Both the productive efficiency and physical health of sika deer are strongly linked to their body conformation phenotypes. Breeding sika deer with excellent growth traits using molecular breeding technologies has become essential. Phenotypic data of 12 body conformation traits were measured for 613 sika deer across three age groups, including body traits and cephalic traits. Genetic typing was performed using the Sika Deer 100K SNP Liquid Chip, and genetic parameters were estimated through animal models to obtain the heritability and genetic correlation of traits within each age group. Single-trait and multi-trait GWASs were conducted, using GEMMA software to identify gene variants significantly associated with sika deer body conformation traits. Most of the 12 body conformation traits exhibited moderate to high heritability. The single-trait GWAS identified 49 SNPs (38 candidate genes), while multi-trait GWAS detected 134 SNPs (80 candidate genes), including 114 novel loci. A total of 163 SNPs and 196 candidate genes were identified, with 17 genes detected by both methods. These genes may be involved in pain perception, cell cycle regulation, immune response, and protein ubiquitination. Two significant KEGG pathways were enriched: steroid hormone biosynthesis and drug metabolism–cytochrome P450. Collectively, these detected loci and genes may serve as potential genetic resources for marker-assisted breeding, contributing to subsequent genetic improvement of body conformation in sika deer.

BACKGROUND:Population genomic analysis can reconstruct the phylogenetic relationship and demographic history, and identify genomic selective signatures of a species. To date, fundamental aspects of population genomic analyses, such as intraspecies taxonomy, evolutionary history, and adaptive evolution, of sika deer have not been systematically investigated. Furthermore, accumulating lines of evidences have illustrated that incorrect species delimitation will mislead conservation decisions, and even lead to irreversible mistakes in threatened species. RESULTS:In this study, we resequenced 81 wild and 71 domesticated sika deer representing 10 main geographic populations and two farms to clarify the species delimitation, demographic and divergence histories, and adaptive evolution of this species. First, our analyses of whole genomes, Y chromosomes and mitochondrial genomes revealed substantial genetic differentiation between the continental and Japanese lineages of sika deer, representing two phylogenetically distinct species. Second, sika deer in Japan were inferred to have experienced a "divergence-mixing-isolation" evolutionary scenario. Third, we identified four candidate genes (XKR4, NPAS3, CTNNA3, and CNTNAP5) possibly involved in body size regulation of sika deer by selective sweep analysis. Furthermore, we also detected two candidate genes (NRP2 and EDIL3) that may be associated with an important economic trait (antler weight) were under selection during the process of domestication. CONCLUSION:Population genomic analyses revealed that the continental and Japanese lineages represent distinct phylogenetic species. Moreover, our results provide insights into the genetic selection signatures related to body size differences and a valuable genomic resource for future genetic studies and genomics-informed breeding of sika deer.

Although Cervus elaphus (Linnaeus, 1758) has been well studied, the subspecific taxonomy of Cervus canadensis populations in Qinghai and Gansu, China, is still controversial, and the mitochondrial characteristics of Cervus elaphus (Linnaeus, 1758) remain incompletely understood. We assembled 89 mitogenomes of C. canadensis from five geographical populations across Qinghai and Gansu. Phylogenetic analysis confirmed that the 89 individuals are taxonomically classified as C. c. kansuensis. Nucleotide compositions showed a higher abundance of adenine and cytosine compared to guanine and thymine in both complete mitogenomes and mitochondrial PCGs. Codon usage analysis revealed a strong preference towards A-ending codons (68.04% of over-represented codons, RSCU > 1.6) in mitochondrial PCGs, with systemic avoidance of G-ending codons (53.30% of unused codons, RSCU = 0). The CAMs of 13 PCGs are reported for the first time. Furthermore, the ENC plot showed that the codon usage of all PCGs was biased except for gene ATP8. The PR2 bias plot showed that gene ND6 exhibited bias towards T3 and G3, whereas the other genes preferred A3 and C3. Both the ENC-plot and PR2 bias plot suggested that natural selection played an important role in the forces driving codon usage bias in mitochondrial PCGs. Our results demonstrate the subspecific status of C. canadensis distributed in Qinghai and Gansu as C. c. kansuensis, and provide insights into the mitochondrial characteristics of C. c. kansuensis. The mitogenome sequences assembled in this study provide valuable data for further understanding of the Cervus elaphus (Linnaeus, 1758) mitogenome.

Antlers, as the only fully regenerable bone tissue in mammals, serve as an exceptional model for investigating bone growth, mineralization, articular cartilage repair, and the pathophysiology of osteoporosis. Nevertheless, the exact molecular mechanisms governing osteogenesis, particularly the dynamic cellular interactions and signaling pathways coordinating these processes, remain poorly characterized. This study used single-cell RNA sequencing (scRNA-seq) on the 10× Genomics Chromium platform, combined with bulk-RNA sequencing results, to comprehensively analyze molecular regulatory mechanisms in rapid antler osteogenesis. The results showed that eight cell types were identified in sika deer antler during the growth and ossification stages: mesenchymal, chondrocyte, osteoblast, pericyte, endothelial, monocyte/macrophage, osteoclast, and NK cells. Chondrocytes were predominantly found during the growth stage, while osteoblasts were more abundant during the ossification stage. Mesenchymal cells were subclassified into three subcategories: MSC_1 (VCAN and SFRP2), MSC_2 (TOP2A, MKI67), and MSC_3 (LYVE1 and TNN). MSC_3 was predominantly present during the growth stage. During the growth stage, MSC_1 and MSC_2 upregulated genes related to vasculature development (COL8A1, NRP1) and cell differentiation (PTN, SFRP2). During the ossification stage, these subcategories upregulated genes involved in the positive regulation of p53 class mediator signal transduction (RPL37, RPL23, RPS20, and RPL26), osteoblast differentiation (SPP1, IBSP, BGLAP), and proton-motive ATP synthesis (NDUFA7, NDUFB3, NDUFA3, NDUFB1). Endothelial cells were categorized into five subpopulations: Enc_1 (SPARCL1, VWF), Enc_2 (MCM5), Enc_3 (ASPM, MKI67), Enc_4 (SAT1, CXCL12), and Enc_5 (ZFHX4, COL6A3). Combined scRNA-seq and bulk RNA-seq analysis revealed that the ossification stage’s upregulation genes included osteoclast- and endothelial cell-specific genes, while the growth stage’s upregulation genes were mainly linked to collagen organization, osteoblast differentiation, mitotic cell cycle, and chondrocyte differentiation. Overall, this study offers a detailed single-cell analysis of gene expression patterns in antlers during the growth and ossification stages, providing insights into the molecular mechanisms driving rapid osteogenesis.

Sika deer (Cervus nippon), a species mainly distributed in the northeast of Asia, hold significant economic value in China due to their contributions to traditional Chinese medicine. A systematic investigation of their genetic structure is needed for population management. In this study, mitochondrial genome and AMELY, DBY, USP9Y, and SRY gene fragments on Y chromosome were used to elucidate the genetic structure of 303 individuals across 8 distinct populations. The mitosome analysis identified 72 haplotypes, with a haplotype diversity (Hd) of 0.917 and nucleotide diversity (π) of 0.0143, respectively. Meanwhile, 13 haplotypes were defined by Y chromosome genes with a Hd of 0.791. Analysis of the mitochondrial control region (CR) revealed subspecies-specific patterns of tandem repeat unit organization between continental and Japanese groups. Y chromosome analyses demonstrated a homogeneous paternal lineage across Japanese populations.

The Tahe red deer is currently the largest breeding population of antlered Cervus elaphus in China. It has unique characteristics such as drought and roughage tolerance, high antler yield and early sexual maturity. It is a high-quality provenance for cultivating high-yield Cervus elaphus breeds and is also the subject of study on the origin, evolution, and classification of Cervus elaphus. The breeding quantity of Tahe red deer has decreased significantly in recent years due to the influence of feeding conditions and consumer market. This has resulted in a serious threat to its genetic resources. To provide a scientific theoretical basis for the protection of the Tahe red deer population, we performed PCR amplification and direct sequencing of the AMELY2, DBY and SRY genes of the Y chromosome, and the ND1, COX1, ATP6, ND5, Cyt b and D-loop regions of the mtDNA, and analysed their genetic diversity and population genetic structure. The results showed high haplotype diversity and low nucleotide diversity in both the Y chromosome and mtDNA genes. The phylogenetic tree and haplotype network diagram, constructed using white-lipped deer as the outgroup, indicate that Tahe red deer has two distinct ancestral types. The phylogenetic tree, based on the Cyt b gene and D-loop region, reveals that the Cervus elaphus/hanglu/canadensis is divided into three clades: western, central, and eastern. Tahe red deer, C.h.bactrianus, and C.h.hanglu are clustered in the central clade. The study results indicate that Tahe red deer has low genetic diversity and two distinct ancestor types. It is speculated that the central clade is either the earliest differentiation from the ancestor species or the closest to it.

Cervidae represents a family that is not only rich in species diversity but also exhibits a wide range of karyotypes. The controversies regarding the phylogeny and classification of Cervidae still persist. The flourishing development of the genomic era has made it possible to address these issues at the genomic level. Here, the genomes of nine species were used to explore the phylogeny and chromosomal evolutionary events of Cervidae. By conducting whole-genome comparisons, we identified single-copy orthologous genes across the nine species and constructed a phylogenetic tree based on the single-copy orthologous genes sequences, providing new insights into the phylogeny of Cervidae, particularly the phylogenetic relationship among sika deer, red deer, wapiti and Tarim red deer. Gene family analysis revealed contractions in the olfactory receptor gene family and expansions in the histone gene family across eight Cervidae species. Furthermore, synteny analysis was used to explore the chromosomal evolutionary events of Cervidae species, revealing six chromosomal fissions during the evolutionary process from Bovidae to Cervidae. Notably, specific chromosomal fusion events were found in four species of Cervus, and a unique chromosomal fusion event was identified in Muntiacus reevesi. Our study further completed the phylogenetic relationship within the Cervidae and demonstrated the feasibility of inferring species phylogeny at the whole-genome level. Additionally, our findings on gene family evolution and the chromosomal evolutionary events in eight Cervidae species lay a foundation for comprehensive research of the evolution of Cervidae.

Mink is a kind of small and precious fur animal resource. In this study, we employed transcriptomics technology to analyze the gene expression profile of mink pectoral muscle tissue, thereby elucidating the regulatory mechanisms underlying mink growth and development. Consequently, a total of 25,954 gene expression profiles were acquired throughout the growth and development stages of mink at 45, 90, and 120 days. Among these profiles, 2607 genes exhibited significant differential expression (|log2(fold change)| ≥ 2 and p_adj < 0.05). GO and KEGG enrichment analyses revealed that the differentially expressed genes were primarily associated with the mitotic cell cycle process, response to growth factors, muscle organ development, and insulin resistance. Furthermore, GSEA enrichment analysis demonstrated a significant enrichment of differentially expressed genes in the p53 signaling pathway at 45 days of age. Subsequent analysis revealed that genes associated with embryonic development (e.g., PEG10, IGF2, NRK), cell cycle regulation (e.g., CDK6, CDC6, CDC27, CCNA2), and the FGF family (e.g., FGF2, FGF6, FGFR2) were all found to be upregulated at 45 days of age in mink, which suggested a potential role for these genes in governing early growth and developmental processes. Conversely, genes associated with skeletal muscle development (PRVA, TNNI1, TNNI2, MYL3, MUSTN1), a negative regulator of the cell cycle gene (CDKN2C), and IGFBP6 were found to be up-regulated at 90 days of age, suggesting their potential involvement in the rapid growth of mink. In summary, our experimental data provide robust support for elucidating the regulatory mechanisms underlying the growth and development of mink.

本研究以丹麦咖啡色水貂和明华黑色水貂为研究对象,通过杂交测定后代生产性能和分析杂交优势率评定杂交效果,以期更好地利用优质水貂遗传资源.结果表明,杂交群针、绒毛长度与自繁组合呈现显著性的差异,杂交群之间差异不明显;杂交群体重和体长与自繁群差异不显著;杂交群群平均成活数明显提高.杂种优势率分析表明,以丹麦咖啡色水貂为父本和明华黑色水貂为母本的杂交后代综合性能较好,效果较为理想.

Breeding ornamental white sika deer is a new notion that can be used to broaden the sika deer industry However, it is very rare for other coat phenotypes to occur, especially white (apart from albinism), due to the genetic stability and homogeneity of its coat color phenotype, making it difficult to breed white sika deer between species. We found a white sika deer and sequenced its whole genome. Then, the clean data obtained were analyzed on the basis of gene frequency, and a cluster of coat color candidate genes containing 92 coat color genes, one SV (structure variation), and five nonsynonymous SNPs (single nucleotide polymorphisms) was located. We also discovered a lack of melanocytes in the skin tissue of the white sika deer through histological examination, initially proving that the white phenotype of sika deer is caused by a 10.099 kb fragment deletion of the SCF gene(stem cell factor). By designing SCF-specific primers to detect genotypes of family members of the white sika deer, and then combining them with their phenotypes, we found that the genotype of the white sika deer is SCF789/SCF789, whereas that of individuals with white patches on their faces is SCF789/SCF1–9. All these results showed that the SCF gene plays an important role in the development of melanocytes in sika deer and is responsible for the appearance of the white coat color. This study reveals the genetic mechanism of the white coat color in sika deer and supplies data as a reference for breeding white ornamental sika deer.

Red deer (Cervus elaphus/hanglu/canadensis) are a group of closely-related species that survived through the Last Glacial period in Eurasia and expanded into North America. The evolutionary modes of red deer in Europe through glacial and interglacial cycles have been established relatively well based on both modern and ancient genomes, while the evolutionary and migratory history of red deer in Asia are not so clear. Northern China, as the southern border of their current distribution, is a significant area for exploring the evolution of wapitoids (Cervus canadensis). Here, we constructed six mitochondrial ge-nomes (lengths of 13,282-16,306 bp) from Late Pleistocene fossil cervid materials in Northeastern China, and four modern complete mitochondrial DNA sequences (16,798-16943 bp) from Northern China. Phylogenetic analyses suggest four clades in wapitoids, with our new individuals clustered into three out of the four clades. BEAST analysis indicates that the divergence between elaphoids (including Cervus elaphus and Cervus hanglu) and the complex of 'wapitoids thorn sika deer (Cervus nippon)' occurred at approximately 1.37 million years ago (Ma, 95%Cl: 1.64-1.15 Ma). Bayesian skyline plots (BSPs) show that the effective maternal population size of wapitoids had a slight and stable expansion between Marine isotope stages (MIS) 7-3, and then shrank from the beginning of the Last Glacial Maximum (LGM). Combined with the divergence dates, population dynamics, and fossil findings in Northern China, we infer that after wapitoids migrated eastward from Central Asia to Northeastern Asia, they had multiple migrations southward into Northern China. The current distribution of wapitoids in Northern China was likely shaped by these migrations as well as habitat fragmentation. Meanwhile, the radiocarbon dates of ancient individuals indicate that the Northeast China Plain, located at the same latitude as European refugia, may have provided a refugium for megafaunal species such as red deer to survive the LGM.(c) 2022 Published by Elsevier Ltd.

Sika deer are known to prefer oak leaves, which are rich in tannins and toxic to most mammals; however, the genetic mechanisms underlying their unique ability to adapt to living in the jungle are still unclear. In identifying the mechanism responsible for the tolerance of a highly toxic diet, we have made a major advancement by explaining the genome of sika deer. We generated the first high-quality, chromosome-level genome assembly of sika deer and measured the correlation between tannin intake and RNA expression in 15 tissues through 180 experiments. Comparative genome analyses showed that the UGT and CYP gene families are functionally involved in the adaptation of sika deer to high-tannin food, especially the expansion of the UGT family 2 subfamily B of UGT genes. The first chromosome-level assembly and genetic characterization of the tolerance to a highly toxic diet suggest that the sika deer genome may serve as an essential resource for understanding evolutionary events and tannin adaptation. Our study provides a paradigm of comparative expressive genomics that can be applied to the study of unique biological features in non-model animals.

This study aimed to explore the genetic diversity of stud Tahe red deer and the ancestral types of Tahe red deer at the molecular level. DNA was extracted from fresh blood which was collected at the sawn antler stage， PCR amplification and direct sequencing were performed to analyze AMELY2， DBY， SRY genes on Y chromosome and ND1， COX1， ATP6， ND5， Cytb genes of mtDNA in 38 stud Tahe red deer species. Base composition， nucleotide diversity （Pi）， mean nucleotide difference （K）， Tajima’D value， haplotype number （H） and haplotype diversity （Hd） were calculated to evaluate the genetic diversity of stud Tahe red deer. The haplotype network diagram was constructed and the genetic distance between each haplotype was calculated. The phylogenetic tree was constructed and white-lipped deer was used as the outgroup to analyze the parental lines of stud Tahe red deer. The results showed that the length of the Y chromosome spliced by comparing the AMELY2， DBY and SRY genes was 3 577 bp， and a total of 17 SNPs polymorphic sites were detected， and 4 haplotypes were defined. The dominant haplotype was Hap-1， accounting for 65.79% of the frequency. The nucleotide diversity and haplotype diversity were 0.00 195 and 0.495 0 respectively， showing a low level of genetic diversity. The phylogenetic tree constructed based on the Y chromosome gene showed the existence of two branches A and B. After comparison of ND1， COX1， ATP6， ND5 and Cytb genes of mtDNA， the splicing length was 6 160 bp， and a total of 41 SNPs polymorphic sites were detected， and 8 haplotypes were defined. The dominant haplotype was Hap-1， accounting for 47.37% of the frequency. The nucleotide diversity and haplotype diversity were 0.001 54 and 0.699 9 respectively， showing an imbalance of high haplotype diversity and low nucleotide diversity， and the genetic diversity was at a low level. The phylogenetic tree constructed based on mtDNA showed the existence of two branches， I and II. The genetic diversity of stud Tahe red deer is at a low level， and there are two ancestral types in the population of Tahe red deer.

Sika deer (Cervus nippon) are large ruminants distributed throughout northeastern Asia. The phylogenetic relationship of the sika deer subspecies remains unclear. The complete mitochondrial genomes of 287 sika deer from eight subspecies (C. n. hortulorum, C. n. sichuanicus, C. n. kopschi, C. n. taiouanus, C. n. yesoensis, C. n. centralis, C. n. nippon and C. n. yakushimae) were obtained. Haplotype network and development of a phylogenetic tree revealed China clusters and Japan clusters that were well separated based on the mitochondrial whole-genome level. Our studies indicate that China sika deer are genetically distinguishable from Japanese samples. Our findings increase the understanding of the phylogenetic relationships of sika deer and could provide useful information for sika deer conservation projects as well as for sika deer genomics, emergence and geographical distribution.

对敖鲁古雅驯鹿SRY(Sex-determiningregion of Y-chromosome)基因的cDNA序列进行测序及初步生物信息学分析.运用RT-PCR技术从敖鲁古雅驯鹿鹿茸组织中扩增SRY基因的cDNA序列,利用生物信息学软件对敖鲁古雅驯鹿SRY基因的cDNA序列及蛋白质结构进行分析,同时构建系统发育树.结果表明:扩增的敖鲁古雅驯鹿SRY基因的cDNA序列与GenBank数据库中登录的驯鹿SRY基因的cDNA序列(登录号AB247629.1)一致,全长690 bp.敖鲁古雅驯鹿SRY基因编码229个氨基酸;敖鲁古雅驯鹿SRY蛋白无信号肽和跨膜结构;结合其二级结构和结构域预测分析,SRY蛋白的二级结构以无规则卷曲和α-螺旋为主,具有HMG结构域.通过构建NJ系统发育树,发现敖鲁古雅驯鹿与白尾鹿之间的亲缘关系相对较近.研究结果为深入探索敖鲁古雅驯鹿SRY基因的生物学功能提供了基础参考.

Antler is the fastest growing and ossifying tissue in animals and it is a valuable model for cartilage/bone development. To understand the molecular mechanisms of chondrogenesis and osteogenesis of antlers, the PacBio Sequel II and Illumina sequencing technology were combined and used to investigate the mRNA expression profiles in antler tip, middle, and base at six different developmental stages, i.e., at 15th, 25th, 45th, 65th, 100th and 130th growth days. Consequently, we identified 24,856 genes (FPKM > 0.1), including 8778 novel genes. Besides, principal component analysis (PCA) revealed a significant separation between the growth stage (25th, 45th and 65th days) and ossification stage (100th and 130th days). COL2A1 gene was significantly abundant in the growth stage, whereas S100A7, S100A12, S100A8, and WFDC18 genes were abundant at the ossification stage. Subsequently screened to 14,765 significantly differentially expressed genes (DEGs), WGCNA and GO functional enrichment analyses revealed that genes related to cell division and chondrocyte differentiation were up-regulated, whereas those with steroid hormone-mediated signaling pathways were down-regulated at ossification stages. Additionally, 25 tumor suppressor genes and 11 oncogenes were identified and were predicted to interact with p53. Co-regulation of tumor suppressor genes and oncogenes is responsible for the special growth pattern of antlers. Together, we constructed the most complete sika deer antler transcriptome database so far. The database provides data support for subsequent studies on the molecular mechanism of sika deer antler chondrogenesis and osteogenesis.

旨在探究梅花鹿鹿茸不同生长时期小分子代谢物表达差异变化,为鹿茸快速生长与骨化分子机制的研究奠定基础.本研究选取体况良好的4岁龄雄性东北梅花鹿,收取生长25、45、65、100、130 d的鹿茸作为试验样品,每个时期3个生物学重复.利用UHPLC-TOF-MS技术对样品进行代谢组学分析.样品主成分分析与层次聚类分析结果显示,5个生长时期可分为鹿茸生长期(25、45与65 d)与鹿茸骨化期(100与130 d)两个阶段.筛选、鉴定到171种显著差异表达代谢物(VIP＞1,P＜0.05,| fold change |＞2),比对到HMDB数据库的112种代谢物,分为7大类;其中L-焦谷氨酸、L-组氨酸、L-肌肽与阿坎酸等有机酸类化合物在100 d含量最高,15-脱氧-Δ12,14-前列腺素J2、前列腺素H2、前列腺素12在130 d显著上调,而其他氨基酸及有机酸类化合物均在生长期表达上调.差异代谢物显著富集到氨酰-tRNA生物合成、组氨酸代谢等13种KEGG代谢通路.本研究从代谢组水平为鹿茸快速生长与骨化分子机制的探究提供数据支撑.

Velvet deer are not only a representative special economic animal but also an important part of livestock. With the increasing awareness of international competition for germplasm resources in China, more and more attention has been paid to the protection and utilization of germplasm resources. However, there is poor understanding about velvet deer resources. Therefore, we are providing a comprehensive introduction of Chinese velvet deer germplasm resources from the aspects of ecological distribution, domestication and breeding.

BACKGROUND:Exploration of adaptive evolution of organisms in response to environmental change can help to understand the evolutionary history of species and the underlying mechanisms of adaptation to local environments, thus guiding future conservation programmes. Before the introduction of Apis mellifera in China, eastern honey bees (Apis cerana) were the only species used for beekeeping in this region. In the mountains of Changbai, populations of A. cerana are considered a distinct ecotype of the species which formed through the distinct selective pressures in this area over time.RESULT:We performed a measure of 300 wing specimens of eastern honey bees and obtained the geometric morphological variation in the wing of A. cerana in Changbai Mountain. A total of 3,859,573 high-quality SNP loci were yielded via the whole-genome resequencing of 130 individuals in 5 geographic regions.CONCLUSION:Corresponding geometric morphology and population genomics confirmed the particularity of the A. cerana in Changbai Mountain. Genetic differentiation at the subspecies level exists between populations in Changbai Mountain and remaining geographic regions, and a significant reduction in the effective population size and an excessive degree of inbreeding may be responsible for a substantial loss of population genetic diversity. Candidate genes potentially associated with cold environmental adaptations in populations under natural selection were identified, which may represent local adaptations in populations. Our study provided insights into the evolutionary history and adaptation of A. cerana in Changbai Mountain, as well as its conservation.

Apis cerana in Changbai Mountain is an ecological type of Apis cerana, which is an excellent breeding material with cold-resistant developed by long-term natural selection under the ecological conditions. However, the physiological and molecular mechanisms of Changbai Mountain population under cold stress are still unclear. In this study, the Nanopore sequencing was carried out for the transcriptome of Apis cerana in Changbai Mountain in the coldest period of overwintering, which will provide a reference to the cold-resistant mechanism. We determined 5,941 complete ORF sequences, 1,193 lncRNAs, 619 TFs, 10,866 SSRs and functional annotations of 11,599 new transcripts. Our results showed that the myosin family and the C2H2 zinc finger protein transcription factor family possibly have significant impacts on the response mechanism of cold stress during overwintering. In addition, the cold environment alters genes expression profiles in honeybees via different AS and APA mechanisms. These altered genes in Hippo, Foxo, and MARK pathways help them counter the stress of cold in overwinter period. Our results might provide clues about the response of eastern honeybees to extreme cold, and reflect the possible genetic basis of physiological changes.