Queen Larvae Research Articles

A larval ‘princess pheromone’ identifies future ant queens based on their juvenile hormone content

Numerous studies have identified cuticular compounds that distinguish adult queens from workers in social insect colonies, but how future queens are identified at the larval stage is poorly understood. Nevertheless, the ability of workers to discriminate queen and worker larvae is necessary for them to regulate caste determination and queen production. In the ant Harpegnathos saltator, workers bite larvae to inhibit queen development, and we used biting as an assay to test how workers identify queens at the larval stage. The transfer of cuticular compounds from queen to worker larvae through direct physical contact (rubbing) or using a hexane extract both elicited biting. Gas chromatography revealed significant differences in cuticular hydrocarbon profiles of queen and worker larvae that could be induced by treatment with a juvenile hormone (JH) analogue. Finally, treatment of male larvae with a JH analogue also elicited worker biting, which suggests a direct connection between JH levels and the production of a larval queen signal. These results demonstrate that workers identify larval caste using a chemical signal present on the cuticle, a ‘princess pheromone’, that reflects endocrine changes associated with queen development. Based on the connection between JH levels and the production of a larval queen signal, we developed a model for caste determination in H. saltator that incorporates endocrine, pheromonal and behavioural control of caste development.

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen.

ABSTRACTViral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway.

Specialized castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3-day-old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen-worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates.

DNA methylation comparison between 4-day-old queen and worker larvae of honey bee

The honey bee is a social insect that is famous for queen-worker differentiation. Numerous studies indicate that queen larvae (QL) and worker larvae (WL) have different expressed genes and proteins. DNA methylation has been found to play an important role in regulating gene expression. To further explore the roles of the methylated genes in queen-worker differentiation, we analyzed DNA methylome profiles of 4-day-old QL and WL (Apis mellifera). The results demonstrated that there were 7.2gigabases of sequence data from six methylated DNA immunoprecipitation libraries, and provided a genome-wide DNA methylation map as well as a gene expression map for 4-day-old QL and WL. The genome coverage of every sample was 4.79. According to CpG representation, all promoters in the A. mellifera genome were classified into high CpG promoters, intermediate CpG promoters and low CpG promoters. The methylated cytosines of larvae were enriched in introns, followed by coding sequence regions, 2K downstream of genes, 5′ untranslated regions (UTRs), 2K upstream of genes, and 3′ UTRs. Compared with 4-day-old WL, a number of genes in QL were down-methylated that were involved in biological regulation, immune system and metabolic regulation. In addition, these DMGs were involved in many signal pathways of caste differentiation such as Mitogen-activated protein kinase (MAPK), Notch, Insulin and Wnt signaling pathways.

First confirmed report of a bacterial brood disease in stingless bees

Susceptibility to brood pathogens in eusocial stingless bees (Meliponini), alternative pollinators to honey bees, is unknown. Brood losses in managed colonies of the Australian stingless bee, Tetragonula carbonaria, were studied over 20months. We isolated a disease-causing bacterium, Lysinibacillus sphaericus (Firmicutes, Bacillaceae), from worker and queen larvae, brood cell provisions and honey stores. Pathogenicity experiments confirmed this bacterium as the causal organism. It took 22days from infection to first appearance of brood disease symptoms. This is the first confirmed record of a brood pathogen in stingless bees.

Rearing Drones in Queen Cells ofApis melliferaHoney Bees

AbstractWe investigated the rearing of drone larvae grafted in queen cells. From the 1200 drone larvae that were grafted during spring and autumn, 875 were accepted (72.9%) and reared as queens. Drone larvae in false queen cells received royal jelly of the same composition and of the same amounts as queen larvae. Workers capped the queen cells as if they were drones, 9-10 days after the egg laying. Out of 60 accepted false queen cells, 21 (35%) were capped. The shape of false queen cells with drone larvae is unusually long with a characteristically elongate tip which is probably due to the falling of larvae. Bees start the destruction of the cells when the larvae were 3 days old and maximised it before and after capping. Protecting false queen cells in the colony by wrapping, reversing them upside down, or placing in a horizontal position, did not help. The only adult drones that emerged from the false queen cells were those protected in an incubator and in push-in cages. Adult drones from false queen cells had smaller wings, legs, and proboscis than regular drones. The results of this study verify previous reports that the bees do not recognise the different sex of the larvae at least at the early stage of larval development. The late destruction of false queen cells, the similarity in quality and quantity of the produced royal jelly, and the bigger drone cells, allow for the use of drone larvae in cups for the production of royal jelly.

Differential gene expression underlying ovarian phenotype determination in honey bee, Apis mellifera L., caste development

Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the final larval instar, when massive programmed cell death leads to the degeneration of 95–99% of the ovariole anlagen in workers. The higher juvenile hormone (JH) levels in queen larvae protect the ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate and worker sterility, we performed a microarray analysis on fourth and early fifth instar queen and worker ovaries. For the fourth instar we found nine differentially expressed genes (DEGs) with log2FC > 1.0, but this number increased to 56 in early fifth-instar ovaries. We selected 15 DEGs for quantitative PCR (RT-qPCR) analysis. Nine differed significantly by the variables caste and/or development. Interestingly, genes with enzyme functions were higher expressed in workers, while those related to transcription and signaling had higher transcript levels in queens. For the RT-qPCR confirmed genes we analyzed their response to JH. This revealed a significant up-regulation for two genes, a short chain dehydrogenase reductase (sdr) and a heat shock protein 90 (hsp90). Five other genes, including hsp60 and hexamerin 70b (hex70b), were significantly down-regulated by JH. The sdr gene had previously come up as differentially expressed in other transcriptome analyses on honey bee larvae and heat shock proteins are frequently involved in insect hormone responses, this making them interesting candidates for further functional assays.

GC-MS investigation of the chemical composition of honeybee drone and queen larvae homogenate

Abstract Honeybee larva homogenate appears to be underrated and insufficiently explored but this homogenate is an exceptionally valuable honeybee product. Drone larva homogenate is very nutritional due to its high content of proteins, free amino acids, lipids, and carbohydrates. Moreover, the biological characteristics of honeybee larvae indicate the presence of chemical substances that may be pharmacologically active. In spite of the above, the chemical composition of honeybee larva has not gained as much attention as that of other bee products. In this study, the chemical composition of honeybee brood homogenate has been investigated using gas chromatography/mass spectrometry. As a result, it was possible to isolate as many as 115 extractive organic compounds from 6 samples of crude queen and 9 samples of drone homogenate. The main groups of substances extracted from either type of homogenate were composed of free amino acids and carbohydrates. The relative content of amino acids in queen homogenate as well as the share of essential amino acids were found to be higher than in the drone homogenate. Disaccharide trehalose was the dominant sugar in the queen larvae, whilst glucose prevailed in the drone larvae. Comparative chemical analyses of honeybee queen and drone larva homogenates have allowed us to make a preliminary inference about a higher overall value of the former.

Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development

The relationship between nutrition and phenotype is an especially challenging question in cases of facultative polyphenism, like the castes of social insects. In the honey bee, Apis mellifera, unexpected modifications in conserved signaling pathways revealed the hypoxia response as a possible mechanism underlying the regulation of body size and organ growth. Hence, the current study was designed to investigate possible causes of why the three hypoxia core genes are overexpressed in worker larvae. Parting from the hypothesis that this has an endogenous cause and is not due to differences in external oxygen levels we investigated mitochondrial numbers and distribution, as well as mitochondrial oxygen consumption rates in fat body cells of queen and worker larvae during the caste fate-critical larval stages. By immunofluorescence and electron microscopy we found higher densities of mitochondria in queen larval fat body, a finding further confirmed by a citrate synthase assay quantifying mitochondrial functional units. Oxygen consumption measurements by high-resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production at lower physiological demand. Finally, the expression analysis of mitogenesis-related factors showed that the honey bee TFB1 and TFB2 homologs, and a nutritional regulator, ERR, are overexpressed in queen larvae. These results are strong evidence that the differential nutrition of queen and worker larvae by nurse bees affects mitochondrial dynamics and functionality in the fat body of these larvae, hence explaining their differential hypoxia response.

Natural Larval Diet Differently Influences the Pattern of Developmental Changes in DNA 5-Methylcytosine Levels in Apis mellifera Queens as Compared with Workers and Drones.

The principal mechanism of gene activation/silencing is DNA 5-methylcytosine methylation. This study was aimed at determining global DNA methylation levels in larvae, prepupae, pupae, and 1-day-old adults of Apis mellifera queens, workers and drones. The Imprint Methylated DNA Quantification Kit MDQ1 was used. Percentages of DNA 5-methylcytosine were low and relatively similar in the larvae of all the castes until 4th day of larval development (3-5%). However, they were higher in the drone and worker larvae than in the queen larvae. Generally, the developmental patterns of changes in the DNA methylation levels were different in the queens in comparison with the drones and workers. While methylation increased in the queens, it decreased in the drones and workers. Methylated DNA methylcytosine percentages and weights in the queen prepupae (15%, 9.18ng) and pupae (21%, 10.74ng) were, respectively, three and four times higher than in the worker/drone brood of the same age (2.5-4%, 0.03-0.07ng). Only in the queens, after a substantial increase, did DNA methylation decrease almost twice between the pupal stage and queen emergence (from 21% and 10.74ng to 12% and 6.78ng). This finding seems very interesting, particularly for experimental gerontology.

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis.

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways.

Age matters: pheromone profiles of larvae differentially influence foraging behaviour in the honeybee, Apis mellifera

How a colony regulates the division of labour to forage for nutritional resources while accommodating for size and demographic composition is a fundamental question in the sociobiology of social insects. In honeybees, Apis mellifera, young and old larvae produce pheromones that differ in composition. Nurses differentially regulate larval nutrition, feeding young worker larvae a surplus diet that parallels queen larvae in protein composition and food availability, while old larvae are restrictively fed a diet with similar sugar content as queens. The presence of larvae affects division of labour, but it is unknown whether foragers regulate resource collection based on larval age or pheromone production in the nest. We studied how larval age demography and the larval pheromone e-beta ocimene affect foraging activity and foraging load. Our results suggest that workers recognize larval age, probably by detecting changes in the pheromones emitted by larvae as they mature, and adjust the foraging division of labour (pollen versus nectar) to meet the nutritional needs of the colony's brood. For younger larvae, this results in a bias towards pollen collection.

Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.

Over-expression and Purification of RNA Dependent RNA Polymerase from Black queen cell virus in Honeybee

Honeybee populations infected by numerous viruses could not easily distinguish from the healthy populations because there are no apparent clinical symptoms and signs of infection. Black queen cell virus (BQCV) is one of the most prevalent viruses, resulting in the death of queen larvae and pupae. Like to other picornaviruses, BQCV has the RNA dependent RNA polymerase (RdRp) that catalyzes the replication of viral RNA. The optimal conditions for expression of MBP-BQCV-RdRp protein were confirmed by an induction with 0.1mM IPTG at 25°C for 6 hours and the MBP-BQCV-RdRp protein was purified using by amylose resin. Total yield of protein reached approximately 5mg per 50ml of induced cells. Due to its property that is an essential enzyme of virus replication, purified recombinant BQCV-RdRp proteins can be provided to the further lead as an important source to study how to control the BQCV viral disease in honeybee, development of a rapid kit for the diagnosis of BQCV infection in field,or finding out the anti-viral chemical compound for the inhibitor assay to prevent BQCV replication in honeybee.

Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease

Previously, we found three enzyme fractions containing activities for the hydrolysis of royal jelly proteins from honeybee queen larvae. In this study, we identified a honeybee chymotrypsin-like protease (HCLPase) by LC-MS/MS and expressed it as a recombinant protein in Escherichia coli. The protease had an estimated molecular weight of around 26 kDa and showed high specificity for succinyl-Ala-Ala-Pro-Phe p-nitroanilide as a proteolytic substrate. Furthermore, the protease had an optimal pH of 9, and the activity was markedly inhibited by phenylmethylsulfonyl fluoride but not tosyl phenylalanyl chloromethyl ketone, both of which are irreversible inhibitors of chymotrypsin-like serine proteases. These results suggested that this recombinant protease, HCLPase, was a chymotrypsin-like serine protease with different characteristics from mammalian chymotrypsin.

Differentially expressed microRNAs between queen and worker larvae of the honey bee (Apis mellifera)

Although the honey bee queen and worker have identical genomes, they exhibit morphological, behavioral, and reproductive differences. Queen larvae (QL) and worker larvae (WL) have different gene and protein expression profiles and different DNA methylation profiles. MicroRNAs (miRNAs) play an important role in regulating gene expression and cell biological processes. To examine the roles of miRNAs in caste differentiation, we analyzed small RNA profiles in 4-day-old QL and WL (Apis mellifera). The results demonstrated that the small RNAs with a length of 22 nt showed significant difference between the two castes. By comparison with mirBase 19.0, we identified 61 known miRNAs in QL and WL; 17, 24 and 20 of these miRNAs were respectively up-regulated, equally expressed, and down-regulated in QL compared with WL. These differentially expressed miRNAs were involved in many signaling pathways related to caste differentiation.

MRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees (Apis mellifera).

In honeybee (Apis mellifera) colonies, queens and workers are alternative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR (target of rapamycin) and Dnmt3 (DNA methyltransferase 3) genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K (similar to RPS6-p70-protein kinase) gene linked closely to organismal growth and development in queen and worker larvae (1-day and 3-day old). Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

Juvenile Hormone Biosynthesis Gene Expression in the corpora allata of Honey Bee (Apis mellifera L.) Female Castes

Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

Comparison between local and commercial royal jelly—use of antioxidant activity and 10-hydroxy-2-decenoic acid as quality parameter

SummaryRoyal jelly (RJ) is secreted by worker bees of Apis mellifera to feed the queen larvae. Believed to be the longevity factor of queen honey bees and due to its variety of health promoting ingredients, RJ is used worldwide as dietary supplement, health product and in cosmetics. To reach best possible effects, application of high quality RJ is absolutely required. Here we show, based on physicochemical properties and antioxidant activity, a comparison between fresh, local RJ samples obtained directly from beekeepers and commercial ones. Analyses revealed a higher content of 10-hydroxy-2-decenoic acid, a quality parameter of RJ, for local compared to commercial RJ. Furthermore, determined ferric reducing antioxidant power (FRAP) correlated with radical scavenging activity (DPPH) and total polyphenols was significantly higher in local RJ samples. Therefore, our results indicate a higher quality for local RJ compared to commercial.

Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family

In the honeybee, Apis mellifera, the queen larvae are fed with a diet exclusively composed of royal jelly (RJ), a secretion of the hypopharyngeal gland of young worker bees that nurse the brood. Up to 15% of RJ is composed of proteins, the nine most abundant of which have been termed major royal jelly proteins (MRJPs). Although it is widely accepted that RJ somehow determines the fate of a female larva and in spite of considerable research efforts, there are surprisingly few studies that address the biochemical characterisation and functions of these MRJPs. Here we review the research on MRJPs not only in honeybees but in hymenopteran insects in general and provide metadata analyses on genome organisation of mrjp genes, corroborating previous reports that MRJPs have important functions for insect development and not just a nutritional value for developing honeybee larvae.