Roles of DNA Methylation in Color Alternation of Eastern Honey Bees (Apis cerana) Induced by the Royal Jelly of Western Honey Bees (Apis mellifera).

Honey bees have a very interesting phenomenon where the larval diets of two different honey bee species are exchanged, resulting in altered phenotypes, namely, a honey bee nutritional crossbreed. This is a classical epigenetic process, but its underlying mechanisms remain unclear. This study aims to investigate the contribution of DNA methylation to the phenotypic alternation of a Apis mellifera-Apis cerana nutritional crossbreed. We used a full nutritional crossbreed technique to rear A. cerana queens by feeding their larvae with A. mellifera royal-jelly-based diets in an incubator. Subsequently, we compared genome-wide methylation sequencing, body color, GC ratio, and the DMRs between the nutritional crossbreed, A. cerana queens (NQs), and control, A. cerana queens (CQs). Our results showed that the NQ's body color shifted to yellow compared to the black control queens. Genome methylation sequencing revealed that NQs had a much higher ratio of mCG than that of CQs. A total of 1020 DMGs were identified, of which 20 DMGs were enriched into key pathways for melanin synthesis, including tryptophan, tyrosine, dopamine, and phenylalanine KEGG pathways. Three key differentially methylated genes [OGDH, ALDH(NAD+) and ALDH7] showed a clear, altered DNA methylation in multiple CpG islands in NQs compared to CQs. Consequently, these findings revealed that DNA methylation participates in A. cerana-A. mellifera nutritional crossbreeding as an important epigenetic modification. This study serves as a model of cross-kingdom epigenetic mechanisms in insect body color induced by environmental factors.

H3K4me1 Modification Functions in Caste Differentiation in Honey Bees.

Honey bees are important species for the study of epigenetics. Female honey bee larvae with the same genotype can develop into phenotypically distinct organisms (sterile workers and fertile queens) depending on conditions such as diet. Previous studies have shown that DNA methylation and histone modification can establish distinct gene expression patterns, leading to caste differentiation. It is unclear whether the histone methylation modification H3K4me1 can also impact caste differentiation. In this study, we analyzed genome-wide H3K4me1 modifications in both queen and worker larvae and found that H3K4me1 marks are more abundant in worker larvae than in queen larvae at both the second and fourth instars, and many genes associated with caste differentiation are differentially methylated. Notably, caste-specific H3K4me1 in promoter regions can direct worker development. Thus, our results suggest that H3K4me1 modification may act as an important regulatory factor in the establishment and maintenance of caste-specific transcriptional programs in honey bees; however, the potential influence of other epigenetic modifications cannot be excluded.

Sublethal fluvalinate negatively affect the development and flight capacity of honeybee (Apis mellifera L.) workers.

Fluvalinate has been heavily used to control the pest Varroa destructor and residues in honeybee colony causing long-term exposure threat for bees. But, little is known about the lifetime trips and homing ability of worker bees under fluvalinate stresses during the development period. In this study, honeybees from 2-day-old larvae to 7-day-old adults were continuously fed with different concentrations of fluvalinate (0, 0.5, 5 and 50 mg/kg) and the effects of fluvalinate on the development of larvae were examined. And then, all the treated bees were reintroduced into the original source colony and were monitored, and the homing ability of 20 days old bees at 1000 and 2000 m away from the beehive were tested using the radio frequency identification (RFID). We found that fluvalinate significantly activates the superoxide dismutase (SOD) activities of larvae and 5 mg/kg fluvalinate reduced the homing rate of workers at 2000 m away from colony. 50 mg/kg fluvalinate reduced proportion of capped worker cells, activated Cytochrome P450 (CYP450) activity of larvae, affected the foraging times, influenced the homing rate and homing time of one trip at 2000 m away from colony. Our results showed that the larvae can activate the activities of SOD and detoxification enzymes in detoxification of fluvalinate and reduce the influence on honeybees. But, when the concentration is higher than 5 mg/kg fluvalinate, it is difficult for bees to detoxify fluvalinate completely, which affect the homing rate. The results reflect the potential risk for honeybees in the development stage continuously exposed to fluvalinate.

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.

Dynamic transcriptome landscape of Asian domestic honeybee (Apis cerana) embryonic development revealed by high-quality RNA sequencing.

BACKGROUND: Honeybee development consists of four stages: embryo, larva, pupa and adult. Embryogenesis, a key process of cell division and differentiation, takes 3 days in honeybees. However, the embryonic transcriptome and the dynamic regulation of embryonic transcription are still largely uncharacterized in honeybees, especially in the Asian honeybee (Apis cerana). Here, we employed high-quality RNA-seq to explore the transcriptome of Asian honeybee embryos at three ages, approximately 24, 48 and 72 h (referred to as Day1, Day2 and Day3, respectively). RESULTS: Nine embryo samples, three from each age, were collected for RNA-seq. According to the staging scheme of honeybee embryos and the morphological features we observed, our Day1, Day2 and Day3 embryos likely corresponded to the late stage four, stage eight and stage ten development stages, respectively. Hierarchical clustering and principal component analysis showed that same-age samples were grouped together, and the Day2 samples had a closer relationship with the Day3 samples than the Day1 samples. Finally, a total of 18,284 genes harboring 55,646 transcripts were detected in the A. cerana embryos, of which 44.5% consisted of the core transcriptome shared by all three ages of embryos. A total of 4088 upregulated and 3046 downregulated genes were identified among the three embryo ages, of which 2010, 3177 and 1528 genes were upregulated and 2088, 2294 and 303 genes were downregulated from Day1 to Day2, from Day1 to Day3 and from Day2 to Day3, respectively. The downregulated genes were mostly involved in cellular, biosynthetic and metabolic processes, gene expression and protein localization, and macromolecule modification; the upregulated genes mainly participated in cell development and differentiation, tissue, organ and system development, and morphogenesis. Interestingly, several biological processes related to the response to and detection of light stimuli were enriched in the first-day A. cerana embryogenesis but not in the Apis mellifera embryogenesis, which was valuable for further investigations. CONCLUSIONS: Our transcriptomic data substantially expand the number of known transcribed elements in the A. cerana genome and provide a high-quality view of the transcriptome dynamics of A. cerana embryonic development.

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.

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.

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.

A descriptive study of the prevalence of parasites and pathogens in Chinese black honeybees.

The Chinese black honey bee is a distinct honey bee subspecies distributed in the Xinjiang, Heilongjiang and Jilin Provinces of China. We conducted a study to investigate the genetic origin and the parasite/pathogen profile on Chinese black honeybees. The phylogenetic analysis indicated that Chinese black honeybees were two distinct groups: one group of bees formed a distinct clade that was most similar to Apis mellifera mellifera and the other group was a hybrid of the subspecies, Apis mellifera carnica, Apis mellifera anatolica and Apis mellifera caucasica. This suggests that the beekeeping practices might have promoted gene flow between different subspecies. Screening for pathogens and parasites showed that Varroa destructor and viruses were detected at low prevalence in Chinese black honeybees, compared with Italian bees. Further, a population of pure breeding black honeybees, A. m. mellifera, displayed a high degree of resistance to Varroa. No Varroa mites or Deformed wing virus could be detected in any examined bee colonies. This finding suggests that a population of pure breeding Chinese black honeybees possess some natural resistance to Varroa and indicated the need or importance for the conservation of the black honeybees in China.

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.

(-)-Gallocatechin Gallate: A Novel Chemical Marker to Distinguish Triadica cochinchinensis Honey.

Triadica cochinchinensis honey (TCH) is collected from the nectar of the medicinal plant T. cochinchinensis and is considered the most important honey variety in southern China. TCH has significant potential medicinal properties and commercial value. However, reliable markers for application in the authentication of TCH have not yet been established. Herein, a comprehensive characterization of the botanical origin and composition of TCH was conducted by determining the palynological characteristics and basic physicochemical parameters. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used to investigate the flavonoid profile composition of TCH, T. cochinchinensis nectar (TCN) and 11 other common varieties of Chinese commercial honey. (-)-Gallocatechin gallate (GCG) was identified as a reliable flavonoid marker for TCH, which was uniquely shared with TCN but absent in the other 11 honey types. Furthermore, the authentication method was validated, and an accurate quantification of GCG in TCH and TCN was conducted. Overall, GCG can be applied as a characteristic marker to identify the botanical origin of TCH.

Revealing Changes in Ovarian and Hemolymphatic Metabolites Using Widely Targeted Metabolomics between Newly Emerged and Laying Queens of Honeybee (Apis mellifera).

The queen bee is a central and pivotal figure within the colony, serving as the sole fertile female responsible for its reproduction. The queen possesses an open circulatory system, with her ovaries immersed in hemolymph. A continuous and intricate transportation and interchange of substances exist between the ovaries and hemolymph of queen bees. To determine the characteristic metabolites in the hemolymph and ovary, as well as understand how their rapid metabolism contributes to the process of egg-laying by queens, we reared Apis mellifera queens from three different age groups: newly emerged queen (NEQ), newly laying queen (NLQ), and old laying queen (OLQ). Using widely targeted metabolomics, our study revealed that the laying queen (NLQ and OLQ) exhibited faster fatty acid metabolism, up-regulated expression of antioxidants, and significant depletion of amino acids compared to the NEQ. This study revealed that the levels of carnitine and antioxidants (GSH, 2-O-α-D-glucopyranosyl-L-ascorbic acid, L-ascorbic acid 2-phosphate, etc.) in the NLQ and OLQ were significantly higher compared to NEQ. However, most of the differentially expressed amino acids, such as L-tryptophan, L-tyrosine, L-aspartic acid, etc., detected in NLQ and OLQ were down-regulated compared to the NEQ. Following egg-laying, pathways in the queens change significantly, e.g., Tryptophan metabolism, Tyrosine metabolism, cAMP signaling pathway, etc. Our results suggest that carnitine and antioxidants work together to maintain the redox balance of the queen. Additionally, various amino acids are responsible for maintaining the queen's egg production.

The Honey Bee Colony's Criterion for Candidate Selection: "Ongoing" or "One-Shot"?

In the honey bee, the queen's death severely threatens the survival of the colony. In an emergency, new queens are reared from young worker larvae, where nepotism is thought to influence the choice of queen candidates by the workers. This article simulates the emergency queen-rearing process in a colony under natural conditions and records the results of colony selection (without nepotism). In queenless colonies, worker larvae aged three days or younger were preferred for queen rearing, and 1-day-old larvae were the first to be selected for the queen-cell cups. In the capping stage, the number of capped queen cells selected from the 1-day-old larvae was much higher than the 3-day-old larvae. On the first day, the number of emerging queens reared from 1-day-old larvae was significantly higher than the queens reared from 2-day-old and 3-day-old larvae. However, there was no significant difference in the birth weights of queens reared from 1-day-old, 2-day-old, or 3-day-old larvae. When the newly emerged queens were introduced into the original queenless colony, 1-day-old larval queens triggered more worker followers than 2-day-old larval queens. The expression of ovarian development-related genes (vg, hex110, and Jh) was higher in queens reared from 1-day-old larvae than those reared from 2-day-old and 3-day-old larvae, indicating that the quality of the queens reared from 1-day-old larvae is superior. This study shows that in the absence of nepotism, the colony selection of queen candidates at the larval stage, capping stage, and emerging stage is not final, but is gradually optimized to maximize colony development through a "quality control" process.

The mechanism of Andrena camellia in digesting toxic sugars.

Camellia oleifera is an economically and medicinally valuable oilseed crop. Honeybee, the most abundant pollinator, rarely visits C. oleifera because of the toxic sugars in the nectar and pollen. These toxic sugars cannot be fully digested by honeybees and inhibit the process of synthesizing trehalose in honeybees. C. oleifera exhibits self-incompatibility, and its pollination heavily depends on Andrena camellia. However, the mechanism by which A. camellia digests toxic sugars in C. oleifera nectar and pollen remains unknown. Consequently, we identified and validated four single-copy genes (α-N-acetyl galactosamine-like, galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose-4'-epimerase, abbreviated as NAGA-like, GALK, GALT, and GALE) essential for detoxifying toxic sugars in vitro. Then, we cloned the four genes into Escherichia coli, and expressed enzyme successfully degraded the toxic sugars. The phylogeny suggests that the genes were conserved and functionally diverged among the evolution. These results provide novel insights into pollinator detoxification during co-evolution.

Gene expression analysis following olfactory learning in Apis mellifera.

The honeybee has a strong learning and memory ability, and is recognized as the best model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the gene expression difference following proboscis extension response-based olfactory learning in the A. mellifera using a tag-based digital gene expression (DGE) method. We obtained about 5.71 and 5.65 million clean tags from the trained group and untrained group, respectively. A total of 259 differentially expressed genes were detected between these two samples, with 30 genes up-regulated and 229 genes down-regulated in trained group compared to the untrained group. These results suggest that bees tend to actively suppress some genes instead of activating previously silent genes after olfactory learning. Our DGE data provide comprehensive gene expression information for olfactory learning, which will facilitate our understanding of the molecular mechanism of honey bee learning and memory.

The adverse impact on lifespan, immunity, and forage behavior of worker bees (Apis mellifera Linnaeus 1758) after exposure to flumethrin.

Several pyrethroids (such as flumethrin and fluvalinate) with low toxicity to honey bees and comparable high toxicity to mites are used worldwide as acaricides. However, flumethrin has been used for a long time in colonies to control Varroa destructor and the honey bees might be exposed to flumethrin cumulatively, which could affect the health of honey bee colonies. This study evaluated the potential adverse effects of direct flumethrin exposure on worker bees under laboratory and colony conditions. Under laboratory conditions, downregulation of genes related to immune was observed when worker bees were exposed to flumethrin above 1/16 LD50; at levels above 1/8 LD50, olfactory learning was impaired, and genes related to learning memory were downregulated; and at >1/4 LD50, their lifespan was shortened. Monitoring with radio frequency identification (RFID) revealed that worker bees in a colony exposed to flumethrin above 1/8 LD50 had a shortened lifespan and reduced foraging ability. When worker bees are exposed to >1/4 LD50 of flumethrin, it can lead to excessive rest day behavior. These results indicate that applying flumethrin in colonies may pose a severe health risk to honey bees and reveal the urgent need to develop non-toxic and highly effective acaricides.

Kaempferitrin: A Flavonoid Marker to Distinguish Camellia oleifera Honey.

C. oleifera is an economically important oilseed crop and medical plant. However, as a characteristic honey resource, the standard protocol used to identify the composition of C. oleifera honey has not been established yet. Previously, distinctive flavonoid has been shown as an effective marker to trace the botanical origin of honey. In this study, we examined the flavonoid types in C. oleifera honey and nine other monofloral honeys by using liquid chromatography tandem-mass spectrometry (LC-MS/MS) and compared the differences and identified eight distinct flavonoids in C. oleifera honey. Then, comparing the 8 flavonoids with the 14 flavonoids common to C. oleifera honey and nectar, two distinct flavonoids were identified in C. oleifera honey and nectar. Finally, we identified kaempferitrin as the distinct flavonoid marker in C. oleifera honey using the degree of influence of the partial least-squares discriminant analysis (PLS-DA) model on C. oleifera honey and ployfloral honey.

Comparison of learning and memory of Apis cerana and Apis mellifera.

The honeybee is an excellent model organism for research on learning and memory among invertebrates. Learning and memory in honeybees has intrigued neuroscientists and entomologists in the last few decades, but attention has focused almost solely on the Western honeybee, Apis mellifera. In contrast, there have been few studies on learning and memory in the Eastern honeybee, Apis cerana. Here we report comparative behavioral data of color and grating learning and memory for A. cerana and A. mellifera in China, gathered using a Y-maze apparatus. We show for the first time that the learning and memory performance of A. cerana is significantly better on both color and grating patterns than that of A. mellifera. This study provides the first evidence of a learning and memory difference between A. cerana and A. mellifera under controlled conditions, and it is an important basis for the further study of the mechanism of learning and memory in honeybees.

Polymorphism analysis of csd gene in six Apis mellifera subspecies.

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