticular compounds.     

Cuticular lipids correlate with age and insemination status in queen honeybees

Eusocial insects exhibit reproductive division of labour, in which one or a few queens perform almost all of the reproduction, while the workers are largely sterile and assist in rearing their siblings. Consequently, many of the colony’s tasks (e.g. nest construction and brood rearing) should be modulated depending on whether the queen is fertile. Here, we tested whether queens’ cuticular lipids could provide reliable signals of fertility in the honeybee Apis mellifera, as they do in other social Hymenoptera. Specifically, we tested whether cuticular lipids differ between virgin queens of different ages, and between queens exposed to different artificial insemination treatments being semen (sperm and seminal fluid), seminal fluid only or saline control. Using gas chromatography–mass spectrometry, we found 27 lipids: 21 different hydrocarbons, namely alkanes, alkenes and dienes, as well as six wax esters. The lipid profile changed dramatically in the first 10 days after eclosion, and there were differences in lipid composition between virgin and artificially inseminated queens. Insemination with semen, seminal fluid or saline did not result in distinct chemical profiles. Our findings indicate that the physical stimulus of insemination was responsible for the observed changes in the cuticular profile in honeybee queens. Our results demonstrate that cuticular lipid profiles encode information on queen age, fertility and mating status, which could in principle be utilised by workers and rival queens.     

Glandular Epithelium as a Possible Source of a Fertility Signal in Ectatomma tuberculatum (Hymenoptera: Formicidae) Queens

The wax layer covering the insect''s cuticle plays an important protective role, as for example, uncontrolled water loss. In social insects, wax production is well-known in some bees that use it for nest building. Curiously, mated-fertile queens of the ant Ectatomma tuberculatum produce an uncommon extra-wax coat and, consequently queens (mated-fertile females) are matte due to such extra cuticular hydrocarbon (CHC) coat that covers the cuticle and masks the brightness of the queens'' cuticle while gynes (virgin-infertile queens) are shiny. In this study, histological analysis showed differences in the epidermis between fertile (i.e., queens or gynes with highly ovarian activity) and infertile females (gynes or workers with non developed ovaries). In fertile females the epidermis is a single layer of cubic cells found in all body segments whereas in infertile females it is a thin layer of flattened cells. Ultrastructural features showed active secretory tissue from fertile females similar to the glandular epithelium of wax-producing bees (type I gland). Different hypotheses related to the functions of the glandular epithelium exclusive to the E. tuberculatum fertile queens are discussed.     

Castes in the neotropical social wasp Leipomeles dorsata (Fabricius) (Hymenoptera: Vespidae): a window for workers achieving a new status in the colony

Morphometric studies performed in several species of Neotropical social wasps from the tribe Epiponini showed that in some species there are marked differences between castes, while other species present highly distinct castes with differences only in ovarian development. This work analyzed females from six colonies of the social wasp Leipomeles dorsata (Fabricius) in which queens (egglayers) and workers showed differences in ovarian development and coloration. We propose that wasps with developed ovaries (egglayers) and coloration similar to those of workers are possibly intermediates that obtained the status of queens in the colony.     

Ovarian activity correlates with extreme changes in cuticular hydrocarbon profile in the highly polygynous ant, Linepithema humile

In social insects, cuticular hydrocarbons of adults may vary with ovarian activity. Such variations are suggested to function in the regulation of reproduction within colonies. The modification of the CH profile with ovarian activity is usually interpreted as a signal of fertility causing workers to refrain from reproducing in the presence of the queen. We examined the effect of ovarian activity on the CH profiles in the Argentine ant Linepithema humile, a species where workers lack ovaries and are completely sterile. Our data show considerable differences in the CH profiles between fertile and infertile individuals. These differences are mainly qualitative. The CH profile strongly changes at the start of egg laying, both in mated and unmated queens. These results show that variation in cuticular hydrocarbons with ovarian activity in ants is not restricted to species with worker reproduction. We propose that in the Argentine ant, the cuticular hydrocarbons of laying queens correspond to a signal of fertility involved in the regulation of various aspects of reproduction, such as the rearing of new sexuals.     

Recognition of social parasites as nest-mates: adoption of colony-specific host cuticular odours by the paper wasp parasite Polistes sulcifer.

Colonies of the polistine wasp Polistes dominulus are parasitized by the permanent worker-less social parasite Polistes sulcifer. After usurpation of the host colony, parasite females are characterized by a change in the relative proportions of their cuticular hydrocarbons to match those of the host species. In this paper we present evidence from field data and laboratory experiments that P. sulcifer females adopt a colony-specific host odour that facilitates their acceptance by host females of the usurped colony. Presentation experiments demonstrate that parasite females are recognized as foreign individuals by workers of other parasitized nests. We show that the modification of parasite cuticular compounds is sufficient for this recognition. This provides evidence that, after invasion, P. sulcifer queens do not require appeasement or propaganda substances for their acceptance by host colonies. Furthermore, multivariate discriminant analysis of the cuticular hydrocarbon proportions of the parasites after usurpation assigns the parasites together with P. dominulus females of their own host colony. To the authors' knowledge, this is the first confirmation that social parasites adopt colony-specific host odours.     

The role of juvenile hormone in dominance behavior,reproduction and cuticular pheromone signaling in the caste-flexible epiponine wasp, <Emphasis Type="Italic">Synoeca surinama</Emphasis>

Background

The popular view on insect sociality is that of a harmonious division of labor among two morphologically distinct and functionally non-overlapping castes. But this is a highly derived state and not a prerequisite for a functional society. Rather, caste-flexibility is a central feature in many eusocial wasps, where adult females have the potential to become queens or workers, depending on the social environment. In non-swarming paper wasps (e.g., Polistes), prospective queens fight one another to assert their dominance, with losers becoming workers if they remain on the nest. This aggression is fueled by juvenile hormone (JH) and ecdysteroids, major factors involved in caste differentiation in most eusocial insects. We tested whether these hormones have conserved aggression-promoting functions in Synoeca surinama, a caste-flexible swarm-founding wasp (Epiponini) where reproductive competition is high and aggressive displays are common.

Results

We observed the behavioral interactions of S. surinama females in field nests before and after we had removed the egg-laying queen(s). We measured the ovarian reproductive status, hemolymph JH and ecdysteroid titers, ovarian ecdysteroid content, and analyzed the cuticular hydrocarbon (CHC) composition of females engaged in competitive interactions in both queenright and queenless contexts. These data, in combination with hormone manipulation experiments, revealed that neither JH nor ecdysteroids are necessary for the expression of dominance behaviors in S. surinama. Instead, we show that JH likely functions as a gonadotropin and directly modifies the cuticular hydrocarbon blend of young workers to match that of a reproductive. Hemolymph ecdysteroids, in contrast, are not different between queens and workers despite great differences in ovarian ecdysteroid content.

Conclusions

The endocrine profile of S. surinama shows surprising differences from those of other caste-flexible wasps, although a rise in JH titers in replacement queens is a common theme. Extensive remodeling of hormone functions is also evident in the highly eusocial bees, which has been attributed to the evolution of morphologically defined castes. Our results show that hormones which regulate caste-plasticity can lose these roles even while caste-plasticity is preserved.

     

Aggression and worker control of caste fate in a multiple-queen wasp, Parachartergus colobopterus

Although famously cooperative, social insect colonies harbour considerable potential for genetic conflict among colonymates. This conflict may be expressed behaviourally as aggression by workers. We investigated aggression in 34 colonies of the wasp Parachartergus colobopterus, by evaluating the characteristics of both instigators and victims of aggressive interactions. We estimated genetic relatedness and queen number using DNA microsatellites and found that workers and emerging females should be most in conflict over the caste of the latter when there are many queens on the nest. We found that aggressive interactions are more likely to involve older workers attacking either males or younger workers, and that victim and aggressor females have more ovarian development than randomly sampled colonymates. Moreover, mated females with low levels of ovarian development relative to active queens were also more likely to be aggressors and victims than were randomly sampled females. Aggression among females supports the hypothesis that older workers use aggression towards younger females as a means of policing the development of emerging females into queens. Workers also may use aggression to suppress the reproduction of some mated females. Our findings thus support the hypothesis that genetic conflicts of interest motivate worker aggression in swarm-founding wasp colonies.     

Chemical correlates of reproduction and worker policing in a myrmicine ant

In a number of wasps, bees, and in particular ponerine ants, quantitative and qualitative variation in the profile of cuticular hydrocarbons is associated with variation in fecundity and is likely to serve for communicating the reproductive status of an individual. Here, we demonstrate that the chemical profile on the cuticle of fertile workers and queens of the myrmicine ant Temnothorax unifasciatus is different from that of non-reproductive workers. Fertility and apparently also cuticular signatures are reversible under the influence of policing by worker aggression. Though no policing by egg-eating occurs in this species, queen and worker laid eggs also differed in their chemical profile.     

Colony dynamics in a primitively eusocial halictine beeLasioglossum (Dialictus) zephyrum (Hymenoptera: Halictidae)

Summary Proximate control of colony dynamics was studied in the primitively eusocial halictine beeLasioglossum (Dialictus) zephyrum using allozyme markers. The results indicate that workers produce on average 15% of the male brood (range=0–50%) in small laboratory colonies made up of unrelated, single-generation, uninseminated females. This proportion is not influenced by colony size, but is influenced by the relative size of the queen. Large queens are more successful in dominating their workers than are small queens, the queen being defined as the female that is the mother of most of the brood produced in the colony. Older and larger females tend to become queens. Thus, while small differences in age (up to 4 days) influence which female becomes a queen, her ability to control her workers is primarily influenced by her relative size. The proportion of reproduction that is co-opted by the queen is negatively correlated with colony reproductivity (the number of males/day/female). Colony reproductivity is also negatively correlated with the standard deviation in size among females.     

Chemical profiles of mated and virgin queens, egg-laying intermorphs and workers of the ant Crematogaster smithi

Many colonies of the North American ant Crematogaster smithi contain a “third female caste” in addition to queens and workers. These “intermorphs” are morphological intermediate of queens and workers and have well-developed ovaries but lack a spermatheca for the storage of sperm. They are specialised for laying large numbers of unfertilised, viable eggs, most of which serve as food for larvae and adults, though a few may eventually develop into males. Based on the assumption that cuticular hydrocarbons (CHCs) in social insects honestly signal the reproductive status of an individual we investigated the CHC of mated mature queens, virgin queens, intermorphs and workers. We expected intermorphs to show chemical profiles intermediate between those of mated queens and non-reproductive workers. A discriminant analysis of the chemical profiles reliably separated queens, virgin queens, and workers, but failed to distinguish between queens and intermorphs even though workers were apparently capable of doing so.     

Development of physogastry in the queen of the fungus-growing termite Macrotermes michaelseni (Isoptera: Macrotermitinae)

Female reproductives of the fungus-growing termite Macrotermes michaelseni never showed signs of physogastry if they were kept in petri dishes to establish an incipient colony. During the 200 days of observation, their corpora allata volume, juvenile hormone titre and number of active ovarioles remained on a more or less constant level, and their gut was filled with soil. In contrast, these parameters are enhanced in partially and especially in fully physogastric queens, and these females had only a transparent liquid in the gut.Application of the juvenile hormone mimic ZR 515 to young females accelerated yolk incorporation, increased the number of active ovarioles and brought about the release of the soil from the gut. The same changes were induced by adding workers to increase the population of incipient colonies from about 45 to 400. This also resulted in an increase of the corpus allatum volume and of the juvenile hormone titre in young females. Their intestine was in this case filled with transparent liquid associated with the increased population of workers. It was, therefore, concluded that the development of physogastry depends on a positive feedback: the more workers are with the young female, the more nutrients are available and the more juvenile hormone the female produces, the more eggs are laid and the more workers can again develop to nurture the queen.     

Virgin wasps develop ovaries on par with mated females, but lay fewer eggs

In the primitively eusocial wasp Ropalidia marginata, mating is not necessary for a female wasp to develop her ovaries, lay eggs, and even to become the sole egg layer of her colony despite the presence of other mated nestmates. Here, we show that virgin wasps do not differ from their mated counterparts in the extent and rapidity of their ovarian development, in the proportion of individuals that build a nest and laid eggs, and in the time taken to do so. However, a significantly larger proportion of virgin females showed resorbing oocytes, and laid fewer eggs as compared to mated individuals. Thus, virgin females have the ability to develop ovaries and lay eggs but also to refrain from necessarily laying all mature eggs produced, before mating opportunities arise. This dual ability would be adaptive in haplodiploid, tropical species with perennial nesting cycles and frequent opportunities for workers to become replacement queens or solitary nest foundresses throughout the year.     

Queen reproduction, chemical signalling and worker behaviour in polygyne colonies of the ant Formica fusca

Potential reproductive conflicts are common in social insects and may occur between and within castes. In multiqueen (polygyne) colonies of ants, reproductive conflicts among coexisting queens may be resolved by aggressive interactions and/or by pheromonal signalling. Pheromonal signals may be directed at workers, which may adjust the reproductive shares of queens behaviourally. Workers are expected to favour a queen that is more likely to be their mother and to produce offspring of close kin to them. We used microsatellite markers to study reproductive partitioning between cobreeding Formica fusca queens in a laboratory experiment where workers received a choice between two queens. We expected queens of this species to communicate their reproductive status by chemical communication, because no aggressive interactions between queens have been observed. We found that queens of different reproductive status (with majority, minority or no production) differed in their cuticular hydrocarbon (CHC) profiles. The fecundity of a queen was associated with worker behaviour; the higher a queen's fecundity, the more attention she received from workers. Our results suggest that a queen fecundity signal is encoded in her CHC profile and acts as a pheromone for workers, which respond to the signal by discriminating between queens. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Nestmate recognition and identification of cuticular hydrocarbons composition in the swarm founding paper wasp Ropalidia opifex

Eusocial polistine wasps comprise species in which new colonies are founded by single wasps or groups of foundresses (independent founding species) or by swarms of queens and workers (swarm founding species). The first ones have relatively small societies generally comprising only one egg-laying queen, while the second ones have larger colonies where several egg-laying queens are usually present. These differences in social organisation are expected to influence the acceptance threshold of conspecifics in the colony and the intra-colony communication modalities. In this paper, we showed by field experiments that Ropalidia opifex (a swarm founding species) presents a good discrimination between alien and nestmate conspecifics. Moreover by GC/MS, we identified 19 cuticular hydrocarbons in this species, and we demonstrated that cuticular signature in this species depends mainly on colony membership, not showing any correlation with fertility.     

Habitat age, breeding system and kinship in the ant Formica fusca

In polygyne ants (multiple queens per colony) factors that affect the distribution and survival of queens may play a key role in shaping the population-wide mating system and colony kin structure. The aim of this paper was to study the breeding system in two populations of different age in the facultatively polygyne ant Formica fusca. Both the observed numbers of queens, and the relatedness patterns among queens, workers and colony fathers were compared in two adjacent populations (ages 17 years and > 100 years) in Southern Finland. The results showed that both the mating system and colony kin structure differed between the study populations. In the old population the relatedness among workers, queens and colony fathers was high. The queens were also related to their mates, resulting in significant inbreeding in workers, but not in queens. Finally, the number of queens per colony fluctuated between years, suggesting queen turnover, and nest-mate queens shared their reproduction unequally (reproductive skew). In the younger population relatedness among queens and workers was lower than in the old population, and the colony fathers were unrelated. Furthermore, inbreeding was absent, and no conclusive evidence was found for reproductive skew among nest-mate queens. Finally, the number of queens per colony appeared more stable between years, although queen turnover occurred also in this population. The observed differences in dispersal and mating behaviour are discussed in the light of a potential connection between population age and habitat saturation.