Honey Bee Genes Research Articles

A field realistic model to assess the effects of pesticides residues and adulterants on honey bee gene expression.

While studies on the sublethal effects of chemical residues in beeswax on adult honey bees are increasing, the study protocols assessing the impacts on honey bee brood in realistic conditions still need to be investigated. Moreover, little is known about the residue's effect on gene expression in honey bee brood. This study reports the effects of chlorpyriphos-ethyl, acrinathrin and stearin worker pupae exposure through contaminated or adulterated beeswax on the gene expression of some key health indicators, using a novel in vivo realistic model. Larvae were reared in acrinathrin (12.5, 25, 10 and 100 ppb) and chlorpyriphos-ethyl (5, 10, 500 and 5000 ppb) contaminated or stearin adulterated beeswax (3, 4, 5, 6 and 9%) in newly formed colonies to reduce the influence of external factors. On day 11, mortality rates were assessed. Honey bee pupae were extracted from the comb after 19 days of rearing and were analysed for the gene expression profile of four genes involved in the immune response to pathogens and environmental stress factors (Imd, dorsal, domeless and defensin), and two genes involved in detoxifications mechanisms (CYP6AS14 and CYP9Q3). We found no linear relation between the increase in the pesticide concentrations and the brood mortality rates, unlike stearin where an increase in stearin percentage led to an exponential increase in brood mortality. The immune system of pupae raised in acrinathrin contaminated wax was triggered and the expression of CYP6AS14 was significantly upregulated (exposure to 12.5 and 25 ppb). Almost all expression levels of the tested immune and detoxification genes were down-regulated when pupae were exposed to chlorpyrifos-contaminated wax. The exposure to stearin triggered the immune system and detoxification system of the pupae. The identification of substance-specific response factors might ultimately serve to identify molecules that are safer for bees and the ecosystem's health.

Effects of abamectin nanocapsules on bees through host physiology, immune function, and gut microbiome

Pesticide usage is a common practice to increase crop yields. Nevertheless, the existence of pesticide residues in the surrounding environment presents a significant hazard to pollinators, specifically the potential undisclosed dangers related to emerging nanopesticides. This study examines the impact of abamectin nanocapsules (AbaNCs), created through electrostatic self-assembly, as an insecticide on honey bees. It was determined that AbaNCs upregulated detoxification genes, including CYP450, as well as antioxidant and immune genes in honey bees. Furthermore, AbaNCs affected the activity of crucial enzymes such as superoxide dismutase (SOD). Although no apparent damage was observed in bee gut tissue, AbaNCs significantly decreased digestive enzyme activity. Microbiome sequencing revealed that AbaNCs disrupted gut microbiome, resulting in a reduction of beneficial bacteria such as Bifidobacterium and Lactobacillus. Additionally, these changes in the gut microbiome were associated with decreased activity of digestive enzymes, including lipase. This study enhances our understanding of the impact of nanopesticides on pollinating insects. Through the revelation of the consequences arising from the utilization of abamectin nanocapsules, we have identified potential stress factors faced by these pollinators, enabling the implementation of improved protective measures.

Genetic Diversity of Kırklareli Honey Bee (Apis mellifera L.) Populations in Thrace Region of Turkey: Identification of Mitochondrial COI and ND5 Gene Regions

The main goal of genetic resource conservation is to keep as much genetic diversity as possible within each species. In this respect, some difficulties in the protection of honey bee gene resources make it necessary to reveal the genetic structures of the subspecies and the genetic relationships between the subspecies. In this study, Kırklareli honey bee populations which were officially registered as an ecotype of Turkey’s honey bee (Apis mellifera L.) gene resources by the Republic of Turkey, Ministry of Agriculture and Forestry, were examined in the COI and ND5 genes of mitochondrial DNA. The restriction fragment length polymorphism (RFLP) together with the polymerase chain reaction (PCR) was used to define Apis mellifera populations. A total of 117 worker bee samples were used which were collected from mostly the Kırklareli province. A newly found single nucleotide polymorphism (SNP), G→A transition in the COI gene region formed a novel NcoI restriction site resulting in a new haplotype. This new haplotype has been abbreviated as haplotype C. As a result of the COI/SspI digestion, the previously reported C haplotype was determined. No restriction was found with the treatment of COI/StyI enzyme. On the other hand, as a result of ND5/AluI restriction, 2 restriction site and previously reported haplotype C was obtained in all of the studied samples. No restriction was screened with ND5/FokI and ND5/HincII enzymes in the whole samples, only a reported uncut B haplotype was observed. Within this study, novel genetic information has been revealed for the Kırklareli honey bee ecotype registered as the Thrace honey bee of Turkey’s honey bee gene resources. Moreover, detailed studies with larger sample sizes should be conducted to characterize the origin and the subspecies of Kırklareli honey bees in detail. It is thought that this study will be useful in the identification and registration of the Kırklareli honey bees to be carried out in the future, and also in the creation of a database.

Monitoring Immune Modulation in Season Population: Identifying Effects and Markers Related to Apis mellifera ligustica Honey Bee Health.

Honey bees play a significant role in ecology, producing biologically active substances used to promote human health. However, unlike humans, the molecular markers indicating honey bee health remain unknown. Unfortunately, numerous reports of honey bee collapse have been documented. To identify health markers, we analyzed ten defense system genes in Apis mellifera ligustica honey bees from winter (Owb) and spring (Fb for foragers and Nb for newly emerged) populations sampled in February and late April 2023, respectively. We focused on colonies free from SBV and DWV viruses. Molecular profiling revealed five molecular markers of honey bee health. Of these, two seasonal molecular markers-domeless and spz genes-were significantly downregulated in Owb compared to Nb and Fb honey bees. One task-related marker gene, apid-1, was identified as being downregulated in Owb and Nb compared to Fb honey bees. Two recommended general health markers, SOD and defensin-2, were upregulated in honey bees. These markers require further testing across various honey bee subspecies in different climatic regions. They can diagnose bee health without colony intervention, especially during low-temperature months like winter. Beekeepers can use this information to make timely adjustments to nutrients or heating to prevent seasonal losses.

Effects of Clothianidin Pesticide Application on the Strength of Honey Bee Colonies and Stress-Related Genes in the Vicinity of Rice Fields in the Republic of Korea

The honey bee, a crucial organism that provides ecological and economic value to humans, is currently facing threats from various environmental factors including pesticides. Numerous studies have been conducted to demonstrate the risks associated with neonicotinoid pesticides, but research on their occurrence in actual field conditions has not been identified. Therefore, in this study, we observed changes in honey bee (Apis mellifera) colonies located near rice cultivation areas as they progressed beyond the rice pesticide application period. Furthermore, we collected honey bees exposed to the clothianidin and analyzed their stress-related gene expression. The results showed that the foraging behavior of honey bee colonies located near rice cultivation areas did not exhibit significant differences between the treatment sites (Cheongyang and Gimje) and the control site (Wanju) during the experimental period. However, it was observed that the expression levels of stress-related genes in honey bees collected from the treatment group were significantly higher than those in the control. Most of the stress-related genes were associated with detoxification processes in response to pesticides. As a result, pesticide treatment in proximity to rice cultivation areas did not cause direct damage to honey bees but had an indirect impact, suggesting the potential for ongoing chronic damage.

Honey bee Apis mellifera L. responses to oxidative stress induced by pharmacological and pesticidal compounds

The western honey bee, Apis mellifera L., is a eusocial insect that plays a significant role in ecosystem balances and the pollination of plants and food crops. Honey bees face multiple biotic and abiotic stressors, such as pathogens, diseases, chemical pesticides, and climate change, which all contribute to honey bee colony loss. This study investigated the impacts of multiple pharmacological and pesticidal molecules on honey bee survival and gene regulation responses. In an 11-day cage experiment, sublethal doses of tunicamycin, thapsigargin, metformin, paraquat, hydrogen peroxide, and imidacloprid were administered to newly emerged sister bees. Daily treatment consumption and mortality were recorded, as well as the transcription expression of 12 major genes (AChE-2, Apisimin, Apidaecin, mrjp1, Sodq, cp450, SelT, SelK, Ire1, Xbp1, Derl-1, and Hsc70), some of which are markers of oxidative and endoplasmic reticulum (ER) stresses in honey bees. At day 9 of the treatments, protein damage was quantified in caged bees. A Kaplan–Meier model indicated significant (p < 0.001) toxicological effects of paraquat, H2O2, and tunicamycin on bee survivorship compared with controls with better survival for other molecules. Post-ingestive aversion responses were recorded only for tunicamycin, hydrogen peroxide, and imidacloprid. Nonetheless, significantly higher protein damage on day 9 was identified only in bees exposed to paraquat and imidacloprid. Some antioxidant genes were significantly regulated vis-à-vis specific treatments. Our results reveal age-related regulation of other major genes with significant inter-gene positive correlations.

Methionine can reduce the sublethal risk of Chlorantraniliprole to honeybees (Apis mellifera L.): Based on metabolomics analysis

Bees, essential for pollination in agriculture and global economic growth. However, the great wax moth (Galleria mellonella, GWM), a Lepidopteran insect, poses a substantial threat to bee colonies, contributing to a global decline in bee populations. Chlorantraniliprole (CH) is one of the primary insecticide used to control GWM due to its efficacy and low toxicity to bees. To improve beekeeping safety and reduce the risk of GWM developing resistance to prolonged use of CH, we investigated the potential of combining methionine (MET) which has been found to have insecticidal activity against certain Lepidoptera pests, with chlorantraniliprole for use in the apiculture industry. This study assessed the combined effects of MET and CH on GWM and honeybees by employing the maximum concentration of MET (1 %, w/w), previously reported as safe for honeybees, and the practical concentration of CH (1 mg/kg) for GWM control. The results revealed limited acute lethal toxicity of MET to GWM and honeybees, whereas the combined chronic exposure of MET and CH (MIX) led to significant synergistic lethal effects on GWM mortality. Nevertheless, the protective effect of MET on honeybees exposed to CH was significant under chronic exposure. Potential mechanisms underlying the synergistic actions of MET and CH may stem from MET-induced protection of the "Cysteine and methionine" and the "Glycine, serine, and threonine" metabolism pathways. Furthermore, immune stress mitigation was also observed in honeybee immune-related gene transcripts treated by the combination of MET and CH under both acute and chronic exposure. The effects of MET on CH activity in GWM and honeybees are likely due to metabolic regulation. This study suggests the potential of developing MET as a promising biopesticide or protective agent in the future.

Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis.

It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.

Transcriptomic Responses Underlying the High Virulence of Black Queen Cell Virus and Sacbrood Virus following a Change in Their Mode of Transmission in Honey Bees (Apis mellifera).

Over the last two decades, honey bees (Apis mellifera) have suffered high rates of colony losses that have been attributed to a variety of factors, chief among which are viral pathogens, such as deformed wing virus (DWV), whose virulence has increased because of vector-based transmission by the invasive, ectoparasitic varroa mite (Varroa destructor). A shift in the experimental mode of transmission of the black queen cell virus (BQCV) and sacbrood virus (SBV) from fecal/food-oral (direct horizontal) to vector-mediated (indirect horizontal) transmission also results in high virulence and viral titers in pupal and adult honey bees. Agricultural pesticides represent another factor that acts independently or in interaction with pathogens, and they are also thought to cause colony loss. Understanding the molecular mechanisms underlying the higher virulence following a vector-based mode of transmission provides deeper insight into honey bee colony losses, as does determining whether or not host-pathogen interactions are modulated by exposure to pesticides. Through an experimental design with controlled laboratory, we investigated the effects of the modes of transmission of BQCV and SBV (feeding vs. vector-mediated via injection) alone or in combination with chronic exposure to sublethal and field-realistic concentrations of flupyradifurone (FPF), a novel agricultural insecticide, on honey bee survival and transcription responses by using high-throughput RNA sequencing (RNA-seq) analysis. Co-exposure to viruses via feeding (VF) or injection (VI) and FPF insecticide had no statistically significant interactive effect on their survival compared to, respectively, VF or VI treatments alone. Transcriptomic analysis revealed a distinct difference in the gene expression profiles of bees inoculated with viruses via injection (VI) and exposed to FPF insecticide (VI+FPF). The number of differentially expressed genes (DEGs) at log2 (fold-change) > 2.0 in VI bees (136 genes) or/and VI+FPF insecticide (282 genes) was very high compared to that of VF bees (8 genes) or the VF+FPF insecticide treatment (15 genes). Of these DEGs, the expression in VI and VI+FPF bees of some immune-related genes, such as those for antimicrobial peptides, Ago2, and Dicer, was induced. In short, several genes encoding odorant binding proteins, chemosensory proteins, odor receptors, honey bee venom peptides, and vitellogenin were downregulated in VI and VI+FPF bees. Given the importance of these suppressed genes in honey bees' innate immunity, eicosanoid biosynthesis, and olfactory associative function, their inhibition because of the change in the mode of infection with BQCV and SBV to vector-mediated transmission (injection into haemocoel) could explain the high virulence observed in these viruses when they were experimentally injected into hosts. These changes may help explain why other viruses, such as DWV, represent such a threat to colony survival when transmitted by varroa mites.

Gut microbiota composition and gene expression changes induced in the Apis cerana exposed to acetamiprid and difenoconazole at environmentally realistic concentrations alone or combined.

Apis cerana is an important pollinator of agricultural crops in China. In the agricultural environment, A. cerana may be exposed to acetamiprid (neonicotinoid insecticide) and difenoconazole (triazole fungicide), alone or in combination because they are commonly applied to various crops. At present, our understanding of the toxicological effects of acetamiprid and difenoconazole on honey bee gut microbiomes is limited. The primary objective of this study was to explore whether these two pesticides affect honey bees' gut microbiota and to analyze the transcriptional effects of these two pesticides on honey bees' head and gut. In this study, adults of A. cerana were exposed to acetamiprid and/or difenoconazole by contaminated syrup at field-realistic concentrations for 10days. Results indicated that acetamiprid and/or difenoconazole chronic exposure did not affect honey bees' survival and food consumption, whereas difenoconazole decreased the weight of honey bees. 16S rRNA sequencing suggested that difenoconazole and the mixture of difenoconazole and acetamiprid decreased the diversity index and shaped the composition of gut bacteria microbiota, whereas acetamiprid did not impact the gut bacterial community. The ITS sequence data showed that neither of the two pesticides affected the fungal community structure. Meanwhile, we also observed that acetamiprid or difenoconazole significantly altered the expression of genes related to detoxification and immunity in honey bees' tissues. Furthermore, we observed that the adverse effect of the acetamiprid and difenoconazole mixture on honey bees' health was greater than that of a single mixture. Taken together, our study demonstrates that acetamiprid and/or difenoconazole exposure at field-realistic concentrations induced changes to the honey bee gut microbiome and gene expression.

The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin (vg) and superoxide-dismutase-1 (sod-1) genes and mortality of bees infected with N. ceranae. Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased (p < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Non-optimal ambient temperatures aggravate insecticide toxicity and affect honey bees Apis mellifera L. gene regulation

In this study, we conducted a transcriptional analysis of five honey bee genes to examine their functional involvement vis-à-vis ambient temperatures and exposure to imidacloprid. In a 15-day cage experiment, three cohorts of one-day-old sister bees emerged in incubators, were distributed into cages, and maintained at three different temperatures (26 °C, 32 °C, 38 °C). Each cohort was fed a protein patty and three concentrations of imidacloprid-tainted sugar (0 ppb, 5 ppb and 20 ppb) ad libitum. Honey bee mortality, syrup and patty consumption were monitored daily over 15 days. Bees were sampled every three days for a total of five time points. RT-qPCR was used to longitudinally assess gene regulation of Vg, mrjp1, Rsod, AChE-2 and Trx-1 using RNA extracted from whole bee bodies. Kaplan–Meier models show that bees kept at both non-optimal temperatures (26 °C and 38 °C) were more susceptible to imidacloprid, with significantly higher mortality (P < 0.001 and P < 0.01, respectively) compared to the control. At 32 °C, no differences in mortality (P = 0.3) were recorded among treatments. In both imidacloprid treatment groups and the control, the expression of Vg and mrjp1 was significantly downregulated at 26 °C and 38 °C compared to the optimal temperature of 32 °C, indicating major influence of ambient temperature on the regulation of these genes. Within the ambient temperature groups, both imidacloprid treatments exclusively downregulated Vg and mrjp1 at 26 °C. AChE-2 and the poorly characterized Rsod gene were both consistently upregulated at the highest temperature (38 °C) compared to the ideal temperature (32 °C) in all treatment groups. Trx-1 showed no effect to both temperature and imidacloprid treatments and was regulated in an age-related manner. Overall, our results indicate that ambient temperatures amplify imidacloprid toxicity and affect honey bee gene regulation.

Comparative transcriptome analysis of adult worker bees under short-term heat stress

High temperature affects behavior, physiology, survival, and the expression of related genes in adult honeybees. Apis mellifera is the common pollinator in greenhouse and is susceptible to high temperature stress. To further explore the molecular basis related to heat stress, we compared the transcriptome profiles of adult worker bees at 25 and 45°C, and detected the expression patterns of some differentially expressed genes (DEGs) in different tissues by q RT-PCR. Differential expression analysis showed that 277 DEGs were identified, including 167 genes upregulated and 110 genes downregulated after heat stress exposure in adult worker bees. In GO enrichment analysis, DEGs were mostly enriched for protein folding, unfold protein binding, and heme binding terms. Protein processing in endoplasmic reticulum and longevity regulating pathway-multiple species were significantly enriched in KEGG. The expression levels of 16 DEGs were consistent with the transcriptome results. The expression patterns of 9 DEGs in different tissues revealed high levels in the thorax, which was supposed that the thorax may be the most important part in the response to heat stress. This study provided valuable data for exploring the function of heat resistance-related genes.

Validation of reference genes for quantitative real‐time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

AbstractIn quantitative real‐time polymerase chain reaction (qRT‐PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up‐ or down‐regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT‐PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT‐PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.

Analysis of Honey Export Potential and Competitiveness of Türkiye

The rich vegetation of Türkiye, different climatic zones and honey bee gene resources make the country`s beekeeping activities advantageous. Türkiye has an important potential with the presence of colonies and honey production. In 2021, 96344 tons of honey was produced with 8733394 colonies, and 9994 tons of honey was exported to 66 countries for 31148000 dollars. This study was designed to reveal the honey export structure and competitiveness of Türkiye in the 2002-2021 period. In Türkiye`s honey trade, the USA (29.64%), Germany (22.32%) and Spain (7.22%) are the main importing countries. It has been determined that Türkiye is a net exporter country in the 2002–2021 periods. Türkiye is predominantly a country with medium and weak comperative advantage. The Revealed Comparative Advantages (RCA), Revealed Symmetric Comparative Advantages (RSCA), Trade Balance (TBI) values calculated with Türkiye`s 2021 data was found 1.13, 0.06 and 0.98 respectively. These values show that Türkiye is a net exporter and a highly competitive country with a declared comparative advantage. Providing Türkiye`s access to rapidly growing markets in direct proportion to its beekeeping capacity and potential will make Türkiye a more competitive country.

Dietary Supplement of Grape Wastes Enhances Honeybee Immune System and Reduces Deformed Wing Virus (DWV) Load.

Ingredients rich in phenolic compounds and antioxidants of winemaking wastes, which play an important role in the prevention of various diseases and the control of viruses, are being explored. Currently, there is a concern about honeybee population loss, with deformed wing virus (DWV) being the most common virus infecting apiaries and one of the main causes of honeybee decline. Hence, the effect of grape pomace powder (GPP) as a dietary supplement to enhance the immune system of honeybees affected by DWV was evaluated. The characteristics of the ingredient GPP, obtained by spray-drying, revealed a high anthocyanin content (1102.45 mg 100 g-1), and it was applied at doses of 0.5, 1, 2.5 and 5% as a dietary supplement for bees infected by DWV. The results showed that the GPP treatments strengthened the immune response of honeybees against DWV. Moreover, the expression of the Relish gene was significantly higher in bees fed with GPP compared to the infected control. This study, which is framed in the search of food waste valorization for environmental sustainability, proves the feasibility of using grape wastes as dietary supplements for pollinators, and provides knowledge of the influence of polyphenols on the expression profiles of immune-related genes in honeybees.

Molecular cloning and characterization of a lipase from the honeybee Apis mellifera

• Am-Lipase protein preferentially degrades triglycerides with long-chain fatty acids. • Am-Lipase is highly expressed in the fat body of foragers in comparison with the nurses. • Am-Lipase plays a role in the fat body for lipid metabolism. Lipids perform essential and important functions, such as serving as an energy source for growth, development, reproduction, flight, starvation, and diapause in insects. Lipases are key enzymes involving in lipid metabolism and have been reported in several insect species. However, the molecular characterization of the pancreatic triacylglycerol lipase-like genes in honeybees has not been elucidated. Here, we describe the cDNA cloning and characterization of lipase from the honeybee Apis mellifera (Am-Lipase). Am-Lipase consists of 321 amino acids, including a Ser-Glu-His catalytic triad and a consensus active site motif GXSXG. Recombinant Am-Lipase protein degrades triglycerides, preferentially catalyzing the hydrolysis of long-chain fatty acids. Furthermore, the expression pattern of Am-Lipase seems to be a fat body-specific lipase, shows higher expression in forager bees, and is decreased under starvation conditions. Thus, our results suggest that Am-Lipase plays a role in the utilization of lipids stored in the fat body for lipid metabolism.

Chemical Stimulants and Stressors Impact the Outcome of Virus Infection and Immune Gene Expression in Honey Bees (Apis mellifera).

Western honey bees (Apis mellifera) are ecologically, agriculturally, and economically important plant pollinators. High average annual losses of honey bee colonies in the US have been partially attributed to agrochemical exposure and virus infections. To examine the potential negative synergistic impacts of agrochemical exposure and virus infection, as well as the potential promise of phytochemicals to ameliorate the impact of pathogenic infections on honey bees, we infected bees with a panel of viruses (i.e., Flock House virus, deformed wing virus, or Sindbis virus) and exposed to one of three chemical compounds. Specifically, honey bees were fed sucrose syrup containing: (1) thyme oil, a phytochemical and putative immune stimulant, (2) fumagillin, a beekeeper applied fungicide, or (3) clothianidin, a grower-applied insecticide. We determined that virus abundance was lower in honey bees fed 0.16 ppb thyme oil augmented sucrose syrup, compared to bees fed sucrose syrup alone. Parallel analysis of honey bee gene expression revealed that honey bees fed thyme oil augmented sucrose syrup had higher expression of key RNAi genes (argonaute-2 and dicer-like), antimicrobial peptide expressing genes (abaecin and hymenoptaecin), and vitellogenin, a putative honey bee health and age indicator, compared to bees fed only sucrose syrup. Virus abundance was higher in bees fed fumagillin (25 ppm or 75 ppm) or 1 ppb clothianidin containing sucrose syrup relative to levels in bees fed only sucrose syrup. Whereas, honey bees fed 10 ppb clothianidin had lower virus levels, likely because consuming a near lethal dose of insecticide made them poor hosts for virus infection. The negative impact of fumagillin and clothianidin on honey bee health was indicated by the lower expression of argonaute-2, dicer-like, abaecin, and hymenoptaecin, and vitellogenin. Together, these results indicate that chemical stimulants and stressors impact the outcome of virus infection and immune gene expression in honey bees.

The Honey Bee Gene Bee Antiviral Protein-1 Is a Taxonomically Restricted Antiviral Immune Gene.

Insects have evolved a wide range of strategies to combat invading pathogens, including viruses. Genes that encode proteins involved in immune responses often evolve under positive selection due to their co-evolution with pathogens. Insect antiviral defense includes the RNA interference (RNAi) mechanism, which is triggered by recognition of non-self, virally produced, double-stranded RNAs. Indeed, insect RNAi genes (e.g., dicer and argonaute-2) are under high selective pressure. Honey bees (Apis mellifera) are eusocial insects that respond to viral infections via both sequence specific RNAi and a non-sequence specific dsRNA triggered pathway, which is less well-characterized. A transcriptome-level study of virus-infected and/or dsRNA-treated honey bees revealed increased expression of a novel antiviral gene, GenBank: MF116383, and in vivo experiments confirmed its antiviral function. Due to in silico annotation and sequence similarity, MF116383 was originally annotated as a probable cyclin-dependent serine/threonine-protein kinase. In this study, we confirmed that MF116383 limits virus infection, and carried out further bioinformatic and phylogenetic analyses to better characterize this important gene-which we renamed bee antiviral protein-1 (bap1). Phylogenetic analysis revealed that bap1 is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach) and that the majority of bap1 amino acids are evolving under neutral selection. This is in-line with the results from structural prediction tools that indicate Bap1 is a highly disordered protein, which likely has relaxed structural constraints. Assessment of honey bee gene expression using a weighted gene correlation network analysis revealed that bap1 expression was highly correlated with several immune genes-most notably argonaute-2. The coexpression of bap1 and argonaute-2 was confirmed in an independent dataset that accounted for the effect of virus abundance. Together, these data demonstrate that bap1 is a taxonomically restricted, rapidly evolving antiviral immune gene. Future work will determine the role of bap1 in limiting replication of other viruses and examine the signal cascade responsible for regulating the expression of bap1 and other honey bee antiviral defense genes, including coexpressed ago-2, and determine whether the virus limiting function of bap1 acts in parallel or in tandem with RNAi.

Halofuginone triggers a transcriptional program centered on ribosome biogenesis and function in honey bees

We previously found that pharmacological inhibition of prolyl-tRNA synthetase by halofuginone has potent activity against Nosema ceranae, an important pathogen of honey bees. However, we also observed that prolyl-tRNA synthetase inhibition is toxic to bees, suggesting further work is necessary to make this a feasible therapeutic strategy. As expected, we found that pharmacological inhibition of prolyl-tRNA synthetase activity resulted in robust induction of select canonical ATF4 target genes in honey bees. However, our understanding of this and other cellular stress responses in general in honey bees is incomplete. Thus, we used RNAseq to identify novel changes in gene expression after halofuginone treatment and observed induction of genes involved in ribosome biogenesis, translation, tRNA synthesis, and ribosome-associated quality control (RQC). These results suggest that halofuginone, potentially acting through the Integrated Stress Response (ISR), promotes a transcriptional response to ribosome functional impairment in honey bees rather than the response designed to oppose amino acid limitation, which has been observed in other organisms after ISR induction. In support of this idea, we found that cycloheximide (CHX) administration also induced all tested target genes, indicating that this gene expression program could be induced by ribosome stalling in addition to tRNA synthetase inhibition. Only a subset of halofuginone-induced genes was upregulated by Unfolded Protein Response (UPR) induction, suggesting that mode of activation and cross-talk with other cellular signaling pathways significantly influence ISR function and cellular response to its activation. Future work will focus on understanding how the apparently divergent transcriptional output of the ISR in honey bees impacts the health and disease of this important pollinator species.