Whole-Genome Resequencing-Based Qualitative Trait Locus Mapping Correlated yellow with the Mutant Color in Honeybees, Apis cerana cerana.

The honeybee, Apis cerana cerana (Ac), is an important pollinator and has adapted to the local ecological environment with relevant coloration. The cuticle coloration of the brown (br) mutant is brown instead of black in wild-type individuals. Therefore, this study aimed to identify and characterize the gene responsible for the br mutation. Genome resequencing with allele segregation measurement using Euclidean distance followed by Lowess regression analysis revealed that the color locus linked to the mutation was located on chromosome 11. A 2-base deletion on exon 4 was identified in the g7628 (yellow) gene after genome assembly and sequence cloning. In addition, the cuticle color of the abdomen of worker bees changed from black to brown when a defect was induced in the yellow gene using short interfering RNA (siRNA); however, the survival rate did not decrease significantly. These results indicate that the yellow gene participated in the body pigmentation, and its defect was responsible for the br mutation. This study promotes the understanding of the molecular basis of body coloration in honeybees, enriching the molecular mechanisms underlying insect pigmentation.

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.

Honey bee maternal effects improve worker performance and reproductive ability in offspring.

Maternal effects are an evolutionary strategy used to improve offspring quality. In an example of maternal effects in honey bees (Apis mellifera), mother queens produce larger eggs in queen cells than in worker cells in order to breed better daughter queens. In our current study, morphological indexes, reproductive tissues, and the egg-laying ability of newly reared queens reared with eggs laid in queen cells (QE), eggs laid in worker cells (WE), and 2-day-old larvae in worker cells (2L) were evaluated. In addition, morphological indexes of offspring queens and working performance of offspring workers were examined. The thorax weight, number of ovarioles, egg length, and number of laid eggs and capped broods of QE were significantly higher than those of WE and 2L, indicating that the reproductive capacity of QE group was better than that of other groups. Furthermore, offspring queens from QE had larger thorax weights and sizes than those from the other two groups. Offspring worker bees from QE also had larger body sizes and greater pollen-collecting and royal jelly-producing abilities than those of other two groups. These results demonstrate that honey bees display profound maternal effects on queen quality that can be transmitted across generations. These findings provide a basis for improving queen quality, with implications in apicultural and agricultural production.

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.

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.

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.

High-Quality Queens Produce High-Quality Offspring Queens.

Honey bees, rather than rear queens with eggs and larvae from worker cells, prefer to rear new queens with eggs form queen cells, if available. This may be a result of long-term evolutionary process for honey bee colonies. However, the exact mechanism of this phenomenon is unclear. In this study, queens were reared with eggs from queen cells (F1-QE), eggs from worker cells (F1-WE), and two-day-old larvae from worker cells (F1-2L). Physiological indexes and the expression of the development-related genes ((Hexamerin (Hex110, Hex70b), Transferrin (Trf), and Vitellogenin (Vg)) of reared F1 generation queens were measured and compared. Furthermore, F2 generation queens were reared with one-day-old larvae from F1 queens, and the weight and ovariole count of reared F2 generation daughter queens were examined. Meanwhile, the expression of the development- and reproduction-related genes (Hex110, Hex70b, Trf, Vg, and Juvenile Hormone (Jh)) and immune detoxication-related genes (Hymenoptaecin, Abeacin, and CytP450) of reared F2 queens were further explored. We found that the F1-QE queens had the highest physiological indexes and higher Hex110 and Trf expression levels, while no significant difference was found in the expression of Hex70b and Vg among the three groups of F1 queens. In addition, the reared queens of F2-QE had the highest quality, with the highest development, reproduction, immune-detoxication genes' expression levels. Our results revealed that the quality of reared offspring queens from high-quality mother queens was also high. These findings inform methods for rearing high-quality queens and highlight that a high-quality queen is essential for offspring colony growth and survival.

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.

Honey bee Apis mellifera larvae gut microbial and immune, detoxication responses towards flumethrin stress.

Mites are considered the worst enemy of honey bees, resulting in economic losses in agricultural production. In apiculture, flumethrin is frequently used to control mites. It causes residues of flumethrin in colonies which may threaten honey bees, especially for larvae. Still, the impact of flumethrin-induced dysbiosis on honey bees larval health has not been fully elucidated, and any impact of microbiota for decomposing flumethrin in honey bees is also poorly understood. In this study, 2-day-old larvae were fed with different flumethrin-sucrose solutions (0, 0.5, 5, 50 mg/kg) and the dose increased daily (1.5, 2, 2.5 and 3 µL) until capped, thereafter the expression level of two immune genes (hymenoptaecin, defensin1) and two detoxication-related genes (GST, catalase) were measured. Meanwhile, the effect of flumethrin on honey bee larvae (Apis mellifera) gut microbes was also explored via 16S rRNA Illumina deep sequencing. We found that flumethrin at 5 mg/kg triggered the over expression of immune-related genes in larvae, while the larval detoxification-related genes were up-regulated when the concentrations reached 50 mg/kg. Moreover, the abundance and diversity of microbes in flumethrin-treated groups (over 0.5 mg/kg) were significantly lower than control group, but it increased with flumethrin concentrations among the flumethrin-treated groups. Our results revealed that microbes served as a barrier in the honey bee gut and were able to protect honey bee larvae to a certain extent, and reduce the stress of flumethrin on honey bee larvae. In addition, as the concentration of flumethrin increases, honey bee larvae activate their immune system then detoxification system to defend against the potential threat of flumethrin. This is the first report on the impact of flumethrin on gut microbiota in honey bees larvae. The findings revealed new fundamental insights regarding immune and detoxification of host-associated microbiota.

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.

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.

[Study on foraging behaviors of honeybee Apis mellifera based on RFID technology].

Honeybee foragers can flexibly adjust their out-hive activities to ensure growth and reproduction of the colony. In order to explore the characteristics of honey bees foraging behaviors, in this study, their flight activities were monitored 24 hours per day for a duration of 38 days, using an radio frequency identification (RFID) system designed and manufactured by the Honeybee Research Institute of Jiangxi Agricultural University in cooperation with the Guangzhou Invengo Information Technology Co., Ltd. Our results indicated that 63.4% and 64.5% of foragers were found rotating more than one day off during the foraging period in two colonies, and 22.5% and 26.4% of the total foraging days were used for rest respectively. Further, although the total foraging time between rotating day-off foragers and continuously working foragers was equal, the former had a significant longer lifespan than the latter. Additionally, the lifespan of the early developed foragers was significantly lower than that of the normally developed foragers. This study enriched the content of foraging behaviors of honey bees, and it could be used as the basis for the further explorations on evolutionary mechanism of foraging behaviors of eusocial insects.

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.