The monofloral honey derived from Bauhinia championii (Benth.) Benth. (MH-Bc) possesses significant nutritional and bioactive value, making it highly suitable for commercial exploitation. However, the poorly defined characteristics and unknown composition have hindered MH-Bc product development. In this study, we employed a combination of untargeted and targeted mass spectrometry analyses to characterize MH-Bc honey. As a result, 4,7,8-trimethoxydibenzo[b,d]furan-3-ol (TDBF) was identified as a robust chemical marker for distinguishing MH-Bc from other types of honey. This specific marker was detected in both MH-Bc and the Bc plant but was absent in other honey varieties. Furthermore, a targeted mass spectrometry quantitative method was developed and validated to accurately determine the content of TDBF in honey samples. Overall, the presence of TDBF serves as a discerning indicator for future commercial MH-Bc products.

Epigenetic modifications facilitate organismal adaptation to environmental toxins. While the toxicity of the acaricide flumethrin toward honeybees has been documented, the mechanisms underlying its effects on epigenetic modifications remain unclear. Risk screening revealed a high prevalence of flumethrin residues in honeybee colonies and notable bioaccumulation in honeybee brains following oral exposure. Flumethrin exposure at 10 μg/L (the minimum detected residue level) significantly altered the global levels of multiple epigenetic modifications-including RNA m6A, Am, m5C, m7G, and DNA 5 mC and 6 mA-along with the expression of related genes. m6A-seq demonstrated that flumethrin significantly altered m6A methylation on transcripts crucial for neuronal activity and memory within the Hedgehog and Hippo signaling pathways. Critically, m6A methylation modulated the transcripts of 12 odorant receptors, correlating with flumethrin-induced olfactory sensitivity deficits confirmed by electroantennography. Additionally, flumethrin altered methylation on transcripts encoding stress sensors and immune modulators in the FoxO and MAPK signaling pathways, triggering defense responses. The induced epigenetic marks and m6A-mediated transcripts, including EDD1, PI3K, Ptc, FoxO, Jra, and Argk, can be regarded as potential biomarkers for neurotoxicity assessment in honeybees. Our findings provide novel mechanistic insights into flumethrin toxicity and support a framework for epigenetic biomarkers in assessing environmental risks to honeybees.

Lespedeza bicolor Turcz. (L. bicolor) honey, a monofloral honey, has garnered increased attention due to its origin in the L. bicolor plant. A previous study has shown that L. bicolor honey can ameliorate inflammation. In this study, we aimed to investigate the effects of L. bicolor honey extract and its biomarker (Trifolin) on DSS-induced ulcerative colitis (UC). Our results demonstrated that L. bicolor honey extract and Trifolin significantly increased the expression levels of the tight junction cytokines Claudin-1 and ZO-1. Additionally, they decreased the pro-inflammatory factors TNF-α and IL-6 and enhanced the antioxidant factors NQO1 and GSTA1. Based on metabolomic analyses, L. bicolor honey extract and Trifolin regulated the progression of UC by inhibiting ferroptosis. Mechanistically, they improved the levels of SOD and iron load, increased the GSH/GSSG ratio, reduced MDA content and ROS release, and upregulated the Nrf2/HO-1 pathway, thereby inhibiting DSS-induced UC. Moreover, the expression levels of ferroptosis-related genes indicated that they decreased FTL, ACSL4, and PTGS2 while increasing SLC7A11 expression to resist ferroptosis. In conclusion, our study found that L. bicolor honey improves DSS-induced UC by inhibiting ferroptosis by activating the Nrf2/HO-1 pathway. These findings further elucidate the understanding of anti-inflammatory and antioxidant activities of L. bicolor honey.

Helvella bachu, an ectomycorrhizal fungus, forms a symbiotic relationship with Populus euphratica, a rare and endangered species crucial to desert riparian ecosystems. In this study, endofungal bacteria (EFBs) within the fruiting bodies of H. bachu were confirmed by a polyphasic approach, including genomic sequencing, real-time quantitative PCR targeting the 16S rRNA gene, full-length and next-generation sequencing (NGS) of the 16S rRNA gene, and culture methods. The genera Stenotrophomonas, Variovorax, Acidovorax, and Pedobacter were abundant in the EFBs of fruiting bodies associated with three Populus hosts and were consistently present across different developmental stages. Notably, S. maltophilia and V. paradoxus were detected in high abundance, as revealed by full-length 16S rRNA sequencing, with S. maltophilia also isolated by culture methods. KO-pathway analysis indicated that pathways related to primary, secondary, and energy metabolism were predominantly enriched, suggesting these bacteria may promote H. bachu growth by producing essential compounds, including sugars, proteins, and vitamins, and secondary metabolites. This study confirmed the presence of EFBs in H. bachu and provided the first comprehensive overview of their structure, functional potential, and dynamic changes throughout fruiting body maturation, offering valuable insights for advancing the artificial domestication of this species.

Cationic surface-active agents (CSAAs) can persist in ambient water, be ingested by bees, and contaminate honey. Residues of CSAAs in honey remains unknown. This study measured the residual levels of five CSAAs in 271 honey samples from China using ultrahigh-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry. Residual benzalkonium chloride-C12 (BAC-C12), BAC-C14, BAC-C16, chlorhexidine (CHG), and 4-chloraniline levels were 0.0098-2.1468, 0.0061-1.7492, 0.0012-1.6305, 0.1576-0.8401, and 0.0019-0.0234 μg kg-1, respectively. CHG and all BAC were detected in 100 % of Z. jujuba, V. negundo var. heterophylla, wildflower, L. chinensis, and D. longan Lour honey; T. tuan honey had the lowest detection rate of any CSAAs. BAC-C16 had the highest residual level among all BAC tested in Central, North China. CHG levels were detected in 91.38 % of samples in North China and 100 % in East China. BAC-C12 was significantly higher in A. cerana versus A. mellifera honey (P < 0.001). Hazard quotient and Hazard index values indicate that CSAAs residuals in honey do not pose a health risk. Correlation analysis revealed a positive correlation between BACs resides in honey and surrounding environment. The findings suggest that continuous monitoring of CSAAs in honey is imperative to ensure its safety for human consumption, while also serving as an effective matrix to assess the environmental pollution of a given region.

A complete telomere-to-telomere (T2T) genome has been a longstanding goal in the field of genomic research. By integrating high-coverage and precise long-read sequencing data using multiple assembly strategies, we present here the first T2T gap-free genome assembly of Ganoderma leucocontextum strain GL72, a Tibetan medicinal mushroom. The T2T genome, with a size of 46.69 Mb, consists 13 complete nuclear chromosomes and typical telomeric repeats (CCCTAA)n were detected at both ends of 13 chromosomes. The high mapping rate, uniform genome coverage, a complete BUSCOs of 99.7%, and base accuracy exceeding 99.999% indicate that this assembly represents the highest level of completeness and quality. Regions characterized by distinct structural attributes, including highest Hi-C interaction intensity, high repeat content, decreased gene density, low GC content, and minimal or no transcription levels across all chromosomes may represent potential centromeres. Sequence analysis revealed the first Copia centromeric retrotransposon in macro-fungi genome. Phylogenomic analysis identified that G. leucocontextum and G. tsugae diverged from the other Ganoderma species approximately 9.8–17.9 MYA. The prediction of secondary metabolic clusters confirmed the capability of this fungus to produce a substantial quantity of metabolites. This T2T gap-free genome will contribute to the genomic ‘dark matter’ elucidation and server as a great reference for genetics, genomics, and evolutionary studies of G. leucocontextum.

Agrocybe chaxingu is a widely cultivated edible fungus in China, which is rich in nutrients and medicinal compounds. However, the lack of a high-quality genome hinders further research. In this study, we assembled the telomere-to-telomere genomes of two sexually compatible monokaryons (CchA and CchB) derived from a primarily cultivated strain AS-5. The genomes of CchA and CchB were 50.60 Mb and 51.66 Mb with contig N50 values of 3.95 Mb and 3.97 Mb, respectively. Each contained 13 complete chromosomes with telomeres at both ends. The high mapping rate, uniform genome coverage, high LAI score, all BUSCOs with 98.5%, and all base accuracy exceeding 99.999% indicated the high level of integrity and quality of these two assembled genomes. Comparison of the two genomes revealed that approximately 30% of the nucleotide sequences between homologous chromosomes were non-syntenic, including 19 translocations, 36 inversions, and 15 duplications. An additional gene CchA_000467 was identified at the Mat A locus of CchA, which was observed exclusively in the Cyclocybe cylindracea species complex. A total of 613 (4.26%) and 483 (3.4%) unique genes were identified in CchA and CchB, respectively, with over 80% of these being hypothetical proteins. Transcriptomic analysis revealed that the expression levels of unique genes in CchB were significantly higher than those in CchA, and both CchA and CchB had unique genes specifically expressed at stages of mycelium and fruiting body. It was indicated that the growth and development of the A. chaxingu strain AS-5 required the coordinated action of two different nuclei, with CchB potentially playing a more significant role. These findings contributed to a more profound comprehension of the growth and developmental processes of basidiomycetes.

Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

Honeybees play a crucial role as pollinators for crops and are regarded as sensitive bioindicators of environmental health. The widespread use of pesticides poses a severe threat to honeybee survival. However, there is limited information available on the specific risks associated with fipronil exposure in honeybees, particularly concerning the impact on RNA methylation throughout their lifespan. This study aimed to evaluate the effects of sublethal concentrations of fipronil on RNA m6A and m5C methylations, along with the associated genes in honeybee larvae and newly emerged adults. LC-MS/MS analysis revealed a notable hypomethylation of m5C in larvae, while hypermethylation of m6A was observed in the adult brain. Significant changes in the expression of genes such as AmWTAP, AmYTHDF, AmALKBH4, AmALKBH6, AmALKBH8, AmNSUN5, AmNOP2, AmTET1, and AmYBX1 were observed in the adult brain, whereas alterations in the expression of AmNSUN2, AmMETTL14, AmALKBH1, AmALKBH4, AmALKBH6 AmALYREF, AmTET1, and AmYBX1 were observed in the larvae. Notably, the expression of AmALKBH1 was not detected in any fipronil-treated larvae, suggesting its potential as an early risk indicator for honeybee larvae in future assessments. This pioneering study provides insights into the effects of fipronil on RNA methylations in honeybees and explores the possibility of employing RNA methylation as a tool for assessing pesticide risks in this important pollinator species. These findings offer new perspectives on honeybee protection and the development of toxicity evaluation systems for pesticides.

蜜蜂是重要的环境污染指示生物.作为氟虫腈的替代药剂,乙虫腈对蜜蜂仍有高风险.本文首次基于DNA和RNA甲基化角度评估了亚致死剂量(10-5、10-4、10-3和10-2 mg·L-1)乙虫腈重复暴露对意大利蜜蜂工蜂幼虫生长发育的影响.结果显示,乙虫腈持续暴露引起了 DNA 5mC甲基化水平显著下降和DNA甲基化酶基因Dnmt3显著下调;与DNA相比,RNA(总RNA和mRNA)甲基化所受影响更为显著,各暴露浓度下,总RNA m5C、m3C甲基化和mRNA m6A、m3C甲基化水平均显著提高,RNA m6A去甲基化酶基因ALKBH1均显著下调(P＜0.01),m5C甲基化酶基因NSUN4的表达均显著上调(P＜0.01).综上,甲基化酶基因Dnmt3、ALKBH1和NSUN4可作为乙虫腈暴露风险评估的潜在标志物.本研究为杀虫剂的风险预警及评估提供了新的科学视角和技术手段.

Although the honey produced by Lespedeza bicolor Turcz. is precious because of its medicinal value, its pharmacological mechanism is still unclear. Here, its anti-inflammatory and antioxidant functions on lipopolysaccharide (LPS)-treated murine RAW 264.7 macrophages were analyzed using targeted and non-targeted metabolomics. Results showed that twelve polyphenols were identified in L. bicolor honey using UHPLC-QQQ-MS/MS. L. bicolor honey extract could scavenge the free radicals DPPH• and ABTS+ and reduce Fe3+. Furthermore, pretreatment with L. bicolor honey extract significantly decreased NO production; suppressed the expression of COX-2, IL-10, TNF-α, and iNOS; and upregulated HO-1′s expression in the cells with LPS application. UHPLC-Q-TOF-MS/MS-based metabolomics results revealed that L. bicolor honey extract could protect against inflammatory damage caused by LPS through the reduced activation of sphingolipid metabolism and necroptosis pathways. These findings demonstrate that L. bicolor honey possesses excellent antioxidant and anti-inflammatory activities.

Astragalus membranaceus var. mongholicus Hsiao (Am) is a widely used traditional Chinese herbal medicine. The monofloral honey from Am plant nectar collected by honeybees (MH-Am) has potential medicinal activities. Quality control of MH-Am requires discovery of characteristic markers. In this study, calycosin and formononetin were identified as reliable chemical markers for MH-Am authentication, which were shared with its plant (P-Am), but absent in other honeys based on untargeted mass spectrometry (MS) analysis. The contents of calycosin and formononetin in MH-Am, other honeys and P-Am were determined through a targeted MS-based quantitative approach. Furthermore, free radical scavenging assays showed that calycosin functioned directly in the antioxidative activity of MH-Am. Thus, calycosin has great potential to be certified as a bioactive marker contributing to future quality control of commercial MH-Am products.

Bee pollen (BP) and royal jelly (RJ) have shown therapeutic effects against colitis, but the functional components contained therein remain elusive. Here, we used an integrated microbiomic-metabolomic strategy to clarify the mechanism by which bee pollen lipid extracts (BPL) and royal jelly lipid extracts (RJL) ameliorated dextran sulfate sodium (DSS)-induced colitis in mice. Lipidomic results showed that levels of ceramide (Cer), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were significantly higher in BPL than in RJL. The anti-inflammatory efficacy of BPL surpassed that of RJL, although both BPL and RJL could attenuate DSS-induced colitis through several mechanisms: reducing the disease activity index (DAI); decreasing histopathological damage; inhibiting the expression of genes encoding proinflammatory cytokines; improving intestinal microbial community structure, and modulating host metabolism. These findings demonstrated that BPL and RJL have great potential as functional ingredients for the production of dietary supplements to prevent early colitis.

One of the impacts of the Coronavirus disease 2019 (COVID-19) pandemic has been a profound increase in the application amounts of disinfectants. Dodecyl dimethyl benzyl ammonium chloride (DDBAC) is a widely used disinfectant, yet its hazards to non-target species remain largely unknown. We are unaware of any studies assessing DDBAC's impacts on honeybee, a pollinator species that is a useful indicator of environmental pollution essential for many forms of agricultural production. Here, we assessed the potentially negative effects of DDBAC on honeybees. After conducting a formal toxicity evaluation of DDBAC on honeybee mortality, we detected an accumulation of DDBAC in the honeybee midgut. We subsequently studied the midgut tissues of honeybees exposed to sub-lethal concentrations of DDBAC: histopathological examination revealed damage to midgut tissue upon DDBAC exposure, microbiome analysis showed a decreased abundance of beneficial midgut microbiota, lipidomics analysis revealed a significant reduction in cell membrane phospholipids with known functions in signal transduction, and a transcriptome analysis detected altered expression of genes involved in calcium signaling pathways (that variously function in calcium absorption, muscle contraction, and neurotransmission). Thus, our study establishes that DDBAC impacts honeybee midgut functions at multiple levels. Our study represents an early warning about the hazards of DDBAC and appeals for the proper stewardship of DDBAC to ensure the protection of our ecological environment.

Bee pollen as a plant-derived food is consumed as nutritional/functional supplements by humans. But it might confer foodborne allergenicity in susceptible populations, limiting its extensive application. In this study, five potential allergens including profilin, cystatin, prolamin, expansin, and alcohol dehydrogenase in bee pollen derived from Brassica campestris (BP-Bc), were identified through mass spectrometry-based proteomic analysis. Moreover, different types of enzymes (cellulases, pectases, and papains) serve biological roles in pollen wall breaking and expansion, but also promote allergen release and degradation. Proteomic analysis showed that profilin, cystatin, and alcohol dehydrogenase were significantly reduced in BP-Bc following joint treatment with three enzymes. Metabolomic characterization of potential enzymatic hydrolysates of these significantly-decreased allergens was performed, which showed nine major oligopeptides and six amino acids at significantly higher levels in the enzyme-treated BP-Bc. These findings clarified the culprit responsible for bee pollen allergy and the mechanism of enzymatic desensitization for its further development.

Honey produced from Lespedeza bicolor Turcz. (L. bicolor) is highly valued and relatively rare, leading to adulterated or ersatz substitutes in the marketplace, with no reliable authentication methods available for enforcement of regulations. Here, we characterize the physicochemical parameters (water content, pH, sugar content, amylase activity, and 5-hydroxymethylfurfural content) in L. bicolor honey and palynological characteristics of L. bicolor pollen as reference for assessment of quality and monoflorality. Mass spectrometry with Orthogonal Partial Least Square Discriminant Analysis of chemical constituent data from L. bicolor, chaste, acacia, jujube, and linden honeys, all commonly sold in China, identified kaempferol-3-O-galactoside as a candidate chemical marker of L. bicolor honey. We validated this screening method and quantified kaempferol-3-O-galactoside in L. bicolor, but not other honeys, at concentrations between 90.2 and 430.1 µg/kg, with high sensitivity (LOD = 0.002 mg/kg), linearity (R2 ≥ 0.99), and recovery (90.2%-110.6%), supporting its use in authenticating L. bicolor honey.

Genetically identical female honeybee larvae with different diets develop into sterile workers or fertile queens. It remains unknown whether the reversible RNA N6-methyladenosine (m6A) mark functionally impact this "caste differentiation." Here, we profile the transcriptome-wide m6A methylome of honeybee queen and worker larvae at three instar stages and discover that m6A methylation dynamics are altered by differential feeding. Multiple methylome comparisons show an obvious increase in m6A marks during larval development and reveal a negative correlation between gene expression and m6A methylation. Notably, we find that worker larvae contain more hypermethylated m6A peaks than do queen larvae, and many caste-differentiation-related transcripts are differentially methylated. Chemical suppression of m6A methylation in worker larvae by 3-deazaadenosine (DAA) reduces overall m6A methylation levels and triggers worker larvae to develop queen caste features. Thus, our study demonstrates that m6A functionally impacts caste differentiation and larval development, yet it does not exclude potential contributions from other factors.

Microplastic contamination is not only a pressing environmental concern in oceans, but also terrestrial ecosystems. However, little is known about its potential impacts on pollinators. Here, we reported the effects of 25 μm-diameter spherical polystyrene microplastics (PS-MPs) alone or in combination with the antibiotic tetracycline on honey bees (Apis mellifera L.) in the laboratory. We noticed that PS-MPs exposure for 14 d had sublethal effects, with low mortalities (up to 1.6 %) across three different treatments (0.5, 5, and 50 mg/L) and no changes to the body weight gains compared to the control bees. Nevertheless, PS-MPs exposure led to significant decreases in the α-diversity of bees’ gut microbiota accompanied by changes to the core microbial population structure. Additionally, PS-MPs lead to alterations in the expression of antioxidative (Cat), detoxification (CypQ1 and GstS3), and immune system-related genes (Domeless, Hopscotch, and Symplekin) in guts. More interestingly, we observed that PS-MPs accumulated and degraded inside of the hindgut and interacted with gut bacteria. The depletion of the normal gut microbiota using tetracycline dramatically increased the lethality of microplastics. These results provide a resource for future research on microplastic-microbiome interactions in other insects and also shed light on understanding the potential effects of microplastics in terrestrial ecosystems.

The invasion of Vespa velutina presents a great threat to the agriculture economy, the ecological environment, and human health. An effective strategy for this hornet control is urgently required, but the limited genome information of Vespa velutina restricts the application of molecular-genomic tools for targeted hornet management. Therefore, we conducted large-scale transcriptome profiling of the hornet brain to obtain functional target genes and molecular markers. Using an Illumina HiSeq platform, more than 41 million clean reads were obtained and de novo assembled into 182,087 meaningful unigenes. A total of 56,400 unigenes were annotated against publicly available protein sequence databases and a set of reliable Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNP) markers were developed. The homologous genes encoding crucial behavior regulation factors, odorant binding proteins (OBPs), and vitellogenin, were also identified from highly expressed transcripts. This study provides abundant molecular targets and markers for invasive hornet control and further promotes the genetic and molecular study of Vespa velutina.

Embryonic development depends on a highly coordinated shift in transcription programs known as the maternal-to-zygotic transition (MZT). It remains unclear how haploid and diploid embryo coordinate their genomic activation and embryonic development during MZT in haplodiploid animals. Here, we applied a single-embryo RNA-seq approach to characterize the embryonic transcriptome dynamics in haploid males vs. diploid females of the haplodiploid insect honeybee (Apis mellifera). We observed typical zygotic genome activation (ZGA) occurred in three major waves specifically in female honeybee embryos; haploid genome activation was much weaker and occurred later. Strikingly, we also observed three waves of transcriptional activation for thousands of long non-coding transcripts (lncRNA), 73% of which are transcribed from intronic regions and 65% were specific to female honeybee embryos. These findings support a model in which introns encode thousands of lncRNAs that are expressed in a diploid-embryo-specific and ZGA-triggered manner that may have potential functions to regulate gene expression during early embryonic development in the haplodiploid insect honeybee.