Egg marking pheromones of anarchistic worker honeybees (Apis mellifera)

In honeybees, worker policing via egg eating enforces functionalworker sterility in colonies with a queen and brood. It is thoughtthat queens mark their eggs with a chemical signal, indicatingthat their eggs are queen-laid. Worker-laid eggs lack this signaland are, therefore, eaten by policing workers. Anarchistic workerhoneybees have been hypothesized to circumvent worker policingby mimicking the queen egg-marking signal. We investigated thisphenomenon by relating chemical profiles of workers and theireggs to egg acceptability. We found that the ability of someworkers (anarchistic workers in queenright colonies and deviantworkers from a queenless colony) to lay more acceptable eggsis due to them producing significant amounts of queen-like estersfrom their Dufour's gland. These esters appear to be transferredto eggs during laying and increase egg survival. However, theseesters cannot be the normal queen egg-marking signal, as theyare generally absent from queen-laid eggs and only increasethe short-term persistence of worker-laid eggs, because only7–30% of anarchistic worker-laid eggs persisted to hatchingversus 91–92% of queen-laid eggs. All workers can producesome esters, but only workers that greatly increase their esterproduction lay more acceptable eggs. The production of estersappears to be a flexible response, as anarchistic workers rearedin queenless colonies did not increase their ester production,while some deviant workers in queenless colonies did increasetheir ester production.     

Several workers lay eggs in the same brood cell in queenless honey bee (Apis mellifera) colonies

Queenless honey bee (Apis mellifera) colonies are often characterized by the presence of multiple eggs in brood cells. This is surprising because only one egg can be reared to maturity per cell. Moreover, worker honey bees cannot produce many eggs per day. There are several reasons that could explain the presence of multiple eggs in single cells: a) workers cannot control how many eggs they release; in this case we would expect all eggs to be from the same mother; b) excess eggs could be provided as food for the first larva to hatch in the absence of adequate brood care and this would again result in all eggs in one cell sharing the same mother; c) the number of cells available for oviposition may be limiting, obliging workers to lay eggs in cells that already contain eggs, resulting in eggs of mixed maternity. Here we show that the majority of brood cells in queenless colonies contain eggs from multiple mothers. Therefore our results suggest that the presence of multiple eggs in brood cells arises from a limitation on the number of suitable cells available for oviposition. Received 29 September 2008; revised 21 November 2008; accepted 24 November 2008.     

Egg discrimination by workers in <Emphasis Type="Italic">Diacamma</Emphasis> sp. from Japan

In eusocial Hymenoptera, workers usually cannot mate but can lay male-destined haploid eggs. In contrast, in many species, worker reproduction is regulated by means of worker policing, which consists of two behavioral categories: aggression towards workers that have developed ovaries, and the destruction of worker-laid eggs. We focused on the latter behavior in the ant Diacamma sp. from Japan, in which a mated worker (gamergate) serves as the functional queen in each colony. An experiment in which we introduced both worker-laid eggs and gamergate-laid eggs from foreign colonies showed that workers preferentially destroyed the worker-laid eggs. This indicates that workers can discriminate worker-laid eggs from gamergate-laid eggs. In contrast, gamergates destroyed all introduced eggs that they discovered. Our chemical analysis of egg surface substances revealed that the worker-laid eggs clearly differed from gamergate-laid eggs in their surface hydrocarbons (SHCs), with the former containing more long-chain components. However, a bioassay to test whether specific components of the SHCs trigger the observed discrimination behavior by workers remains to be done.     

Nepotism and brood reliability in the suppression of worker reproduction in the eusocial Hymenoptera

In many eusocial Hymenoptera, workers prevent each other from producing male offspring by destroying worker-laid eggs. Kin selection theory predicts that such 'worker policing' behaviour can evolve by increasing the average relatedness between workers and their male brood. Alternatively, if worker-laid eggs are of low relative viability, their replacement would increase the developmental reliability of the brood. Less colony investment in terms of time and resources would be lost on poor males. This gain is independent of the relatedness of the males. Unfortunately, both nepotistic and group efficiency benefits can simultaneously accrue with the replacement of worker-laid eggs. Therefore, worker behaviour towards eggs cannot completely resolve whether both processes have been equally evolutionarily important. Adequate resolution requires the presentation of worker-produced brood of various ages. The stage at which brood are replaced can discriminate whether worker policing occurs owing to a preference for closer genetic kin, a preference for the more reliable brood or both.     

Interaction between the solitary bee Chelostoma florisomne and its nest parasite Sapyga clavicornis − empty cells reduce the impact of parasites

1. Nesting behaviour and interactions between the bee Chelostoma florisomne (L.) (Megachilidae) and its nest parasite Sapyga clavicornis (L.) (Sapygidae) were studied through continual observations of individuals and dissections of bee nests. Protection of bee offspring is based on (1) the bee’s discovery and removal of parasite eggs deposited prior to the construction of a cell closure, (2) minimising the time when fully provisioned cells might be parasitised successfully, and (3) the construction of empty cells in front of brood cells. 2. An empty cell was found in front of 64.4% of all brood cells and, if the outermost brood cell in a nest was excluded, in front of 74.3% of inner brood cells. A vestibule closure is most often constructed in front of the outermost brood cell. 3. Following oviposition, the bee made only five flights, which together lasted 6–13 min, to construct a cell closure. A cell closure does not prevent the nest parasite from oviposition inside the brood cell, however, and parasite eggs deposited through the cell closure are not detected and removed by the bee. Only an additional cell closure, i.e. the formation of an empty cell, may protect a brood cell when the bee is not in the nest. The nest parasite often oviposited through the additional cell closure but its offspring were then trapped in the empty cell and starved to death. 4. Only 5.4% of the inner brood cells that were protected by an empty cell were parasitised, compared with 28.9% of those without an anterior, empty cell; 27.4% of the empty cells contained dead parasite offspring (eggs and larvae). Thus, the empty cells provided significant protection and, combined with additional means of protection of brood cells, led to a low degree of parasitism. More than 77% of the wasp offspring died at an early stage due to intraspecific interference competition within brood cells and as result of the wasps’ oviposition into empty cells.     

Effect of queen excluders on ovary activation in workers of the Eastern honeybee Apis cerana

Honeybee workers generally refrain from personal reproduction when a queen is present. Workers discern the presence and fecundity of the queen via volatile pheromones that permeate throughout the colony. Pheromones are emitted both by the queen herself and by the brood that she produces. If pheromone production is disrupted, some workers initiate egg laying. The Eastern honeybee Apis cerana is unusual in that workers have high levels of ovary activation even in the presence of a queen. To investigate the effect of disruption to pheromone dispersal, we fitted three A. cerana colonies with vertical queen excluders, thus splitting the colonies into a half containing a queen and a half without a queen. We regularly sampled adult workers from both sides of the excluder for 3 weeks. We also sampled workers from three control colonies that did not contain excluders. We found a significant increase in worker ovary activation 3 days after addition of excluders, suggesting that the reduced dispersal of pheromones allowed some workers to become reproductively active. Workers attempted to rear queen cells on the queenless halves of all three colonies. Queen-rearing ceased on day 9, at which time no queen-laid brood remained on the queenless halves of the colonies. Ovary activation rates continued to climb until day 9 and then gradually began to decline. With the exception of one egg, we did not observe worker-laid brood on the queenless side of the colonies, suggesting that workers continued policing eggs laid by workers. We conclude that if the distribution of brood pheromone is impeded, workers prepare to supersede their queen, accompanied by high levels of worker ovary activation. However, because workers continue to police each other, high ovary activation does not result in worker-produced drones.     

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Abstract Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers’ ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti‐egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.     

Monthly changes in various drone characteristics of Apis mellifera ligustica and Apis mellifera syriaca

This study was conducted to investigate drone rearing activity and semen production of Apis mellifera ligustica and Apis mellifera syriaca . Tendency of worker bees of both subspecies towards egg laying under semiarid conditions were also monitored in the experiments. Differences were not observed in drone brood production between both honeybee subspecies throughout the investigation. Worker bees of both subspecies needed a significantly shorter time to start egg laying during February and March in comparison with the time those workers needed for laying eggs during the remaining months of the study. Syrian bee workers started egg laying earlier than Italian bee workers. Drones from laying workers were much smaller and produced less sperms with more abnormalities than normal drones. Drones produced from queens in May were heavier and produced more sperms with less abnormalities than those produced during the other months. The drone brood rearing of both subspecies tended to follow the same general cycle in 2005 and 2006. The study suggests that virgin queens have a better chance to receive adequate viable sperm amounts from drones in April and May in semiarid Mediterranean conditions.     

Worker policing persists in a hopelessly queenless honey bee colony (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Summary In queenright colonies of Apis mellifera, worker policing normally eliminates worker-laid eggs thereby preventing worker reproduction. However, in queenless colonies that have failed to rear a replacement queen, worker reproduction is normal. Worker policing is switched off, many workers have active ovaries and lay eggs, and the colony rears a last batch of male brood before dying out. Here we report a colony which, when hopelessly queenless, did not stop policing although a high proportion of workers had active ovaries (12.6%) and many eggs were laid. However, all these eggs and also worker-laid eggs transferred from another colony were policed. This unusual pattern was repeated eight weeks later by a second queenless colony made using worker bees from the same mother colony, which strongly suggests genetic determination.Received 19 May 2003; revised 11 September 2003; accepted 23 September 2003.     

Egg‐size plasticity in Apis mellifera: Honey bee queens alter egg size in response to both genetic and environmental factors

Social evolution has led to distinct life‐history patterns in social insects, but many colony‐level and individual traits, such as egg size, are not sufficiently understood. Thus, a series of experiments was performed to study the effects of genotypes, colony size and colony nutrition on variation in egg size produced by honey bee (Apis mellifera) queens. Queens from different genetic stocks produced significantly different egg sizes under similar environmental conditions, indicating standing genetic variation for egg size that allows for adaptive evolutionary change. Further investigations revealed that eggs produced by queens in large colonies were consistently smaller than eggs produced in small colonies, and queens dynamically adjusted egg size in relation to colony size. Similarly, queens increased egg size in response to food deprivation. These results could not be solely explained by different numbers of eggs produced in the different circumstances but instead seem to reflect an active adjustment of resource allocation by the queen in response to colony conditions. As a result, larger eggs experienced higher subsequent survival than smaller eggs, suggesting that honey bee queens might increase egg size under unfavourable conditions to enhance brood survival and to minimize costly brood care of eggs that fail to successfully develop, and thus conserve energy at the colony level. The extensive plasticity and genetic variation of egg size in honey bees has important implications for understanding life‐history evolution in a social context and implies this neglected life‐history stage in honey bees may have trans‐generational effects.     

Worker reproductive parasitism in naturally orphaned colonies of the Asian red dwarf honey bee, Apis florea

The truce between honey bee (Apis spp.) workers over reproduction is broken in the absence of their queen. Queenright workers generally abstain from personal reproduction, raising only the queen’s offspring. Queenless workers activate their ovaries, produce eggs, and reduce the rate at which they destroy worker-laid eggs, so that some eggs are reared to maturity. Reduced policing of worker-laid eggs renders queenless nests vulnerable to worker reproductive parasitism (WRP), and may result in the colony raising eggs of unrelated (non-natal) workers that parasitize it. Queenless colonies of A. florea are heavily parasitized with the eggs of non-natal workers. However, queenless colonies often abscond upon disturbance and build a small comb in which to rear their own male offspring. We investigated three naturally occurring orphaned colonies to determine if they are also parasitized. We show that WRP is present in orphaned colonies, and non-natal workers have significantly higher rates of ovary activation than natal workers. In contrast to experimentally manipulated colonies, in our samples, natal and non-natal workers had statistically equal reproductive success, but this may have been due to the small number of non-natals present.     

Colony social organization of <Emphasis Type="Italic">Lasioglossum malachurum</Emphasis> Kirby (Hymenoptera,Halictidae) in southern Greece

Summary We studied the nesting and social biology of two aggregations of the obligately eusocial halictine bee Lasioglossum malachurum at Agios Nikolaos Monemvasias (ANM) in southern Greece. Observations and nest excavations carried out from May to June 2000, revealed social and demographic variation between aggregations and years at ANM, as well as notable differences between these and other European populations. In southern Greece, the colony cycle includes multiple broods: the first two broods comprise only workers, whereas the third brood comprises workers, males, and gynes. Although pleometrosis is unknown in other populations, in the ANM region, as many as 10% of nests have more than one foundress. Newly emerged workers and gynes exhibit non-overlapping size distributions, but a few queens are worker-sized, indicating that workers occasionally overwinter and become foundresses. Although the vast majority of workers are unmated and most exhibit no ovarian development, an increase in worker ovarian development at the time of male production suggests that many males may develop from worker-laid eggs. Worker reproduction seems to be inhibited by the presence of queens, and annual variation in queen mortality may underlie annual variation in worker ovarian development. Across Europe, the major demographic and social differences among L. malachurum populations are in the number of worker broods and the extent of worker ovarian development. This contrasts with the results of a principal components analysis of social traits among 15 social L. (Evylaeus) populations, which shows that interspecific social variation is defined by the proportion of males in the early brood, the proportion of workers mated, queen-worker size dimorphism, gyne overwintering locale, and the proportion of workers with developed ovaries.Received 17 June 2002; revised 16 January 2002; accepted 27 January 2003.     

Do policing honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) workers target eggs in drone comb?

Summary. Honey bee (Apis mellifera) workers police each other's reproduction by eating worker-laid eggs while preferentially leaving queen-laid eggs. Policing effort is presumably associated with biological costs, including those of search time and of errors of identification of eggs so that the wrong eggs are retained or eaten. Selection should act to minimise these costs. One way costs can be reduced is to focus policing effort on eggs that are most likely to be worker laid. We show here, in accordance with this prediction, that policing effort is focussed on drone cells where workers are more likely to lay eggs, rather than on worker cells which are more likely to contain queen-laid eggs.     

The evolution of worker sterility in honeybees: the genetic basis of failure of worker policing

Worker honeybees (Apis mellifera) usually only lay eggs when their colony is queenless. However, an extremely rare ''anarchistic'' phenotype occurs, in which workers develop functional ovaries and lay large numbers of haploid eggs which develop into adult drones despite the presence of the queen. Studies of such colonies can give important insights into the mechanisms by which worker sterility is maintained in normal colonies. Here we report on the results of a breeding programme which enhanced the frequency of the anarchistic phenotype. Colonies derived from queens inseminated only by worker-laid males showed up to 9% of workers with highly developed ovaries. In these colonies a large proportion of males arose from worker-laid eggs. Colonies headed by queens inseminated with 50% worker-laid drones and 50% queen-laid drones showed variable phenotypes. In most such colonies there was no worker reproduction. In some, many workers had highly developed ovaries, but no worker-laid eggs were reared. In one colony, many worker-laid eggs were reared to maturity. The results suggest that the anarchy phenotype results from a complex interaction of queen genotype, the worker genotype of subfamilies that successfully reproduce and of those that do not, and the external environment.     

Worker policing by egg eating in the ponerine ant Pachycondyla inversa

We investigated worker policing by egg eating in the ponerine ant Pachycondyla inversa, a species with morphologically distinct queens and workers. Colonies were split into one half with the queen and one half without. Workers in queenless colony fragments started laying unfertilized male eggs after three weeks. Worker-laid eggs and queen-laid eggs were introduced into five other queenright colonies with a single queen and three colonies with multiple queens, and their fate was observed for 30 min. Significantly more worker-laid eggs (range of 35-62%, mean of 46%) than queen-laid eggs (range of 5-31%, mean of 15%) were eaten by workers in single-queen colonies, and the same trend was seen in multiple-queen colonies. This seems to be the first well-documented study of ants with a distinct caste polymorphism to show that workers kill worker-laid eggs in preference to queen-laid eggs. Chemical analyses showed that the surfaces of queen-laid and worker-laid eggs have different chemical profiles as a result of different relative proportions of several hydrocarbons. Such differences might provide the information necessary for differential treatment of eggs. One particular alkane, 3,11-dimeC27, was significantly more abundant on the surfaces of queen-laid eggs. This substance is also the most abundant compound on the cuticles of egg layers.     

Maternity of emergency queens in the Cape honey bee,Apis mellifera capensis

During reproductive swarming, some workers of the Cape honey bee, Apis mellifera capensis, lay eggs in queen cells, many of which are reared to maturity. However, it is unknown if workers are able to lay in queen cells immediately after queen loss during an episode of emergency queen rearing. In this study we experimentally de‐queened colonies and determined the maternity of larvae and pupae that were reared as queens. This allowed us to determine how soon after queen loss workers contribute to the production of new queens. We were further interested to see if workers would preferentially raise new queens from queen‐laid brood if this was introduced later. We performed our manipulations in two different settings: an apiary setting where colonies were situated close together and a more natural situation in which the colonies were well separated. This allowed us to determine how the vicinity of other colonies affects the presence of parasites. We found that workers do indeed contribute to queen cell production immediately after the loss of their queen, thus demonstrating that some workers either have activated ovaries even when their colony has a queen or are able to activate their ovaries extremely rapidly. Queen‐laid brood introduced days after queen loss was ignored, showing that workers do not prefer to raise new queens from queen brood when given a choice. We also detected non‐natal parasitism of queen cells in both settings. We therefore conclude that some A. m. capensis genotypes specialize in parasitizing queen cells.     

Worker policing in the common wasp Vespula vulgaris is not aimed at improving colony hygiene

In insect societies, eggs laid by workers are frequently killed by other workers – a behaviour known as “worker policing”. The traditional explanation of worker policing is that it is a mechanism to resolve intracolony conflict, and maintain the reproductive monopoly of the queen. Recently, Pirk et al. (2004) proposed that worker policing instead is aimed at removing unviable worker-laid eggs and is ultimately just another example of hygienic behaviour. Here we test this hypothesis for the common wasp Vespula vulgaris, a species with highly effective worker policing. We show that worker-laid eggs from queenless colonies have a lower hatch rate (68%) than queen-laid eggs (82%). Analysis of egg laying rates of queens and workers, however, shows that the difference is not big enough to explain the apparent absence of adult worker-derived males in this species. Received 30 January 2006; revised 2 May 2006; accepted 5 May 2006.     

Which egg features predict egg rejection responses in American robins? Replicating Rothstein's (1982) study

Rothstein (Behavioral Ecology and Sociobiology, 11, 1982, 229) was one of the first comprehensive studies to examine how different egg features influence egg rejection behaviors of avian brood parasite–hosts. The methods and conclusions of Rothstein (1982) laid the foundation for subsequent experimental brood parasitism studies over the past thirty years, but its results have never been evaluated with replication. Here, we partially replicated Rothstein's (1982) experiments using parallel artificial model egg treatments to simulate cowbird (Molothrus ater) parasitism in American robin (Turdus migratorius) nests. We compared our data with those of Rothstein (1982) and confirmed most of its original findings: (1) robins reject model eggs that differ from the appearance of a natural robin egg toward that of a natural cowbird egg in background color, size, and maculation; (2) rejection responses were best predicted by model egg background color; and (3) model eggs differing by two or more features from natural robin eggs were more likely to be rejected than model eggs differing by one feature alone. In contrast with Rothstein's (1982) conclusion that American robin egg recognition is not specifically tuned toward rejection of brown‐headed cowbird eggs, we argue that our results and those of other recent studies of robin egg rejection suggest a discrimination bias toward rejection of cowbird eggs. Future work on egg recognition will benefit from utilizing a range of model eggs varying continuously in background color, maculation patterning, and size in combination with avian visual modeling, rather than using model eggs which vary only discretely.     

Morphology of reproductive and trophic eggs and their controlled release by workers in Trigona (Tetragonisca) angustula llliger (Apidae, Meliponinae)

Abstract. To study the reproductive potential of workers in the stingless bee Trigona (Tetragonisca) angustula Illiger (Apidae, Meliponinae), we examined the morphological quality of their eggs. Worker-eggs were all placed on the inside of the upper wall of brood cells. Normally, such eggs are consumed by the queen and are therefore referred to as trophic. The provisioning and oviposition processes in queenright colonies are characterized by the occurrence of circular aggregations of workers, 'rosettes', around the broodcell opening immediately after the release of a worker-egg. The ovaries of 35% of these rosette workers contained mature, chorionated eggs. In a single worker ovary, always only one mature egg was found. Some of the eggs, dissected from the ovarioles, showed a reticulate chorion pattern. Worker-eggs which lacked this pattern were significantly bigger than patterned eggs. Scanning electron micrographs revealed that the patterned worker-eggs are similar in appearance to queen-eggs.
After a worker-egg had been removed experimentally from a broodcell, the same cell could be oviposited by a worker again. Light microscopic analysis revealed that all these worker-eggs lacked the reticulate chorion pattern and were very similar in their morphology. In a colony without a laying queen workers laid eggs which had a reticulate pattern on the chorion. Since these eggs developed into males, we assume that the pattern on the chorion is characteristic for reproductive eggs. We also assume that the queen prevents the release of reproductive eggs by the workers. However, she does not inhibit the development of this type of egg.     

中华蜜蜂卵表面微观结构及化学成分初步研究

为认识蜜蜂的工蜂监督机制,利用扫描电镜分别观察了中华蜜蜂Apis cerana cerana不同类型卵（蜂王产的受精卵、未受精卵以及工蜂产的未受精卵）的表面超微结构,同时用气 质联用技术测定了各类型卵新产卵（0 h）和5 h卵的表面化学信息素成分。结果表明：中华蜜蜂3种卵表面均覆有六边形的结构,结构内充满小突起,3种卵的大小及表面超微结构无显著差异。受精卵表面化学信息素种类比未受精卵更为丰富,C27∶2和C27∶1可能是卵带有的标记化学信息素。