Identification of the cytochrome P450 gene AccCYP6A13 in Apis cerana cerana and its response to environmental stress.

Cytochrome P450 plays a crucial role in regulating insect growth, development, and resisting a variety of stresses. Insect metamorphosis and response to external stress are altered by deleting CYP450 genes. In this study, we identified and analyzed a novel gene of CYP450 family, AccCYP6A13, from Apis cerana cerana, and explored its role in the response of Apis cerana cerana to adverse external stressors. It was found that the expression of AccCYP6A13 was spatiotemporal specificity. The expression level increased with age and reached its highest value in the adult stage. The primarily expressiong location were legs, brain, and epidermis of honeybees. Stress conditions can affect the expression of AccCYP6A13 depending on treatment times. RNA interference experiments have shown that knocking down AccCYP6A13 reduces antioxidant activity and deactivates detoxification enzymes, resulting in oxidative damage accumulation and a decline in detoxification capability in bees, as well as inhibiting numerous antioxidant genes. Additionally, knockdown of the AccCYP6A13 gene in Apis cerana cerana resulted in increased sensitivity to pesticides and increased mortality when treated with neonicotinoid pesticides such as thiamethoxam. AccCYP6A13 overexpression in a prokaryotic system further confirmed its role in resistance to oxidative stress. To summarize, AccCYP6A13 may play an essential role in the normal development and response to environmental stress in Apis cerana cerana. Furthermore, this study contributed to the theoretical understanding of bee resistance biology.

Whole-genome resequencing reveals genetic diversity and adaptive evolution in Chinese honeybee (Apis cerana cerana) in Guizhou, China.

Introduction: Guizhou Province, characterized by complex and diverse geographic and climatic environments, has rich genetic resources for the Chinese honeybee (Apis cerana cerana) and is one of the main bee-producing areas in China. However, research on the genetic diversity of Chinese honeybee in the Guizhou region is very limited, despite implications for conservation of biodiversity. Methods: In this study, we analyzed the genetic diversity, differentiation, and selection signals based on 116 Chinese honeybees from 12 regions in Guizhou Province using whole-genome sequencing. Results: We identified 1,400,430 high-quality SNPs across all samples. A population structure analysis revealed two independent genetic subgroups of Chinese honeybees in Guizhou, a Yunnan-Guizhou Plateau population in western Guizhou and a hilly-mountainous population in eastern Guizhou. The average nucleotide diversity (Pi) ranged from 0.00138 to 0.00161 and average expected heterozygosity (He) ranged from 0.2592 to 0.2604. The average genetic differentiation index (F ST) for Chinese honeybees in pairwise comparisons of 12 regions ranged from 0.0094 to 0.0293. There was clear genetic differentiation between the western plateau and the eastern hilly mountainous areas of Guizhou; however, F ST values between the eastern and western populations ranged from 0.0170 to 0.0293, indicating a low degree of differentiation. A genome-wide scan revealed a number of genes under selection in the Yunnan-Guizhou Plateau environment. These genes were related to growth and development, reproduction, and cold resistance, and several candidate genes involved in environmental adaptation were identified, including CTR, MAPK, MAST, HSF, and MKKK. Discussion: The results of the present study provide important theoretical bases for the conservation, evaluation, development, and utilization of genetic resources for Chinese honeybees in the Guizhou region and for further investigations of environmental adaptation and underlying mechanisms in the species.

Decline in Honeybees and Its Consequences for Beekeepers and Crop Pollination in Western Nepal.

In understudied regions of the world, beekeeper records can provide valuable insights into changes in pollinator population trends. We conducted a questionnaire survey of 116 beekeepers in a mountainous area of Western Nepal, where the native honeybee Apis cerana cerana is kept as a managed bee. We complemented the survey with field data on insect-crop visitation, a household income survey, and an interview with a local lead beekeeper. In total, 76% of beekeepers reported declines in honeybees, while 86% and 78% reported declines in honey yield and number of beehives, respectively. Honey yield per hive fell by 50% between 2012 and 2022, whilst the number of occupied hives decreased by 44%. Beekeepers ranked climate change and declining flower abundance as the most important drivers of the decline. This raises concern for the future food and economic security of this region, where honey sales contribute to 16% of total household income, and where Apis cerana cerana plays a major role in crop pollination, contributing more than 50% of all flower visits to apple, cucumber, and pumpkin. To mitigate further declines, we promote native habitat and wildflower preservation, and using well-insulated log hives to buffer bees against the increasingly extreme temperature fluctuations.

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.

Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection.

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs' expression between uninoculated and A. apis-inoculated larval guts were confirmed based on molecular methods. These findings not only enrich our understanding of bee host-fungal pathogen interactions but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. c. cerana larvae against A. apis invasion. KEY POINTS: • The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection. • Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis-challenged host guts.

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.

Isolation of the AccCDK8 gene of Apis cerana cerana and its functional analysis under pesticide and heavy metal stress.

Environmental pollution has gained negative attention in recent years. The pesticides and heavy metals are top list of environmental toxicants directly endangering the survival and development of Apis cerana cerana. Cyclin-dependent kinases (CDKs) are heteromeric serine/threonine kinases that participate in cell cycle regulation and have a vital role in pesticide and heavy metal stress in Apis cerana cerana. In this experiment, we filtered out CDK8 gene from Apis cerana cerana (AccCDK8) and investigated its functions of pesticide and heavy metals resistance. Sequence analysis indicated that AccCDK8 is highly homologous to multiple CDK8s and contains a highly conserved CDK active site sequence. Phylogenetic analysis showed that AmCDK8 and AccCDK8 were closely related evolutionarily in Apis mellifera. Transcriptome analysis revealed that AccCDK8 expression was differentially affected after exposure to pesticide and heavy metal stresses. This indicates that AccCDK8 has a significant role in the resistance of Apis cerana cerana to pesticide and heavy metal stresses. It has implications for studying the function of CDK in other insects in response to stress.

The gene AccCyclin H mitigates oxidative stress by influencing trehalose metabolism in Apis cerana cerana.

BACKGROUND: Environmental stress can induce oxidative stress in Apis cerana cerana, leading to cellular oxidative damage, reduced vitality, and even death. Currently, owing to an incomplete understanding of the molecular mechanisms by which A. cerana cerana resists oxidative damage, there is no available method to mitigate the risk of this type of damage. Cyclin plays an important role in cell stress resistance. The aim of this study was to explore the in vivo protection of cyclin H against oxidative damage induced by abiotic stress in A. cerana cerana and clarify the mechanism of action. We isolated and identified the AccCyclin H gene in A. cerana cerana and analysed its responses to different exogenous stresses. RESULTS: The results showed that different oxidative stressors can induce or inhibit the expression of AccCyclin H. After RNA-interference-mediated AccCyclin H silencing, the activity of antioxidant-related genes and related enzymes was inhibited, and trehalose metabolism was reduced. AccCyclin H gene silencing reduced A. cerana cerana high-temperature tolerance. Exogenous trehalose supplementation enhanced the total antioxidant capacity of A. cerana cerana, reduced the accumulation of oxidants, and improved the viability of A. cerana cerana under high-temperature stress. CONCLUSION: Our findings suggest that trehalose can alleviate adverse stress and that AccCyclin H may participate in oxidative stress reactions by regulating trehalose metabolism. © 2023 Society of Chemical Industry.

Diverse Regulatory Manners and Potential Roles of lncRNAs in the Developmental Process of Asian Honey Bee (Apis cerana) Larval Guts.

Long non-coding RNAs (lncRNAs) are crucial modulators in a variety of biological processes, such as gene expression, development, and immune defense. However, little is known about the function of lncRNAs in the development of Asian honey bee (Apis cerana) larval guts. Here, on the basis of our previously obtained deep-sequencing data from the 4-, 5-, and 6-day-old larval guts of A. cerana workers (Ac4, Ac5, and Ac6 groups), an in-depth transcriptome-wide investigation was conducted to decipher the expression pattern, regulatory manners, and potential roles of lncRNAs during the developmental process of A. cerana worker larval guts, followed by the verification of the relative expression of differentially expressed lncRNAs (DElncRNAs) and the targeting relationships within a competing endogenous RNA (ceRNA) axis. In the Ac4 vs. Ac5 and Ac5 vs. Ac6 comparison groups, 527 and 498 DElncRNAs were identified, respectively, which is suggestive of the dynamic expression of lncRNAs during the developmental process of larval guts. A cis-acting analysis showed that 330 and 393 neighboring genes of the aforementioned DElncRNAs were respectively involved in 29 and 32 functional terms, such as cellular processes and metabolic processes; these neighboring genes were also respectively engaged in 246 and 246 pathways such as the Hedgehog signaling pathway and the Wnt signaling pathway. Additionally, it was found that 79 and 76 DElncRNAs as potential antisense lncRNAs may, respectively, interact with 72 and 60 sense-strand mRNAs. An investigation of competing endogenous RNA (ceRNA) networks suggested that 75 (155) DElncRNAs in the Ac4 vs. Ac5 (Ac5 vs. Ac6) comparison group could target 7 (5) DEmiRNAs and further bind to 334 (248) DEmRNAs, which can be annotated to 33 (29) functional terms and 186 (210) pathways, including 12 (16) cellular- and humoral-immune pathways (lysosome pathway, necroptosis, MAPK signaling pathway, etc.) and 11 (10) development-associated signaling pathways (Wnt, Hippo, AMPK, etc.). The RT-qPCR detection of five randomly selected DElncRNAs confirmed the reliability of the used sequencing data. Moreover, the results of a dual-luciferase reporter assay were indicative of the binding relationship between MSTRG.11294.1 and miR-6001-y and between miR-6001-y and ncbi_107992440. These results demonstrate that DElncRNAs are likely to modulate the developmental process of larval guts via the regulation of the source genes' transcription, interaction with mRNAs, and ceRNA networks. Our findings not only yield new insights into the developmental mechanism underlying A. cerana larval guts, but also provide a candidate ceRNA axis for further functional dissection.

Functional analysis of AccCDK2-like and AccCINP-like genes in Apis cerana cerana under pesticide and heavy metal stress.

Heavy metals and pesticides represent prominent sources of pollution in the natural habitat of Apis cerana cerana, potentially endangering their health through the induction of oxidative stress reactions. This study aimed to address this issue by isolating AccCDK2-like and AccCINP-like proteins from Apis cerana cerana and investigating their functional roles in honey bee resistance against pesticide and heavy metal stresses. Bioinformatics analysis revealed significant homology of these proteins with those found in other species. Functional studies confirmed their participation in interaction with each other, alongside demonstrating distinct patterns of expression and localization. Specifically, AccCDK2-like exhibited higher expression levels in prepupae and muscle tissues, while AccCINP-like showed maximal expression in brown pupae and abdomen. Furthermore, the expression levels of these proteins were found to be modulated in response to pesticide and heavy metal stresses. Notably, overexpression of AccCDK2-like and AccCINP-like led to a noticeable alteration in E. coli's ability to withstand external stresses. Additionally, silencing of the AccCDK2-like and AccCINP-like genes resulted in a significant reduction in antioxidant enzyme activity and the expression levels of genes related to antioxidant function. Consequently, the mortality rate of Apis cerana cerana under pesticide and heavy metal stresses conspicuously increased. Hence, our findings suggest that AccCDK2-like and AccCINP-like proteins potentially play a crucial role in the response of Apis cerana cerana to pesticide and heavy metal stress, likely by modulating the antioxidant pathway.

Regulatory Roles of Long Non-Coding RNAs Relevant to Antioxidant Enzymes and Immune Responses of Apis cerana Larvae Following Ascosphaera apis Invasion.

Long non-coding RNAs (lncRNAs) play an essential part in controlling gene expression and a variety of biological processes such as immune defense and stress-response. However, whether and how lncRNAs regulate responses of Apis cerana larvae to Ascosphaera apis invasion has remained unclear until now. Here, the identification and structural analysis of lncRNAs in the guts of A. cerana worker larvae were conducted, and the expression profile of larval lncRNAs during the A. apis infection process was then analyzed, followed by an investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in the host response. In total, 76 sense lncRNAs, 836 antisense lncRNAs, 184 intron lncRNAs, 362 bidirectional lncRNAs, and 2181 intron lncRNAs were discovered in the larval guts. Additionally, 30 known and 9 novel lncRNAs were potential precursors for 36 and 11 miRNAs, respectively. In the three comparison groups, 386, 351, and 272 DElncRNAs were respectively identified, indicating the change in the overall expression pattern of host lncRNAs following the A. apis invasion. Analysis of cis-acting effect showed that DElncRNAs in the 4-, 5-, and 6-day-old comparison groups putatively regulated 55, 30, and 20 up- and down-stream genes, respectively, which were involved in a series of crucial functional terms and pathways, such as MAPK signaling pathway, and cell process. Analysis showed that 31, 8, and 11 DElncRNAs as potential antisense lncRNAs may interact with 26, 8, and 9 sense-strand mRNAs. Moreover, investigation of the competing endogenous RNA (ceRNA) network indicated that 148, 283, and 257 DElncRNAs were putatively regulated. The expression of target genes by targeting corresponding DEmiRNAs included those associated with antioxidant enzymes and immune responses. These results suggested that DElncRNAs played a potential part in the larval guts responding to the A. apis infection through a cis-acting manner and ceRNA mechanisms. Our findings deepen our understanding of interactions between A. cerana larvae and A. apis and offer a basis for clarifying the DElncRNA-mediated mechanisms underlying the host response to fungal invasion.

Expression Profile, Regulatory Network, and Putative Role of microRNAs in the Developmental Process of Asian Honey Bee Larval Guts.

MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs' differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the "Ac4 vs. Ac5" comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the "Ac5 vs. Ac6" comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the "Ac4 vs. Ac5" comparison group and 31 targets in the "Ac5 vs. Ac6" comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

Nano-pesticide carrier O-Carboxymethyl chitosan is indigestible in Apis cerana cerana and affects intestinal flora.

O-Carboxymethyl chitosan nanoparticles (O-CMC-NPs), which are organic pesticide carriers, have excellent application potential. Exploring the effects of O-CMC-NPs on non-target organisms, such as Apis cerana cerana, is critical for their effective application; however, such studies are limited. This study investigated the stress response of A. cerana Fabricius after O-CMC-NPs ingestion. The administration of high O-CMC-NP concentrations enhanced the activities of antioxidant and detoxifying enzymes in A. cerana, with the activity of glutathione-S-transferase increasing by 54.43 %-64.33 % after one day. The transit of O-CMC-NPs into the A. cerana midgut resulted in their deposition and adherence to the intestinal wall, as they cluster and precipitate in acidic conditions. The population of Gillianella bacteria in the middle intestine was remarkably reduced after 6 d of administration of high O-CMC-NP concentrations. Contrastingly, the abundance of Bifidobacteria and Lactobacillus in the rectum significantly increased. These results indicate that the intake of high concentrations of O-CMC-NPs causes a stress response in A. cerana and affects the relative abundance of crucial intestinal flora, which may pose a potential risk to the colony. This implies that even nanomaterials with favorable biocompatibility should be applied reasonably within a specific range to avoid adverse effects on the environment and non-target organisms in the context of large-scale research and promotion of nanomaterials.

Discovery of SNP Molecular Markers and Candidate Genes Associated with Sacbrood Virus Resistance in Apis cerana cerana Larvae by Whole-Genome Resequencing.

Sacbrood virus (SBV) is a significant problem that impedes brood development in both eastern and western honeybees. Whole-genome sequencing has become an important tool in researching population genetic variations. Numerous studies have been conducted using multiple techniques to suppress SBV infection in honeybees, but the genetic markers and molecular mechanisms underlying SBV resistance have not been identified. To explore single nucleotide polymorphisms (SNPs), insertions, deletions (Indels), and genes at the DNA level related to SBV resistance, we conducted whole-genome resequencing on 90 Apis cerana cerana larvae raised in vitro and challenged with SBV. After filtering, a total of 337.47 gigabytes of clean data and 31,000,613 high-quality SNP loci were detected in three populations. We used ten databases to annotate 9359 predicted genes. By combining population differentiation index (FST) and nucleotide polymorphisms (π), we examined genome variants between resistant (R) and susceptible (S) larvae, focusing on site integrity (INT < 0.5) and minor allele frequency (MAF < 0.05). A selective sweep analysis with the top 1% and top 5% was used to identify significant regions. Two SNPs on the 15th chromosome with GenBank KZ288474.1_322717 (Guanine > Cytosine) and KZ288479.1_95621 (Cytosine > Thiamine) were found to be significantly associated with SBV resistance based on their associated allele frequencies after SNP validation. Each SNP was authenticated in 926 and 1022 samples, respectively. The enrichment and functional annotation pathways from significantly predicted genes to SBV resistance revealed immune response processes, signal transduction mechanisms, endocytosis, peroxisomes, phagosomes, and regulation of autophagy, which may be significant in SBV resistance. This study presents novel and useful SNP molecular markers that can be utilized as assisted molecular markers to select honeybees resistant to SBV for breeding and that can be used as a biocontrol technique to protect honeybees from SBV.

AccsHSP21.7 enhances the antioxidant capacity of Apis cerana cerana.

BACKGROUND: The widespread use of glyphosate has many adverse effects on Apis cerana cerana. Due to the incomplete understanding of the molecular mechanisms of glyphosate toxicity, there are no available methods for mitigating the threat of glyphosate to Apis cerana cerana. Small heat shock proteins (sHSPs) play an important role in resisting oxidative stress, but their mechanism of action in Apis cerana cerana remains unclear. RESULTS: In this experiment, we cloned and identified AccsHSP21.7. Studies have shown that AccsHSP21.7 contains binding motifs for various transcription factors related to oxidative stress. Abiotic stresses induced the expression of AccsHSP21.7. Bacteriostatic testing of a recombinant AccsHSP21.7 protein proved that Escherichia coli overexpressing AccsHSP21.7 showed increased resistance to oxidative stress. Knocking down the AccsHSP21.7 gene caused significant damage to midgut cells, which seriously disrupted the antioxidant system in Apis cerana cerana and greatly increased mortality under glyphosate stress. CONCLUSION: This study investigated the relationship between antioxidant regulation and the AccsHSP21.7 gene at the molecular level, and the results have guiding significance for the improvement of stress resistance in Apis cerana cerana. © 2023 Society of Chemical Industry.

Identification of the AccCDK7 and AccCDK9 genes and their involvement in the response to resist external stress in Apis cerana cerana.

Previous studies examining the functions of cyclin-dependent kinases (CDKs) have mainly focused on the regulation of the cell cycle. Recent studies have found that cyclin-dependent kinase 7 (CDK7) and cyclin-dependent kinase 9 (CDK9) play important roles in cell stress, metabolism of toxic substances and maintaining the stability of the internal environment. Here, we found that under stress conditions, the transcription and protein expression of AccCDK7 and AccCDK9 were induced to varying degrees. Meanwhile, the silencing of AccCDK7 and AccCDK9 also affected the expression of antioxidant genes and the activity of antioxidant enzymes, and reduced the survival rate of bees under high temperature stress. Furthermore, the exogenous overexpression of AccCDK7 and AccCDK9 improved the viability of yeast under stress conditions. Therefore, AccCDK7 and AccCDK9 may play roles in A.cerana cerana resistance to oxidative stress caused by external stimuli, potentially revealing a new mechanism of the honeybee response to oxidative stress.

Fox transcription factor AccGRF1 in response to glyphosate stress in Apis cerana cerana.

Glyphosate is an herbicide commonly used in agriculture, and its widespread use has adversely affected the survival of nontarget organisms. Among these organisms, bees in particular are important pollinators, and declining bee populations have severely affected crop yields around the world. However, the molecular mechanism by which glyphosate harms bees remains unclear. In our experiment, we screened and cloned a glyphosate-induced gene in Apis cerana cerana (A. c. cerana) and named glyphosate response factor 1 (AccGRF1). Sequence analysis showed that AccGRF1 contains a winged-helix DNA binding domain, which suggests that it belongs to the Forkhead box (Fox) protein family. qRT-PCR and heterologous expression in Escherichia coli and yeast showed that AccGRF1 can respond to glyphosate and oxidative stress. After AccGRF1 knockdown by means of RNA interference (RNAi), the resistance of A. c. cerana to glyphosate stress improved. The results suggested that AccGRF1 is involved in A. c. cerana glyphosate stress tolerance. This study reveals the functions of Fox transcription factors in response to glyphosate stress and provides molecular insights into the regulation of glyphosate responses in honeybees.

Systematic Characterization and Regulatory Role of lncRNAs in Asian Honey Bees Responding to Microsporidian Infestation.

Long noncoding RNAs (lncRNAs) are pivotal regulators in gene expression and diverse biological processes, such as immune defense and host-pathogen interactions. However, little is known about the roles of lncRNAs in the response of the Asian honey bee (Apis cerana) to microsporidian infestation. Based on our previously obtained high-quality transcriptome datasets from the midgut tissues of Apis cerana cerana workers at 7 days post inoculation (dpi) and 10 dpi with Nosema ceranae (AcT7 and AcT10 groups) and the corresponding un-inoculated midgut tissues (AcCK7 and AcCK10 groups), the transcriptome-wide identification and structural characterization of lncRNAs were conducted, and the differential expression pattern of lncRNAs was then analyzed, followed by investigation of the regulatory roles of differentially expressed lncRNAs (DElncRNAs) in host response. Here, 2365, 2322, 2487, and 1986 lncRNAs were, respectively, identified in the AcCK7, AcT7, AcCK7, and AcT10 groups. After removing redundant ones, a total of 3496 A. c. cerana lncRNAs were identified, which shared similar structural characteristics with those discovered in other animals and plants, such as shorter exons and introns than mRNAs. Additionally, 79 and 73 DElncRNAs were screened from the workers' midguts at 7 dpi and 10 dpi, respectively, indicating the alteration of the overall expression pattern of lncRNAs in host midguts after N. ceranae infestation. These DElncRNAs could, respectively, regulate 87 and 73 upstream and downstream genes, involving a suite of functional terms and pathways, such as metabolic process and Hippo signaling pathway. Additionally, 235 and 209 genes co-expressed with DElncRNAs were found to enrich in 29 and 27 terms, as well as 112 and 123 pathways, such as ABC transporters and the cAMP signaling pathway. Further, it was detected that 79 (73) DElncRNAs in the host midguts at 7 (10) dpi could target 321 (313) DEmiRNAs and further target 3631 (3130) DEmRNAs. TCONS_00024312 and XR_001765805.1 were potential precursors for ame-miR-315 and ame-miR-927, while TCONS_00006120 was the putative precursor for both ame-miR-87-1 and ame-miR-87-2. These results together suggested that DElncRNAs are likely to play regulatory roles in the host response to N. ceranae infestation through the regulation of neighboring genes via a cis-acting effect, modulation of co-expressed mRNAs via trans-acting effect, and control of downstream target genes' expression via competing endogenous RNA networks. Our findings provide a basis for disclosing the mechanism underlying DElncRNA-mediated host N. ceranae response and a new perspective into the interaction between A. c. cerana and N. ceranae.

Role of the tyrosine aminotransferase AccTATN gene in the response to pesticide and heavy metal stress in Apis cerana cerana.

Tyrosine aminotransferase (TATN) is the first enzyme involved in the metabolic degradation of tyrosine, and it plays an important role in tyrosine detoxification and helps the body resist oxidative damage. However, the function of TATN in Apis cerana cerana (A. c. cerana) remains unclear. To explore the role of TATN in the response to pesticide and heavy metal stress in A. c. cerana, AccTATN was isolated and identified. AccTATN was highly expressed in the integument and the adult stage. Exposure to multiple pesticides and heavy metal stress upregulated AccTATN expression. RNA interference experiments showed that silencing AccTATN reduced the resistance of A. c. cerana to glyphosate and avermectins stress. The expression of antioxidant-related genes and the activity of antioxidant enzymes were reduced after AccTATN was silenced, leading to the accumulation of oxidative damage. Overexpression of the recombinant AccTATN protein in a prokaryotic system also confirmed its role in heavy metal stress and improved antioxidant capacity. Our study showed that AccTATN may promote resistance to pesticide and heavy metal stress by regulating the antioxidant capacity of A. c. cerana. This study provides a valuable theoretical basis for A. c. cerana conservation.

RNAi-mediated silencing of AccCYP6k1 revealed its role in the metabolic detoxification of Apis cerana cerana.

Insect cytochrome P450 monooxygenases (P450s or CYPs) perform important functions in the metabolic detoxification of both endogenous and exogenous substrates. However, the mechanism of action of the P450 genes in bees is unclear. In this study, we investigated the effects of AccCYP6k1 on the metabolism and detoxification of Apis cerana cerana. Spatiotemporal expression profiling revealed that the expression of AccCYP6k1 was the highest in foragers (A15) and was mainly expressed in the leg, midgut and head. RT-qPCR results showed that AccCYP6k1 exhibited different expression patterns following exposure to xenobiotics. In addition, silencing AccCYP6k1 increased the pesticides sensitivity and affected the detoxification system and antioxidant process of A. cerana cerana. In brief, the induced expression of AccCYP6k1 is related to the resistance of A. cerana cerana, while knockdown AccCYP6k1 affect the pesticides resistance and metabolic detoxification system of A. cerana cerana. These findings not only support the theoretical basis of metabolic detoxification in bees but also provide a better understanding of P450-mediated resistance to pesticides in insects.