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Honey bees provides valuable pollination services for world food crops and wild flowering plants which are habitats of many animal species and remove carbon dioxide from the atmosphere, a powerful tool in the fight against climate change. Nevertheless, the honey bee population has been declining and the majority of colony losses occur during the winter.The goal of this study was to understand the mechanisms underlying overwinter colony losses and develop novel therapeutic strategies for improving bee health.First, pathogen prevalence in overwintering bees were screened between 2015 and 2018. Second, RNA sequencing (RNA-Seq) for transcriptional profiling of overwintering honey bees was conducted and qRT-PCR was performed to confirm the results of the differential expression of selected genes. Lastly, laboratory bioassays were conducted to measure the effects of cold challenges on bee survivorship and stress responses and to assess the effect of a novel medication for alleviating cold stress in honey bees.We identified that sirtuin signaling pathway is the most significantly enriched pathway among the down-regulated differentially expressed genes (DEGs) in overwintering diseased bees. Moreover, we showed that the expression of SIRT1 gene, a major sirtuin that regulates energy and immune metabolism, was significantly downregulated in bees merely exposed to cold challenges, linking cold stress with altered gene expression of SIRT1 . Furthermore, we demonstrated that activation of SIRT1 gene expression by SRT1720, an activator of SIRT1 expression, could improve the physiology and extend the lifespan of cold-stressed bees.Our study suggests that increased energy consumption of overwintering bees for maintaining hive temperature reduces the allocation of energy toward immune functions, thus making the overwintering bees more susceptible to disease infections and leading to high winter colony losses. The novel information gained from this study provides a promising avenue for the development of therapeutic strategies for mitigating colony losses, both overwinter and annually.

The remarkably adaptive mite

The most important ectoparasite of the honeybee is the remarkably adaptive mite Varroa destructor. The Varroa mite is a competent vector of Deformed Wing Virus (DWV), the Varroa-virus complex is one of the main factors associated with elevated annual honey bee colony mortality. Micro-RNAs (miRNAs) are small, non-coding RNAs of ~22-24 nucleotides, produced by all plants, animals, and viruses that influence biological processes through post-transcriptional regulation of gene expression. Knowledge of miRNAs and their functional role in mite biology remains limited. This study developed small RNA libraries from male and female V. destructor by utilizing Illumina's small RNA-Seq platform. A total of 101,913,208 and 91,904,732 small RNA reads (>18 nucleotides) from male and female mites were analyzed using the mirDeep2 algorithm. A conservative approach predicted a total of 306 miRNAs, of which 18 were upregulated and 13 were downregulated in female V. destructor compared to males. A qPCR assay validated the expression of selected differentially expressed female Varroa miRNAs. This dataset provides a list of potential miRNA targets involved in regulating vital Varroa biological processes and for deciphering new targets against Varroa and honey bee viruses they carry.

The main objective of this study was to test comparatively the effects of two common insecticides on honey bee Apis mellifera worker’s lifespan, food consumption, mortality, and expression of antioxidant genes. Newly emerged worker bees were exposed to organophosphate insecticide coumaphos, a neonicotinoid imidacloprid, and their mixtures. Toxicity tests were conducted along with bee midgut immunohistological TUNEL analyses. RT-qPCR assessed the regulation of 10 bee antioxidant genes linked to pesticide toxicity. We tested coumaphos at 92,600 ppb concentration, in combination with 5 and 20 ppb imidacloprid. Coumaphos induced significantly higher bee mortality, which was associated with down regulation of catalase compared to coumaphos and imidacloprid (5/20 ppb) mixtures, whereas, both imidacloprid concentrations independently had no effect on bee mortality. Mixture of coumaphos and imidacloprid reduced daily bee consumption of a control food patty to 10 mg from a coumaphos intake of 14.3 mg and 18.4 and 13.7 mg for imidacloprid (5 and 20) ppb, respectively. While coumaphos and imidacloprid mixtures induced down-regulation of antioxidant genes with noticeable midgut tissue damage, imidacloprid induced intensive gene up-regulations with less midgut apoptosis.

Sixteen honey bee (Apis mellifera L.) colonies were placed in four different agricultural landscapes to study the effects of agricultural landscape and exposure to pesticides on honey bee health. Colonies were located in three different agricultural areas with varying levels of agricultural intensity (AG areas) and one nonagricultural area (NAG area). Colonies were monitored for their performance and productivity for one year by measuring colony weight changes, brood production, and colony thermoregulation. Palynological and chemical analyses were conducted on the trapped pollen collected from each colony and location. Our results indicate that the landscape’s composition significantly affected honey bee colony performance and development. Colony weight and brood production were significantly greater in AG areas compared to the NAG area. Better colony thermoregulation in AG areas’ colonies was also observed. The quantities of pesticides measured in the trapped pollen were relatively low compared to their acute toxicity. Unexplained queen and colony losses were recorded in the AG areas, while colony losses because of starvation were observed in the NAG area. Our results indicate that landscape with high urban activity enhances honey bee brood production, with no significant effects on colony weight gain. Our study indicates that agricultural crops provide a valuable resource for honey bee colonies, but there is a trade-off with an increased risk of exposure to pesticides.

In this study, we present phenotypic and genetic data characterizing the impact of imidacloprid and caging stress on honey bee Apis mellifera physiological responses and regulation of 45 genes using targeted-RNA seq. The term ‘caging stress’ characterizes the effects of depriving honey bees of all hive aspects and conditions. Two cohorts of 1 day old sister bees were subjected to different conditions. One cohort was caged and fed different imidacloprid-tainted sugar solutions and the second was marked and introduced back to its natal hive. Physiological bee parameters and diet behavior were monitored daily for caged bees over several weeks. Bee samples from both cohorts were sampled weekly for RNA sequencing and oxidative stress analyses. Imidacloprid induced significant protein damage and post-ingestive aversion responses in caged bees, leading to lower tainted syrup consumption and higher water intake compared with the controls. No differentially expressed genes were observed among caged bees in regards to imidacloprid treatment. However, significant upregulation in antioxidant genes was recorded in caged bees as compared with hive bees, with overwhelming downregulation in all gene categories in caged bees at week 4. We identified two sets of genes that were constantly regulated in caged bees, including Rsod with unknown function in insects that could potentially characterize caging stress in honey bees.

Twenty‐two honeybee (Apis mellifera) colonies were placed in four different cornfield areas in order to study the potential in situ effects of seed‐coated systemic neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Two apiaries were located in two independent neonicotinoid‐treated cornfield areas and two others in two independent untreated cornfield areas used as controls. These experimental hives were extensively monitored for their performance and health traits over a period of one year. Trapped pollen was collected and microscopically identified to define the visited flowers and the amount of corn pollen collected by bees. Liquid chromatography–mass spectrometry was performed to detect pesticide residues in honeybee foragers and trapped pollen. Honeybee colonies located in neonicotinoid‐treated cornfields expressed significantly higher varroa mite loads than those in untreated cornfields. However, brood production and colony weight were less disturbed by the treatment factor. Sublethal doses of neonicotinoids were detected in the trapped corn pollen and none in bee foragers. Overall, our results show that forager bees collected 20% of corn pollen containing variable concentrations of neonicotinoids. Colonies located in treated cornfields expressed higher varroa loads and long‐term mortality than those in untreated cornfields. On the other hand, no significant differences were observed regarding the brood production and colony weight.

In this study, we present phenotypic and genetic data characterizing the impact of imidacloprid and caging stress on honey bee Apis mellifera physiological responses and regulation of 45 genes using targeted-RNA seq. The term ‘caging stress’ characterizes the effects of depriving honey bees of all hive aspects and conditions. Two cohorts of 1 day old sister bees were subjected to different conditions. One cohort was caged and fed different imidacloprid-tainted sugar solutions and the second was marked and introduced back to its natal hive. Physiological bee parameters and diet behavior were monitored daily for caged bees over several weeks. Bee samples from both cohorts were sampled weekly for RNA sequencing and oxidative stress analyses. Imidacloprid induced significant protein damage and post-ingestive aversion responses in caged bees, leading to lower tainted syrup consumption and higher water intake compared with the controls. No differentially expressed genes were observed among caged bees in regards to imidacloprid treatment. However, significant upregulation in antioxidant genes was recorded in caged bees as compared with hive bees, with overwhelming downregulation in all gene categories in caged bees at week 4. We identified two sets of genes that were constantly regulated in caged bees, including Rsod with unknown function in insects that could potentially characterize caging stress in honey bees.

In order to study the in situ effects of the agricultural landscape and exposure to pesticides on honey bee health, sixteen honey bee colonies were placed in four different agricultural landscapes. Those landscapes were three agricultural areas with varying levels of agricultural intensity (AG areas) and one non-agricultural area (NAG area). Colonies were monitored for different pathogen prevalence and pesticide residues over a period of one year. RT-qPCR was used to study the prevalence of seven different honey bee viruses as well as Nosema sp. in colonies located in different agricultural systems with various intensities of soybean, corn, sorghum, and cotton production. Populations of the parasitic mite Varroa destructor were also extensively monitored. Comprehensive MS-LC pesticide residue analyses were performed on samples of wax, honey, foragers, winter bees, dead bees, and crop flowers for each apiary and location. A significantly higher level of varroa loads were recorded in colonies of the AG areas, but this at least partly correlated with increased colony size and did not necessarily result from exposure to pesticides. Infections of two viruses (deformed wing virus genotype a (DWVa) and acute bee paralysis virus (ABPV)) and Nosema sp. varied among the four studied locations. The urban location significantly elevated colony pathogen loads, while AG locations significantly benefited and increased the colony weight gain. Cotton and sorghum flowers contained high concentrations of insecticide including neonicotinoids, while soybean and corn had less pesticide residues. Several events of pesticide toxicity were recorded in the AG areas, and high concentrations of neonicotinoid insecticides were detected in dead bees.

The efficacies of various acaricides in order to control a parasitic mite, the Varroa mite, Varroa destructor, of honey bees, were measured in two different settings, namely, in laboratory caged honey bees and in queen-right honey bee colonies. The Varroa infestation levels before, during, and after the acaricide treatments were determined in two ways, namely: (1) using the sugar shake protocol to count mites on bees and (2) directly counting the dead mites on the hive bottom inserts. The acaricides that were evaluated were coumaphos, tau-fluvalinate, amitraz, thymol, and natural plant compounds (hop acids), which were the active ingredients. The acaricide efficacies in the colonies were evaluated in conjunction with the final coumaphos applications. All of the tested acaricides significantly increased the overall Varroa mortality in the laboratory experiment. Their highest efficiencies were recorded at 6 h post-treatment, except for coumaphos and thymol, which exhibited longer and more consistent activity. In the honey bee colonies, a higher Varroa mortality was recorded in all of the treatments, compared with the natural Varroa mortality during the pretreatment period. The acaricide toxicity to the Varroa mites was consistent in both the caged adult honey bees and workers in the queen-right colonies, although, two of these acaricides, coumaphos at the highest doses and hop acids, were comparatively more toxic to the worker bees.

The honey bee, Apis mellifera L., is a major pollinator insect that lacks novel “selenoprotein genes”, rendering it susceptible to elevated levels of Selenium (Se) occurring naturally in the environment. We investigated the effects of two inorganic forms of Se on biological traits, oxidative stress, and gene regulation. Using bioassay arenas in the laboratory, one-day old sister bees were fed ad libitum 4 different concentrations of selenate and selenite, two common inorganic forms of Se. The transcription levels of 4 honey bee antioxidant genes were evaluated, and three putative selenoprotein-like genes (SELENOT, SELENOK, SELENOF) were characterized as well as Sbp2, a Selenium binding protein required for the translation of selenoproteins mRNA. Oxidative stress and Se residues were subsequently quantified in honey bee bodies throughout the experiment. Se induced higher oxidative stress in treated honey bees leading to a significantly elevated protein carbonyl content, particularly at the highest studied concentrations. Early upregulations of Spb2 and MsrA were identified at day 2 of the treatment while all genes except SELENOT were upregulated substantially at day 8 to alleviate the Se-induced oxidative stress levels. We determined that doses between 60 and 600 mg.Se.L−1 were acutely toxic to bees (< 48 h) while doses between 0.6 and 6 mg.Se.L−1 led to much lower mortality (7–16)%. Furthermore, when fed ad libitum, Se residue data indicated that bees tolerated accumulation up to 0.12 μg Se bee−1 for at least 8 days with a Se LC50 of ~6 mg/L, a field realistic concentration found in pollen of certain plants in a high Se soil environment., Graphical Abstract

International audience; AbstractThe aim of the present work was to assess the effects of landscape and pesticides on honey bee survival and physiological stress. Integrated use of acetylcholinesterase and detoxification enzymes was tested on honey bee brains for detecting possible exposure to pesticides. Foragers were tracked in agricultural and non-agricultural landscapes in West Tennessee (USA) and then recovered for molecular and chemical analyses. In addition, four honey bee cohorts were fed imidacloprid in the laboratory ad libitum for several weeks and were analyzed by RT-qPCR for gene expression. Pesticides were identified at different concentrations in both crop flowers and recovered foragers. No significant differences in foragers’ mortality were found among locations. Acetylcholinesterase and detoxification genes showed no response to exposure to pesticides except for GstS3 and GstS4. Our results suggest that none of the studied genes make suitable biomarkers for honey bee exposed to pesticides.

The black honeybee (Apis mellifera mellifera; M lineage) which is affected by widespread colony losses, colony introgression and population shifts within its natural range of distribution, needs proper protection and management planning. The DraI COI-COII test discriminates between and within honeybee lineages (M, C, A and O). With this test, a global assessment of the diversity found in the M lineage (i.e. description of haplotype frequencies, fragment length and size, nucleotide sequences) is made with colonies newly sampled in France and Belgium (n = 3,261 colonies) and previously described in the literature (n = 3,367). A total of 91 M haplotypes were described including 52 new ones. The haplotypes M4 and M4’ were the most frequent followed by M17, M7, M6, M19, M8, M17’ and by many rare ones. Finally, these data were used to infer hypotheses on the putative origin of the M haplotypic diversity and to determine a protocol for the establishment of protected areas of black honeybee colonies. This protocol is based on the DraI COI-COII test and on case-studies of protected areas of black honeybees from France.

In an effort to identify landscapes that provide honey bee (Apis mellifera L.) colonies with the most sustainable pollen sources, we conducted an extensive palynological analysis on trapped pollen collected from honey bee hives located in various agricultural landscapes. Collected pollen, honey bee preferences, and potential exposure to pesticides through collected crop pollen were investigated. We collected 128 trapped pollen samples throughout the 2015 season from 16 honey bee colonies distributed in four different agricultural landscapes: a high agricultural (High-AG) area, a moderate agricultural (Mod-AG) area, a low agricultural (Low-AG) area with high levels of urban activity, and a nonagricultural (Non-AG) area. We carried out comprehensive pesticide residue analyses on representative sets of pollen samples. Our results indicate that the Low-AG area with high levels of urban activity had significantly greater pollen diversity than other locations and had the least fluctuation in pollen types across the season. We recorded the lowest pollen diversity in both the High-AG and Non-AG areas with only 23 pollen types, suggesting that both settings lack a diversity of plant species attractive to honey bees. Noncrop pollen collected by honey bees largely exceeded the crop pollen in number and amount, indicating that bees' pollen-diet relies on a large spectrum of noncrop flowers. None of the pesticide residues identified in the pollen samples approached the honey bee 50% lethal dose concentrations.

International audience; AbstractThe genetic diversity and structure of 414 honeybee workers from eight different populations in Algeria were analyzed using 14 polymorphic DNA microsatellite loci. The results showed that the honeybee populations were characterized by substantial genetic variation in terms of the average number of alleles and the degree of heterozygosity. Most populations were at Hardy–Weinberg equilibrium. Phylogenetic and population structure analyses confirmed the African origin of the studied Algerian populations and clustered them in a group distinct from evolutionary lineages West Mediterranean (M), North Mediterranean (C), and Oriental (O). Structure analyses revealed weak allelic introgression from both lineages M and C. High genetic variability was found within the Algerian populations. Two honeybee subspecies, Apis mellifera intermissa and Apis mellifera sahariensis, were present. However, to delimit the natural spread area of A. mellifera sahariensis, more samples from southern Algerian are needed.

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile-de-France, France. CANIF's honeybee colonies were intensively studied over a 3-year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI-COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.

International audience; Honeybees (Apis mellifera) from Syria (n = 1,624), Lebanon (n = 177), and Iraq (n = 35) were collected for genetic analysis. These samples were analyzed by the cleaved amplified polymorphisms technique using mitochondrial DNA (mtDNA) as a molecular marker in their COI-COII intergenic region (Cytochrome Oxidase I and II). Polymerase chain reaction amplicons (500-950 bp) were subjected to restriction analysis by DraI enzyme. The restriction profiles were analyzed and compared with the existing mtDNA markers on polyacrylamide gels. Sixteen new haplotypes were identified, 14 of them on the African lineage A, one new haplotype on the North Mediterranean lineage C and one other on the West Mediterranean lineage M. DNA sequences of all these haplotypes were subsequently determined. We also identified five other haplotypes that had been described in a previous study. These five haplotypes were renamed and grouped more appropriately with the subgroup Z, rather than with the Oriental lineage O. The sequence structures of these novel haplotypes clearly display the specific characteristics of the African branch (A). However, they differ from the African haplotypes by a new restriction site; therefore, they have been moved into a new subgroup. We conclude that A. mellifera syriaca subspecies does not belong to the Oriental lineage O, but is situated geographically in an area of contact between the A and O lineages.

Background Hygienic behavior is a complex, genetically-based quantitative trait that serves as a key defense mechanism against parasites and diseases in Apis mellifera. Yet, the genomic basis and functional pathways involved in the initiation of this behavior are still unclear. Deciphering the genomic basis of hygienic behavior is a prerequisite to developing an extensive repertoire of genetic markers associated to the performance level of this quantitative trait. To fill this knowledge gap, we performed an RNA-seq on brain samples of 25 honeybees per hives from five hygienic and three non-hygienic hives. Results This analysis revealed that a limited number of functional genes are involved in honeybee hygienic behavior. The genes identified, and especially their location in the honeybee genome, are consistent with previous findings. Indeed, the genomic sequences of most differentially expressed genes were found on the majority of the QTL regions associated to the hygienic behavior described in previous studies. According to the Gene Ontology annotation, 15 genes are linked to the GO-terms DNA or nucleotide binding, indicating a possible role of these genes in transcription regulation. Furthermore, GO-category enrichment analysis revealed that electron carrier activity is over-represented, involving only genes belonging to the cytochrome P450. Cytochrome P450 enzymes’ overexpression can be explained by a disturbance in the regulation of expression induced by changes in transcription regulation or sensitivity to xenobiotics. Over-expressed cytochrome P450 enzymes could potentially degrade the odorant pheromones or chemicals that normally signal the presence of a diseased brood before activation of the removal process thereby inhibit hygienic behavior. Conclusions These findings improve our understanding on the genetics basis of the hygienic behavior. Our results show that hygienic behavior relies on a limited set of genes linked to different regulation patterns (expression level and biological processes) associated with an over-expression of cytochrome P450 genes.

Background Apiculture has been practiced in North Africa and the Middle-East from antiquity. Several thousand years of selective breeding have left a mosaic of Apis mellifera subspecies in the Middle-East, many uniquely adapted and survived to local environmental conditions. In this study we explore the genetic diversity of A. mellifera from Syria (n = 1258), Lebanon (n = 169) and Iraq (n = 35) based on 14 short tandem repeat (STR) loci in the context of reference populations from throughout the Old World (n = 732). Results Our data suggest that the Syrian honeybee Apis mellifera syriaca occurs in both Syrian and Lebanese territories, with no significant genetic variability between respective populations from Syria and Lebanon. All studied populations clustered within a new fifth independent nuclear cluster, congruent with an mtDNA Z haplotype identified in a previous study. Syrian honeybee populations are not associated with Oriental lineage O, except for sporadic introgression into some populations close to the Turkish and Iraqi borders. Southern Syrian and Lebanese populations demonstrated high levels of genetic diversity compared to the northern populations. Conclusion This study revealed the effects of foreign queen importations on Syrian bee populations, especially for the region of Tartus, where extensive introgression of A. m. anatolica and/or A. m. caucasica alleles were identified. The policy of creating genetic conservation centers for the Syrian subspecies should take into consideration the influence of the oriental lineage O from the northern Syrian border and the large population of genetically divergent indigenous honeybees located in southern Syria.