The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees.

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

Phenotypic dimorphism between honeybee queen and worker is regulated by complicated epigenetic modifications.

Phenotypic dimorphism between queens and workers is an important biological characteristic of honeybees that has been the subject of intensive research. The enormous differences in morphology, lifespan, physiology, and behavior between queens and workers are caused by a complicated set of factors. Epigenetic modifications are considered to play an important role in this process. In this study, we analyzed the differences in chromosome interactions and H3K27ac and H3K4me1 modifications between the queens and workers using high-throughput chromosome conformation capture (Hi-C) and Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) technologies. We found that the queens contain more chromosome interactions and more unique H3K27ac modifications than workers; in contrast, workers have more H3K4me1 modifications than queens. Moreover, we identified Map3k15 as a potential caste gene in queen-worker differentiation. Our results suggest that chromosomal conformation and H3K27ac and H3K4me1 modifications are involved in regulating queen-worker differentiation, which reveals that the queen-worker phenotypic dimorphism is regulated by multiple epigenetic modifications.

The diverging epigenomic landscapes of honeybee queens and workers revealed by multiomic sequencing.

The role of the epigenome in phenotypic plasticity is unclear presently. Here we used a multiomics approach to explore the nature of the epigenome in developing honey bee (Apis mellifera) workers and queens. Our data clearly showed distinct queen and worker epigenomic landscapes during the developmental process. Differences in gene expression between workers and queens become more extensive and more layered during the process of development. Genes known to be important for caste differentiation were more likely to be regulated by multiple epigenomic systems than other differentially expressed genes. We confirmed the importance of two candidate genes for caste differentiation by using RNAi to manipulate the expression of two genes that differed in expression between workers and queens were regulated by multiple epigenomic systems. For both genes the RNAi manipulation resulted in a decrease in weight and fewer ovarioles of newly emerged queens compared to controls. Our data show that the distinct epigenomic landscapes of worker and queen bees differentiate during the course of larval development.

The involvement of a floral scent in plant-honeybee interaction.

Volatile odors from flowers play an important role in plant-pollinator interaction. The honeybee is an important generalist pollinator of many plants. Here, we explored whether any components of the odors of a range of honeybee-pollinated plants are commonly involved in the interaction between plants and honeybees. We used a needle trap system to collect floral odors, and GC-MS analysis revealed nonanal was the only component scent detected in 12 different honeybee-pollinated flowers and not present in anemophilous plant species. For Ligustrum compactum, blooming flowers released significantly more nonanal than buds and faded flowers. For Sapium sebiferum, nonanal release through the day correlated with nectar secretion. Experimentally increasing nectar load in flowers of Sapium sebiferum, Ligustrum compactum, and Castanea henryi increased nonanal levels also. Nonanal was also detected in flower nectar and honeys from experimental colonies. Electroantennogram recordings and behavioral observations showed that untrained honeybees could detect and were strongly attracted to nonanal. We argue that nonanal persists in both honey and nectar odors facilitating a learned association between nonanal and food reward in honeybees.

Extent and complexity of RNA processing in honey bee queen and worker caste development.

The distinct honeybee (Apis mellifera) worker and queen castes have become a model for the study of genomic mechanisms of phenotypic plasticity. Here we performed a nanopore-based direct RNA sequencing with exceptionally long reads to compare the mRNA transcripts between queen and workers at three points during their larval development. We found thousands of significantly differentially expressed transcript isoforms (DEIs) between queen and worker larvae. These DEIs were formatted by a flexible splicing system. We showed that poly(A) tails participated in this caste differentiation by negatively regulating the expression of DEIs. Hundreds of isoforms uniquely expressed in either queens or workers during their larval development, and isoforms were expressed at different points in queen and worker larval development demonstrating a dynamic relationship between isoform expression and developmental mechanisms. These findings show the full complexity of RNA processing and transcript expression in honey bee phenotypic plasticity.

Honey Bee Habitat Sharing Enhances Gene Flow of the Parasite Nosema ceranae.

Host-parasite co-evolution is a process of reciprocal, adaptive genetic change. In natural conditions, parasites can shift to other host species, given both host and parasite genotypes allow this. Even though host-parasite co-evolution has been extensively studied both theoretically and empirically, few studies have focused on parasite gene flow between native and novel hosts. Nosema ceranae is a native parasite of the Asian honey bee Apis cerana, which infects epithelial cells of mid-guts. This parasite successfully switched to the European honey bee Apis mellifera, where high virulence has been reported. In this study, we used the parasite N. ceranae and both honey bee species as model organisms to study the impacts of two-host habitat sharing on parasite diversity and virulence. SNVs (Single Nucleotide Variants) were identified from parasites isolated from native and novel hosts from sympatric populations, as well as novel hosts from a parapatric population. Parasites isolated from native hosts showed the highest levels of polymorphism. By comparing the parasites isolated from novel hosts between sympatric and parapatric populations, habitat sharing with the native host significantly enhanced parasite diversity, suggesting there is continuing gene flow of parasites between the two host species in sympatric populations.

Identification of novel miRNAs from the microsporidian parasite Nosema ceranae.

Previously, six miRNAs were identified from the microsporidian parasite Nosema ceranae. By taking advantage of the recently updated N. ceranae and honey bee genome assemblies, we re-analyzed the deep sequencing datasets. Three novel miRNAs were identified, which were further validated by plasmid cloning and sequencing. The miRNAs correlated with significantly higher number of genes from the parasite than the host. Our data suggest the parasitic miRNAs are involved in self-regulation during the proliferation.

Haplotype Analysis of Varroa destructor and Deformed Wing Virus Using Long Reads.

As a phoretic parasite and virus vector, the mite Varroa destructor and the associated Deformed wing virus (DWV) form a lethal combination to the honey bee, Apis mellifera. Routine acaricide treatment has been reported to reduce the diversity of mites and select for tolerance against these treatments. Further, different DWV strains face selective pressures when transmitted via mites. In this study, the haplotypes of Varroa mites and associated DWV variants were quantified using long reads. A single haplotype dominated the mite mitochondrial gene cytochrome oxidase subunit I, reflecting an ancient bottleneck. However, highly polymorphic genes were present across the mite genome, suggesting the diversity of mites could be actively maintained at a regional level. DWV detected in both mites and honey bees show a dominant variant with only a few low-frequency alternate haplotypes. The relative abundances of DWV haplotypes isolated from honey bees and mites were highly consistent, suggesting that some variants are favored by ongoing selection.

Transgenerational accumulation of methylome changes discovered in commercially reared honey bee (Apis mellifera) queens.

Whether a female honey bee (Apis mellifera) develops into a worker or a queen depends on her nutrition during development, which changes the epigenome to alter the developmental trajectory. Beekeepers typically exploit this developmental plasticity to produce queen bee by transplanting worker larvae into queen cells to be reared as queens, thus redirecting a worker developmental pathway to a queen developmental pathway. We studied the consequences of this manipulation for the queen phenotype and methylome over four generations. Queens reared from worker larvae consistently had fewer ovarioles than queens reared from eggs. Over four generations the methylomes of lines of queens reared from eggs and worker larvae diverged, accumulating increasing differences in exons of genes related to caste differentiation, growth and immunity. We discuss the consequences of these cryptic changes to the honey bee epigenome for the health and viability of honey bee stocks.

A Chromosome-Scale Assembly of the Asian Honeybee Apis cerana Genome.

Apis cerana is one of the main honeybee species in artificial farming, which is widely distributed in Asian countries. The genome of A. cerana has been sequenced by several different research groups using second generation sequencing technologies. However, it is still necessary to obtain more complete and accurate genome sequences. Here we present a chromosome-scale assembly of the A. cerana genome using single-molecule real-time (SMRT) Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated assembly is 215.67 Mb in size with a contig N50 of 4.49 Mb, representing an 212-fold improvement over the previous Illumina-based version. Hi-C scaffolding resulted in 16 pseudochromosomes occupying 97.85% of the assembled genome sequences. A total of 10,741 protein-coding genes were predicted and 9,627 genes were annotated. Besides, 314 new genes were identified compared to the previous version. The improved high-quality A. cerana reference genome will provide precise sequence information for biological research of A. cerana.

Deltamethrin Impairs Honeybees (Apis mellifera) Dancing Communication.

As a commonly used pyrethroid insecticide, deltamethrin is very toxic to honeybees, which seriously threatens the managed and feral honeybee population. Because deltamethrin is a nerve agent, it may interfere with the nervous system of honeybees, such as dance behavior and memory-related characteristics. We found that the waggle dances were less precise in honeybees that consumed syrup containing deltamethrin (pesticide group) than those that consumed normal sucrose syrup (control group). Compared with the control group, honeybees of the pesticide group significantly increased number of circuits per 15 s, the divergence angle, return phases in waggle dances, as well as the crop content of the dance followers. Furthermore, six learning and memory-related genes were significantly interfered with the gene expression levels. Our data suggest that the sublethal dose of deltamethrin impaired the honeybees' learning and memory and resulted in cognitive disorder. The novel results assist in establishing guidelines for the risk assessment of pesticide to honeybee safety and prevention of nontarget biological agriculture pesticide poisoning.

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 3 days 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.

Differential protein expression analysis following olfactory learning in Apis cerana.

Studies of olfactory learning in honeybees have helped to elucidate the neurobiological basis of learning and memory. In this study, protein expression changes following olfactory learning in Apis cerana were investigated using isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 2406 proteins were identified from the trained and untrained groups. Among these proteins, 147 were differentially expressed, with 87 up-regulated and 60 down-regulated in the trained group compared with the untrained group. These results suggest that the differentially expressed proteins may be involved in the regulation of olfactory learning and memory in A. cerana. The iTRAQ data can provide information on the global protein expression patterns associated with olfactory learning, which will facilitate our understanding of the molecular mechanisms of learning and memory of honeybees.

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana).

BACKGROUND: Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager). RESULTS: Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs. CONCLUSIONS: In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

Cross-modal interaction between visual and olfactory learning in Apis cerana.

The power of the small honeybee brain carrying out behavioral and cognitive tasks has been shown repeatedly to be highly impressive. The present study investigates, for the first time, the cross-modal interaction between visual and olfactory learning in Apis cerana. To explore the role and molecular mechanisms of cross-modal learning in A. cerana, the honeybees were trained and tested in a modified Y-maze with seven visual and five olfactory stimulus, where a robust visual threshold for black/white grating (period of 2.8°-3.8°) and relatively olfactory threshold (concentration of 50-25%) was obtained. Meanwhile, the expression levels of five genes (AcCREB, Acdop1, Acdop2, Acdop3, Actyr1) related to learning and memory were analyzed under different training conditions by real-time RT-PCR. The experimental results indicate that A. cerana could exhibit cross-modal interactions between visual and olfactory learning by reducing the threshold level of the conditioning stimuli, and that these genes may play important roles in the learning process of honeybees.

[Clinical analysis of acute zonal occult outer retinopathy masquerading as optic neuropathy].

OBJECTIVE: To analyze the clinical features of acute zonal occult outer retinopathy (AZOOR) and the reasons for misdiagnosing as optic neuropathy or intracranial lesions. METHODS: Case series study. Twenty patients (23 eyes) who were all initially diagnosed as optic neuropathy or intracranial lesions were recruited in the ophthalmologic department of Beijing Tongren Hospital from November 2003 to June 2012, but they were ultimately diagnosed as AZOOR after the examinations including regular eye examination, fundus fluorescein angiography (FFA), optical coherence tomography (OCT), automatic perimetry, flash electroretinogram (F-ERG),multifocal electroretinogram (mfERG),systemic rheumatism immunological examination and neuroimaging. RESULTS: These 20 patients with an average age of 32.9 years (range 15 to 55 years) consisted of 17 females (85%) and 3 males (15.0%). Among them, 17 patients (85.0%) were affected unilaterally, while 3 patients (15.0%) were affected bilaterally. Sixteen of 23 eyes (69.6%) had myopia. Ten eyes (58.8%) showed relative afferent papillary defect. Inflammatory response was not found by slit lamp and fundus examination. Besides the other abnormal fundus changes of high myopia, fundus angioid streaks were found in 4 eyes and retinal pigment epithelium dystrophy was found in 3 eyes by fundus examination. FFA revealed that there was mild fluorescein leakage around the retinal vascular arch in one eye. The patterns of visual field detect in these patients were various, while temporal scotoma and blind spot enlargement were most common. Twelve of 14 patients who had been performed F-ERG showed abnormal F-ERG. mfERG of all patients showed the decreased amplitude densities of the retina in accordance with the areas of visual field defect. IS/OS layer of 17 patients who went through OCT examination were found abnormal in the corresponding areas of visual field defect. Neuroimaging examination revealed there was nothing abnormal in the patients who were diagnosed as intracranial lesions. Immunological parameters in these patients were almost normal except for one patient with an increased level of rheumatoid factor. CONCLUSIONS: AZOOR patients in this study rarely has ocular or systemic inflammatory. AZOOR occurs predominantly in the unilateral eye of young female patients, and are easily misdiagnosed as optic neuropathy due to the almost normal appearances of fundus. The examinations of retinal functional and morphological changes are the key to its diagnosis and differential diagnosis.

Clinical features of retinal diseases masquerading as retrobulbar optic neuritis.

BACKGROUND: Managements of optic neuritis (ON) included high-dose corticosteroids or combined with systemic immunomodulatory agents. It was important to make a correct diagnosis of ON before initiation of treatment. The purpose of the study was to report and analyze the clinical features of retinal diseases in patients who were misdiagnosed as having retrobulbar ON. METHODS: Retrospective review of 26 patients (38 eyes) initially diagnosed with retrobulbar ON but were ultimately diagnosed with retinal or macular diseases. Data obtained from fundus examination, fluorescence fundus angiography (FFA), automated static perimetry, full-field electroretinogram (ffERG), multifocal electroretinogram (mfERG), and optical coherence tomography (OCT) were evaluated. RESULTS: Thirty-eight eyes of 26 patients were found to have misdiagnosis of retrobulbar ON, based on normal or slight abnormal fundus findings and abnormal visual evoked potentials (VEP). The mean age of the patients was 34 years and the correct diagnosis of the patients included acute zonal occult outer retinopathy (AZOOR, 15 eyes, 14 patients), occult macular dystrophy (OMD, 8 eyes, 4 patients), cone or cone-rod dystrophy (10 eyes, 5 patients), acute macular neuroretinopathy (AMNR, 3 eyes, 2 patients), and cancer-associated retinopathy (CAR, 2 eyes, 1 patient). CONCLUSION: When attempting to diagnose retrobulbar ON in clinical practice, it is crucial to carry out necessary examinations of the retinal function and morphology to decrease misdiagnosis.

A comparison of digital gene expression profiling and methyl DNA immunoprecipitation as methods for gene discovery in honeybee (Apis mellifera) behavioural genomic analyses.

The honey bee has a well-organized system of division of labour among workers. Workers typically progress through a series of discrete behavioural castes as they age, and this has become an important case study for exploring how dynamic changes in gene expression can influence behaviour. Here we applied both digital gene expression analysis and methyl DNA immunoprecipitation analysis to nurse, forager and reverted nurse bees (nurses that have returned to the nursing state after a period spent foraging) from the same colony in order to compare the outcomes of these different forms of genomic analysis. A total of 874 and 710 significantly differentially expressed genes were identified in forager/nurse and reverted nurse/forager comparisons respectively. Of these, 229 genes exhibited reversed directions of gene expression differences between the forager/nurse and reverted nurse/forager comparisons. Using methyl-DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) we identified 366 and 442 significantly differentially methylated genes in forager/nurse and reverted nurse/forager comparisons respectively. Of these, 165 genes were identified as differentially methylated in both comparisons. However, very few genes were identified as both differentially expressed and differentially methylated in our comparisons of nurses and foragers. These findings confirm that changes in both gene expression and DNA methylation are involved in the nurse and forager behavioural castes, but the different analytical methods reveal quite distinct sets of candidate genes.

Genomewide analysis indicates that queen larvae have lower methylation levels in the honey bee (Apis mellifera).

The honey bee is a social insect characterized by caste differentiation, by which a young larva can develop into either a queen or a worker. Despite possessing the same genome, queen and workers display marked differences in reproductive capacity, physiology, and behavior. Recent studies have shown that DNA methylation plays important roles in caste differentiation. To further explore the roles of DNA methylation in this process, we analyzed DNA methylome profiles of both queen larvae (QL) and worker larvae (WL) of different ages (2, 4, and 6 day old), by using methylated DNA immunoprecipitation-sequencing (meDIP-seq) technique. The global DNA methylation levels varied between the larvae of two castes. DNA methylation increased from 2-day- to 4-day-old QL and then decreased in 6-day-old larvae. In WL, methylation levels increased with age. The methylcytosines in both larvae were enriched in introns, followed by coding sequence (CDS) regions, CpG islands, 2 kbp downstream and upstream of genes, and 5' and 3' untranslated regions (UTRs). The number of differentially methylated genes (DMGs) in 2-, 4-, and 6-day-old QL and WL was 725, 3,013, and 5,049, respectively. Compared to 4- and 6-day-old WL, a large number of genes in QL were downmethylated, which were involved in many processes including development, reproduction, and metabolic regulation. In addition, some DMGs were concerned with caste differentiation.

Transcriptome analysis of the Asian honey bee Apis cerana cerana.

BACKGROUND: The Eastern hive honey bee, Apis cerana cerana is a native and widely bred honey bee species in China. Molecular biology research about this honey bee species is scarce, and genomic information for A. c. cerana is not currently available. Transcriptome and expression profiling data for this species are therefore important resources needed to better understand the biological mechanisms of A. c. cerana. In this study, we obtained the transcriptome information of A. c. cerana by RNA-sequencing and compared gene expression differences between queens and workers of A. c. cerana by digital gene expression (DGE) analysis. RESULTS: Using high-throughput Illumina RNA sequencing we obtained 51,581,510 clean reads corresponding to 4.64 Gb total nucleotides from a single run. These reads were assembled into 46,999 unigenes with a mean length of 676 bp. Based on a sequence similarity search against the five public databases (NR, Swissport, GO, COG, KEGG) with a cut-off E-value of 10(-5) using BLASTX, a total of 24,630 unigenes were annotated with gene descriptions, gene ontology terms, or metabolic pathways. Using these transcriptome data as references we analyzed the gene expression differences between the queens and workers of A. c. cerana using a tag-based digital gene expression method. We obtained 5.96 and 5.66 million clean tags from the queen and worker samples, respectively. A total of 414 genes were differentially expressed between them, with 189 up-regulated and 225 down-regulated in queens. CONCLUSIONS: Our transcriptome data provide a comprehensive sequence resource for future A. c. cerana study, establishing an important public information platform for functional genomic studies in A. c. cerana. Furthermore, the DGE data provide comprehensive gene expression information for the queens and workers, which will facilitate our understanding of the molecular mechanisms of the different physiological aspects of the two castes.