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.

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.

Feeding Asian honeybee queens with European honeybee royal jelly alters body color and expression of related coding and non-coding RNAs.

Background and aims: The Asian honeybee (Apis cerana) and the European honeybee (Apis mellifera) are reproductively isolated. Previous studies reported that exchanging the larval food between the two species, known as nutritional crossbreeding, resulted in obvious changes in morphology, physiology and behavior. This study explored the molecular mechanisms underlying the honeybee nutritional crossbreeding. Methods: This study used full nutritional crossbreeding technology to rear A. cerana queens by feeding them with an A. mellifera royal jelly-based diet in an incubator. The body color and the expression of certain genes, microRNA, lncRNA, and circRNA among nutritional crossbred A. cerana queens (NQ), and control A. cerana queens (CQ) were compared. The biological functions of two target genes, TPH1 and KMO, were verified using RNA interference. Results: Our results showed that the NQ's body color turned yellow compared to the black control queens. Whole transcriptome sequencing results showed that a total of 1484, 311, 92, and 169 DEGs, DElncRNAs, DEmiRNAs, and DEcircRNAs, respectively, were identified in NQ and CQ, in which seven DEGs were enriched for three key pathways (tryptophan, tyrosine, and dopamine) involved in melanin synthesis. Interestingly, eight DElncRNAs and three DEmiRNAs were enriched into the key pathways regulating the above key DEGs. No circRNAs were enriched into these key pathways. Knocking down two key genes (KMO and TPH1) resulted in altered body color, suggesting that feeding NQ's an RNAi-based diet significantly downregulated the expression of TPH1 and KMO in 4-day-old larvae, which confirmed the function of key DEGs in the regulation of honeybee body color. Conclusion: These findings reveal that the larval diets from A. mellifera could change the body color of A. cerana, perhaps by altering the expression of non-coding RNAs and related key genes. This study serves as a model of epigenetic regulation in insect body color induced by environmental factors.

Signatures of Positive Selection in the Genome of Apis mellifera carnica: A Subspecies of European Honeybees.

The technology of long reads substantially improved the contingency of the genome assembly, particularly resolving contiguity of the repetitive regions. By integrating the interactive fragment using Hi-C, and the HiFi technique, a solid genome of the honeybee Apis mellifera carnica was assembled at the chromosomal level. A distinctive pattern of genes involved in social evolution was found by comparing it with social and solitary bees. A positive selection was identified in genes involved with cold tolerance, which likely underlies the adaptation of this European honeybee subspecies in the north hemisphere. The availability of this new high-quality genome will foster further studies and advances on genome variation during subspeciation, honeybee breeding and comparative genomics.

A Comparison of RNA Interference via Injection and Feeding in Honey Bees.

RNA interference (RNAi) has been used successfully to reduce target gene expression and induce specific phenotypes in several species. It has proved useful as a tool to investigate gene function and has the potential to manage pest populations and reduce disease pathogens. However, it is not known whether different administration methods are equally effective at interfering with genes in bees. Therefore, we compared the effects of feeding and injection of small interfering RNA (siRNA) on the messenger RNA (mRNA) levels of alpha-aminoadipic semialdehyde dehydrogenase (ALDH7A1), 4-coumarate-CoA ligase (4CL), and heat shock protein 70 (HSP70). Both feeding and injection of siRNA successfully knocked down the gene but feeding required more siRNA than the injection. Our results suggest that both feeding and injection of siRNA effectively interfere with brain genes in bees. The appropriateness of each method would depend on the situation.

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.

Female developmental environment delays development of male honeybee (Apis mellifera).

BACKGROUND: Nutrition and cell size play an important role in the determination of caste differentiation in queen and worker of honeybees (Apis mellifera), whereas the haploid genome dominates the differentiation of drones. However, the effects of female developmental environment on the development of males remain unclear. In this study, young drone larvae were transferred into worker cells (WCs) or remained in drone cells (DCs) to rear drones. The drone larvae were also grafted into queen cells (QCs) for 48 h and then transplanted into drone cells until emerging. Morphological indexes and reproductive organs of these three types of newly emerged drones were measured. Newly emerged drones and third instar drone larvae from WCs, DCs and QCs were sequenced by RNA sequencing (RNA-Seq). RESULTS: The amount of food remaining in cells of the QC and WC groups was significantly different to that in the DC group at the early larval stage. Morphological results showed that newly emerged DC drones had bigger body sizes and more well-developed reproductive tissues than WC and QC drones, whereas the reproductive tissues of QC drones were larger than those of WC drones. Additionally, whole body gene expression results showed a clear difference among three groups. At larval stage there were 889, 1761 and 1927 significantly differentially expressed genes (DEGs) in WC/DC, QC/DC and WC/QC comparisons, respectively. The number of DEGs decreased in adult drones of these three comparisons [678 (WC/DC), 338 (QC/DC) and 518 (WC/QC)]. A high number of DEGs were involved in sex differentiation, growth, olfaction, vision, mammalian target of rapamycin (mTOR), Wnt signaling pathways, and other processes. CONCLUSIONS: This study demonstrated that the developmental environment of honeybee females can delay male development, which may serve as a model for understanding the regulation of sex differentiation and male development in social insects by environmental factors.

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.

Transcriptomic, Morphological, and Developmental Comparison of Adult Honey Bee Queens (Apis mellifera) Reared From Eggs or Worker Larvae of Differing Ages.

Queens and workers are very distinct phenotypes that develop from the same genome. Larvae from worker cells up to 3.5 d old can be transferred to larger queen cells and will subsequently be reared as queens and develop into functional queens. This has become a very popular queen rearing practice in contemporary apiculture. Here we used RNA-Seq to study the consequences of rearing queens from transplanted worker larvae on the transcriptome of the adult queens. We found that queens reared from transferred older larvae developed slower, weighted less, and had fewer ovarioles than queens reared from transferred eggs, indicating queens were cryptically intercaste. RNA-Seq analysis revealed differentially expressed genes between queens reared from transferred larvae compared with queens reared from transferred eggs: the older the larvae transferred, the greater the number of differentially expressed genes. Many of the differentially expressed genes had functions related to reproduction, longevity, immunity, or metabolism, suggesting that the health and long-term viability of queens was compromised. Our finds verify the previous studies that adult queens reared from older transferred larvae were of lower quality than queens reared from transferred eggs or younger larvae.

Clinical outcomes of revision with retrograde intermedullary nailing for failed plating of distal femoral fractures: a retrospective study.

PURPOSE: To assess the feasibility and effectiveness of retrograde intramedullary nail (RIN) revision surgeries for locking compression plate (LCP) failure in distal femoral fractures. METHODS: This retrospective study included 13 patients who suffered from metalwork failures after they initially underwent open reduction and LCP fixation. In patients who eventually underwent RIN revision from January 2014 to December 2016, range of motion (ROM) and Hospital for Special Surgery (HSS) scores obtained before surgery and at the final follow-up time were analysed. RESULTS: The average operative time was 155 minutes (range, 120-210 minutes), and the average blood loss volume was 650 ml (range, 200-1350 ml). There were two cases of complications (15.38%): one was calf muscle vein thrombosis, and the other was a superficial infection. No deep tissue infection or deep vein thrombosis was observed post-operatively. The average follow-up time was 16 months (range, 12-24 months). All fractures healed in a mean of 6.5 months (range, 4-12 months), and one patient underwent an additional bone graft surgery that did not involve a bone graft during the RIN revision operation (this eventually healed at 12 months post-operatively). The mean ROM before the operation was 86.92 ± 12.34°. At the final follow-up, the mean ROM was 112.69 ± 9.27°. There was a significant difference between pre-operative and post-operative ROM (P < 0.01). The mean HSS score improved significantly from 38.85 ± 9.62 points pre-operatively to 79.62 ± 5.42 points post-operatively. There was a significant difference between pre-operative and post-operative HSS scores (P < 0.01). CONCLUSIONS: RIN revision surgery achieved excellent clinical results in patients with LCP failure.

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.

A Maternal Effect on Queen Production in Honeybees.

Influences from the mother on offspring phenotype, known as maternal effects, are an important cause of adaptive phenotypic plasticity [1, 2]. Eusocial insects show dramatic phenotypic plasticity with morphologically distinct reproductive (queen) and worker castes [3, 4]. The dominant paradigm for honeybees (Apis mellifera) is that castes are environmentally rather than genetically determined, with the environment and diet of young larvae causing caste differentiation [5-9]. A role for maternal effects has not been considered, but here we show that egg size also influences queen development. Queens laid significantly bigger eggs in the larger queen cells than in the worker cells. Eggs laid in queen cells (QE), laid in worker cells (WE), and 2-day old larvae from worker cells (2L) were transferred to artificial queen cells to be reared as queens in a standardized environment. Newly emerged adult queens from QE were heavier than those from the other two groups and had more ovarioles, indicating a consequence of egg size for adult queen morphology. Gene expression analyses identified several significantly differentially expressed genes between newly emerged queens from QE and those from the other groups. These included a disproportionate number of genes involved in hormonal signaling, body development, and immune pathways, which are key traits differing between queens and workers. That egg size influences emerging queen morphology and physiology and that queens lay larger eggs in queen cells demonstrate both a maternal effect on the expression of the queen phenotype and a more active role for the queen in gyne production than has been realized previously.

High-Efficiency CRISPR/Cas9-Mediated Gene Editing in Honeybee (Apis mellifera) Embryos.

The honeybee (Apis mellifera) is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the Mrjp1 gene. Two batches of honeybee embryos were collected and injected with Mrjp1 sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, Pax6, and injected Pax6 sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for Mrjp1) and 76.9% (for Pax6) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.

A comparison of honeybee (Apis mellifera) queen, worker and drone larvae by RNA-Seq.

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNA-Seq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.

Molecular cloning and expression analysis of the tyramine receptor genes in Apis cerana cerana.

Tyramine is a biological polyamine, which serves important functions as neurotransmitters, neuromodulators and neurohormone of the central nervous system. It participates in the regulation of various behavior and physiological processes in insects. For example, tyramine and its receptor genes are involved in the regulation of learning and memory in the animals. In this study, the full-length cDNA sequences of the tyramine receptor genes (Actyr1 and Actyr2) of the Chinese honeybee, Apis cerana cerana, were cloned and sequenced for the first time. Their expression patterns were examined in different tissues by qRT-PCR and localized in the head by in situ hybridization with digoxigenin (DIG)-labeled RNA probes. The full-length cDNAs of Actyr1 and Actyr2 are 1241 bp (GenBank accession no. KC814693) and 1270 bp (GenBank accession no.KC814693) in length and encode 297 amino acids and 399 amino acids, respectively. qRT-PCR results showed that the expression levels of both Actyr1 and Actyr2 were the highest in the head, followed by the abdomen, then the antennae and the lowest in the thorax. The expression level in the head was significantly higher than that in other tissues. Moreover, in situ hybridization showed that the expression of Actyr1 and Actyr2 genes were mainly localized to the Kenyon cells of the mushroom bodies and cells around the antennal lobes. These observations suggest that some interactions between these two genes in certain cells could be important in regulating various biological functions, such as learning and memory, in the honeybee.

Short-Term Exposure to Lambda-Cyhalothrin Negatively Affects the Survival and Memory-Related Characteristics of Worker Bees Apis mellifera.

Pesticides are considered one of the major contemporary stressors of honey bee health. In this study, the effects of short-term exposure to lambda-cyhalothrin on lifespan, learning, and memory-related characteristics of Apis mellifera were systematically examined. Short-term exposure to lambda-cyhalothrin in worker bees reduced lifespan, affected learning and memory performance, reduced the homing ability, and influenced the expression levels of two learning and memory-related genes of A. mellifera. This research identifies the nature of the sublethal effects of lambda-cyhalothrin on bees and the level of exposure that can be harmful to bee health. This new information will assist in establishing guidelines for the safe use of lambda-cyhalothrin in the field.

Genome-wide DNA methylation changes associated with olfactory learning and memory in Apis mellifera.

The honeybee is a model organism for studying learning and memory formation and its underlying molecular mechanisms. While DNA methylation is well studied in caste differentiation, its role in learning and memory is not clear in honeybees. Here, we analyzed genome-wide DNA methylation changes during olfactory learning and memory process in A. mellifera using whole genome bisulfite sequencing (WGBS) method. A total of 853 significantly differentially methylated regions (DMRs) and 963 differentially methylated genes (DMGs) were identified. We discovered that 440 DMRs of 648 genes were hypermethylated and 274 DMRs of 336 genes were hypomethylated in trained group compared to untrained group. Of these DMGs, many are critical genes involved in learning and memory, such as Creb, GABA B R and Ip3k, indicating extensive involvement of DNA methylation in honeybee olfactory learning and memory process. Furthermore, key enzymes for histone methylation, RNA editing and miRNA processing also showed methylation changes during this process, implying that DNA methylation can affect learning and memory of honeybees by regulating other epigenetic modification processes.