Virgin Queens Research Articles

RFID Technology Serving Honey Bee Research: A Comprehensive Description of a 32-Antenna System to Study Honey Bee and Queen Behavior

The fields of electronics and information technology have witnessed rapid development during the last decades, providing significant technical support to the field of biological sciences. Radio-Frequency Identification (RFID) technology has been used to automate the monitoring of animal location and behaviors in a wide range of vertebrate and invertebrate species, including social insects such as ants and honey bees (Apis mellifera L.). This technology relies on electromagnetic fields to identify and track transponders attached to objects automatically. Implementing new technologies to serve research purposes could be time consuming and require technical expertise from entomologists and researchers. Herein, we present a detailed description on how to harness RFID technology to serve honey bee research effectively. We describe how to build and operate a 32-antenna RFID system used to monitor various honey bee behaviors such as foraging, robbing, and queen and drone mating, which can be used in other social insects as well. Preliminary data related to queen nuptial flights were obtained using this unit and presented in this study. Virgin queens labeled with ≈5 mg transponders performed multiple (one to four) nuptial/orientation flights a day (9 a.m. to 5 p.m.) ranging from 8 to 145 s each. Contrary to virgin queens, no hive exit was recorded for mated queens. At full capacity, this unit can monitor up to 32 honey bee colonies concurrently and is self-sustained by a solar panel to work in remote areas. All materials, hardware, and software needed to build and operate this unit are detailed in this study, offering researchers and beekeepers a practical solution and a comprehensive source of information enabling the implementation of RFID technology in their research perspective.

Effects of Heat Stress on Mating Behavior and Colony Development in Bombus terrestris (Hymenoptera: Apidae)

Climate change is related to an increase in the frequency and intensity of extreme events such as heatwaves. In insect pollinators, heat exposure is associated with direct physiological perturbations, and in several species, could lead to a decrease of fitness related to a decrease in fertility. Here we developed a new experimental protocol in controlled conditions to assess if the exposure to high temperatures could modify the attractiveness and fertility of Bombus terrestris males. Our results show that virgin queens of B. terrestris do not have preferences between the pheromonal secretions of heat-exposed and control males. Moreover, mating with a heat-exposed male has no impact on the copulation behavior and the development of the nest (brood composition). We advise to extend trials to cover a range of wild and heat-sensitive species on multiple generations to better understand the impact of heat waves on the bumblebee communities.

Colony fitness increases in the honey bee at queen mating frequencies higher than genetic diversity asymptote

Across the eusocial Hymenoptera, a queen’s mating frequency is positively associated with her workers’ genetic diversity and colony’s fitness. Over 90% of a colony’s diversity potential is achieved by its mother’s tenth effective mating (me); however, many females mate at levels of me > 10, a zone we here call hyperpolyandry. We compared honey bee colony fitness at mating levels near and above this genetic diversity asymptote. We were interested in how hyperpolyandry affects colony phenotypes arising from both common tasks (brood care) and rare specialized tasks (parasite resistance). We used an unselected wild line of bees and a Varroa Sensitive Hygiene (VSH) line selected to resist the parasite Varroa destructor. Virgin queens were instrumentally inseminated to replicate the following queen/colony conditions: (1) VSH semen/low polyandry (observed mating number = mo = 9), (2) VSH semen/high polyandry (mo = 54), (3) wild type semen/low polyandry, or (4) wild semen/high polyandry. There was a positive effect of polyandry on brood survival, an outcome of common tasks, with highest values at mo = 54. There was an interaction between polyandry and genetics such that differences between genetic lines expressed only at mo = 54, with fewer mites in VSH colonies. These results are consistent with two hypotheses for the evolution of mating levels in excess of the genetic diversity asymptote: hyperpolyandry improves colony fitness by (1) optimizing genotype compositions for common tasks and (2) by capturing rare specialist allele combinations, resisting cliff-edge ecological catastrophes.Significance statementPolyandry is a female’s practice of mating with several males, storing their sperm, and using it to produce one or more clutches of genetically diverse offspring. In the social Hymenoptera, polyandry increases the genetic diversity and task efficiency of workers, leading to improved colony fitness. Over 90% of the increase in a colony’s diversity potential is achieved by its mother’s tenth mating; however, many females practice hyperpolyandry, a term we reserve here for mating levels above this genetic diversity asymptote. We show that a token of colony fitness arising from common tasks, brood survival, improves universally as one moves from sub- to hyperpolyandrous mating levels. However, a colony phenotype arising from a rare parasite resistance task is only expressed in the presence of the controlling alleles and under conditions of hyperpolyandry. These results suggest adaptive mechanisms by which hyperpolyandry could evolve.

Upregulation of Transferrin and Major Royal Jelly Proteins in the Spermathecal Fluid of Mated Honeybee (Apis mellifera) Queens.

Simple SummaryTo understand the mechanisms underlying long-term storage and survival of sperm in honeybee Apis mellifera queens, previous studies have elucidated the components of honeybee spermathecal fluid. However, the expression profiles of transferrin (Tf) and major royal jelly proteins 1–9 (MRJPs 1–9) in the spermatheca and spermathecal fluid of mated honeybee queens have still not been characterized. In this study, we confirmed upregulation of Tf and MRJPs in the spermatheca and spermathecal fluid of mated honeybee queens by using RNA sequencing, reverse transcription-polymerase chain reaction, and Western blot analyses. The levels of Tf and antioxidant enzymes were elevated in the spermathecal fluid of the mated queens, paralleling the levels of reactive oxygen species, H2O2, and iron. The increased levels of MRJPs, especially MRJP1, MRJP4, and MRJP6, in the spermathecal fluid of mated queens may be responsible for energy provision during sperm storage in honeybee queens. Overall, our findings indicate that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens, providing a novel insight into antioxidant defense and energy metabolism for stored sperm in honeybee queens.Sperm storage in the spermathecae of honeybee (Apis mellifera) queens is vital for reproduction of honeybees. However, the molecular mechanisms whereby queens store sperm in a viable state over prolonged periods in the spermatheca are not fully understood. Here, we conducted RNA sequencing analysis of the spermathecae in both virgin and mated A. mellifera queens 24 h after mating and observed that the genes encoding transferrin (Tf) and major royal jelly proteins (MRJPs) were differentially expressed in the spermathecae of mated queens. The concentrations of Tf and antioxidant proteins such as superoxide dismutase 1, catalase, and glutathione peroxidase as well as the levels of reactive oxygen species, H2O2, and iron were higher in the spermathecal fluid of the mated queens than in virgin queens. Tf upregulation is likely to perform a protective role against the Fenton reaction occurring between iron and H2O2 in the antioxidant pathway in the mated queen’s spermathecal fluid. Furthermore, MRJPs—especially MRJP1, MRJP4, and MRJP6—were upregulated in the mated queen’s spermathecal fluid, indicating that they may serve as antimicrobial and antioxidant agents as well as an energy source for stored sperm in the spermathecal fluid of honeybee queens. Together, our findings show that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens.

Cuticular hydrocarbons as cues of caste and sex in the German wasp Vespula germanica

The complex system of communication used by social insects is responsible for their success on Earth. Cuticular hydrocarbons (CHCs) are considered the most commonly used form of communication among social insects. The best-known function of CHCs is to distinguish nestmates, providing cues to the whole colony to identify potential threats. In some species, CHCs function as queen pheromones, which contribute to the queens’ ability to regulate the reproductive division of labor. Additionally, differences of CHCs were described in castes and they are also important in sex recognition. Here, we reinvestigated the chemical composition on the cuticle of German wasp Vespula germanica and we found that proportions of linear alkanes were more abundant in the cuticle of queens than other castes. Chemical analysis between caste and sex, including data of virgin queens and males, showed that each group possesses different compositions on their cuticle, being mostly quantitative differences. Thus, individuals of V. germanica may recognize and discriminate caste and sex through subtle chemical variations, or alternatively, based on templates used in nestmate recognition. The chemical analysis provides correlative evidence of CHCs being used as cues in the chemical communication of this species.

Comprehensive analysis of male-free reproduction in Monomorium triviale (Formicidae: Myrmicinae).

We report comprehensive evidence for obligatory thelytokous parthenogenesis in an ant Monomorium triviale. This species is characterized by distinct queen-worker dimorphism with strict reproductive division of labor: queens produce both workers and new queens without mating, whereas workers are completely sterile. We collected 333 nests of this species from 14 localities and three laboratory-reared populations in Japan. All wild queens dissected had no sperm in their spermathecae. Laboratory observation confirmed that virgin queens produced workers without mating. Furthermore, microsatellite genotyping showed identical heterozygous genotypes between mothers and their respective daughters, suggesting an extremely low probability of sexual reproduction. Microbial analysis detected no bacterial genera that are known to induce thelytokous parthenogenesis in Hymenoptera. Finally, the lack of variation in partial sequences of mitochondrial DNA among individuals sampled from across Japan suggests recent rapid spread or selective sweep. M. triviale would be a promising model system of superorganism-like adaptation through comparative analysis with well-studied sexual congeners, including the pharaoh ant M. pharaonis.

Морфо-этологическая характеристика медоносных пчел Apis mellifera L. Пермского края

Abstract. The European dark bees (Apis mellifera mellifera L.) divided into many populations occupying a wide range and living in different climatic zones. The research of morphological features of all castes of bee colony, and the description of their ethological features makes it possible to more fully characterize and distinguish a particular group of bees. The aim of the research was to describe the main morphological and some ethological features of queen artificial reproduction, drones, and worker of the European dark bees of the Perm region. Methods. The research was carried out on one of the breeding apiaries of the Perm region on colony of the European dark bees. Morphometric studies were performed according to the standard technique with the use of computer technology. Scientific novelty. For the first time, the morphological and ethological characteristics of honeybees of the Perm region are described most fully. Also experimental study was carried out on the use of a new technology for reproduction of virgin queen bees. Research results and practical significance. In the long-term dynamics, it is shown that the main breed-defining morphometric and ethological characteristics of the studied individuals of all castes of the colony of bee belong to the European dark breed. The results obtained can be used in the identification of bees in the Permian population and the implementation of the selection process in apiaries in the region. New method of artificial breeding of virgin queens allowed to obtain queens corresponding to the standard of the European dark bees and can be applied in practical breeding. Observations were made on the flight activity of drones and flight timing of virgin queen bees, the results of which can be used in practice in the process of obtaining mated queens.

Differential expression of aquaporin genes during ovary activation in the honeybee Apis mellifera (Hymenoptera: Apidae) queens.

Aquaporins are membrane proteins responsible for the rapid transport of water and solutes through the plasma membrane in almost all organisms including microorganisms, animals and plants. The knowledge about aquaporins in insects has advanced in the last years, especially in organs such as the gut and Malpighian tubules, but their roles in ovaries are poorly understood. This study reports the occurrence and the expression of gene profiles of predicted aquaporins in the ovaries of virgin and mated queens of the honeybee Apis mellifera. The expression of six aquaporin genes (Am-DRIP, Am-PRIP, Am-Eglp1, Am-Eglp2, Am-Eglp3 and Am-BIB) was investigated in the ovaries of queens of different ages and physiological status. The Am-Eglp3 has the higher level of transcript in newly emerged virgin queens. The Am-PRIP and Am-Eglp2 have a higher level of transcripts in 4days-old virgin queens, aged expected to mate. In mated queens, only the Am-Eglp2 show high level of transcripts. Aquaporin was immunolocalized in the follicular cells of both nurse and oocytic chambers of the previtellogenic and vitellogenic follicles. This is the first report for the presence of aquaporins in the ovaries of bees.

A methodology to assist in locating drone congregation area using remote sensing technique

The mating of honey bees, Apis mellifera L. occurs in the air at specific locations called drone congregation areas (DCAs). The identification of these areas is important for beekeepers especially breeders of queens to ensure the mating of their virgin queens, and for studies on the mating behaviour of honey bee queens. In this study, remote sensing and geographical information systems were used for the first time to develop a methodology that can assist in locating these DCAs. The methodology included analysing landscape characteristics around the apiary to exclude unsuitable areas as DCAs, and finally to limit the search to a smaller area. An isolated apiary was used to develop the methodology and the actual location of the DCA was confirmed using field observations. The results of this study indicated that remote sensing can assist in locating DCAs.

Differential Viral Distribution Patterns in Reproductive Tissues of Apis mellifera and Apis cerana Drones.

Honeybee drones are male bees that mate with virgin queens during the mating flight, consequently transferring their genes to offspring. Therefore, the health of drones affects the overall fitness of the offspring and ultimately the survivability of the colony. Honeybee viruses are considered to be a major threat to the health of honeybees. In the present study, we demonstrated the pattern of common honeybee viruses in various tissues of drones in the western honeybee, Apis mellifera, and the eastern honeybee, Apis cerana. Drones were collected during the mating flight and analyzed using quantitative real-time (qRT-PCR) to detect the presence of seven honeybee viruses. The qRT-PCR result revealed that three honeybee viruses, namely Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Chinese Sacbrood Virus (CSBV), were detected in the reproductive tissues of A. mellifera and A. cerana drones. The results from qRT-PCR showed that the Israeli Acute Paralysis Virus (IAPV) was only detected in A. mellifera drone body tissues. Moreover, the prevalence of DWV and BQCV in the drones collected from A. mellifera colonies was significantly higher than that of A. cerana. In addition, virus multiple infections were higher in A. mellifera drones compared to those in A. cerana. CSBV was found predominantly in the reproductive tissues of A. cerana drones. This study is the first report describing the presence of the CSBV in reproductive tissues of A. mellifera drones. Our results may reflect the preference of honeybee viruses in honeybee species and may provide a piece of interesting evidence for understanding the virus transmission in A. cerana.

Effect of the Bacterium Paenibacillus larvae larvae on Vitellogenin Gene Expression of the Queen Honey Bee Apis mellifera L.

Virgin queens of the honey bee Apis mellifera L. were assessed following feeding in fourth larval instar with a sublethal dose of bacterium Paenibacillus larvae larvae (LD20 = 1.07 × 102 colony forming units (CFU)/queen), the causative agent of American foulbrood disease (AFB). The extraction of ovary proteins showed a highly significant increase for treated queens. The bacterium P. l. larvae was detected by conventional PCR-technique within treated queens ovaries with molecular size of 550 bp. Vitellogenin gene has been expressed in healthy and treated queens using RT-PCR technique, up-regulation took place in treated queens, vitellogenin was found with a high level of gene expression (∼20-fold). Finally, this study could contribute to initial efforts to help attempts to breed honey bee queens that are resistant to the pathogen.

Origin, behaviour, and genetics of reproductive workers in an invasive ant

BackgroundWorker reproduction has an important influence on the social cohesion and efficiency of social insect colonies, but its role in the success of invasive ants has been neglected. We used observations of 233 captive colonies, laboratory experiments, and genetic analyses to investigate the conditions for worker reproduction in the invasive Anoplolepis gracilipes (yellow crazy ant) and its potential cost on interspecific defence. We determined the prevalence of worker production of males and whether it is triggered by queen absence; whether physogastric workers with enlarged abdomens are more likely to be reproductive, how normal workers and physogastric workers compare in their contributions to foraging and defence; and whether worker-produced males and males that could have been queen- or worker-produced differ in their size and heterozygosity.ResultsSixty-six of our 233 captive colonies produced males, and in 25 of these, some males could only have been produced by workers. Colonies with more workers were more likely to produce males, especially for queenless colonies. The average number of days between the first appearance of eggs and adult males in our colonies was 54.1 ± 10.2 (mean ± SD, n = 20). In our laboratory experiment, queen removal triggered an increase in the proportion of physogastric workers. Physogastric workers were more likely to have yolky oocytes (37–54.9%) than normal workers (2–25.6%), which is an indicator of fertile or trophic egg production. Physogastric workers were less aggressive during interspecific aggression tests and foraged less than normal workers. The head width and wing length of worker-produced males were on average 4.0 and 4.3% greater respectively than those of males of undetermined source. Our microsatellite DNA analyses indicate that 5.5% of worker-produced males and 14.3% of males of undetermined source were heterozygous, which suggests the presence of diploid males and/or genetic mosaics in A. gracilipes.ConclusionsOur experimental work provides crucial information on worker reproduction in A. gracilipes and its potential cost to colony defence. The ability of A. gracilipes workers to produce males in the absence of queens may also contribute to its success as an invasive species if intranidal mating can take place between virgin queens and worker-produced males.

Male-biased dispersal in a fungus-gardening ant symbiosis.

For nearly all organisms, dispersal is a fundamental life‐history trait that can shape their ecology and evolution. Variation in dispersal capabilities within a species exists and can influence population genetic structure and ecological interactions. In fungus‐gardening (attine) ants, co‐dispersal of ants and mutualistic fungi is crucial to the success of this obligate symbiosis. Female‐biased dispersal (and gene flow) may be favored in attines because virgin queens carry the responsibility of dispersing the fungi, but a paucity of research has made this conclusion difficult. Here, we investigate dispersal of the fungus‐gardening ant Trachymyrmex septentrionalis using a combination of maternally (mitochondrial DNA) and biparentally inherited (microsatellites) markers. We found three distinct, spatially isolated mitochondrial DNA haplotypes; two were found in the Florida panhandle and the other in the Florida peninsula. In contrast, biparental markers illustrated significant gene flow across this region and minimal spatial structure. The differential patterns uncovered from mitochondrial DNA and microsatellite markers suggest that most long‐distance ant dispersal is male‐biased and that females (and concomitantly the fungus) have more limited dispersal capabilities. Consequently, the limited female dispersal is likely an important bottleneck for the fungal symbiont. This bottleneck could slow fungal genetic diversification, which has significant implications for both ant hosts and fungal symbionts regarding population genetics, species distributions, adaptive responses to environmental change, and coevolutionary patterns.

Sex Investment Ratio and Split Sex Ratio in the Fission-Performing Ant Cataglyphis tartessica

Inclusive fitness theory predicts that the sex investment ratio should be female biased in social insects with haplodiploidy-generated relatedness asymmetry (between females and males and also among females). However, this ratio should become male biased if related females are competing with each other for resources, as it is predicted by local resource competition hypothesis. This specific situation is expected to occur in ant species that reproduce by fission, where young queens leave their mother colonies on foot to found daughter colonies. We tested this idea using the ant Cataglyphis tartessica in Donana National Park (Spain). This species is monogynous (colonies headed by a single queen), monandrous (queen mate with only one male), and reproduces by fission. Given the similarity between foraging and colony fission distances in this species, a strong local competition for resources should be expected between mother and daughter colonies. In this study, we considered investment in females the sum of the investment in gynes (virgin queens) plus the investment in the worker force that leaves with them to found a new colony. Our results show a male-biased sex investment ratio in C. tartessica of 1:8 (numerical ratio gynes/males 1:56), suggesting queen control over sexual production. In addition, we found that, during its reproductive period, this species exhibits split sex (gyne-male) production as well as split male-worker production, without any evidence of variation in relatedness asymmetry between its colonies, at the population level. An interaction between local resources competition and colony size could explain our results.

Transcriptomic analysis of the honey bee (Apis mellifera) queen spermathecae reveals genes that may be involved in sperm storage after mating.

Honey bee (Apis mellifera) queens have a remarkable organ, the spermatheca, which successfully stores sperm for years after a virgin queen mates. This study uniquely characterized and quantified the transcriptomes of the spermathecae from mated and virgin honey bee queens via RNA sequencing to identify differences in mRNA levels based on a queen’s mating status. The transcriptome of drone semen was analyzed for comparison. Samples from three individual bees were independently analyzed for mated queen spermathecae and virgin queen spermathecae, and three pools of semen from ten drones each were collected from three separate colonies. In total, the expression of 11,233 genes was identified in mated queen spermathecae, 10,521 in virgin queen spermathecae, and 10,407 in drone semen. Using a cutoff log2 fold-change value of 2.0, we identified 212 differentially expressed genes between mated and virgin spermathecal queen tissues: 129 (1.4% of total) were up-regulated and 83 (0.9% of total) were down-regulated in mated queen spermathecae. Three genes in mated queen spermathecae, three genes in virgin queen spermathecae and four genes in drone semen that were more highly expressed in those tissues from the RNA sequencing data were further validated by real time quantitative PCR. Among others, expression of Kielin/chordin-like and Trehalase mRNAs was highest in the spermathecae of mated queens compared to virgin queen spermathecae and drone semen. Expression of the mRNA encoding Alpha glucosidase 2 was higher in the spermathecae of virgin queens. Finally, expression of Facilitated trehalose transporter 1 mRNA was greatest in drone semen. This is the first characterization of gene expression in the spermathecae of honey bee queens revealing the alterations in mRNA levels within them after mating. Future studies will extend to other reproductive tissues with the purpose of relating levels of specific mRNAs to the functional competence of honey bee queens and the colonies they head.

The neuroethology of olfactory sex communication in the honeybee Apis mellifera L.

The honeybee Apis mellifera L. is a crucial pollinator as well as a prominent scientific model organism, in particular for the neurobiological study of olfactory perception, learning, and memory. A wealth of information is indeed available about how the worker bee brain detects, processes, and learns about odorants. Comparatively, olfaction in males (the drones) and queens has received less attention, although they engage in a fascinating mating behavior that strongly relies on olfaction. Here, we present our current understanding of the molecules, cells, and circuits underlying bees' sexual communication. Mating in honeybees takes place at so-called drone congregation areas and places high in the air where thousands of drones gather and mate in dozens with virgin queens. One major queen-produced olfactory signal-9-ODA, the major component of the queen pheromone-has been known for decades to attract the drones. Since then, some of the neural pathways responsible for the processing of this pheromone have been unraveled. However, olfactory receptor expression as well as brain neuroanatomical data point to the existence of three additional major pathways in the drone brain, hinting at the existence of 4 major odorant cues involved in honeybee mating. We discuss current evidence about additional not only queen- but also drone-produced pheromonal signals possibly involved in bees' sexual behavior. We also examine data revealing recent evolutionary changes in drone's olfactory system in the Apis genus. Lastly, we present promising research avenues for progressing in our understanding of the neural basis of bees mating behavior.

Praising Elizabeth I in Latin at Norwich (1578)

Abstract When Elizabeth I visited the city of Norwich, she was publicly praised as a virgin queen for the first time in her reign. Although this image of Elizabeth becomes important to later historiography, this essay argues that there is a more sustained strand of royal myth-making in this visit that gives her even greater independent and specific political authority: that of an educated queen. At Norwich, Elizabeth was addressed more frequently in Latin than during any other visit during her reign, except for her visits to the universities. This essay analyzes the Latin texts to show how Norwich’s civic officials used this image to praise Elizabeth as a queen so individually powerful that she should commit more firmly to remaining a distinctly unmarried goddess of wisdom, a champion of Norwich, and the Nurse of God’s True (Protestant) Church. What goes unspoken is that she has no need for a foreign Catholic husband in the French Duke of Anjou—the context that underwrites the praise of her as a virgin queen. These Latin texts convey Elizabeth as a queen who has already the specific authority and nurturing care that give her distinctly peaceful nation all it needs to remain strong, prosperous, and religiously unified.

Food storage and morphological divergence between worker and soldier castes in a subterranean myrmicine ant, Carebara perpusilla

ABSTRACT Food reserves and trophallaxis help insect colonies regulate brood development and survive starvation events. Unlike wasps and bees, ants do not store food in external structures within the nest. Therefore, an ant colony’s ability to store food is greatly enhanced by the development of highly dimorphic workers and soldiers that are able to store food within their bodies. Carebara perpusilla (subfamily Myrmicinae, Emery, 1895) has a subterranean lifestyle and three castes: workers, soldiers with a proportionally larger head and gaster, and large winged queens. Workers are minute (1.5 mm long) and can hunt solitarily on tiny arthropods such as springtails and scavenge in groups on dead insects of various sizes. Rather than carrying big corpses to the nest, they bury it with soil and only retrieve haemolymph and small solid pieces. Moreover, some soldiers and virgin queens have a distended gaster and function as repletes. Food dyes revealed the storage of fat in the crop. Using dissections supplemented by micro-CT visualisation, we compared the crop of workers, soldiers and virgin queens and its capacity for considerable expansion. We discuss the morphological specialisation of repletes in ants that do not collect honeydew. Natural history data of more subterranean ants are needed to understand the adaptive context of foraging and other behaviours.

Metabolite Support of Long-Term Storage of Sperm in the Spermatheca of Honeybee (Apis mellifera) Queens.

The polyandrous mating system of honeybees (Apis mellifera L.) has garnered widespread attention. Long-lived honeybee queens only mate early in maturation, and the sperm obtained from the aerial mating is stored in the spermatheca. The maintenance of sperm viability in the spermatheca is an intriguing and complex process. However, the key physiological and biochemical adaptations underlying the long-term storage of sperm remain unclear. Analysis of the metabolite profile could help better understand the biology of the spermatheca and offer insights into the breeding and conservation of honeybees and even pest control strategies. Here, the changes in metabolites in the spermatheca were quantified between virgin queens and new-laying queens (with stored sperm) via liquid chromatography–mass spectrometry. Compared with virgin queens, changes occurred in lipids and lipid-like molecules, including fatty acyls and glycerophospholipids (GPL), prenol lipids, and sterol lipids, during storage of sperm in new-laying honeybee queens. Furthermore, the metabolic pathways that were enriched with the differentially expressed metabolites were identified and included GPL metabolism, biosynthesis of amino acids, and the mTOR signaling pathway. The likely roles of the pathways in the maintenance and protection of sperm are discussed. The study identifies key metabolites and pathways in the complex interplay of substances that contribute to the long-term storage of sperm and ultimately reproductive success of honeybee queens.

The Effect of the Queen's Presence on Thermal Behavior and Locomotor Activity of Small Groups of Worker Honey Bees.

We examined effects of the queen’s presence on diurnal rhythms of temperature preference (TP) and locomotor activity (LA) in worker honeybees’ groups. TP and LA of six queenless and six queenright (with the queen) groups of bees, consisting of 7–8 worker bees, were recorded in a thermal gradient system for four days, under light to darkness (LD) 12:12 photoperiod. The same experiments were conducted on five virgin queens (of the same age as those in the queenright groups), which were placed individually in the gradient chambers. The single virgin queens showed signs of distress and no rhythms of TP and LA. In contrast, there were diurnal rhythms of TP and LA in both group variants with daytime activity and nighttime rest. However, the queen’s presence exerted a strong calming effect, reducing LA of bees both at day- and nighttime. The nighttime minimum LA of queenright groups was five times lower than that in queenless groups. Moreover, there was a reversal of the diurnal pattern of TP in queenright groups. The results are discussed in terms of the bee colony organization as a superorganism.