Determination of bisphenols in beeswax based on sugaring out-assisted liquid-liquid extraction: Method development and application in survey, recycling and degradation studies.

The methodology of sugaring out-assisted liquid-liquid extraction (SULLE) coupled with high-performance liquid chromatography-fluorescence detection was devised for quantifying bisphenol A (BPA) and bisphenol B (BPB) in beeswax. The effectiveness of SULLE was methodically explored and proved superior to the salting out-assisted liquid-liquid extraction approach for beeswax sample preparation. The analytical performance underwent comprehensive validation, revealing detection limits of 10 µg/kg for BPA and 20 µg/kg for BPB. The method developed was employed to analyse commercial beeswax (n = 15), beeswax foundation (n = 15) and wild-build comb wax (n = 26) samples. The analysis revealed BPA presence in four commercial beeswax samples and three beeswax foundation samples, with the highest detected residue content being 88 ± 7 µg/kg. For BPB, two beeswax foundation samples were positive, with concentrations below the limits of quantification and 85 ± 4 µg/kg, respectively. No bisphenols were detected in wild-build comb wax. Furthermore, the bisphenol removal efficacy of two recycling methods-boiling in water and methanol extraction-was assessed. The findings indicated that after four recycling cycles using water boiling, 9.6% of BPA and 29.2% of BPB remained in the beeswax. Whereas methanol extraction resulted in approximately 7% residual after one recycling process. A long-term study over 210 days revealed the slow degradation of bisphenols in comb beeswax. This degradation fitted well with a first-order model, indicating half-lives (DT50) of 139 days for BPA and 151 days for BPB, respectively. This research provides the first report on bisphenol contamination in beeswax. The low removal rate during the recycling process and the gradual degradation in beeswax underscore the significance of bisphenol contamination and migration in bee hives along with their potential risk to pollinators warranting concern. Furthermore, the developed SULLE method shows promise in preparing beeswax samples to analyse other analytes.

Variation in the physiological response of adult worker bees of different ages (Apis mellifera L.) to pyraclostrobin stress.

The social division of labor within the honeybee colony is closely related to the age of the bees, and the age structure is essential to the development and survival of the colony. Differences in tolerance to pesticides and other external stresses among worker bees of different ages may be related to their social division of labor and corresponding physiological states. Pyraclostrobin was widely used to control the fungal diseases of nectar and pollen plants, though it was not friend to honey bees and other pollinators. This work aimed to determine the effects of field recommended concentrations of pyraclostrobin on the activities of protective and detoxifying enzymes, on the expression of genes involved in nutrient metabolism, and immune response in worker bees of different ages determined to investigate the physiological and biochemical differences in sensitivity to pyraclostrobin among different age of worker bees. The result demonstrates that the tolerance of adult worker bees to pyraclostrobin was negatively correlated with their age, and the significantly reduced survival rate of forager bees (21 day-old) with continued fungicide exposure. The activities of protective enzymes (CAT and SOD) and detoxifying enzymes (CarE, GSTs and CYP450) in different ages of adult worker bees were significantly altered, indicating the physiological response and the regulatory capacity of worker bees of different ages to fungicide stress was variation. Compared with 1 and 8 day-old worker bees, the expression of nutrient-related genes (ilp1 and ilp2) and immunity-related genes (apidaecin and defensin1) in forager bees (21 day-old) was gradually downregulated with increasing pyraclostrobin concentrations. Moreover, the expression of vitellogenin and hymenoptaecin in forager bees (21 day-old) was also decreased in high concentration treatment groups (250 and 313 mg/L). The present study confirmed the findings of the chronic toxicity of pyraclostrobin on the physiology and biochemistry of worker bees of different ages, especially to forager bees (21 day-old). These results would provide important physiological and biochemical insight for better understanding the potential risks of pyraclostrobin on honeybees and other non-target pollinators.

Salivary Cystatin-L2-like of Varroa destructor Causes Lower Metabolism Activity and Abnormal Development in Apis mellifera Pupae.

Varroa destructor injects a salivary secretion into honeybees during their feeding process. The salivary secretion plays a vital role in mite-bee interactions and is the main cause of honeybee illness. To determine the biological function of cystatin-L2-like, one of the components of V. destructor salivary secretion, its gene expression in mites during the reproductive phase and dispersal phase was quantified using RT-qPCR, respectively. Moreover, the E. coli-expressed and -purified cystatin was injected into the white-eyed honeybee pupae, and its effects on the survival, the weight of the newly emerged bee, and the transcriptome were determined. The results showed that cystatin was significantly upregulated in mites during the reproductive phase. Cystatin significantly shortened the lifespan of pupae and decreased the weight of the newly emerged bees. Transcriptome sequencing showed that cystatin upregulated 1496 genes and downregulated 1483 genes in pupae. These genes were mainly enriched in ATP synthesis, the mitochondrial respiratory chain, and cuticle structure and function. Cystatin comprehensively downregulated the metabolism of carbohydrates, fatty acids, and amino acids, and energy production in the pupae. The downregulation of metabolic activity could save more nutrients and energy for V. destructor, helping it to maximize its reproduction potential, implying that the mite could manipulate the metabolism of host bees through the injected salivary secretion. The results provide new insights into mite-bee interactions, providing a basis for related studies and applications.

A DNA Plasmid-Based Approach for Efficient Synthesis of Sacbrood Virus Infectious Clones within Host Cells.

RNA viruses are often cited as a significant factor affecting the populations of both domestic honey bees and wild pollinators. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail the in vitro transcription of the viral genome RNA in 8-10 kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of the Chinese variant sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a widely used lepidopteran immortal cell line (Sf9) and honey bee pupae. Furthermore, the negative strand of the clone was detected in both Sf9 cells and honey bee pupae, indicating active infection and replication. However, the transfection of Sf9 cells was observed in only a limited proportion (less than 10%) of the cells, and the infection did not appear to spread to adjacent cells or form infective virions. The injection of honey bee pupae with 2500 ng of the IE1-SBV plasmid resulted in high infection rates in Apis cerana pupae but low rates in A. mellifera pupae, although the dosage was comparatively high compared with other studies using in vitro transcribed viral RNA. Our findings suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, albeit requiring additional optimization. However, this method holds substantial potential for facilitating the production of clones with various sequence modifications, enabling the exploration of viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid form may foster collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately offering a comprehensive approach to managing virus diseases in the future.

Exploring RNA methylation as a promising biomarker for assessing sublethal effects of fipronil on honeybees (Apis mellifera L.).

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.

Field recommended concentrations of pyraclostrobin exposure disturb the development and immune response of worker bees (Apis mellifera L.) larvae and pupae.

The strobilurin fungicide pyraclostrobin is widely used to prevent and control the fungal diseases of various nectar and pollen plants. Honeybees also directly or indirectly contact this fungicide with a long-term exposure period. However, the effects of pyraclostrobin on the development and physiology of Apis mellifera larvae and pupae during continuous exposure have been rarely known. To investigate the effects of field-realistic concentrations of pyraclostrobin on honeybee survival and development, the 2-day-old larvae were continuously fed with different pyraclostrobin solutions (100 mg/L and 83.3 mg/L), and the expression of development-, nutrient-, and immune-related genes in larvae and pupae were examined. The results showed that two field-realistic concentrations of pyraclostrobin (100 and 83.3 mg/L) significantly decreased the survival and capped rate of larvae, the weight of pupae and newly emerged adults, and such decrease was a positive correlation to the treatment concentrations. qPCR results showed that pyraclostrobin could induce the expression of Usp, ILP2, Vg, Defensin1, and Hymenoptaecin, decrease the expression of Hex100, Apidaecin, and Abaecin in larvae, could increase the expression of Ecr, Usp, Hex70b, Vg, Apidaecin, and Hymenoptaecin, and decreased the expression of ILP1, Hex100 and Defensin1in pupae. These results reflect pyraclostrobin could decrease nutrient metabolism, immune competence and seriously affect the development of honeybees. It should be used cautiously in agricultural practices, especially in the process of bee pollination.

Person Re-Identification with Improved Performance by Incorporating Focal Tversky Loss in AGW Baseline.

Person re-identification (re-ID) is one of the essential tasks for modern visual intelligent systems to identify a person from images or videos captured at different times, viewpoints, and spatial positions. In fact, it is easy to make an incorrect estimate for person re-ID in the presence of illumination change, low resolution, and pose differences. To provide a robust and accurate prediction, machine learning techniques are extensively used nowadays. However, learning-based approaches often face difficulties in data imbalance and distinguishing a person from others having strong appearance similarity. To improve the overall re-ID performance, false positives and false negatives should be part of the integral factors in the design of the loss function. In this work, we refine the well-known AGW baseline by incorporating a focal Tversky loss to address the data imbalance issue and facilitate the model to learn effectively from the hard examples. Experimental results show that the proposed re-ID method reaches rank-1 accuracy of 96.2% (with mAP: 94.5) and rank-1 accuracy of 93% (with mAP: 91.4) on Market1501 and DukeMTMC datasets, respectively, outperforming the state-of-the-art approaches.

A Simple High-Throughput Field Sample Preparation Method Based on Matrix-Induced Sugaring-Out for the Simultaneous Determination of 5-Hydroxymethylfurfural and Phenolic Compounds in Honey.

In the present work, a high-throughput field sample preparation method was reported for the simultaneous determination of 5-hydroxymethylfurfural and phenolic compounds in honey. Combining a simple and green homogenous liquid−liquid extraction, matrix-induced sugaring-out, with the use of a 96-deepwell plate and multichannel pipette, the proposed method showed its merits in instrument-free and high-throughput preparation. Due to the high-throughput property, the parameters of the method were rapidly and systematically studied using a constructed 4 × 2 × 4 × 3 array (sample amount × ratio of ACN:H2O × standing time × replicates) in a 96-deepwell plate. Analytical performance was fully validated, and the limits of detection and limits of quantification were in the range of 0.17−1.35 µg/g and 0.51−4.14 µg/g, respectively. Recoveries were between 83.98 and 117.11%, and all the precisions were <5%. Furthermore, the developed method was successfully applied in the outdoor preparation of commercial honey samples and the in-field preparation of raw honey samples in apiary. The current work presented a simple, rapid, and high-throughput method for the field sample preparation of honey and provides a valuable strategy for the design of field and on-site sample preparation.

Dipicolinic Acid-Tb3+/Eu3+ Lanthanide Fluorescence Sensor Array for Rapid and Visual Discrimination of Botanical Origin of Honey.

In the present study, a lanthanide fluorescence sensor array was developed for the discrimination of honey's botanical origin. Dipicolinic acid (DPA) was used as the antenna ligand for sensitizing the fluorescence of Tb3+ and Eu3+ to prepare the DPA-Tb3+/Eu3+ complex. This lanthanide fluorescence sensor showed a cross-reactive response to the major constituents of honey, which led to the result that different classes of honey solution exhibited distinct quenching effects on the fluorescence of the DPA-Tb3+/Eu3+ complex. Furthermore, a fluorescence sensor array composed of ten sensors was constructed by adjusting the pH and the component of the DPA-Tb3+/Eu3+ complex to show multivariate responses towards honey. The visual fluorescence image of the sensor array was recorded by using a smartphone under excitation with portable UV lamp. Results indicated that the pattern of the visual image was related with the botanical origin. After extracting the RGB value of each sensor in 96-well plate, the ratio of R/G was used for principal component analysis (PCA). The results showed that three classes of honey (astragalus, logan, and litchi) were well distinguished. Moreover, the value of principal component 1 (PC1) showed good linearity with the composition of mixing honey and could be used for semi-quantitative analysis. The proposed lanthanide fluorescence sensor array presents a visual and portable method for the discrimination of a honey's origin without the use of analytical instruments, and might provide a novel and simple strategy for the measurement of food origin.

Extremely Low-Frequency Electromagnetic Field Impairs the Development of Honeybee (Apis cerana).

Increasing ELF-EMF pollution in the surrounding environment could impair the cognition and learning ability of honeybees, posing a threat to the honeybee population and its pollination ability. In a social honeybee colony, the numbers of adult bees rely on the successful large-scale rearing of larvae and continuous eclosion of new adult bees. However, no studies exist on the influence of ELF-EMFs on honeybee larvae. Therefore, we investigated the survival rate, body weight, and developmental duration of first instar larvae continuously subjected to ELF-EMF exposure. Moreover, the transcriptome of fifth instar larvae were sequenced for analyzing the difference in expressed genes. The results showed that ELF-EMF exposure decreases the survival rate and body weight of both white-eye pupae and newly emerged adults, extends the duration of development time and seriously interferes with the process of metamorphosis and pupation. The transcriptome sequencing showed that ELF-EMF exposure decreases the nutrient and energy metabolism and impedes the degradation of larvae tissues and rebuilding of pupae tissues in the metamorphosis process. The results provide an experimental basis and a new perspective for the protection of honeybee populations from ELF-EMF pollution.

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.

Procedures and potential pitfalls for constructing a bee-infecting RNA virus clone.

Viruses are factors that can fluctuate insect populations, including honey bees. Most honey bee infecting viruses are single positive-stranded RNA viruses that may not specifically infect honey bees and can be hazardous to other pollinator insects. In addition, these viruses could synergize with other stressors to worsen the honey bee population decline. To identify the underlying detailed mechanisms, reversed genetic studies with infectious cDNA clones of the viruses are necessary. Moreover, an infectious cDNA clone can be applied to studies as an ideal virus isolate that consists of a single virus species with a uniform genotype. However, only a few infectious cDNA clones have been reported in honey bee studies since the first infectious cDNA clone was published four decades ago. This article discusses steps, rationales, and potential issues in bee-infecting RNA virus cloning. In addition, failed experiences of cloning a Deformed wing virus isolate that was phylogenetically identical to Kakugo virus were addressed. We hope the information provided in this article can facilitate further developments of reverse-genetic studies of bee-infecting viruses to clarify the roles of virus diseases in the current pollinator declines.

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.

Sublethal effects of Isoclast™ Active (50% sulfoxaflor water dispersible granules) on larval and adult worker honey bees (Apis mellifera L.).

Sulfoxaflor is a novel sulfoximine insecticide which is widely used to control crop pests. Risk assessments have reported its high toxicity to pollinators. However, sulfoxaflor is not persistent in the environment and few studies have addressed its negative effects on larval and newly emerged honeybees at environmentally relevant concentrations. In the present study, the sublethal effects of a sulfoxaflor commercial product, Isoclast™ Active, were evaluated in the laboratory using larvae and newly emerged worker honeybees. The results of 96-h acute toxicity showed that Isoclast is moderately toxic to adult bees, and it could induce significant death and growth failure of larvae after continuous dietary intake. In addition, Isoclast induced significant changes in antioxidative (SOD, CAT), lipid peroxidation (POD, LPO, MDA), detoxification (GST, GR, GSH) and signal transduction-related (AChE, ACh) enzymes or products both in larvae and adult honey bees under residue levels. Here we firstly reported the lethal and sublethal effects of commercial sulfoxaflor to honeybees' larvae and young workers. All these findings revealed the potential risks of sulfoxaflor residue in environment to honey bees, and may also to other pollinators. This is a laboratory mimic studies, and further studies are still needed to investigate the risks and in-depth mechanisms of sulfoxaflor to bees in field.

A novel method for the detection and diagnosis of virus infections in honey bees.

In terms of infectious diseases caused by a variety of microorganisms, the ability to promptly and accurately identify the causative agents is the first step on the path to all types of effective management of such infections. Among the various factors that are affecting global bee health, viruses have often been linked to honey bee colony losses and they pose a serious threat to the fraction of agriculture that depends on the service of pollinators. Over the past few decades, PCR-based molecular methods have provided powerful tools for rapid, specific, and sensitive detection and the quantification of difficult-to-grow pathogenic microorganisms such as viruses in honey bees. However, PCR-based methods require nucleic acid extraction and purification, which can be quite laborious and time-consuming and they involve the use of organic solvents and chaotropic agents like phenol and chloroform which are volatile and highly toxic. In response, we developed a novel and non-sacrificial method for detecting viral infections in honey bees. As little as 1 µl of hemolymph was collected from adult workers, larvae, and queens of bee colonies by puncturing the soft inter-tergal integument between the second and third dorsal tergum with a fine glass capillary. The hemolymph was then diluted and subjected to RT-PCR analysis directly. The puncture wound caused by the glass capillary was found to heal automatically and rapidly without any trouble and the lifespan of the experimental workers remained unaffected. Using this method, we detected multiple viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV), and Sacbrood virus (SBV) in infected bees. Furthermore, expressed transcripts that indicate the induction of innate immune response to the virus infections were also detected in the hemolymph of infected bees. The simplicity and cost-effectiveness of this innovative approach will allow it to be a valuable, time-saving, safer, and more environmentally friendly contribution to bee disease management programs.

Antibiotic Treatment Decrease the Fitness of Honeybee (Apis mellifera) Larvae.

Symbiotic bacteria could increase the nutrient provision, regulate the physiological state, and promote immunity in their insect host. Honeybee larvae harbor plenty of bacteria in their gut, but their functions are not well studied. To determine their effect on honeybee larvae, the 1-day-old larvae were grafted on to 24-well plates from the comb and artificially reared in the lab. They were treated with penicillin-streptomycin to remove the gut symbiotic bacteria. Then, the 5-day-old larvae and the newly emerged adults were weighted. The developmental periods to pupae and eclosion were investigated, respectively. The bacterial amount, expression of developmental regulation genes (ecr and usp), nutrient metabolism genes (ilp1, ilp2, hex 70a, hex 70b, hex 70c, and hex 110), and immunity genes (apidaecin, abaecin, defensin-1, and hymenoptaecin) were determined by qRT-PCR. The result showed that the antibiotics-treated larvae have significantly lower body weights in the 5-day-old larvae and the emerged bees. The expression of ilp2 and hex 70c in 5-day-old larvae was down-regulated. The usp was down-regulated in 5-day-old larvae, but increased in 7-day-old larvae, which disturbed the normal developmental process and caused the extension of eclosion. Moreover, antibiotics treatment significantly decreased the expression of apidaecin and abaecin in 5-day-old larvae, and defensin-1 and hymenoptaecin in 7-day-old larvae, respectively. These results showed that antibiotics could weaken the nutrient metabolism, disturb the development process, and decrease the immune competence of honeybee larvae, indicating the vital roles of gut bacteria in bee larvae fitness, so the antibiotics should be avoided to control microbial disease in honeybee larvae.

Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Deep Learning-Based Real-Time Multiple-Person Action Recognition System.

Action recognition has gained great attention in automatic video analysis, greatly reducing the cost of human resources for smart surveillance. Most methods, however, focus on the detection of only one action event for a single person in a well-segmented video, rather than the recognition of multiple actions performed by more than one person at the same time for an untrimmed video. In this paper, we propose a deep learning-based multiple-person action recognition system for use in various real-time smart surveillance applications. By capturing a video stream of the scene, the proposed system can detect and track multiple people appearing in the scene and subsequently recognize their actions. Thanks to high resolution of the video frames, we establish a zoom-in function to obtain more satisfactory action recognition results when people in the scene become too far from the camera. To further improve the accuracy, recognition results from inflated 3D ConvNet (I3D) with multiple sliding windows are processed by a nonmaximum suppression (NMS) approach to obtain a more robust decision. Experimental results show that the proposed method can perform multiple-person action recognition in real time suitable for applications such as long-term care environments.

Iterative Pose Refinement for Object Pose Estimation Based on RGBD Data.

Accurate estimation of 3D object pose is highly desirable in a wide range of applications, such as robotics and augmented reality. Although significant advancement has been made for pose estimation, there is room for further improvement. Recent pose estimation systems utilize an iterative refinement process to revise the predicted pose to obtain a better final output. However, such refinement process only takes account of geometric features for pose revision during the iteration. Motivated by this approach, this paper designs a novel iterative refinement process that deals with both color and geometric features for object pose refinement. Experiments show that the proposed method is able to reach 94.74% and 93.2% in ADD(-S) metric with only 2 iterations, outperforming the state-of-the-art methods on the LINEMOD and YCB-Video datasets, respectively.

Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein.

Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3' untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3'-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.