Honey bees are important for agriculture (e.g., pollination and honey production). Additionally, honey bees are an important insect model species, especially as model social insects. The Japanese honey bee, Apis cerana japonica (a subspecies of the Asian honey bee, Apis cerana ), is a Japanese domestic honey bee, which has several subspecies-specific traits. We previously constructed the draft genome sequence data of A. cerana japonica , but it needed to be improved considering the use of the genome sequence data for genome structural analysis and repetitive region analysis, as well as the availability of chromosome-level genome data of A. mellifera and A. cerana . In this study, we constructed the improved A. cerana japonica genome data and new gene set data with functional annotations. The constructed genome data, including 16 pseudochromosomes, was found to be highly contiguous and complete, and the gene set data covered most of the core genes in the BUSCO database. Thus, the constructed genome and gene set data have become more suitable as the reference data of A. cerana japonica .

Transposable elements (TEs) are grouped into several families with diverse sequences. Owing to their diversity, studies involving the detection, classification, and annotation of TEs are difficult tasks. Moreover, simple comparisons of TEs among different species with different methods can lead to misinterpretations. The genome data of several honey bee (Apis) species are available in public databases. Therefore, we conducted a meta-analysis of TEs, using 11 sets of genome data for Apis species, in order to establish data of "landscape of TEs". Consensus TE sequences were constructed and their distributions in the Apis genomes were determined. Our results showed that TEs belonged to four to seven TE families among 13 and 15 families of TEs detected in classes I and II respectively mainly consisted of Apis TEs and that more DNA/TcMar-Mariner consensus sequences and copies were present in all Apis genomes tested. In addition, more consensus sequences and copy numbers of DNA/TcMar-Mariner were detected in Apis mellifera than in other Apis species. These results suggest that TcMar-Mariner might exert A. mellifera-specific effects on the host A. mellifera species. In conclusion, our unified approach enabled comparison of Apis genome sequences to determine the TE landscape, which provide novel evolutionary insights into Apis species.

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

繁殖隔離が不完全なミツバチの小集団における選抜反応と近交係数を予測する理論を開発した．得られた理論を用いて，国内の養蜂家が保有する蜂群規模を想定した選抜計画に適用して育種の効率を評価した．数値計算の結果，繁殖隔離が不完全であっても雄の90%以上が隔離できれば，顕著な遺伝的改良量が得られることが示された．また，養蜂場外からの雄の流入は，近交係数の上昇を抑える上で有効であることも示された．

Sequences of transposase coding regions plus flanking 500bp on both sides of the long MLEs. Predicted TIR sequences are shown lower cases.

The pyrethroid τ-fluvalinate is widely used to control Varroa destructor Anderson and Trueman (Acari: Varroidae), a pest mite that can cause colony loss for the western honey bee Apis mellifera Linnaeus (Hymenoptera: Apidae). However, τ-fluvalinate resistance has been reported in V. destructor populations worldwide, and mutations in voltage-gated sodium channels (VGSCs) at L1002 (equivalent to L925 in the house fly) are thought to be a major cause. τ-fluvalinate is commonly used in Japanese apiaries for controlling V. destructor; however, a detailed investigation of VGSC mutation(s) has not been yet conducted. The polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis is used to detect VGSC mutations. Because Varroa mites are haplodiploid with sib-mating, the inbred offspring of heterozygote mothers could be L925 homozygotes. In this study, we optimized PCR–RFLP with two sets of primers to detect L925 alleles precisely. The method could detect L925 homo- and heterozygotes in the Varroa mites in Japanese apiaries. The sequencing of PCR amplicons confirmed these mites had an L925M mutation, which has been reported to cause τ-fluvalinate resistance. Our report of incidences of the L925M mutation suggests that some mites may be resistant to this pyrethroid. (190/200 words)

Alleviating nutritional stress in European honeybee hives helps to increase resilience to parasite infections and reduces the interactive effects of pesticides. Here, we used a field experiment to evaluate the effectiveness of floral enhancement in reducing bee exposure to insecticides. A mass-flowering crop, white mustard, was cultivated in a small patch near an experimental apiary comprising 10 hives. To assess the frequency of floral patch use by bees in each hive, we attached electronic tags to bees foraging on white mustard flowers and then recorded the number of tagged bees in each hive a day before insecticide spraying in the adjacent paddy fields. The number of corpses around hive entrances increased within a day after the spraying but varied among hives. There was a significant negative correlation between the number of tagged bees and the cumulative number of corpses at each hive. We suggest that attracting foraging bees to mass-flowering resources near an apiary helps to reduce insecticide exposure risk.

The ectoparasitic miteVarroa destructorAnderson and Trueman (Acari: Varroidae) is a pest that severely damages honey bee health worldwide. This mite is considered a major reason for winter colony loss of the western honey beeApis melliferaLinnaeus (Hymenoptera: Apidae). Monitoring ofVarroamites in honey bee colonies is required for effective management ofVarroainfestations. The mite is dislodged from adult bees when the bees roll in powdered sugar. The sugar roll test is one of the major methods used to surveyV. destructorinfestations in honeybee colonies. However, powdered sugar agglomerates during commodity distribution to the customers or under humid conditions. In this study, roasted soybean flour was evaluated as a powder forVarroamite detection, because this powder rarely forms a hard agglomerate. Roasted soybean flour may eliminateVarroamites from adult bees as well as powdered sugar. Therefore, the roasted soybean flour can be a useful powder for convenientVarroamonitoring under humid conditions.

Varroa destructor Anderson and Trueman (Acari: Varroidae) are ectoparasitic mites found in the western honeybee Apis mellifera Linnaeus (Hymenoptera: Apidae). Varroa destructor is classified into two haplotypes, i.e., Korea (K) and Japan (J), based on mtDNA sequences. Among these, V. destructor K haplotype is possibly a more severe threat to A. mellifera colonies. Previous studies collected both V. destructor haplotypes from honeybee colonies in Japan. However, no detailed surveillance of infestation of Japanese apiaries by V. destructor or identification of their genetic structure has been conducted to date. We surveyed V. destructor at 15 different Japanese apiaries of A. mellifera. Varroa destructor was collected from 14 Japanese apiaries, and all mites were classified as V. destructor K haplotype. Varroa destructor infestation of the Japanese honeybee A. cerana japonica Radoszkawsi (Hymenoptera: Apidae) was also analyzed. Varroa destructor K haplotype was predominant in A. cerana colonies. Despite the different host species, all collected V. destructor K haplotype samples were classified into a single haplogroup, i.e., K1-1/K1-2. These results indicate that A. mellifera and A. cerana were infested by the same V. destructor haplogroup. This is the first report detailing a survey on V. destructor prevalence and haplogroups among Japanese apiaries.

Radio-frequency identification (RFID) is both a commonly used method and a promising tool for investigating the foraging activities of bees. To prevent loss of electronic tags from individual bees, they must be immobilized long enough for adhesives to dry, but because narcosis can negatively affect bee health and behaviors, a very brief narcosis period is recommended. The present study assessed the impact of 30 min of cold narcosis, compared with carbon dioxide or no narcosis, on foraging activities using RFID monitoring. From each of three hives, 8 to 10 bees were placed in each of three narcosis treatments (n = 85), then tagged and monitored for homing success, activity after returning to the hive, and duration of flights outside the hive. Bees showed no significant differences in homing success and flight duration between cold narcosis treatment and the control (no narcosis), whereas bees narcotized by carbon dioxide showed significantly lower homing success and shorter flight duration than control bees. We conclude that for observing bee foraging behaviors using RFID systems, the effects of cold narcosis on ice for 30 min on foraging behaviors are acceptable, and cold narcosis is more practical for tagging many bees at once than physical immobilization using plunger cages.

RepeatMasker out file of the Apis species. Meanings of each column is described in RepeatMasker homepage (http://www.repeatmasker.org/webrepeatmaskerhelp.html#reading).

Mariner and mariner-like elements (MLEs) are distributed in various species and their sequences are highly diverse. In previous studies, transposable elements in Apis species mainly consisted of mariner and MLE. However, Apis MLE evolution has not yet been elucidated in detail. For it, MLEs were searched for in multiple Apis species, and those detected were then classified. More MLEs were detected in the A. mellifera genome than in other Apis species genomes, and this was not attributed to the domestication of A. mellifera . The MLEs detected were classified into 31 Drosophila MLE classes. In this classification, almost all MLEs were classified into the three classes belonging to mellifera subfamilies. A phylogenetic analysis of MLEs in the three classes revealed two types of clusters, one of which consist of multiple Apis MLEs and the other of only A. mellifera MLEs. The results obtained provided the “whole picture” of MLEs in Apis species. Furthermore, an analysis of long MLEs (>1 kbp or encoding full-length transposases) from the two types of clusters indicated that only one long MLE encoding a complete transposase settled in Apis species and burst in the A. mellifera genome, while the other long MLEs invaded the A. mellifera genome by horizontal transfer after A. mellifera speciation and burst in the A. mellifera genome, which may also induce the bursting of “settled” MLEs. To the best of our knowledge, this is the first study to provide evolutionary insights into MLEs between multiple related species.

All Dromar hmmer files were merged to a single file to use for nhmmer.

Transposable elements (TEs) are grouped into several classes with diverse sequences. Owing to their diversity, studies involving the detection, classification, and annotation of TEs are difficult tasks. Moreover, simple comparisons of TEs among different species with different methods can lead to misinterpretations. The genome data of several honey bee ( Apis ) species are available in public databases. Therefore, we conducted a meta-analysis of TEs, using 11 sets of genome data for Apis species, in order to establish the basal TE data (termed here as the ‘landscape of TEs’). Consensus TE sequences were constructed and their distributions in the Apis genomes were determined. Our results showed that TEs from several limited families mainly consisted of Apis TEs and that more DNA/TcMar-Mariner consensus sequences and copies were present in all Apis genomes tested. In addition, more consensus sequences and copy numbers of DNA/TcMar-Mariner were detected in Apis mellifera than in other Apis species. These results suggest that TcMar-Mariner might exert A. mellifera -specific effects on the host A. mellifera species. In conclusion, our unified approach enabled comparison of Apis genome sequences to determine the TE landscape, which provide novel evolutionary insights into Apis species. Simple Summary Studies on the detection of transposable elements and their annotations have posed several challenges. For example, simple comparisons of transposable elements in different species using different methods can lead to misinterpretations. Thus, assembling data for transposable elements analyzed by unified methods is important for comparison purposes. Therefore, we performed a meta-analysis of transposable elements identified using genome datasets from 5 Apis species (11 sets of genome data) and specific software to detect the transposable elements, which revealed the landscapes of transposable elements. We examined the types and locations of transposable elements in the Apis genomes. The landscapes of transposable elements showed that several limited transposable element families consisted mainly of Apis -associated transposable elements. These limited families include DNA/TcMar-Mariner and DNA/CMC-EnSpm. In addition, more DNA/TcMar-Mariner consensus sequences and copies were detected in Apis mellifera than in other Apis species. These data suggest that TcMar-Mariner might exert A. mellifera -specific effects in the host A. mellifera species. Our landscape data provide new insights into Apis transposable elements; furthermore, detailed analyses of our data could pave the way for new biological insights in this field.

The waggle dances of European honeybees provide important information that can be used to estimate forage areas and identify food resource limitations. However, manually decoding these dances is labor-intensive. This study develops an automatic waggle decoding method applicable to video recordings taken in field apiaries using a generic camcorder with a normal frame rate. Particle image velocimetry was used to detect the typical characteristics of abdominal waggling in bees. We demonstrated our proposed method using video recordings taken at three hives in field apiaries. The decoded information was used to estimate forage area, which was compared against estimates obtained from manual decoding. For all three video recordings, we obtained a 78–87% overlap in the probable forage regions estimated using automatic and manual decoding. Our results suggest that our automatic decoding method is comparable to manual interpretation for the purposes of forage area estimation.

The European honeybee, Apis mellifera L. (Hymenoptera: Apidae), is the most important crop pollinator, and there is an urgent need for a sustained supply of honeybee colonies. Understanding the availability of pollen resources around apiaries throughout the brood-rearing season is crucial to increasing the number of colonies. However, detailed information on the floral resources used by honeybees is limited due to a scarcity of efficient methods for identifying pollen species composition. Therefore, we developed a DNA barcoding method for identifying the species of each pollen pellet and for quantifying the species composition by summing the weights of the pellets for each species. To establish the molecular biological protocol, we analyzed 1008 pellets collected between late July and early September 2016 from five hives placed in a forest/agricultural landscape of Hokkaido, northern Japan. Pollen was classified into 31 plant taxa, of which 29 were identified with satisfactory discrimination (25 species and 4 genera) using trnL-trnF and ITS2 as DNA barcoding regions together with available floral and phenological information. The remaining two taxa were classified to the species level using other DNA barcoding regions. Of the 1008 pollen pellets tested, 1005 (99.7%) were successfully identified. As an example of the use of this method, we demonstrated the change in species composition of pollen pellets collected each week for 9 weeks from the same hive.

Honey bees are not only important for honey production but also as pollinators of wild and cultivated plants. The Eastern honeybee (Apis cerana) is more resistant to several pathogens than the Western honeybee (Apis mellifera), and the genomes of two strains of the nominotypical subspecies, A. cerana cerana, northern (Korea) and southern (China) strains, have been sequenced. Apis cerana japonica, another subspecies of A. cerana, shows many specific features (e.g. mildness, low honey production and frequently absconds) and it is important to study the molecular biological and genetic aspects of these features. To accelerate the genetic research on A. cerana japonica, we sequenced the genome of this subspecies. The draft genome sequence of A. cerana japonica presented here is of high quality in terms of basic genome status (e.g. N50 is 180 kbp, total length is 211 Mbp, and largest contig length is 1.31 Mbp) and BUSCO results. The gene set of A. cerana japonica was predicted using AUGUSTUS software and the set of genes was annotated using Blastp and InterProScan, and GO terms were added to each gene. The number of genes is higher than in A. mellifera and in the two strains of A. cerana cerana sequenced previously. A small number of transposable elements and repetitive regions were found in A. cerana japonica, which are also in the genomes of A. mellifera and the northern and southern strains of A. cerana cerana. Apis cerana is resistant to several pathogens that seriously damage A. mellifera. We searched for 41 orthologs related to the IMD and Toll pathways, which have key roles in the immune reaction to invading pathogens. Some orthologs were not identified in the genome of the northern strain of A. cerana cerana. This indicates that the Toll and IMD pathways function in the same way as in A. mellifera and Drosophila melanogaster. Use of the draft genome sequence of A. cerana japonica provided herein and those of the other Apis (sub)species may help to accelerate comparative research on the genome of honey bees.

Honeybees are important in terms of pollinators and bee products. However, honeybee colonies keep declining due to colony collapse disorder, loss of food sources, environmental deterioration, pesticide exposure and various diseases, as well as the interaction of these causes. Pathogenic infections are high because there is an antagonistic coevolution between host and pathogen/parasite, which leads to a reciprocal adaptation, as well as the weakening of the immune system of the hosts by other (including the above mentioned) compounding factors. Since many antibiotics have been used in apiaries, there is growing public health concern about antibiotic residues in food, while the spread of antibiotic resistant isolates of pathogens is of concern. This review focusses on a management system for apiary hygiene and antimicrobial compounds from natural products. Important traits for apiary management are the habitat quality, landscape heterogeneity, climate, management and health. Successful management can be determined by many criteria, such as the level of honey production/harvesting, the diversity of honey types, disease control and pesticide accumulation. Furthermore, attention to land cover is important because it provides the quality and quantity of nutrients. High elevations and slopes can influence the bee health, as can human actions, including global trade and hobby bee keepers. In order to reduce the use of chemicals and antibiotics in apiaries, the use of alternative compounds from natural products are still required. Compounds inhibiting Paenibacillus larvae larvae, the cause of American foulbrood disease and Varroa mites are very challenging because the bacteria can produce resistant spores and the mites are vectors for many viruses and pathogens. Bee products and plants, both crude and purified forms, can be alternative sources for honeybee disease control. Several substances, like propolis, have already been applied in field experiments. Various crude extracts, volatile compounds and pure compounds appear to have potential in honeybee disease prevention and treatment. It is concluded that the apiary management system and use of suitable alternative compounds from natural products can improve the health and decrease the loss of honeybees.