Large cells suppress the reproduction of Varroa destructor.

BACKGROUND: The parasitic mite, Varroa destructor has posed a threat to the health and survival of European honey bees, Apis mellifera worldwide. There is a prevailing belief that small comb cells could provide a management tool against Varroa mites. However, the hypothesis that smaller cells can impede Varroa reproduction has not been fully tested. Here, we tested this hypothesis under laboratory conditions by using two distinct Varroa in vitro rearing systems: one involved gelatin capsules of different sizes, specifically size 00 (0.95 mL) versus size 1 (0.48 mL), and the second consisted of brood comb cells drawn on 3D printed foundations with varying cell sizes, ranging from 5.0 mm to 7.0 mm at 0.5 mm intervals. RESULTS: The results showed that mother mites in size 00 cells had significantly lower fecundity and fertility compared to those in size 1 cells. Interestingly, the reproductive suppression in larger cells could be reversed by adding an extra worker larva. Similarly, gonopore size of mother mites was smaller in size 00 cells, but restored with another host larva. Furthermore, both the fecundity and fertility of mother mites decreased linearly with the size of brood comb cells. CONCLUSIONS: Our results suggest that the reproduction of V. destructor is hindered by larger cells, possibly because larger brood cells disperse or weaken host volatile chemical cues that are crucial for Varroa reproduction. The insights derived from this study are expected to hold significant implications for the implementation of Varroa management programs. © 2024 Society of Chemical Industry.

Response to comment on "Food Wanting is Mediated by Transient Activation of Dopaminergic Signaling in the Honeybee Brain".

In a technical comment, Barron et al. (1) criticized the work of Huang et al. (2) putting the accent on the quantification of dopamine levels via high-performance liquid chromatography (HPLC), yet also including data interpretation through alternative hypotheses aimed at invalidating the original ones proposed by Huang et al. We thank the authors of this technical comment, which allows us to clarify technical aspects of our work that may have been unclear, and for promoting discussion around the conclusions of our work. Below we provide answers to the points raised in their comment.

Survey Results of Honey Bee Colony Losses in Winter in China (2009-2021).

There is growing concern that massive loss of honey bees can cause serious negative effects on biodiversity and ecosystems. Surveys of colony losses have been performed worldwide to monitor the dynamic changes and health status of honey bee colonies. Here, we present the results of surveys regarding winter colony losses from 21 provinces in China from 2009 to 2021, with a total of 1,744,324 colonies managed by 13,704 beekeepers. The total colony losses were low (9.84%; 95% Confidence Interval (CI): 9.60-10.08%) but varied among years, provinces, and scales of apiaries. As little is known about the overwintering mortality of Apis cerana, in this study, we surveyed and compared the loss rates between Apis mellifera and A. cerana in China. We found colonies of A. mellifera suffered significantly lower losses than A. cerana in China. Larger apiaries resulted in higher losses in A. mellifera, whereas the opposite was observed in A. cerana. Furthermore, we used generalized linear mixed-effects models (GLMMs) to evaluate the effects of potential risk factors on winter colony losses and found that the operation size, species, migration, migration×species interaction, and queen problems were significantly related to the loss rates. New queens can increase their colony overwintering survival. Migratory beekeepers and large operations reported lower loss rates.

Differential Brain Expression Patterns of microRNAs Related to Olfactory Performance in Honey Bees (Apis mellifera).

MicroRNAs (miRNAs) play a vital role in the nerve regulation of honey bees (Apis mellifera). This study aims to investigate the differences in expression of miRNAs in a honey bee's brain for olfactory learning tasks and to explore their potential role in a honey bee's olfactory learning and memory. In this study, 12 day old honey bees with strong and weak olfactory performances were utilized to investigate the influence of miRNAs on olfactory learning behavior. The honey bee brains were dissected, and a small RNA-seq technique was used for high-throughput sequencing. The data analysis of the miRNA sequences revealed that 14 differentially expressed miRNAs (DEmiRNAs) between the two groups, strong (S) and weak (W), for olfactory performance in honey bees were identified, which included seven up-regulated and seven down-regulated. The qPCR verification results of the 14 miRNAs showed that four miRNAs (miR-184-3p, miR-276-3p, miR-87-3p, and miR-124-3p) were significantly associated with olfactory learning and memory. The target genes of these DEmiRNAs were subjected to the GO database annotation and KEGG pathway enrichment analyses. The functional annotation and pathway analysis showed that the neuroactive ligand-receptor interaction pathway, oxidative phosphorylation, biosynthesis of amino acids, pentose phosphate pathway, carbon metabolism, and terpenoid backbone biosynthesis may be a great important pathway related to olfactory learning and memory in honey bees. Our findings together further explained the relationship between olfactory performance and the brain function of honey bees at the molecular level and provides a basis for further study on miRNAs related to olfactory learning and memory in honey bees.

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.

Chronic Cadmium Exposure Induces Impaired Olfactory Learning and Altered Brain Gene Expression in Honey Bees (Apis mellifera).

The honey bee (Apis mellifera) plays vital ecological roles in the pollination of crops and the maintenance of ecological balance, and adult honey bees may be exposed to exogenous chemicals including heavy metals during their foraging activities. Cadmium (Cd) is regarded as a nonessential toxic metal and is readily accumulated in plants; honey bees can therefore acquire Cd through the collection of contaminated nectar. In the present study, honey bees were chronically exposed to Cd to investigate the effects of sublethal cadmium doses on the olfactory learning and brain gene expression profiles of honey bees. The results showed that Cd-treated bees exhibited significantly impaired olfactory learning performances in comparison with control bees. Moreover, the head weight was significantly lower in Cd-treated bees than in control bees after chronic exposure to Cd. Gene expression profiles between the Cd treatment and the control revealed that 79 genes were significantly differentially expressed. Genes encoding chemoreceptors and olfactory proteins were downregulated, whereas genes involved in response to oxidative stress were upregulated in Cd-treated bees. The results suggest that Cd exposure exerts oxidative stress in the brain of honey bees, and the dysregulated expression of genes encoding chemoreceptors, olfactory proteins, and cytochrome P450 enzymes is probably associated with impaired olfactory learning in honey bees.

Pharmacological effects and mechanisms of bee venom and its main components: Recent progress and perspective.

Bee venom (BV), a type of defensive venom, has been confirmed to have favorable activities, such as anti-tumor, neuroprotective, anti-inflammatory, analgesic, anti-infectivity effects, etc. This study reviewed the recent progress on the pharmacological effects and mechanisms of BV and its main components against cancer, neurological disorders, inflammatory diseases, pain, microbial diseases, liver, kidney, lung and muscle injury, and other diseases in literature during the years 2018-2021. The related target proteins of BV and its main components against the diseases include Akt, mTOR, JNK, Wnt-5α, HIF-1α, NF-κB, JAK2, Nrf2, BDNF, Smad2/3, AMPK, and so on, which are referring to PI3K/Akt/mTOR, MAPK, Wnt/ß-catenin, HIF-1α, NF-κB, JAK/STAT, Nrf2/HO-1, TrkB/CREB/BDNF, TGF-ß/Smad2/3, and AMPK signaling pathways, etc. Further, with the reported targets, the potential effects and mechanisms on diseases were bioinformatically predicted via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, disease ontology semantic and enrichment (DOSE) and protein-protein interaction (PPI) analyses. This review provides new insights into the therapeutic effects and mechanisms of BV and its main components on diseases.

The in vitro and in vivo antibacterial activities of uniflorous honey from a medicinal plant, Scrophularia ningpoensis Hemsl., and characterization of its chemical profile with UPLC-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Compendium of Materia Medica, honey has been used as a traditional medicine in treatment against mucositis, tinea, hemorrhoids and psoriasis. In complementary medicine, due to its significant antimicrobial activity, honey has been widely used as a remedy for skin wounds and gastrohelcosis for thousands of years. AIM OF THE STUDY: This study is aimed at exploring the antimicrobial activity and mechanisms of honey sourced from medicinal plants, and revealing the composition-activity relationship, to facilitate their complementary and alternative application in the therapy of bacterial infectious diseases. MATERIALS AND METHODS: Eight kinds of medicinal plant-derived uniflorous honey, native to China, were gathered. Their antimicrobial activities were evaluated in vitro, and then in vivo with the systemically infected mouse model and the acute skin infection model. SYTOX uptake assay, scanning electron microscopy, DNA binding assay, and quantitative real-time PCR, were carried out to elucidate the antibacterial mechanisms. This was followed by an investigation of the componential profile with the UPLC-MS/MS technique. RESULTS: It was found that Scrophularia ningpoensis Hemsl. (figwort) honey (S. ningpoensis honey) exhibited broad-spectrum and the strongest antibacterial potency (MICs of 7.81-125.00%, w/v), comparable to manuka honey. In the in vivo assays, S. ningpoensis honey significantly decreased the bacterial load of the muscles under the acute MRSA-infected skin wounds; the sera level of TNF-α in the S. aureus and P. aeruginosa-infected mice decreased by 45.38% and 51.75%, respectively, after the treatment of S. ningpoensis honey (125 mg/10 g). It was capable of killing bacteria through disrupting the cell membranes and the genomic DNA, as well as down-regulating the expression of genes associated with virulence, biofilm formation and invasion, including icaA, icaD, eno, sarA, agrA, sigB, fib and ebps in S. aureus, and lasI, lasR, rhlI, rhlR and algC in P. aeruginosa. Apart from H2O2, some other nonperoxide compounds such as adenosine, chavicol, 4-methylcatechol, trehalose, palmitoleic acid and salidroside, might play a vital role in the antibacterial properties of S. ningpoensis honey. CONCLUSIONS: This is the first study to thoroughly investigate the antibacterial activity, mode of action, and componential profile of S. ningpoensis honey. It suggested that S. ningpoensis honey might be a potential supplement or substitute for manuka honey, for the prevention or treatment of bacterial infections. It will facilitate the precise application of medicinal plant-sourced honey, provide a new thread for the development of antibacterial drugs, and assist in the distinction of different kinds of honey.

Food wanting is mediated by transient activation of dopaminergic signaling in the honey bee brain.

The biological bases of wanting have been characterized in mammals, but whether an equivalent wanting system exists in insects remains unknown. In this study, we focused on honey bees, which perform intensive foraging activities to satisfy colony needs, and sought to determine whether foragers leave the hive driven by specific expectations about reward and whether they recollect these expectations during their waggle dances. We monitored foraging and dance behavior and simultaneously quantified and interfered with biogenic amine signaling in the bee brain. We show that a dopamine-dependent wanting system is activated transiently in the bee brain by increased appetite and individual recollection of profitable food sources, both en route to the goal and during waggle dances. Our results show that insects share with mammals common neural mechanisms for encoding wanting of stimuli with positive hedonic value.

Understanding of Waggle Dance in the Honey Bee (Apis mellifera) from the Perspective of Long Non-Coding RNA.

The ethological study of dance behaviour has yielded some findings since Karl Von Frisch discovered and interpreted the 'dance language' in the honey bee. However, the function and role of long non-coding RNAs on dance behaviour are hardly known until now. In this study, the differential expression patterns of lncRNAs in the brains of waggling dancers and non-dancing bees were analysed by RNA sequencing. Furthermore, lncRNA-mRNA association analysis was constructed to decipher the waggle dance. The results of RNA sequencing indicated that a total of 2877 lncRNAs and 9647 mRNAs were detected from honey bee brains. Further comparison analysis displayed that two lncRNAs, MSTRG.6803.3 and XR_003305156.1, may be involved in the waggle dance. The lncRNA-mRNA association analysis showed that target genes of differentially expressed lncRNAs in the brains between waggling dancers and non-dancing bees were mainly annotated in biological processes related to metabolic process, signalling and response to stimulus and in molecular function associated with signal transducer activity, molecular transducer activity and binding. Nitrogen metabolism was likely implicated in the modulation of the waggle dance. Our findings contribute to further understanding the occurrence and development of waggle dance.

Differential gene expression analysis following olfactory learning in honeybee (Apis mellifera L.).

Insects change their stimulus-response through the perception of associating these stimuli with important survival events such as rewards, threats, and mates. Insects develop strong associations and relate them to their experiences through several behavioral procedures. Among the insects, Apis species, Apis mellifera ligustica are known for their outstanding ability to learn with tremendous economic importance. Apis mellifera ligustica has a strong cognitive ability and promising model species for investigating the neurobiological basis of remarkable olfactory learning abilities. Here we evaluated the olfactory learning ability of A. mellifera by using the proboscis extension reflex (PER) protocol. The brains of the learner and failed-learner bees were examined for comparative transcriptome analysis by RNA-Seq to explain the difference in the learning capacity. In this study, we used an appetitive olfactory learning paradigm in the same age of A. mellifera bees to examine the differential gene expression in the brain of the learner and failed-learner. Bees that respond in 2nd and 3rd trials or only responded to 3rd trials were defined as learned bees, failed-learner individuals were those bees that did not respond in all learning trials The results indicate that the learning ability of learner bees was significantly higher than failed-learner bees for 12 days. We obtained approximately 46.7 and 46.4 million clean reads from the learner bees failed-learner bees, respectively. Gene expression profile between learners' bees and failed-learners bees identified 74 differentially expressed genes, 57 genes up-regulated in the brains of learners and 17 genes were down-regulated in the brains of the bees that fail to learn. The qRT-PCR validated the differently expressed genes. Transcriptome analyses revealed that specific genes in learner and failed-learner bees either down-regulated or up-regulated play a crucial role in brain development and learning behavior. Our finding suggests that down-regulated genes of the brain involved in the integumentary system, storage proteins, brain development, sensory processing, and neurodegenerative disorder may result in reduced olfactory discrimination and olfactory sensitivity in failed-learner bees. This study aims to contribute to a better understanding of the olfactory learning behavior and gene expression information, which opens the door for understanding of the molecular mechanism of olfactory learning behavior in honeybees.

Lotus Bee Pollen Extract Inhibits Isoproterenol-Induced Hypertrophy via JAK2/STAT3 Signaling Pathway in Rat H9c2 Cells.

Bee pollen possesses an anti-cardiomyocyte injury effect by reducing oxidative stress levels and inhibiting inflammatory response and apoptosis, but the possible effect mechanism has rarely been reported. This paper explores the effect of the extract of lotus bee pollen (LBPE) on cardiomyocyte hypertrophy (CH) and its mechanism. The main components of LBPE were identified via UPLC-QTOF MS. An isoproterenol-induced rat H9c2 CH model was subsequently used to evaluate the protection of LBPE on cells. LBPE (100, 250 and 500 µg∙mL-1) reduced the surface area, total protein content and MDA content, and increased SOD activity and GSH content in CH model in a dose-dependent manner. Meanwhile, quantitative real-time PCR trials confirmed that LBPE reduced the gene expression levels of CH markers, pro-inflammatory cytokines and pro-apoptosis factors, and increased the Bcl-2 mRNA expression and Bcl-2/Bax ratio in a dose-dependent manner. Furthermore, target fishing, bioinformatics analysis and molecular docking suggested JAK2 could be a pivotal target protein for the main active ingredients in the LBPE against CH. Ultimately, Western blot (WB) trials confirmed that LBPE can dose-dependently inhibit the phosphorylation of JAK2 and STAT3. The results show that LBPE can protect against ISO-induced CH, possibly via targeting the JAK2/STAT3 pathway, also suggesting that LBPE may be a promising candidate against CH.

Royal jelly from different floral sources possesses distinct wound-healing mechanisms and ingredient profiles.

In recent years, population aging together with the increased prevalence of diabetes and obesity has fuelled a surge in the instances of cutaneous non-healing wounds. Royal jelly (RJ) is a traditional remedy for wound repair; however, the subjacent mechanisms and ingredient profiles are still largely unknown. Our previous study found that Castanea mollissima Bl. RJ (CmRJ-Zj) possessed superior wound healing-promoting effects on both the in vivo and in vitro models than Brassica napus L. RJ (BnRJ-Zj). This study conducted an in-depth investigation on the wound-repairing mechanisms of CmRJ-Zj and BnRJ-Zj to explain the previously observed phenomenon and also comprehensively characterized their constituents. It was found that chestnut RJ could enhance cutaneous wound healing by boosting the growth and mobility of keratinocytes, modulating the expression of aquaporin 3 (AQP3), regulating MAPK and calcium pathways, and mediating inflammatory responses. By employing LC-MS/MS-based proteomic and metabolomic techniques, the comprehensive molecules present in CmRJ-Zj and BnRJ-Zj were elucidated, resulting in a clear discrimination from each other. A total of 15 and 631 differential proteins and compounds were identified, and 217 proteins were newly found in RJ proteome. With bioinformatic functional analysis, we speculated that some differential components were responsible for the wound-healing properties of CmRJ-Zj. Therefore, this study provides an insight into the wound-healing mechanisms of RJ and is the first to explore the compositions of RJ from different nectar plants. It will facilitate the development of therapeutic agents from RJ to treat difficult-to-heal wounds and the distinction of different RJ categories.

Expansion of CRISPR Targeting Sites Using an Integrated Gene-Editing System in Apis mellifera.

CRISPR/Cas9, a predominant gene-editing tool, has been utilised to dissect the gene function in Apis mellifera. However, only the genomic region containing NGG PAM could be recognised and edited in A. mellifera, seriously hampering the application of CRISPR technology in honeybees. In this study, we carried out the bioinformatics analysis for genome-wide targeting sites of NGG, TTN, and NNGRRT to determine the potential expansion of the SpCas9, SaCas9, Cpf1, and it was found that the targetable spectrum of the CRISPR editing system could be markedly extended via the integrated gene manipulation system. Meanwhile, the single guide RNA (sgRNA)/crRNA of different novel gene editing systems and the corresponding CRISPR proteins were co-injected into honeybee embryos, and their feasibility was tested in A. mellifera. The sequencing data revealed that both SaCas9 and Cpf1 are capable of mediating mutation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our results provide the first demonstration that SaCas9 and Cpf1 can function to induce genome sequence alternation, which extended the editing scope to the targets with TTN and NNGRRT and enabled CRISPR-based genome research in a broader range in A. mellifera.

Vairimorpha (Nosema) ceranae Infection Alters Honey Bee Microbiota Composition and Sustains the Survival of Adult Honey Bees.

Vairimorpha (Nosema) ceranae is the most common eukaryotic gut pathogen in honey bees. Infection is typically chronic but may result in mortality. Gut microbiota is a factor that was recently noted for gut infectious disease development. Interestingly, studies identified positive, instead of negative, associations between core bacteria of honey bee microbiota and V. ceranae infection. To investigate the effects of the positive associations, we added isomaltooligosaccharide (IMO), a prebiotic sugar also found in honey, to enhance the positive associations, and we then investigated the infection and the gut microbiota alterations using qPCR and 16S rRNA gene sequencing. We found that infected bees fed IMO had significantly higher V. ceranae spore counts but lower mortalities. In microbiota comparisons, V. ceranae infections alone significantly enhanced the overall microbiota population in the honey bee hindgut and feces; all monitored core bacteria significantly increased in the quantities but not all in the population ratios. The microbiota alterations caused by the infection were enhanced with IMO, and these alterations were similar to the differences found in bees that naturally have longer lifespans. Although our results did not clarify the causations of the positive associations between the infections and microbiota, the associations seemed to sustain the host survival and benefit the pathogen. Enhancing indigenous gut microbe to control nosema disease may result in an increment of bee populations but not the control of the pathogen. This interaction between the pathogen and microbiota potentially enhances disease transmission and avoids the social immune responses that diseased bees die prematurely to curb the disease from spreading within colonies.

Adipokinetic hormone (AKH), energy budget and their effect on feeding and gustatory processes of foraging honey bees.

The adipokinetic hormone (AKH) of insects is considered an equivalent of the mammalian hormone glucagon as it induces fast mobilization of carbohydrates and lipids from the fat body upon starvation. Yet, in foraging honey bees, which lack fat body storage for carbohydrates, it was suggested that AKH may have lost its original function. Here we manipulated the energy budget of bee foragers to determine the effect of AKH on appetitive responses. As AKH participates in a cascade leading to acceptance of unpalatable substances in starved Drosophila, we also assessed its effect on foragers presented with sucrose solution spiked with salicin. Starved and partially-fed bees were topically exposed with different doses of AKH to determine if this hormone modifies food ingestion and sucrose responsiveness. We found a significant effect of the energy budget (i.e. starved vs. partially-fed) on the decision to ingest or respond to both pure sucrose solution and sucrose solution spiked with salicin, but no effect of AKH per se. These results are consistent with a loss of function of AKH in honey bee foragers, in accordance with a social life that implies storing energy resources in the hive, in amounts that exceed individual needs.

Transcriptional Profiles of Diploid Mutant Apis mellifera Embryos after Knockout of csd by CRISPR/Cas9.

In honey bees, complementary sex determiner (csd) is the primary signal of sex determination. Its allelic composition is heterozygous in females, and hemizygous or homozygous in males. To explore the transcriptome differences after sex differentiation between males and females, with genetic differences excluded, csd in fertilized embryos was knocked out by CRISPR/Cas9. The diploid mutant males at 24 h, 48 h, 72 h, and 96 h after egg laying (AEL) and the mock-treated females derived from the same fertilized queen were investigated through RNA-seq. Mutations were detected in the target sequence in diploid mutants. The diploid mutant drones had typical male morphological characteristics and gonads. Transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, such as takeout and apolipophorin-III-like protein (A4), had higher expressions in the diploid mutant males. Weighted gene co-expression network analysis (WGCNA) indicated that there might be interactions between csd and fruitless (fru), feminizer (fem) and hexamerin 70c (hex70c), transformer-2 (tra2) and troponin T (TpnT). The information provided by this study will benefit further research on the sex dimorphism and development of honey bees and other insects in Hymenoptera.

CRISPR/Cas9 mediated knockout of Amyellow-y gene results in melanization defect of the cuticle in adult Apis mellifera.

Visible genetic markers are critical to gene function studies using genome editing technology in insects. However, there is no report about visible phenotypic markers in Apis mellifera, which extremely influences the application of genomic editing in honey bees. Here, we cloned and characterized the Amyellow-y gene in A. mellifera. Stage expression profiles showed that Amyellow-y gene was highly expressed in 2-, 4-day-old pupae, and newly emerged bees, and a high expression level was detected in the leg, thorax, wing and sting. To understand its functional role in pigmentation, Amyellow-y edited honeybees were created using CRISPR/Cas9, and it was found that the black pigment was decreased in the cuticle of mosaic workers and mutant drones. In particular, mutant drones manifested an overall appearance of yellowish cuticle in the body and appendages, including antennae, wings and legs, indicating that mutagenesis induced by disruption of Amyellow-y with CRISPR/Cas9 are heritable. Furthermore, the expression levels of genes associated with melanin pigmentation was investigated in mutant and wild-type drones using quantitative reverse transcription PCR. Transcription levels of Amyellow-y and aaNAT decreased markedly in mutant drones than that in wild-type ones, whereas laccase 2 was significantly up-regulated. Our results provide the first evidence, to our knowledge, that CRISPR/Cas9 edited G1 mutant drones of A. mellifera have a dramatic body pigmentation defect that can be visualized in adults, suggesting that Amyellow-y may serve as a promising visible phenotypic marker for genome editing in honey bees.

The in vitro and in vivo wound-healing effects of royal jelly derived from Apis mellifera L. during blossom seasons of Castanea mollissima Bl. and Brassica napus L. in South China exhibited distinct patterns.

BACKGROUND: Non-healing wounds have been a severe issue in the global healthcare system. Regrettably, royal jelly, a traditional remedy for various skin injuries, has not been widely applied in cutaneous wounds in clinical practice nowadays, which may be due to the confusion and the lack of knowledge about the efficacies of different types of royal jelly, the bioactive constituents, and the precise mechanisms underlying the wound repairing activity. Since the compositions and bioactivities of royal jelly are predominantly influenced by nectar plants, this study aims to explore the differences in the wound-healing properties of royal jelly produced by Apis mellifera L. during the blossom seasons of different floral sources, to provide guidelines for the future rational application of royal jelly in cutaneous wounds, and to promote the further discovery of wound repair-promoting substances. METHODS: Royal jelly samples were harvested during flowering seasons of Castanea mollissima Bl. (chestnut) and Brassica napus L. (rapeseed) in South China, from which hydrophilic and lipophilic fractions were extracted. The in vivo wound-healing potential was preliminarily assessed in Wistar rats' excisional full-thickness wounds, followed by investigating the mechanisms of action through in vitro assays with human epidermal keratinocytes and LPS-stimulated inflammation in macrophages. RESULTS: The results indicated that different royal jelly samples exhibited distinct wound-healing potential, in which Castanea mollissima Bl. royal jelly was more potent. It sped up wound closure between day 2 and day 4 to 0.25 cm2/day (p < 0.05), and could accelerate wound repair by enhancing the proliferative and migratory capabilities of keratinocytes by 50.9% (p < 0.001) and 14.9% (p < 0.001), modulating inflammation through inhibiting nitric oxide production by 46.2% (p < 0.001), and promoting cell growth through increasing the secretion of transforming growth factor-ß by 44.7% (p < 0.001). In contrast, Brassica napus L. royal jelly could regulate inflammation by reducing the amount of tumour necrosis factor-α by 21.3% (p < 0.001). CONCLUSIONS: The present study improves the application of royal jelly for curing difficult-to-heal wounds, in which the hydrosoluble extract of Castanea mollissima Bl. royal jelly promises the greatest potential. It also provides clues which may lead towards the identification of substances derived from royal jelly to treat wounds.

Degradation of an appetitive olfactory memory via devaluation of sugar reward is mediated by 5-HT signaling in the honey bee.

Conditioned taste aversion (CTA) learning induces the devaluation of a preferred food through its pairing with a stimulus inducing internal illness. In invertebrates, it is still unclear how this aversive learning impairs the memories of stimuli that had been associated with the appetitive food prior to its devaluation. Here we studied this phenomenon in the honey bee and characterized its neural underpinnings. We first trained bees to associate an odorant (conditioned stimulus, CS) with appetitive fructose solution (unconditioned stimulus, US) using a Pavlovian olfactory conditioning. We then subjected the bees that learned the association to a CTA training during which the antennal taste of fructose solution was contingent or not to the ingestion of quinine solution, which induces malaise a few hours after ingestion. Only the group experiencing contingent fructose stimulation and quinine-based malaise exhibited a decrease in responses to the fructose and a concomitant decrease in odor-specific retention in tests performed 23 h after the original odor conditioning. Furthermore, injection of dopamine- and serotonin-receptor antagonists after CTA learning revealed that this long-term decrease was mediated by serotonergic signaling as its blockade rescued both the responses to fructose and the odor-specific memory 23 h after conditioning. The impairment of a prior CS memory by subsequent CTA conditioning confirms that bees retrieve a devaluated US representation when presented with the CS. Our findings further highlight the importance of serotonergic signaling in aversive learning in the bee and uncover mechanisms underlying aversive memories induced by internal illness in invertebrates.