What can laboratory studies tell us about potential effects of pesticides on nontarget arthropods populations and communities in the field?

Over the past decades, concern has been increasing over reported declines in aboveground biodiversity on farmland. In many regions, data on the toxicity of pesticides to honeybees (Apis mellifera), but not wider nontarget arthropod (NTA) data, are required for pesticide registration. In Europe, the effects of pesticides on NTAs and honeybees have been the subject of regulatory risk assessment for more than 30 years, resulting in a large database. Although insecticides may be expected to affect NTA populations, solely identifying insecticidal modes of action for further NTA testing would result in redundancy among low-risk testing products and may also exclude other modes of action with potential effects in the field. This study assessed whether the honeybee acute risk assessment could provide any indication of the potential impact and recovery time of NTAs in cropped areas at the field scale and, if so, how it might be used in a tiered testing approach. The hazard quotients (HQs; foliar application rate/LR50) were derived for 151 active substances (32% insecticides, 28% fungicides, 38% herbicides, 2% plant growth regulators) for which toxicity data for established EU Tier 1 NTA indicator species (Typhlodromus pyri, Aphidius rhopalosiphi) and application rate data were available. These HQs were compared with published NTA HQ thresholds indicating the time to recovery of NTA populations and communities in field studies (>1 to >12 months). Using the same application rate data, honeybee acute risk quotient (RQ) and HQ were also determined and compared with NTA HQs and honeybee regulatory thresholds. These comparisons demonstrated that, where required, the current regulatory honeybee acute RQ of 0.4 or honeybee HQ of 50 can provide an efficient screening tier to target NTA testing at those products and uses with potential effects in the field where recovery may exceed 12 months. Integr Environ Assess Manag 2024;00:1-8. © 2024 SETAC.

Liposome-based RNAi delivery in honeybee for inhibiting parasite Nosema ceranae.

Nosema ceranae, a parasite that parasitizes and reproduces in the gut of honeybees, has become a serious threat to the global apiculture industry. RNA interference (RNAi) technology can be used to inhibit N. ceranae growth by targeting silencing the thioredoxin reductase (TrxR) in N. ceranae. However, suitable carriers are one of the reasons limiting the application of RNAi due to the easy degradation of dsRNA in honeybees. As a vesicle composed of a lipid bilayer, liposomes are a good carrier for nucleic acid delivery, but studies in honeybees are lacking. In this study, liposomes were used for double-stranded RNA (dsRNA) dsTrxR delivery triggering RNAi to inhibit the N. ceranae growth in honeybees. Compared to naked dsTrxR, liposome-dsTrxR reduced N. ceranae numbers in the midgut and partially restored midgut morphology without affecting bee survival and gut microbial composition. The results of this study confirmed that liposomes could effectively protect dsRNA from entering the honeybee gut and provide a reference for using RNAi technology to suppress honeybee pests and diseases.

Brain energy metabolism as an underlying basis to slow-fast cognitive phenotypes in honeybees.

In the context of slow-fast behavioral variation, fast individuals are hypothesized to be those who prioritize speed over accuracy while slow individuals are those which do the opposite. Since energy metabolism is a critical component of neural and cognitive functioning, this predicts such differences in cognitive style to be reflected at the level of the brain. We tested this idea in honeybees by first classifying individuals into slow and fast cognitive phenotypes based on a learning assay and then measuring their brain respiration with high-resolution respirometry. Our results broadly show that interindividual differences in cognition are reflected in differences in brain mass and accompanying energy use at the level of the brain and the whole animal. Bigger brains had lower mass-specific energy usage and those bees with bigger brains had a higher metabolic rate. These differences in brain respiration and brain mass were in turn associated with cognitive differences such that fast cognitive phenotypes were those bees with bigger brains while slow cognitive phenotypes were those with smaller brains. We discuss these results in the context of the role of energy in brain functioning and slow-fast decision making and speed accuracy tradeoff.

Review: Nutritional Needs of Honeybees and Legislation on Apiculture By-Products in Animal Nutrition.

Honeybees are some of the smallest farmed animals, and apiculture by-products, e.g., honey, beeswax, propolis, royal jelly, and pollen contribute to animal nutrition. For the effective production of these by-products, the optimal development and nutrient supply of the honeybee is required. Beginning with the development of the mouth and anal pores on the second day of embryonic development, the digestive tract differentiates into the mouth and fore-, mid-, and hindgut during the pupal stage. The various glands within the oral cavity are particularly important, secreting enzymes and substances that are crucial for digestion and hive nutrition, e.g., invertase and royal jelly. Honeybees rely on a specialized caste system, with worker bees collecting nectar, pollen, water, and resin for the nutrition of the entire hive. Macronutrients, including proteins, carbohydrates, and lipids, obtained primarily from pollen and nectar, are essential for the growth and development of larvae and the overall health of the colony. Inadequate nutrient intake can lead to detrimental effects on larval development, prompting cannibalism within the hive. Apiculture by-products possess unique nutritional and therapeutic properties, leading to a growing interest in the use of honey, beeswax, propolis, and pollen as a feed additive. In recent years, the use of apicultural by-products in animal nutrition has been primarily limited to in vivo studies, which have demonstrated various positive impacts on the performance of farm animals. Honey, beeswax, propolis, royal jelly, and pollen are listed feed stuffs according to Regulation (EC) No. 68/2013. However, for animal nutrition there is not any specific legal definition for these products and no legal requirements regarding their ingredients as given for honey or beeswax in European food law.

Wing vein abnormality analysis in honeybee (Apis mellifera L. 1758) populations from Iran.

The insect wing is one of the most important characteristics that allowed insects to occupy most of the habitats on the planet. Honeybee wings has been the subject of studies on the venation abnormalities. A total of 424 honeybees from 14 locations were collected and all four wings were removed and examined for 19 abnormalities on the forewings and 6 abnormalities on the hindwings. In general, supernumerary veins were the most common abnormalities seen and abnormalities no. 23, 2, 6, 1, 5, 21, 10, 13 had the highest and abnormalities no. 11, 17, 18, 19, 20, and 25 had the lowest frequencies. All of the abnormalities had similar frequencies in the right and left wings in the population. In terms of correlation between 25 abnormalities, abnormality pairs AB3-AB13, AB6-AB7, AB7-AB8, AB10-AB12, AB16-AB17 on the forewing and AB2-AB23, AB12-AB20, AB12-AB24, AB13-AB21, AB16-AB25, and AB17-AB25 between the forewing and hindwing show significant positive correlations and abnormality pairs AB4-AB5, AB7-AB15 and AB8-AB9 on the forewing show significant negative correlations with each other. In terms of the differential occurrence of abnormalities , a few locations differed significantly from other locations. This study provides some insights into the nature of these abnormalities on the honeybee wings.

Lactobacillus Firm-5-derived succinate prevents honeybees from having diabetes-like symptoms.

The gut microbiome plays an important role in honeybee hormonal regulation and growth, but the underlying mechanisms are poorly understood. Here, we showed that the depletion of gut bacteria resulted in reduced expression of insulin-like peptide gene (ilp) in the head, accompanied by metabolic syndromes resembling those of Type 1 diabetes in humans: hyperglycemia, impaired lipid storage, and decreased metabolism. These symptoms were alleviated by gut bacterial inoculation. Gut metabolite profiling revealed that succinate, produced by Lactobacillus Firm-5, played deterministic roles in activating ilp gene expression and in regulating metabolism in honeybees. Notably, we demonstrated that succinate modulates host ilp gene expression through stimulating gut gluconeogenesis, a mechanism resembling that of humans. This study presents evidence for the role of gut metabolite in modulating host metabolism and contributes to the understanding of the interactions between gut microbiome and bee hosts.

Honeybee gut bacterial strain improved survival and gut microbiota homeostasis in Apis mellifera exposed in vivo to clothianidin.

Pesticides are causing honeybee mortality worldwide. Research carried out on honeybees indicates that application of pesticides has a significant impact on the core gut community, which ultimately leads to an increase in the growth of harmful pathogens. Disturbances caused by pesticides also affect the way bacterial members interact, which results in gut microbial dysbiosis. Administration of beneficial microbes has been previously demonstrated to be effective in treating or preventing disease in honeybees. The objective of this study was to measure under in vivo conditions the ability of two bacterial strains (the Enterobacter sp. and Pantoea sp.) isolated from honeybee gut to improve survival and mitigate gut microbiota dysbiosis in honeybees exposed to a sublethal clothianidin dose (0.1 ppb). Both gut bacterial strains were selected for their ability to degrade clothianidin in vitro regardless of their host-microbe interaction characteristics (e.g., beneficial, neutral, or harmful). To this end, we conducted cage trials on 4- to 6-day-old newly emerging honeybees. During microbial administration, we jointly monitored the taxonomic distribution and activity level of bacterial symbionts quantifying 16S rRNA transcripts. First, curative administration of the Pantoea sp. strain significantly improved the survival of clothianidin-exposed honeybees compared to sugar control bees (i.e., supplemented with sugar [1:1]). Second, curative administration of the Enterobacter sp. strain significantly mitigated the clothianidin-induced dysbiosis observed in the midgut structural network, but without improving survival. IMPORTANCE: The present work suggests that administration of bacterial strains isolated from honeybee gut may promote recovery of gut microbiota homeostasis after prolonged clothianidin exposure, while improving survival. This study highlights that gut bacterial strains hold promise for developing efficient microbial formulations to mitigate environmental pesticide exposure in honeybee colonies.

From data to harvest: Leveraging ensemble machine learning for enhanced crop yield predictions across Canada amidst climate change.

Accurate crop yield predictions are crucial for farmers and policymakers. Despite the widespread use of ensemble machine learning (ML) models in computer science, their application in crop yield prediction remains relatively underexplored. This study, conducted in Canada, aims to assess the potential of five distinct ensemble ML models-Adaptive Boosting (AdaBoost), Gradient Boosting Machine (GBM), XGBoost, LightGBM, and Random Forest (RF)-in predicting crop yields chosen for their ability to manage complex datasets and their strong performance potential. The study integrated various factors, including climate variables, satellite-derived vegetation indices, soil characteristics, and honeybee census data. Data preparation comprised two main steps: first, climate variables were interpolated and averaged for croplands in ArcGIS Pro, along with averaging vegetation indices and soil characteristics. Honeybee census data was also incorporated. Second, the data was organized in Python to create a structured format for models' input. The models' accuracy was assessed using Root Mean Squared Error (RMSE), R-squared, and Mean Absolute Error (MAE). XGBoost emerged as the most accurate model, with the lowest MAE (68.70 for canola and 39.47 for soybeans), lowest RMSE (119.48 for canola and 102.39 for soybeans), and highest R-squared values (0.95 for canola and 0.96 for soybeans) on the test dataset. The study also assessed crop yields under various climate change scenarios, finding minimal variations across the scenarios, but significant negative impacts on canola and soybean yields across Canada. Honeybee colonies were identified as the most influential factor on crop yields, contributing 52.34 % to canola and 57.18 % to soybean yields. This research provides detailed crop yield maps of canola and soybeans at the Census Consolidated Subdivisions (CCS) level across Canada's agricultural landscape, offering valuable forecasts for localized decision-making. Additionally, it offers a proactive strategy for climate change preparedness, assisting farmers and stakeholders optimise resource allocation and manage risks effectively.

Exploring bee venom and silver nanoparticles for controlling foulbrood pathogen and enhancing lifespan of honeybees.

The beekeeping industry plays a crucial role in local economies, contributing significantly to their growth. However, bee colonies often face the threat of American foulbrood (AFB), a dangerous disease caused by the Gram-positive bacterium Paenibacillus larvae (P. l.). While the antibiotic Tylosin has been suggested as a treatment, its bacterial resistance necessitates the search for more effective alternatives. This investigation focused on evaluating the potential of bee venom (BV) and silver nanoparticles (Ag NPs) as antibacterial agents against AFB. In vitro treatments were conducted using isolated AFB bacterial samples, with various concentrations of BV and Ag NPs (average size: 25nm) applied individually and in combination. The treatments were administered under both light and dark conditions. The viability of the treatments was assessed by monitoring the lifespans of treated bees and evaluating the treatment's efficiency within bee populations. Promising results were obtained with the use of Ag NPs, which effectively inhibited the progression of AFB. Moreover, the combination of BV and Ag NPs, known as bee venom/silver nanocomposites (BV/Ag NCs), significantly extended the natural lifespan of bees from 27 to 40 days. Notably, oral administration of BV in varying concentrations (1.53, 3.12, and 6.25 mg/mL) through sugary syrup doubled the bees' lifespan compared to the control group. The study established a significant correlation between the concentration of each treatment and the extent of bacterial inhibition. BV/Ag NCs demonstrated 1.4 times greater bactericidal efficiency under photo-stimulation with visible light compared to darkness, suggesting that light exposure enhances the effectiveness of BV/Ag NCs. The combination of BV and Ag NPs demonstrated enhanced antibacterial efficacy and prolonged honeybee lifespan. These results offer insights that can contribute to the development of safer and more efficient antibacterial agents for maintaining honeybee health.

Tissue-Specific Accumulation of Orally Administered Short- and Medium-Chain Chlorinated Paraffins in Honeybees (Apis mellifera L.).

The prevalence and distribution of chlorinated paraffins (CPs) have been extensively studied in various matrices and organisms; however, there is a lack of information about insects, particularly in honeybees. To address this gap, we studied young honeybee workers exposed to short- and medium-chain CPs (SCCPs and MCCPs) at an environmentally relevant concentration of 10 mg/L for 7 days, followed by a 7-day elimination period. Results indicated that CPs could transfer into the head after oral consumption and SCCPs and MCCPs exhibited clear bioaccumulation trends: midgut > hindgut > head. An evaluation of congener group distribution patterns demonstrated that the dominant congener groups in all target tissues were C11-13Cl7-8 and C14Cl7-8 for SCCPs and MCCPs, respectively, consistent with the treated CP standards. In honeybees, a significant negative relationship was observed for the log concentration of MCCP congener groups and their log KOW, but not with their log KOA. Conversely, no such correlation was found for SCCPs. These findings suggest that honeybees have a high potential to bioaccumulate MCCPs, particularly those with a low log KOW, and exhibit weak selectivity for SCCPs.

Gut microbiota from patients with Parkinson's disease causes motor deficits in honeybees.

Objective: Parkinson's disease (PD) is possibly caused by genetic factors, environmental factors, and gut microbiota dysbiosis. This study aims to explore whether the microbiota contributes to the behavior abnormalities of PD. Methods: We transplanted gut microbiota from patients with PD or healthy controls (HC) into microbiota-free honeybees. We also established two more groups, namely the rotenone (ROT) group, in which PD-like symptoms of honeybees were induced by rotenone, and the conventional (CV) group, in which honeybees were colonized with conventional gut microbiota. The climbing assay was performed to assess the motor capabilities of honeybees. Histopathological examination was conducted to evaluate the integrity of gut mucosa. Tyrosine hydroxylase (TH) gene expression levels and dopamine (DA) concentrations in the brain were also examined. Additionally, metagenomics and full-length 16S rRNA analyses were performed to identify alterations in gut microbiota profiles, both in PD patients and honeybees. Results: Honeybees in the PD and ROT groups exhibited slower climbing speeds, downregulated TH gene expression, and impaired gut barriers. Both the HC and PD groups of honeybees successfully harbored a portion of gut microbiota from corresponding human donors, and differences in microbial composition were identified. Morganella morganii and Erysipelatoclostridium ramosum exhibited significantly increased relative abundance in the HC group, while Dorea longicatena, Collinsella aerofaciens, Lactococcus garvieae, Holdemanella biformis, Gemmiger formicilis, and Blautia obeum showed significantly increased relative abundance in the PD group. Functional predictions of microbial communities in the PD group indicated an increased synthesis of hydrogen sulfide and methane. Conclusion: A novel PD model was induced in honeybees with rotenone and gut microbiota from PD patients. This study linked PD-related behaviors to altered gut microbiota, highlighting a potential gut microbiota-brain axis involvement in PD pathogenesis. We identify previously unrecognized associations of Dorea longicatena, Collinsella aerofaciens, Lactococcus garvieae, Holdemanella biformis, Gemmiger formicilis, and Blautia obeum with PD. Additionally, pathways related to hydrogen sulfide and methane synthesis have been previously suggested as potential contributors to the development of PD, and our research further supports this hypothesis.

The effect of pollen monodiets on fat body morphology parameters and energy substrate levels in the fat body and hemolymph of Apis mellifera L. workers.

Human activities associated with large-scale farms and the monocultures expose honey bees to one type of food. Moreover, there is an ongoing decline of plant species producing pollen and nectar in Europe. A poorly balanced diet affects a number of processes occurring in a bee's body. The fat body and hemolymph are the tissues that participate in all of them. Therefore, the aim of our study was to determine the effect of hazel, pine, rapeseed, buckwheat, phacelia and goldenrod pollen on the morphological parameters of fat body trophocytes, the diameters of cell nuclei in oenocytes and the concentrations of compounds involved in energy metabolism (glucose, glycogen, triglycerides and protein). In the cage tests, the bees were fed from the first day of life with sugar candy (control group) or candy with a 10% addition of one of the 6 pollen types. Hemolymph and fat body from various locations were collected from 1-, 7- and 14-day-old workers. Pollen produced by plant species such as hazel and pine increased glucose concentrations in the bee tissues, especially in the hemolymph. It can therefore be concluded that they are valuable sources of energy (in the form of simple carbohydrates) which are quickly used by bees. Pollen from plants blooming in the summer and autumn increased the concentrations of proteins, glycogen and triglycerides in the fat body, especially that from the third tergite. The accumulation of these compounds was associated with an increased the length and width of trophocytes as well as with enhanced metabolic activity, which was evidenced in the increasing diameter of oenocyte cell nuclei. It seems a balanced multi-pollen diet is more valuable for bees, but it is important to understand the effects of the particular pollen types in the context of a mono-diet. In the future, this will make it possible to produce mixtures that can ensure homeostasis in the apian body.

Nasally delivered chitosan-coated poly(lactide-co-glycolide) encapsulating honeybee venom enhances T helper 1-related immunity against Salmonella Typhimurium infection in pigs.

OBJECTIVE: Salmonella Typhimurium is a significant zoonotic concern for human food poisoning and a substantial economic burden in the swine industry. We previously reported that nasally delivered chitosan-coated poly(lactide-co-glycolide) (PLGA) encapsulating honeybee venom (CP-HBV) could enhance CD4+ T helper 1 (Th1)-related immune responses in healthy pigs. Building upon these findings, the current study aimed to investigate the protective immune enhancement by nasally delivered CP-HBV in pigs challenged with S Typhimurium. ANIMALS: 36 healthy, 4-week-old, female, Landrace X Yorkshire X Duroc pigs. METHODS: 36 pigs were allocated into 3 groups: CP-HBV (n = 16), control (n = 16), and healthy baseline control (n = 4). CP-HBV and control groups were challenged with S Typhimurium 7 days post-treatment. Pigs from the healthy control group were sacrificed at 0 days postinfection (DPI), and 4 pigs from each of the control and CP-HBV groups were sacrificed at 1, 2, 4, and 7 DPI. Salmonella shedding, immune cell frequencies, cytokines, and transcriptional factor expression levels were measured. RESULTS: The CP-HBV group exhibited lower bacterial shedding and an enhanced Th1-related immune response characterized by an upregulation of CD4+ T cells and CD4+ Interferon-γ+ T cells, accompanied by increased expression of Th1-related cytokines and reduced expression of regulatory T cells and immunosuppressive cytokines compared to the control group. CLINICAL RELEVANCE: CP-HBV is a promising strategy for controlling Salmonella infections in pigs and improving public health.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

Managing Microbiota Activity of Apis mellifera with Probiotic (Bactocell®) and Antimicrobial (Fumidil B®) Treatments: Effects on Spring Colony Strength.

Against a backdrop of declining bee colony health, this study aims to gain a better understanding of the impact of an antimicrobial (Fumidil B®, Can-Vet Animal Health Supplies Ltd., Guelph, ON, Canada) and a probiotic (Bactocell®, Lallemand Inc., Montreal, QC, Canada) on bees' microbiota and the health of their colonies after wintering. Therefore, colonies were orally exposed to these products and their combination before wintering in an environmental room. The results show that the probiotic significantly improved the strength of the colonies in spring by increasing the total number of bees and the number of capped brood cells. This improvement translated into a more resilient structure of the gut microbiota, highlighted by a more connected network of interactions between bacteria. Contrastingly, the antimicrobial treatment led to a breakdown in this network and a significant increase in negative interactions, both being hallmarks of microbiota dysbiosis. Although this treatment did not translate into a measurable colony strength reduction, it may impact the health of individual bees. The combination of these products restored the microbiota close to control, but with mixed results for colony performance. More tests will be needed to validate these results, but the probiotic Bactocell® could be administrated as a food supplement before wintering to improve colony recovery in spring.

Being Stung Once or Twice by Bees (Apis mellifera L.) Slightly Disturbed the Serum Metabolome of SD Rats to a Similar Extent.

In most cases, the number of honeybee stings received by the body is generally small, but honeybee stings can still cause serious allergic reactions. This study fully simulated bee stings under natural conditions and used 1H Nuclear Magnetic Resonance (1H NMR) to analyze the changes in the serum metabolome of Sprague-Dawley (SD) rats stung once or twice by honeybees to verify the impact of this mild sting on the body and its underlying mechanism. The differentially abundant metabolites between the blank control rats and the rats stung by honeybees included four amino acids (aspartate, glutamate, glutamine, and valine) and four organic acids (ascorbic acid, lactate, malate, and pyruvate). There was no separation between the sting groups, indicating that the impact of stinging once or twice on the serum metabolome was similar. Using the Principal Component Discriminant Analysis ( PCA-DA) and Variable Importance in Projection (VIP) methods, glucose, lactate, and pyruvate were identified to help distinguish between sting groups and non-sting groups. Metabolic pathway analysis revealed that four metabolic pathways, namely, the tricarboxylic acid cycle, pyruvate metabolism, glutamate metabolism, and alanine, aspartate, and glutamate metabolism, were significantly affected by bee stings. The above results can provide a theoretical basis for future epidemiological studies of bee stings and medical treatment of patients stung by honeybees.

Caught in the act: the invasion of a viral vector changes viral prevalence and titre in native honeybees and bumblebees.

Novel transmission routes change pathogen landscapes and may facilitate disease emergence. The varroa mite is a virus vector that switched to western honeybees at the beginning of the last century, leading to hive mortality, particularly in combination with RNA viruses. A recent invasion of varroa on the French island of Ushant introduced vector-mediated transmission to one of the last varroa-naive native honeybee populations and caused rapid changes in the honeybee viral community. These changes were characterized by a drastic increase in deformed wing virus type B prevalence and titre in honeybees, as well as knock-on effects in bumblebees, particularly in the year following the invasion. Slow bee paralysis virus also appeared in honeybees and bumblebees, with a 1 year delay, while black queen cell virus declined in honeybees. This study highlights the rapid and far-reaching effects of vector-borne transmission that can extend beyond the directly affected host species, and that the direction of the effect depends on the pathogen's virulence.

Toxic effects of acaricide fenazaquin on development, hemolymph metabolome, and gut microbiome of honeybee (Apis mellifera) larvae.

Fenazaquin, a potent insecticide widely used to control phytophagous mites, has recently emerged as a potential solution for managing Varroa destructor mites in honeybees. However, the comprehensive impact of fenazaquin on honeybee health remains insufficiently understood. Our current study investigated the acute and chronic toxicity of fenazaquin to honeybee larvae, along with its influence on larval hemolymph metabolism and gut microbiota. Results showed that the acute median lethal dose (LD50) of fenazaquin for honeybee larvae was 1.786 µg/larva, and the chronic LD50 was 1.213 µg/larva. Although chronic exposure to low doses of fenazaquin exhibited no significant effect on larval development, increasing doses of fenazaquin resulted in significant increases in larval mortality, developmental time, and deformity rates. At the metabolic level, high doses of fenazaquin inhibited nucleotide, purine, and lipid metabolism pathways in the larval hemolymph, leading to energy metabolism disorders and physiological dysfunction. Furthermore, high doses of fenazaquin reduced gut microbial diversity and abundance, characterized by decreased relative abundance of functional gut bacterium Lactobacillus kunkeei and increased pathogenic bacterium Melissococcus plutonius. The disrupted gut microbiota, combined with the observed gut tissue damage, could potentially impair food digestion and nutrient absorption in the larvae. Our results provide valuable insights into the complex and diverse effects of fenazaquin on honeybee larvae, establishing an important theoretical basis for applying fenazaquin in beekeeping.

Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

Identification of the mutual gliding locus as a factor for gut colonization in non-native bee hosts using the ARTP mutagenesis.

BACKGROUND: The gut microbiota and their hosts profoundly affect each other's physiology and evolution. Identifying host-selected traits is crucial to understanding the processes that govern the evolving interactions between animals and symbiotic microbes. Current experimental approaches mainly focus on the model bacteria, like hypermutating Escherichia coli or the evolutionary changes of wild stains by host transmissions. A method called atmospheric and room temperature plasma (ARTP) may overcome the bottleneck of low spontaneous mutation rates while maintaining mild conditions for the gut bacteria. RESULTS: We established an experimental symbiotic system with gnotobiotic bee models to unravel the molecular mechanisms promoting host colonization. By in vivo serial passage, we tracked the genetic changes of ARTP-treated Snodgrassella strains from Bombus terrestris in the non-native honeybee host. We observed that passaged isolates showing genetic changes in the mutual gliding locus have a competitive advantage in the non-native host. Specifically, alleles in the orphan mglB, the GTPase activating protein, promoted colonization potentially by altering the type IV pili-dependent motility of the cells. Finally, competition assays confirmed that the mutations out-competed the ancestral strain in the non-native honeybee gut but not in the native host. CONCLUSIONS: Using the ARTP mutagenesis to generate a mutation library of gut symbionts, we explored the potential genetic mechanisms for improved gut colonization in non-native hosts. Our findings demonstrate the implication of the cell mutual-gliding motility in host association and provide an experimental system for future study on host-microbe interactions. Video Abstract.