Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Regulatory roles of long non-coding RNAs in short-term heat stress in adult worker bees.

Long non-coding RNAs (lncRNAs) are crucial modulators of post-transcriptional gene expression regulation, cell fate determination, and disease development. However, lncRNA functions during short-term heat stress in adult worker bees are poorly understood. Here, we performed deep sequencing and bioinformatic analyses of honeybee lncRNAs. RNA interference was performed by using siRNA targeting the most highly expressed lncRNA. The silencing effect on lncRNA and the relative expression levels of seven heat shock protein (HSP) genes, were subsequently examined. Overall, 7,842 lncRNAs and 115 differentially expressed lncRNAs (DELs) were identified in adult worker bees following heat stress exposure. Structural analysis revealed that the overall expression abundance, length of transcripts, exon number, and open reading frames of lncRNAs were lower than those of mRNAs. GO analysis revealed that the target genes were mainly involved in "metabolism," "protein folding," "response to stress," and "signal transduction" pathways. KEGG analysis indicated that the "protein processing in endoplasmic reticulum" and "longevity regulating pathway-multiple species" pathways were most enriched. Quantitative real-time polymerase chain reaction (qRT-PCR) detection of the selected DELs confirmed the reliability of the sequencing data. Moreover, the siRNA experiment indicated that feeding siRNA yielded a silencing efficiency of 77.51% for lncRNA MSTRG.9645.5. Upon silencing this lncRNA, the expression levels of three HSP genes were significantly downregulated (p < 0.05), whereas those of three other HSP genes were significantly upregulated (p < 0.05). Our results provide a new perspective for understanding the regulatory mechanisms of lncRNAs in adult worker bees under short-term heat stress.

Insulin receptor participates in the peripheral olfactory processes of honey bees (Apis cerana cerana).

Insulin receptors (InR) are an integral component of the insulin/insulin-like growth factor signaling pathway, which plays a vital role in insect development, lifespan, reproduction, and olfactory sensitivity. However, whether InR participate in the peripheral olfactory system of insects remains unclear. Recently, we found that 2-heptanone (2-HT) affects AcerInR expression, the gene for an InR protein, in Apis cerana cerana. We then examined the spatiotemporal expression profile of the gene in A. cerana cerana. The mRNA of AcerInR was primarily expressed in the antennae, wings, and legs of forager bees, which are probable chemosensory tissues. The results of fluorescence competitive binding assays, combined with site-directed mutagenesis, demonstrated that AcerOBP6 and AcerOBP14 exhibit strong binding affinities to 2-HT. Furthermore, after foragers were fed with double-stranded AcerInR, the expression levels of AcerOBP6 and AcerOBP14 decreased significantly, as did the electroantennogram responsiveness to 2-HT and some other odorants. In conclusion, our findings provide a foundation for understanding the involvement of AcerInR in the odor perception of A. cerana cerana. Moreover, they offer novel insights into the olfactory recognition mechanism in insects.

Propolis Ethanolic Extract Attenuates D-gal-induced C2C12 Cell Injury by Modulating Nrf2/HO-1 and p38/p53 Signaling Pathways.

Previous study has shown that propolis ethanolic extract (PEE) has a protective effect on aging skeletal muscle atrophy. However, the exact molecular mechanism remains unclear. This study aimed to investigate the effect of PEE on D-galactose (D-gal)-induced damage in mouse C2C12 cells. The results revealed that PEE increased the viability of senescent C2C12 cells, decreased the number of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells and promoted the differentiation of C2C12 cells. PEE resisted oxidative stress caused by D-gal by activating the Nrf2/HO-1 signaling pathway and maintained the differentiation ability of C2C12 cells. PEE inhibited apoptosis by suppressing p38 phosphorylation and reducing p53 expression. In summary, our findings reveal the molecular mechanism by which PEE protects D-gal-induced C2C12 cells, providing a theoretical basis for the development of PEE for the alleviation of muscle atrophy.

Honeybees (Hymenoptera: Apidae) Adapt to the Shock of High Temperature and High Humidity Through Changes in Sugars and Polyols and Free Amino Acids.

Temperature and humidity are important factors affecting the honeybees physiological metabolism. When honeybees are stressed by high temperature and high humidity, various physiological stress mechanisms evolved by bees are activated in response to injury. The accumulation of some sugars, polyols, and free amino acids can effectively protect cell structure stability and resist temperature stress. In this study, the changes of glucose, trehalose, cholesterol, sorbitol, sorbitol dehydrogenase, mannitol, and free amino acids content of worker honeybees [Apis cerana cerana Fabricius and Apis mellifera Ligustica (Hymenoptera: Apidae)] under different temperature and humidity conditions were measured. Our research results show that high temperature has an important impact on the metabolism of honeybees. Heat stress can cause the accumulation of various antistress substances in worker. The contents of sugars, polyols, and some free amino acids accumulated in high temperature were significantly higher than those in the control, while the influence of high humidity was less. Although high humidity was improved compared with the control, the difference was not obvious. It provides a theoretical basis for exploring the physiological mechanism of individual heat resistance of honeybees.

Electrophysiological and Behavioral Responses of Apis mellifera and Bombusterrestris to Melon Flower Volatiles.

As important pollinators, honeybees and bumblebees present a pollination behavior that is influenced by flower volatiles through the olfactory system. In this study, volatile compounds from melon flowers were isolated and identified by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), and their effects on Apis mellifera and Bombus terrestris were investigated by electroantennogram (EAG) and behavior tests (Y-tube olfactometer). The results showed that 77 volatile compounds were detected in melon flowers, among which the relative content of aldehydes was the highest (61.34%; 82.09%). A. mellifera showed a strong EAG response to e-2-hexenal, e-2-octenal, and 1-nonanal. B. terrestris showed a strong EAG response to e-2-hexenal, e-2-octenal, 2,5-dimethyl-benzaldehyde, benzaldehyde and benzenepropanal. In behavior tests, the volatiles with the highest attractive rate to A. mellifera were e-2-hexenal (200 µg/µL, 33.33%) and e-2-octenal (300 µg/µL, 33.33%), and those to B. terrestris were e-2-hexenal (10 µg/µL, 53.33%) and 2,5-dimethyl-benzaldehyde (100 µg/µL, 43.33%). E-2-hexenal and e-2-octenal were more attractive to A. mellifera than B. terrestris, respectively (10 µg/µL, 10 µg/µL, 200 µg/µL). In conclusion, the volatiles of melon flowers in facilities have certain effects on the electrophysiology and behavior of bees, which is expected to provide theoretical and technical support for the pollination of A. mellifera and B. terrestris in facilities.

Differences in EAG Response and Behavioral Choices between Honey Bee and Bumble Bee to Tomato Flower Volatiles.

Bumble bees and honey bees are of vital importance for tomato pollination, although honey bees are less attracted to tomato flowers than bumble bees. Little is known about how tomato flower volatile compounds influence the foraging behaviors of honey bees and bumble bees. In this study, compounds of tomato flower volatiles were detected by gas chromatography-mass spectrometry. Electroantennography (EAG) and a dynamic two-choice olfactometer were used, respectively, to compare the differences of antennal and behavioral responses between Apis mellifera and Bombus terrestris towards selected volatile compounds. A total of 46 compounds were detected from the tomato flower volatiles. Of the 16 compounds tested, A. mellifera showed strong antennal responses to 3 compounds (1-nonanal, (+)-dihydrocarvone, and toluene) when compared with a mineral oil control, and B. terrestris showed 7 pronounced EAG responses (1,3-xylene, (+)-dihydrocarvone, toluene, piperitone, eucarvone, 1-nonanal, and ß-ocimene). Additionally, 1-nonanal and (+)-dihydrocarvone elicited significant avoidance behavior of A. mellifera, but not of B. terrestris. In conclusion, bumble bees are more sensitive to the compounds of tomato flower volatiles compared to honey bees, and honey bees showed aversion to some compounds of tomato flower volatiles. The findings indicated that compounds of flower volatiles significantly influenced bee foraging preference for tomato.

A Combined Proteomic and Metabolomic Strategy for Allergens Characterization in Natural and Fermented Brassica napus Bee Pollen.

Bee pollen is consumed for its nutritional and pharmacological benefits, but it also contains hazardous allergens which have not been identified. Here, we identified two potential allergens, glutaredoxin and oleosin-B2, in Brassica napus bee pollen using mass spectrometry-based proteomics analyses, and used bioinformatics to predict their antigenic epitopes. Comparison of fermented (by Saccharomyces cerevisiae) and unfermented bee pollen samples indicated that glutaredoxin and oleosin-B2 contents were significantly decreased following fermentation, while the contents of their major constituent oligopeptides and amino acids were significantly increased based on metabolomics analyses. Immunoblot analysis indicated that the IgE-binding affinity with extracted bee pollen proteins was also significantly decreased after fermentation, suggesting a reduction in the allergenicity of fermented bee pollen. Furthermore, fermentation apparently promoted the biosynthesis of L-valine, L-isoleucine, L-tryptophan, and L-phenylalanine, as well as their precursors or intermediates. Thus, fermentation could potentially alleviate allergenicity, while also positively affecting nutritional properties of B. napus bee pollen. Our findings might provide a scientific foundation for improving the safety of bee pollen products to facilitate its wider application.

The Transcriptomic Landscape of Molecular Effects after Sublethal Exposure to Dinotefuran on Apis mellifera.

The decreasing number of bees is a global ecological problem. With the advancement of agricultural modernisation, the large-scale use of neonicotinoid insecticides is one of the main factors leading to the decline of bees. The aim of the present study was to investigate the effect and the mechanisms underlying bees impaired by dinotefuran. Acute (48 h) oral toxicity tests showed that a 5% lethal concentration (LC5) was 0.220 mg/L, and a 20% lethal concentration (LC20) was 0.458 mg/L. The gene expression profile shows that when compared with the control group, the LC5 group induced 206 significantly upregulated, differentially expressed genes (DEGs) and 363 significantly downregulated DEGs, while the LC20 group induced 180 significantly upregulated DEGs and 419 significantly downregulated DEGs. Significantly, transcriptomic analysis revealed DEGs involved in immunity, detoxification, and the nervous system, such as antimicrobial peptides, vitellogenin, synaptotagmin-10, AChE-2, and nAChRa9. Furthermore, Gene Ontology (GO) annotation and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs were enriched in amino acid and fatty acid biosynthesis and metabolism pathways. Collectively, our findings will help clarify the deleterious physiological and behavioural impacts of dinotefuran on bees and provide a basis for future research on the mechanisms underlying bees impaired by dinotefuran.

Pollen Source Affects Development and Behavioral Preferences in Honey Bees.

With the availability of various plants in bloom simultaneously, honey bees prefer to collect some pollen types over others. To better understand pollen's role as a reward for workers, we compared the digestibility and nutritional value of two pollen diets, namely, pear (Pyrus bretschneideri Rehd.) and apricot (Armeniaca sibirica L.). We investigated the visits, pollen consumption, and pollen extraction efficiency of caged Apis mellifera workers. Newly emerged workers were reared, and the effects of two pollen diets on their physiological status (the development of hypopharyngeal glands and ovaries) were compared. The choice-test experiments indicated a significant preference of A. mellifera workers for apricot pollen diets over pear pollen diets (number of bees landing, 29.5 ± 8.11 and 9.25 ± 5.10, p < 0.001 and pollen consumption, 0.052 ± 0.026 g/day and 0.033 ± 0.013 g/day, p < 0.05). Both pollen diets had comparable extraction efficiencies (67.63% for pear pollen and 67.73% for apricot pollen). Caged workers fed different pollen diets also exhibited similar ovarian development (p > 0.05). However, workers fed apricot pollen had significantly larger hypopharyngeal glands than those fed pear pollen (p < 0.001). Our results indicated that the benefits conferred to honey bees by different pollen diets may influence their foraging preference.

Transcriptomic analysis reveals Apis mellifera adaptations to high temperature and high humidity.

Temperature and humidity are the most important factors affecting the growth, reproduction, and survival of bees. Apis mellifera are important pollinating bees that are widely used in agricultural systems. However, the higher temperatures and humidity in greenhouses are not conducive to the survival of bees. Although previous research has revealed the behavioral responses and physiological mechanisms of honeybees to adapt to high temperature and humidity, there are few data on the exact molecular mechanisms involved. In our study, we investigated gene expression in A. mellifera under different temperature and humidity treatments, using transcriptomic analysis to identify differentially expressed genes (DEGs) and relevant biological processes. Based on the transcriptomic results, we selected several genes with significant differences in expression, and detected the expression patterns of these genes at different temperatures or humidity or different treatment times by q-RT PCR. In the high temperature treatments, 434 DEGs were identified; in the high humidity treatments, 86 DEGs were identified; in the combined high temperature and humidity treatments, 266 DEGs were identified. Analysis results showed that DEGs were enriched in pathways related to amino acid and fatty acid biosynthesis and metabolism under each treatment. In addition, heat shock proteins, zinc finger proteins, serine/threonine-protein kinases, and antioxidase were differentially expressed between the different treatments. The results of the q-RT PCR showed that the expression levels of these genes increased with increasing temperature and over treatment time. Our findings provide a general expression profile of the adaptive expression of heat-resistance genes responding to high temperature and high humidity in A. mellifera, including the expression patterns of several DEGs. Our data provide a basis for future research on the mechanisms underlying the adaptation of insects to high temperature and humidity.

Tolerance and response of two honeybee species Apis cerana and Apis mellifera to high temperature and relative humidity.

The ambient temperature and relative humidity affect the metabolic and physiological responses of bees, thus affecting their life activities. However, the physiological changes in bee due to high temperature and high humidity remain poorly understood. In this study, we explored the effects of higher temperature and humidity on the epiphysiology of bees by evaluating the survival, tolerance and body water loss in two bee species (Apis cerana and Apis mellifera). We also evaluated the changes in the activity of antioxidant and detoxification enzymes in their body. We observed that under higher temperature and humidity conditions, the survival rate of A. mellifera was higher than that of A. cerana. On the other hand, a comparison of water loss between the two species revealed that A. mellifera lost more water. However, under extremely high temperature conditions, A. cerana was more tolerant than A. mellifera. Moreover, under higher temperature and humidity conditions, the activity of antioxidant and detoxification enzymes in bees was significantly increased. Overall, these results suggest that high temperatures can adversely affect bees. They not only affect the survival and water loss, but also stimulate oxidative stress in bees. However, unlike our previous understanding, high humidity can also adversely affect bees, although its effects are lower than that of temperature.

Contact Chemosensory Genes Identified in Leg Transcriptome of Apis cerana cerana (Hymenoptera: Apidae).

Correct gustatory recognition and selection of foods both within and outside the hive by honey bee workers are fundamental to the maintenance of colonies. The tarsal chemosensilla located on the legs of workers are sensitive to nonvolatile compounds and proposed to be involved in gustatory detection. However, little is known about the molecular mechanisms underlying the gustatory recognition of foods in honey bees. In the present study, RNA-seq was performed with RNA samples extracted from the legs of 1-, 10-, and 20-d-old workers of Apis cerana cerana Fabricius, a dominant indigenous crop pollinator with a keen perception ability for phytochemicals. A total of 124 candidate chemosensory proteins (CSPs), including 15 odorant-binding proteins (OBPs), 5 CSPs, 7 gustatory receptors (GRs), 2 sensory neuron membrane proteins (SNMPs), and 95 odorant receptors (ORs), were identified from the assembled leg transcriptome. In silico analysis of expression showed that 36 of them were differentially expressed among the three different ages of A. c. cerana workers. Overall, the genes encoding OBPs and CSPs had great but extremely variable FPKM values and thus were highly expressed in the legs of workers, whereas the genes encoding ORs, GRs, and SNMPs (except SNMP2) were expressed at low levels. Tissue-specific expression patterns indicated that two upregulated genes, AcerOBP15 and AcerCSP3, were predominately expressed in the legs of 20-d-old foragers, suggesting they may play an essential role in gustatory recognition and selection of plant nectars and pollens. This study lays a foundation for further research on the feeding preferences of honey bees.

First Record of the Velvet Ant Mutilla europaea (Hymenoptera: Mutillidae) Parasitizing the Bumblebee Bombus breviceps (Hymenoptera: Apidae).

Mutillid wasps are ectoparasitic insects that parasitize the enclosed developmental stages of their hosts. Adults are sexually dimorphic, with brilliantly colored and hardened cuticles. The biology of parasitic mutillid wasps has rarely been addressed. Here, we investigated the parasitization by Mutilla europaea on an important pollinator, Bombus breviceps. The parasitic biology and dispersal ability of M. europaea were observed and tested under experimental conditions. We provide the first record of M. europaea parasitizing B. breviceps in southwestern China. As is the case with other bumblebee species, M. europaea mainly parasitized the puparia of males. The dispersal and invasion ability of this parasite under experimental conditions indicates that it spreads rapidly, as far as 20 m in one week, and invades different hosts (B. breviceps and Bombus haemorrhoidalis). This report not only clarifies the parasitic relationship between M. europaea and B. breviceps, but also has important ecological implications for the conservation of bumblebees in China.

Expressional and functional interactions of two Apis cerana cerana olfactory receptors.

Apis cerana cerana relies on its sensitive olfactory system to perform foraging activities in the surrounding environment. Olfactory receptors (ORs) are a primary requirement for odorant recognition and coding. However, the molecular recognition of volatile compounds with ORs in A. cerana cerana is still not clear. Hence, in the present study, we achieved transient transfection and cell surface expression of A. cerana cerana ORs (AcerOr1 and AcerOr2; AcerOr2 is orthologous to the co-receptor) in Spodoptera frugiperda (Sf9) cells. AcerOr2 narrowly responded to N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl) thio) acetamide (VUAA1), whereas AcerOr1 was sensitive to eugenol, lauric acid, ocimene, 1-nonanol, linolenic acid, hexyl acetate, undecanoic acid, 1-octyl alcohol, and nerol. Of the compounds tested, AcerOr1 showed the highest sensitivity to these odorants with EC50 values of 10-7 and 10-8 M, and AcerOr2 recognized VUAA1 with higher sensitivity [EC50 = (6.621 ± 0.26) × 10-8]. These results indicate that AcerOr2 is an essential gene for olfactory signaling, and AcerOr1 is a broadly tuned receptor. We discovered ligands that were useful for probing receptor activity during odor stimulation and validated three of them by electroantennography. The response increased with concentration of the odorant. The present study provides insight into the mechanism of olfactory discrimination in A. cerana cerana.

Expression Characterization and Localization of the foraging Gene in the Chinese Bee, Apis cerana cerana (Hymenoptera: Apidae).

In social insects, the foraging gene (for) regulates insect age- and task-based foraging behaviors. We studied the expression and localization of the for gene (Acfor) in Apis cerana cerana workers to explore whether the differential regulation of this gene is associated with the behaviors of nurses and foragers. The expression profiles of Acfor in different tissues and at different ages were examined using real-time quantitative reverse transcription polymerase chain reaction. Cellular localization in the brain was detected using in situ hybridization. Acfor transcripts in different ages workers showed that Acfor expression was detected in all the heads of 1- to 30-d-old worker bees. Acfor expression reached a peak at 25 d of age, and then declined with increasing age. The results showed that Acfor gene expression in five tissues was respectively significantly higher in foragers than in nurses. In nurses, the relative expression of Acfor was the highest in the antennae. There was a highly significant difference in expression between antennae, legs, and the other three tissues. In foragers, Acfor expression was the highest in the thorax, which was significantly different from all other tissues. In situ hybridization showed that Acfor was highly expressed in the lamina of the optic lobes, in a central column of Kenyon cells in the mushroom bodies of the brain of workers, and in the antennal lobes. This suggested that Acfor expression affects age-related foraging behavior in Apis cerana cerana, and that it may be related to flight activity.

Odorant receptor might be related to sperm DNA integrity in Apis cerana cerana.

Olfactory receptors (ORs) are important for insects to recognize and discriminate odorants in the environment and are mainly expressed in olfactory and gustatory organs. Little is known about the potential OR functions in non-olfactory tissues. In the present study, we evaluated the possibility of odorant receptors AcerOr1 and AcerOr2 (AcerOr2 is orthologous to the co-receptor) mediating sperm DNA integrity, and the relationship between sperm DNA integrity and semen parameters in Apis cerana cerana. Based on previous findings in mammals, we speculated that the Ca2+/calmodulin (CaM)/CaM-dependent protein kinase II (CaMKII) signaling pathway might be involved in the regulation of sperm motility in A. cerana cerana. The results showed that both AcerOr1 and AcerOr2 are expressed in the sperms and testis, that components associated with the putative Ca2+/CaM/CaMKII signaling pathway are present in A. cerana cerana sperms, and that at least CaM and CaMKII are localized in the sperms and testis. The AcerOr2 agonist VUAA1 significantly improved sperm motility parameters and apoptosis of sperm cells effect DNA integrity, whereas the CaM inhibitor W7 decreased sperm motility parameters and apoptosis of sperm cells, which affects DNA integrity. We also found a positive correlation between sperm DNA integrity and semen quality. These results indirectly as well as directly suggest that OR-mediated sperm responses and the Ca2+/CaM/CaMKII signaling pathway might affect semen quality and might be useful in regulating insect reproduction in future.

Comparative antennal transcriptome of Apis cerana cerana from four developmental stages.

Apis cerana cerana, an important endemic honey bee species in China, possesses valuable characteristics such as a sensitive olfactory system, good foraging ability, and strong resistance to parasitic mites. Here, we performed transcriptome sequencing of the antenna, the major chemosensory organ of the bee, using an Illumina sequencer, to identify typical differentially expressed genes (DEGs) in adult worker bees of different ages, namely, T1 (1 day); T2 (10 days); T3 (15 days); and T4 (25 days). Surprisingly, the expression levels of DEGs changed significantly between the T1 period and the other three periods. All the DEGs were classified into 26 expression profiles by trend analysis. Selected trend clusters were analyzed, and valuable information on gene expression patterns was obtained. We found that the expression levels of genes encoding cuticle proteins declined after eclosion, while those of immunity-related genes increased. In addition, genes encoding venom proteins and major royal jelly proteins were enriched at the T2 stage; small heat shock proteins showed significantly higher expression at the T3 stage; and some metabolism-related genes were more highly expressed at the T4 stage. The DEGs identified in this study may serve as a valuable resource for the characterization of expression patterns of antennal genes in A. cerana cerana. Furthermore, this study provides insights into the relationship between labor division in social bees and gene function.

Correction: Transcriptomic analysis to uncover genes affecting cold resistance in the Chinese honey bee (Apis cerana cerana).

[This corrects the article DOI: 10.1371/journal.pone.0179922.].

Transcriptomic analysis to uncover genes affecting cold resistance in the Chinese honey bee (Apis cerana cerana).

The biological activity and geographical distribution of honey bees is strongly temperature-dependent, due to their ectothermic physiology. In China, the endemic Apis cerana cerana exhibits stronger cold hardiness than Western honey bees, making the former species important pollinators of winter-flowering plants. Although studies have examined behavioral and physiological mechanisms underlying cold resistance in bees, data are scarce regarding the exact molecular mechanisms. Here, we investigated gene expression in A. c. cerana under two temperature treatments, using transcriptomic analysis to identify differentially expressed genes (DEGs) and relevant biological processes, respectively. Across the temperature treatments, 501 DEGs were identified. A gene ontology analysis showed that DEGs were enriched in pathways related to sugar and amino acid biosynthesis and metabolism, as well as calcium ion channel activity. Additionally, heat shock proteins, zinc finger proteins, and serine/threonine-protein kinases were differentially expressed between the two treatments. The results of this study provide a general digital expression profile of thermoregulation genes responding to cold hardiness in A. c. cerana. Our data should prove valuable for future research on cold tolerance mechanisms in insects, and may be beneficial in breeding efforts to improve bee hardiness.