Melittin, the principal active peptide of bee venom, exhibits potent cytotoxicity against cancer cells. However, its lipid-level mechanisms remain unclear. Here, we present the first untargeted lipidomic dataset that reveals melittin-induced lipid remodeling in triple-negative breast cancer (TNBC) cells (MDA-MB-231). Cells were exposed to 4 mu g/mL of melittin for 15 min, and lipid extracts were analyzed by employing high-resolution LC-MS/MS in both ion modes. Data were processed with XCMS and metaX for peak extraction, normalization, and metabolite annotation, followed by multivariate and KEGG pathway analyses. The results highlight significant alterations in phospholipids, sphingolipids, and acylglycerols, indicative of melittin-mediated disruption of membrane integrity and lipid metabolism. All raw and processed data are publicly accessible at NGDC (accession number PRJCA048975). This dataset not only serves as a comprehensive resource for investigating lipid-based mechanisms underlying melittin's anticancer effects but also supports its potential in lipid-targeted therapeutic strategies for TNBC. Dataset: The dataset can be accessed through the NGDC website by searching with the BioProject accession number PRJCA048975. Reviewers may use this link for anonymous access during the review process. Direct URL to data: Genome Sequence Archive-CNCB-NGDC (https://ngdc.cncb.ac.cn/gsa/, accessed on 21 May 2026).

Ascosphaera apis, an obligate lethal fungal pathogen that infects bee larvae, and causes chalkbrood disease, poses a significant threat to the global beekeeping industry. Long non-coding RNAs (lncRNAs) are employed by pathogens to enhance infectivity and evade host immunity. Here, lncRNAs in A. apis spores (AaCK group) and the guts of 4-, 5-, and 6-day-old Apis cerana cerana worker larvae inoculated with A. apis spores (AaT1, AaT2, and AaT3 groups) were identified, characterized, and validated. Additionally, the expression pattern of fungal lncRNAs during infection was analyzed, followed by an investigation of the regulatory manners and roles of differentially expressed lncRNAs (DElncRNAs). A total of 1379 lncRNAs were identified in AaCK, AaT1, AaT2, and AaT3 groups using bioinformatics, involving various types such as sense lncRNAs, antisense lncRNAs, bidirectional lncRNAs, intergenic lncRNAs, and intronic lncRNAs. Additionally, 4, 9, and 75 up-regulated lncRNAs as well as 2, 1, and 15 down-regulated ones were identified in the 4-, 5-, and 6-day-old larval guts following A. apis inoculation. Fifteen DElncRNAs as potential antisense lncRNAs may interact with 15 sense-strand mRNAs in the AaCK vs. AaT3 comparison group. Cis-acting analysis identified 10, 16, and 136 upstream and downstream genes of DElncRNAs in the aforementioned comparison groups, involving a series of GO terms and KEGG pathways like metabolic process and biosynthesis of secondary metabolites. Following the trans-acting investigation, 752, 821, and 1327 co-transcribed genes with DElncRNAs were discovered, spanning an array of functional terms and pathways such as biological processes and glycerophospholipid metabolism. Analysis of a competing endogenous RNA (ceRNA) network indicated that 1 and 5 DElncRNAs in the AaCK vs. AaT1 and AaCK vs. AaT3 comparison groups potentially targeted 1 and 2 miRNAs, further targeting 208 and 286 mRNAs, respectively. Further analysis identified one ceRNA axis relevant to the MAPK signaling pathway and several ceRNA networks associated with the biosynthesis of secondary metabolites. Finally, RT-qPCR results confirmed that the expression trends of six randomly selected DElncRNAs were consistent with those in the transcriptome data. These findings not only offer a foundation for elucidating the mechanisms underlying DElncRNA-mediated A. apis infection but also enrich our understanding of honeybee host-fungal pathogen interactions.

Chalkbrood disease caused by Ascosphaera apis threatens honey bee brood, yet the transcriptional mechanisms that coordinate antifungal immune responses in Apis cerana remain unclear. Here, RNA interference (RNAi) was used to examine two Dorsal homologs and selected dorsal1 transcript isoforms during larval infection. A. apis inoculation increased dorsal1, dorsal2, and five antimicrobial peptide (AMP) transcripts in larval midguts, consistent with an infection-associated humoral response. Gene-level RNAi produced selective transcript responses: dorsal1 knockdown was accompanied by reduced apidaecin and defensin1 expression, whereas dorsal2 knockdown was accompanied principally by reduced defensin1 expression. abaecin, defensin2, and hymenoptaecin transcripts were not significantly altered after dorsal knockdown under the tested conditions. Isoform-targeted RNAi of RNA9886, RNA9888, and RNA9890 was likewise associated with distinct AMP transcript responses, with RNA9888 and RNA9890 more closely associated with defensin1. These transcript-level data support selective and partially overlapping Dorsal-associated regulation during the A. cerana larval response to A. apis, while direct differences in Dorsal protein abundance or activity remain to be established.

Abstract Melittin, a potent amphipathic cationic peptide derived from bee venom, exhibits broad-spectrum antineoplastic efficacy, notably against cervical carcinoma. Despite its established therapeutic potential, the global transcriptional reprogramming orchestrating its acute multi-pathway cytotoxicity remains incompletely understood. To bridge this knowledge gap, we generated the first comprehensive, untargeted RNA-seq dataset profiling the acute phase of melittin-induced cell death in murine cervical carcinoma U14 cells (exposed to 4 μg/mL melittin for 20 minutes) alongside untreated controls. Utilizing deep sequencing and rigorous bioinformatics workflows, we quantified genome-wide mRNA abundances and mapped a distinct transcriptomic shift, identifying 254 significantly differentially expressed genes, comprising 158 up- and 96 down-regulated transcripts. Validated by stringent quality control metrics, exceptional genomic mapping rates, and comprehensive functional annotations via the GO and KEGG databases, this high-resolution transcriptomic resource provides a systems-level map of early molecular alterations. All raw and processed sequencing data are publicly available. This transcriptomic resource provides a valuable foundation for elucidating the acute regulatory networks underlying melittin-induced anti-cervical cancer effects. Dataset The dataset can be accessed through the NGDC website by searching with the BioProject accession number PRJCA068439. Reviewers may use this link for anonymous access during the review process. Direct URL to data: Genome Sequence Archive-CNCB-NGDC

Abstract Melittin exhibits antitumor activity in cervical cancer models, yet the long non-coding RNA (lncRNA) response and associated regulatory networks remain poorly understood. Here, strand-specific RNA-seq data from melittin-treated and untreated U14 murine cervical cancer cells were analyzed to characterize melittin-responsive lncRNAs and explore their potential functional associations. A total of 28,162 lncRNAs were identified, including 27,307 known and 855 novel transcripts. Differential expression analysis revealed 404 differentially expressed lncRNAs (DElncRNAs), comprising 191 upregulated and 213 downregulated lncRNAs, most of which were predicted to localize to the cytoplasm or nucleus. Cis -target analysis identified 52 neighboring mRNAs as putative targets of 46 DElncRNAs. Functional enrichment highlighted mitochondrial electron transfer and redox-related processes, including the mitochondrial electron transfer flavoprotein complex, electron-transferring-flavoprotein dehydrogenase activity, ubiquinone binding, and quinone binding. In parallel, melittin induced mitochondrial membrane depolarization and increased intracellular reactive oxygen species accumulation in U14 cells. Co-expression analysis further identified 138 lncRNAs coexpressed with 161 mRNAs, which were enriched in chromatin remodeling, DNA replication, and DNA repair. EdU incorporation decreased with increasing melittin concentrations, indicating suppression of DNA synthesis and proliferative activity. RT-qPCR analysis confirmed the expression trends of selected DElncRNAs. Collectively, these findings demonstrate extensive remodeling of the lncRNA landscape in melittin-treated U14 cells and suggest that melittin-responsive lncRNA-mRNA networks are associated with mitochondrial redox disruption and impaired DNA synthesis. This study provides a transcriptomic framework for identifying candidate lncRNA-mRNA regulatory axes underlying the antitumor response to melittin.

Vairimorpha ceranae, an obligate intracellular parasitic fungus, infects the midgut of worker Apis cerana cerana, disrupts nutritional metabolism and immune homeostasis of hosts, and poses severe threats to honeybee health and the sustainable development of apiculture. To date, the expression dynamics of circular RNAs (circRNAs) during pathogen infection and their regulatory functions as competing endogenous RNAs (ceRNAs) remain poorly characterized. In this study, circRNA sequencing was performed on purified V. ceranae spores (VcCK), as well as the midguts of A. cerana cerana worker bees at 7 days post-infection (VcT1) and 10 days post-infection (VcT2). A total of 8,986,470 and 315 circRNAs were identified from the three groups, with their lengths predominantly ranging from 200 to 600 nt. Differential expression analysis screened 575 and 594 differentially expressed circRNAs (DEcircRNAs) from the VcCK vs. VcT1 and VcCK vs. VcT2 comparison groups, respectively. The host genes generating these DEcircRNAs were annotated to 316 and 310 GO terms, alongside 167 KEGG pathways. CeRNA network analysis revealed that DEcircRNAs including novel_circ_006653 target multiple miRNAs, and their downstream target mRNAs are significantly enriched in energy metabolism pathways such as carbon metabolism and glycolysis/gluconeogenesis. RT-qPCR validation exhibited high consistency with transcriptome sequencing data. Further analyses demonstrated that V. ceranae infection drastically remodels the circRNA expression landscape: the total number of circRNAs was sharply reduced, and the dominant circRNA subtype shifted from antisense circRNAs to single-exon circRNAs. Several DEcircRNAs (novel_circ_006653, novel_circ_004839, novel_circ_005596, etc.) may participate in pathogen infection progression via the ceRNA regulatory axis by modulating host energy metabolism, immune responses and cellular biological processes. This study provides novel insights into the molecular interaction mechanisms between microsporidian parasites and their honeybee hosts, and identifies potential molecular targets for the prevention and control of microsporidiosis.

While high-throughput sequencing and bioinformatic predictions have identified numerous circRNAs in honeybees, research into their functional roles and molecular mechanisms remains limited. This study aims to characterize the regulatory functions of a previously identified circRNA (novel_circ_002651, ac2651) and its key target miRNA (ace-miR-6001-y), in eastern honeybee worker larvae responding to infection by A. apis, a causative fungal pathogen for chalkbrood disease. Here, RT-qPCR detection showed that the expression of ac2651 was significantly down-regulated in the 6-d-old larval gut, while the ace-miR-6001-y expression exhibited an opposite trend. RNAi-based interference of ac2651 significantly increased the expression of both host immune genes and fungal proliferation-associated genes. Additionally, silencing ac2651 resulted in a reduction of host survival rate and a significant elevation of chalkbrood incidence. Overexpression and knockdown experiments demonstrated that ace-miR-6001-y negatively regulated the expression of dorsal in the larval guts and affected the expression of mat1-2-1, Ste11-like, and Htf in A. apis. Dual-luciferase reporter assay confirmed direct interactions between ac2651 and ace-miR-6001-y as well as between ace-miR-6001-y and Ac14-3-3ζ. It was validated that simultaneous ac2651 silencing and ace-miR-6001-y knockdown reversed the upregulation of Ac14-3-3ζ induced by ace-miR-6001-y knockdown alone. These results delineate a novel regulatory axis wherein ac2651 sponges ace-miR-6001-y to alleviate its repression of Ac14-3-3ζ, thereby activating the host immune defense against the A. apis infection. Our findings not only offer novel insights into the interaction between honeybee larvae and fungal pathogen but also provide promising biomarkers and targets for the diagnosis and treatment of chalkbrood.

Melittin-treated murine cervical cancer U14 cells have been widely recognized as a classic cellular model for anti-tumor research in cervical cancer. This article contains metabolomic data of U14 cell lysates from both melittin-treated and control groups. Untargeted metabolomic profiling was carried out by liquid chromatography-mass spectrometry (LC-MS) to systematically elucidate the global metabolic disturbances in cervical cancer cells upon melittin intervention. LC-MS raw data were processed for peak extraction and alignment using XCMS software, followed by quality control normalization with metaX software. Metabolite annotation was performed against the HMDB and KEGG databases as well as an in-house MS/MS spectral library, yielding metabolite feature data including mass-to-charge ratio (m/z), retention time (RT), and MS/MS-identified metabolites (MS2). A total of 22,976 metabolic ions were detected in this study, among which 16,176 were assigned Level 1 annotations and 1114 were identified with high confidence at Level 2. All raw and processed data are publicly accessible at NGDC (accession number PRJCA065444). This untargeted LC-MS-based metabolomic dataset not only provides a comprehensive resource for elucidating metabolism-related anticancer mechanisms of melittin in murine U14 cervical cancer cells but also supports the development of targeted therapeutic strategies against cervical cancer.

The Ascosphaera apis infects the honeybee larval gut and causes chalkbrood disease, which impacts colony health and beekeeping production. Currently, the innate immune mechanisms by which honeybee larvae resist A. apis infection remain unclear. This study utilized nanopore sequencing technology to analyze differentially expressed transcripts (DETs) in A. apis-infected and uninfected Apis cerana cerana larval guts, and identified alternative splicing (AS) and alternative polyadenylation (APA) in honeybee genes. The results showed that 1,642, 1,281, and 1,377 DETs were detected on 1-3 days post-infection, respectively. Ten DETs were randomly selected from each group for RT-qPCR validation, and 26 DETs exhibited expression trends consistent with the nanopore sequencing results. A total of 5,476 AS events from 2,430 genes were identified in the larval guts during 1-3 days post-infection, with the number of AS events in the A. apis-infected group being higher than in the control group. Five randomly validated AS events matched the sequencing results. Additionally, 7,310 genes containing APA sites were identified, with the majority having more than five APA sites. Three genes were randomly selected, and their APA sites were validated. These findings provide preliminary insights into the roles of AS and APA at the transcript level in honeybee responses to A. apis infection.

Chalkbrood is a fungal disease caused by the infection of honeybee larvae by Ascosphaera apis. Currently, effective methods for controlling chalkbrood are lacking. Fungal non-coding RNAs play a critical role in enhancing infectivity and pathogenicity, making them potential high-efficacy targets for suppressing A. apis infection. Based on full-length transcriptome data comparing spores and mycelium of A. apis, and the midgut of honeybees (Apis mellifera) infected by A. apis, a specifically expressed long non-coding RNA 6140 (lnc6140) in A. apis was identified. The study demonstrated that lnc6140 in A. apis positively regulates ATPase expression through milR5658-x, thereby influencing pathogen infectivity and chalkbrood development. In this study, after A. apis infection, the expression levels of lnc6140, milR5658-x, and ATPase increased over the infection period. Interference with lnc6140 suppressed the expression of genes associated with A. apis infection and proliferation-Chi3, GriF, AdmB, Pkia, Ste11, and Dmap1-and significantly inhibited chalkbrood incidence. Dual-luciferase assays demonstrated that lnc6140 positively regulates the expression of milR5658-x, and milR5658-x positively regulates the expression of ATPase in A. apis. Similarly, reducing the RNA level of milR5658-x also significantly inhibited chalkbrood incidence. Concurrent reduction of lnc6140 and milR5658-x levels suppressed ATPase expression and markedly decreased chalkbrood occurrence. The result indicated that lnc6140 of A. apis plays a key role in pathogen infection by regulating the milR5658-x-ATPase axis. Suppressing lnc6140 and milR5658-x of A. apis may serve as a crucial strategy for reducing chalkbrood incidence. These findings provide new candidate targets for understanding the pathogenic mechanisms of A. apis and for the prevention and control of chalkbrood.

Long non-coding RNAs (lncRNAs) participate in insect immune regulation, but their relationship with antioxidant responses during fungal infection remains unclear. Here, we examined a candidate regulatory relationship among lncRNA1386.1, novel-miR0032-5p, and the catalase gene (CAT) in Apis cerana worker larvae infected with Ascosphaera apis. Dual-luciferase reporter assays showed that M-miR0032-5p reduced the activity of reporters containing the predicted miRNA response element within lncRNA1386.1 or CAT, whereas mutation of these sequences weakened or abolished the response. In infected larvae, lncRNA1386.1 silencing and novel-miR0032-5p overexpression reduced CAT transcript abundance and CAT protein concentration, and increased dihydroethidium (DHE) fluorescence intensity. These treatments were also accompanied by alterations in the transcript abundance of the host genes Dorsal1 and Relish and the fungal genes Chit3 and STE11-like. Novel-miR0032-5p inhibition generally produced opposite changes in CAT-related measurements, DHE fluorescence intensity, and the selected host and fungal transcripts. lncRNA1386.1 silencing was associated with increased larval survival, whereas novel-miR0032-5p inhibition was associated with a higher hazard of first visible external mycelial growth without significantly affecting survival. These findings support a negative regulatory role of novel-miR0032-5p in CAT expression and indicate that lncRNA1386.1 participates in a candidate shared miRNA-responsive regulatory relationship. This candidate regulatory relationship was associated with CAT-related antioxidant regulation, superoxide-associated DHE fluorescence, immune-related transcription, selected fungal transcriptional responses, and visible external mycelial growth during A. apis infection.

Melittin, the principal bioactive peptide of bee venom, exhibits promising antitumor activity, whereas its molecular mechanisms in cervical cancer remain incompletely un derstood. In this study, the biological effects and molecular responses of melittin in U14 cervical cancer cells were investigated using Astral data-independent acquisition (As tral DIA) based quantitative proteomics combined with molecular validation. The effects of melittin on cell migration, invasion, and cell death were evaluated by Transwell assays and PI/Hoechst staining. Differentially expressed proteins (DEPs) were screened and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein protein interaction (PPI) analyses. Representative oxidative stress-related genes and proteins were further validated by RT-qPCR and Western blotting. Melittin significantly inhibited the migration and invasion of U14 cervical cancer cells and in-creased cell death. Quantitative proteomics identified 9,782 protein groups and 187 DEPs, including 71 up and 116 down-regulated proteins. KEGG pathway enrichment analysis revealed oxidative phosphorylation (OXPHOS) as the most significantly enriched pathway, together with glutathione metabolism, ferroptosis-related pathways, reactive oxygen species signaling, and mitophagy. GO term enrichment analysis indicated that DEGs were mainly engaged in mitochondrial function, electron transport, oxidoreductase activity, and energy metabolism. RT qPCR assay demonstrated altered expression of Duox1, Gpx4, Gsx2, Nfe2l2, and Gstp2. Additionally, PPI analysis identified Gstp2 and ODC1 as representative hub proteins involved in redox regulation and metabolic ad-aptation. Furthermore, Western blotting confirmed increased GSTP2 expression following melittin treatment. Overall, these findings provide a comprehensive proteomic landscape of melittin-treated U14 cervical cancer cells and suggest that mitochondrial OXPHOS remodeling and redox associated pathways may contribute to the antitumor activity of melittin.

Beyond their classical role in preserving germline genome integrity via transposon silencing, piRNAs are increasingly recognized as pivotal regulators in somatic tissues. In these contexts, piRNAs orchestrate essential processes such as gut growth, gene expression regulation, metabolism, and the maintenance of intestinal homeostasis. Based on deep sequencing and bioinformatics, piR-ame-1128833 was previously detected to be significantly up-regulated. Here, we characterized the regulatory function of piR-ame-1128833 during the larval development of the western honeybee (Apis mellifera) worker. Integrative analysis of expression profiles and target predictions identified a significant negative correlation between piR-ame-1128833 and its predicted key target gene, AmYAP1. Dual-luciferase reporter assays confirmed their direct interaction. Additionally, overexpression (knockdown) of piR-ame-1128833 resulted in marked down-regulation (up-regulation) AmYAP1 in larval guts. Functional experiments demonstrated that piR-ame-1128833 significantly impacted the expression of key developmental genes ultraspiracle protein (USP), Ecdysone receptor (EcR), and Wnt-1 as well as metabolic markers alpha glucosidase 2 (AGLU2), ADP/ATP translocase (Ant), and vitellogenin (Vg), consequently impacting the larval viserosomatic index. Moreover, RNAi-mediated knockdown of AmYAP1 similarly led to not only dynamic change of the expression of aforementioned genes associated with gut development and metabolism but also a substantial reduction in the viserosomatic index. Collectively, these data suggest that piR-ame-1128833/AmYAP1 axis acts as a molecular switch that orchestrates the expression of downstream developmental and metabolic genes, ultimately influencing physiological parameters such as the viserosomatic index. Findings from this current work illustrate the molecular mechanism underlying piR-ame-1128833-modulated development of bee gut and deepen our understanding of piRNA as a novel multifunctional regulator in insects.

Vairimorpha ceranae (formerly Nosema ceranae) is an obligate intracellular parasite that poses a major threat to the health of the honey bee. Circular RNAs (circRNAs) have been recognized as key regulators in gene expression and pathogen-host interactions. However, their expression patterns and regulatory roles in V. ceranae infection remain largely unexplored. In this study, we performed circRNA profiling in V. ceranae spores (NcCK) and the midguts of Apis mellifera ligustica workers at 7 d post inoculation (dpi) and 10 dpi (Nc7T and Nc10T) based on transcriptome sequencing, followed by in-depth investigation of the regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 243 circRNAs were identified in V. ceranae, with lengths predominantly ranging from 201 to 400 nucleotides. Comparative analysis screened 70 and 192 DEcircRNAs in the NcCK vs. Nc7T and NcCK vs. Nc10T comparison groups, respectively, with a significant majority being downregulated. The parental genes of these DEcircRNAs were significantly enriched in fundamental cellular processes and critical pathways such as protein processing in the endoplasmic reticulum and ribosome biogenesis. Additionally, we constructed a competing endogenous RNA network, suggesting that DEcircRNAs could potentially interact with DEmiRNAs to modulate mRNAs associated with fungal proliferation-relevant signaling pathways like MAPK, PI3K-Akt, and cAMP. Moreover, numerous DEcircRNAs were predicted to contain internal ribosome entry site elements, indicative of their potential for protein coding. The back-splicing junctions and expression trends of selected DEcircRNAs were successfully validated by RT-PCR and qRT-PCR. Our data not only offer a valuable resource for future functional studies but also provide a basis for elucidating the circRNA-mediated mechanisms underlying microsporidian pathogenesis and host-pathogen interactions.

Vairimorpha ceranae is a fungal pathogen that infects the honeybee midgut and poses a serious threat to colony health. However, the role of long noncoding RNAs (lncRNAs) of V. ceranae in its infection of the host remains poorly understood. Using lncRNA-seq data from the midguts of Apis mellifera workers at 7 and 10 days post-inoculation with V. ceranae (NcT1L and NcT2L groups), along with controls inoculated with spores (NcCKL group), we performed transcriptome-wide identification and structural characterization of lncRNAs. We identified lncRNAs in V. ceranae and analyzed the regulatory network of the differentially expressed lncRNAs (DElncRNAs). A total of 27 V. ceranae lncRNAs were identified in the midguts. The 19, 21, and 4 DElncRNAs were identified in the NcCKL vs. NcT1L, NcCKL vs. NcT2L, and NcT1L vs. NcT2L comparison groups. These DElncRNAs were predicted to regulate 26, 27, and 2 upstream/downstream genes. Furthermore, 15, 23, and 4 DElncRNAs were found to target 195, 211, and 94 miRNAs, which in turn targeted 204, 216, and 73 mRNAs into the respective comparisons. The ceRNA network prediction revealed that DElncRNAs, miRNAs and mRNAs form a complex regulatory network. This study presents the expression profile of lncRNAs during V. ceranae infection and highlights their potential regulatory functions in pathogenesis. Our findings provide new molecular insights into host-pathogen interactions at the RNA level and establish a foundation for developing targeted strategies to control nosemosis.

Microsporidia rely extensively on host resources, yet how parasite-derived microRNAs coordinate infection remains poorly understood. Here, we investigated the function of nce-miR-12220, a miRNA identified in Nosema ceranae spores, during infection of Apis mellifera workers. Target prediction, dual-luciferase assays, and fluorescence in situ hybridization were combined with RNA interference and miRNA gain- and loss-of-function experiments. nce-miR-12220 interacted sequence-specifically with binding regions in ATP-A and γ-tubulin and was detected in infected honeybee midgut epithelial cells. Silencing either target gene reduced expression of the N. ceranae virulence-associated gene NcRBL and improved worker survival relative to the scramble control. In infected workers, nce-miR-12220 overexpression increased endogenous ATP-A and γ-tubulin transcript abundance, whereas inhibition produced the opposite effect. Overexpression also reduced expression of the Toll pathway-associated genes Cactus and dorsal and the antimicrobial peptide genes Defensin and Hymenoptaecin , increased N. ceranae spore load and sucrose consumption, and decreased midgut ATP content. Inhibition of nce-miR-12220 reversed these responses and reduced parasite burden. Survival after nce-miR-12220 manipulation changed in the predicted direction but did not reach statistical significance. Together, these findings identify nce-miR-12220 as a parasite-derived regulator that promotes N. ceranae proliferation while reshaping host immune and energetic responses, and suggest that this microRNA and its target network may provide candidates for controlling bee nosemosis.

Chalkbrood fungus Ascosphaera apis (A. apis) produces miRNA-like small RNAs (milRNAs) that may modulate honey bee (Apis mellifera) larval biology through cross-kingdom RNA interference (ckRNAi). We identified 380, 106, 107, and 110 highly expressed milRNAs (HmilRNAs) in pure spores and infected larval guts at days 4, 5, and 6, respectively. Eighteen HmilRNAs were conserved across all groups, forming a complex regulatory network predicted to target 188 host genes (6,057 mRNAs) associated with critical signaling pathways, including Wnt, Hippo, mTOR, MAPK, and Toll/Imd. Among these, aap-milR-11980-x was validated to directly target the ecdysone-inducible gene E75 and the transcriptional repressor tramtrack (TTK). Dual-luciferase assays confirmed that aap-milR-11980-x represses these targets via specific binding to their 3'-UTR. In vivo modulation of aap-milR-11980-x in 6-day-old larval guts using agomirs and antagomirs successfully altered E75, TTK, and antimicrobial peptide (AMP) transcript levels. However, despite significant molecular regulation, no statistically significant difference in larval survival was observed between treatment groups (P > 0.05). This discrepancy suggests that while aap-milR-11980-x effectively rewires host gene expression, its primary function may be to induce sub-lethal physiological changes, such as developmental delay or immune modulation, rather than acute lethality. These findings indicate that A. apis HmilRNAs fine-tune host cellular responses, potentially to extend the temporal window for fungal development or resource acquisition, highlighting a sophisticated layer of molecular manipulation in the A. apis-honey bee interaction.

DNA methylation is a key epigenetic modification involved in various biological processes. However, its role in Nosema ceranae remains poorly understood. In this study, we employed Oxford Nanopore Technology sequencing to identify whole-genome DNA methylation patterns in N. ceranae. A total of 140,711 CpG sites, 170,035 CHG sites, and 1,053,635 CHH sites were detected. Methylation was also observed at varying levels in repetitive genomic regions and different gene regions. Furthermore, three 5mC motifs were identified. These findings provide a foundation for investigation the epigenetic regulatory roles and mechanisms of DNA methylation in N. ceranae.

The western honey bee is a typical social insect and a vital pollinator for numerous wild plants and crops. Chalkbrood is a fungal disease in honey bees caused by Ascosphaera apis. We recently investigated the role of piR-ame-1128833 in regulating the response of A. mellifera worker larvae to A. apis infection. Herein, we assessed the interaction between piR-ame-1128833 and its potential target gene Apis mellifera lachesin (Amlachesin) and investigated their regulatory relationship by inducing overexpression and knockdown of piR-ame-1128833. Furthermore, RNA interference was used to analyze the regulatory function of Amlachesin in the gut tissue of larvae infected with A. apis. Results revealed that the piR-ame-1128833/Amlachesin axis served as a core regulatory pathway in the immune defense of A. mellifera worker larval guts against A. apis infection. These findings not only provide novel insights into piRNA-mediated epigenetic regulation in honey bees but also improve the understanding of bee-pathogen interactions.