Medium for development of bee cell cultures (<Emphasis Type="Italic">Apis mellifera</Emphasis>: Hymenoptera: Apidae)

A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert–Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21–23°C ranging from 9–15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders.     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

Polymorphism analysis of <Emphasis Type="Italic">csd</Emphasis> gene in six <Emphasis Type="Italic">Apis mellifera</Emphasis> subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.     

Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

Reproductive biology of <Emphasis Type="Italic">Varroa destructor</Emphasis> in Africanized honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Since its first contact with Apis mellifera, the population dynamics of the parasitic mite Varroa destructor varies from one region to another. In many regions of the world, apiculture has come to depend on the use of acaricides, because of the extensive damage caused by varroa to bee colonies. At present, the mite is considered to contribute to the recent decline of honey bee colonies in North America and Europe. Because in tropical climates worker brood rearing and varroa reproduction occurs all year round, it could be expected that here the impact of the parasite will be even more devastating. Yet, this has not been the case in tropical areas of South America. In Brazil, varroa was introduced more than 30 years ago and got established at low levels of infestation, without causing apparent damage to apiculture with Africanized honey bees (AHB). The tolerance of AHB to varroa is apparently attributable, at least in part, to resistance in the bees. The low fertility of this parasite in Africanized worker brood and the grooming and hygienic behavior of the bees are referred as important factors in keeping mite infestation low in the colonies. It has also been suggested that the type of mite influences the level of tolerance in a honey bee population. The Korea haplotype is predominant in unbalanced host-parasite systems, as exist in Europe, whereas in stable systems, as in Brazil, the Japan haplotype used to predominate. However, the patterns of varroa genetic variation have changed in Brazil. All recently sampled mites were of the Korea haplotype, regardless whether the mites had reproduced or not. The fertile mites on AHB in Brazil significantly increased from 56% in the 1980s to 86% in recent years. Nevertheless, despite the increased fertility, no increase in mite infestation rates in the colonies has been detected so far. A comprehensive literature review of varroa reproduction data, focusing on fertility and production of viable female mites, was conducted to provide insight into the Africanized bee host-parasite relationship.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

Clique formation of super-sister honeybee workers (<Emphasis Type="Italic">Apis mellifera</Emphasis>) in experimental groups

Summary One day old honeybee workers (Apis mellifera) were observed in small experimental groups (10 workers per group). These groups were either composed of offspring workers of singly inseminated queens (super-sister groups) or multiply inseminated queens (mixed groups). The groups thus consisted of either super-sisters or a mix of super- and halfsisters. The positions of the individually labelled workers were observed with infrared sensitive video equipment over a 24 h period and analysed with digital image analysis. The spatial distribution of workers in super-sister and mixed groups differed significantly. The distance between super-sister workers was significantly less than in mixed groups (n = 339; p < 0.01). Also the distance of the workers from the group centre was significantly less in super-sister groups as compared to mixed groups (n = 3440, p<0.05). The super-sisters thus formed tighter groups than the groups including half-sisters. A genotypic analysis of the mixed groups with microsatellite DNA markers revealed that workers were significantly more frequently observed next to a super-sister rather than a half-sister (p < 0.001). Irrespective whether this results from kin recognition or not, we expect clique formation to be an important factor for the development of task specialisation among worker bees.Received 26 August 2002; revised 22 April 2003; accepted 25 July 2003.     

Genetic structure of <Emphasis Type="Italic">Varroa destructor</Emphasis> populations infesting <Emphasis Type="Italic">Apis mellifera</Emphasis> colonies in Argentina

Although mitochondrial DNA mapping of Varroa destructor revealed the presence of several haplotypes, only two of them (Korean and Japanese haplotypes) were capable to infest Apis mellifera populations. Even though the Korean haplotype is the only one that has been reported in Argentina, these conclusions were based on mites sampled in apiaries from a specific geographical place (Buenos Aires province). To study mites from several sites of Argentina could reveal the presence of the Japanese genotype, especially considering sites near to Brazil, where Japanese haplotype was already detected. The aim of this work was to study the genetic structure of V. destructor populations from apiaries located in various provinces of Argentina, in order to determine the presence of different haplotypes. The study was carried out between January 2006 and December 2009. Phoretic adult Varroa mites were collected from honey bee workers sampled from colonies of A. mellifera located in Entre Ríos, Buenos Aires, Corrientes, Río Negro, Santa Cruz and Neuquén provinces. Twenty female mites from each sampling site were used to carry out the genetic analysis. For DNA extraction a nondestructive method was used. DNA sequences were compared to Korean haplotype (AF106899) and Japanese haplotype (AF106897). All DNA sequences obtained from mite populations sampled in Argentina, share 98% of similitude with Korean Haplotype (AF106899). Taking into account these results, we are able to conclude that Korean haplotype is cosmopolite in Argentina.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

Morphological changes in the cephalic salivary glands of females and males of <Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Scaptotrigona postica</Emphasis> (Hymenoptera,Apidae)

The cephalic salivary glands of some species of bees are exclusive and well developed only in Apinae. These glands were studied with light and scanning electron microscopy in workers, queens and males from the honey bee Apis mellifera, and the stingless bee Scaptotrigona postica in different life phases. The results show that the cephalic salivary glands are present in females of both the species, and in males of S. postica. Nevertheless, they are poorly developed in young males of A. mellifera. In both species, gland growth is progressive from the time of emergence to the oldest age but, in A. mellifera males, the gland degenerates with age. Scanning electron microscopy shows that the secretory units of newly emerged workers are collapsed while in older workers they are turgid. Some pits on the surface of the secretory units correspond to open intercellular spaces. The possible functions of these glands in females and males of both species are discussed.     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

Comparison of Defense Body Movements of <Emphasis Type="Italic">Apis laboriosa</Emphasis>, <Emphasis Type="Italic">Apis dorsata dorsata</Emphasis> and <Emphasis Type="Italic">Apis dorsata breviligula</Emphasis> Honey Bees

Defense behavior of three, free living giant (Megapis) honey bee subspecies, Apis laboriosa, A. dorsata dorsata and A. dorsata breviligula, was compared. Disturbed worker bees responded with characteristic dorso-ventral defense body twisting (DBT). Workers of A. laboriosa twisted the thorax by 55°, and the two other A. dorsata subspecies by about 10° more. A. laboriosa workers raised the tip of the abdomen by 90° and workers of the two other bee subspecies by about 20° higher. Differences in those traits were highly significant between A. laboriosa and both A. dorsata subspecies, but were not significant between those two subspecies. The whole cycle of DBT was the most vigorous in A. d. breviligula (0.11 s), and it was twice as vigorous as in A. d. dorsata (0.26 s) and trice as in A. laboriosa (0.32 s). A. laboriosa twisted the body together with wings folded over the abdomen, while the two A. dorsata subspecies raised the abdomen between spread wings. This supports the opinion to treat A. laboriosa as a separate species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence of <Emphasis Type="Italic">Ascosphaera apis</Emphasis>, the causative agent of chalkbrood disease,in honeybees <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae) in Japan

The presence of Ascosphaera apis, a fungus that is the causative agent of chalkbrood disease, was surveyed in Japan using a diagnostic polymerase chain reaction (PCR). A total of 336 individual European honeybees Apis mellifera were taken from 25 different apiaries in various regions of Japan. Of the 112 colonies surveyed, A. apis was detected in 27 colonies (24.1%). Positive results by PCR were obtained from 49 out of 336 surveyed individuals (14.6%). Based on these results, the distribution of A. apis in A. mellifera is widespread across Japan and does not exhibit significant differences between geographic areas. DNA sequences of the ITS and 5.8S rRNA region from all 17 isolates of A. apis were identical, even though they were from geographically distinct areas in Japan. It is suggested that no intra-species variation may be due to a recent bottleneck effect probably caused by humans before geographical expansion of the fungus.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

The parasitic mite <Emphasis Type="Italic">Varroa destructor</Emphasis> affects non-associative learning in honey bee foragers, <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

The parasitic mite Varroa destructor influences flight behavior, orientation and returning success of forager honeybees (Apis mellifera) infested as adults. As impaired orientation toward the nest entrance might be due to deficiency in recognition and responsiveness to stimuli in the environment, we examined effects of V. destructor on sensory responsiveness, non-associative and associative learning of honey bee foragers by using proboscis extension reaction paradigm (PER). Although infested and uninfested workers were initially equally responsive to different concentrations of sugar water, we found differences in non-associative learning. In habituation, PER to repeated sugar stimulation of the antennae occurred faster in infested foragers compared to uninfested foragers. In sensitization, infested foragers showed a lower response to an odor stimulus following sugar stimulation than non-infested foragers. Differences in non-associative paradigms were more pronounced in bees with lower responsiveness to sucrose. In conditioning learning experiments, a significant reduction in proboscis extension response was found 1 min but not 12 min after a single conditioning trial indicating that V. destructor predominantly affects the non-associative components of learning and its underlying neural and molecular processes. Jasna Kralj and Axel Brockmann have contributed equally to this study.     

Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

Modulation of social interactions by immune stimulation in honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>, workers

Background  

Immune response pathways have been relatively well-conserved across animal species, with similar systems in both mammals and invertebrates. Interestingly, honey bees have substantially reduced numbers of genes associated with immune function compared with solitary insect species. However, social species such as honey bees provide an excellent environment for pathogen or parasite transmission with controlled environmental conditions in the hive, high population densities, and frequent interactions. This suggests that honey bees may have developed complementary mechanisms, such as behavioral modifications, to deal with disease.     

Nestmate Recognition Differences between Honeybee Colonies of <Emphasis Type="BoldItalic">Apis cerana</Emphasis> and <Emphasis Type="BoldItalic">Apis mellifera</Emphasis>

Nestmate recognition in Apis cerana and Apis mellifera was studied by introducing sealed queen cells heterospecifically between queenless colonies. No A. cerana queens were accepted by queenless A. mellifera; but A. mellifera queens were accepted in queenless A. cerana colonies. A. mellifera queens oviposited in queenless A. cerana colonies, but A. cerana workers removed most eggs. In time, egg removals declined, and some A. mellifera larvae that hatched from these eggs reached adulthood, and eventually about half of the workers were newly emerged A. mellifera. Eventually, the colonies consisted only of A. mellifera after A. cerana workers died by attrition. A. mellifera workers are more sensitive to nestmate recognition and killed the A. cerana virgin queens. In mixed-species colonies, after newly emerged A. mellifera workers matured, they removed eggs laid by the A. cerana queens until there were no workers to replace the old ones.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.