Thirty-two honeybee (Apis mellifera) colonies were studied in order to detect and measure potential in vivo effects of neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Honeybee colonies were randomly split on four different agricultural cornfield areas located near Quebec City, Canada. Two locations contained cornfields treated with a seed-coated systemic neonicotinoid insecticide while the two others were organic cornfields used as control treatments. Hives were extensively monitored for their performance and health traits over a period of two years. Honeybee viruses (brood queen cell virus BQCV, deformed wing virus DWV, and Israeli acute paralysis virus IAPV) and the brain specific expression of a biomarker of host physiological stress, the Acetylcholinesterase gene AChE, were investigated using RT-qPCR. Liquid chromatography-mass spectrometry (LC-MS) was performed to detect pesticide residues in adult bees, honey, pollen, and corn flowers collected from the studied hives in each location. In addition, general hive conditions were assessed by monitoring colony weight and brood development. Neonicotinoids were only identified in corn flowers at low concentrations. However, honeybee colonies located in neonicotinoid treated cornfields expressed significantly higher pathogen infection than those located in untreated cornfields. AChE levels showed elevated levels among honeybees that collected corn pollen from treated fields. Positive correlations were recorded between pathogens and the treated locations. Our data suggests that neonicotinoids indirectly weaken honeybee health by inducing physiological stress and increasing pathogen loads.

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.Apis mellifera syriaca / CAPS / haplotype / honeybee / intergenic region COI-COII / Cyt b / mtDNA

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.

BackgroundHygienic 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.ResultsThis 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.ConclusionsThese 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.

BackgroundApiculture 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).ResultsOur 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.ConclusionThis 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.

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.

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.

Few studies of honey bee colonies exist where varroa mite control is achieved by integrating broodless conditions, through either total brood removal or queen caging, in combination with oxalic acid (OA) applications. We observed significant varroa mortality after total brood removal or caging the queens and OA applications in broodless colonies, as well as in colonies with brood that received four consecutive OA applications. In laboratory tests, we recorded higher mortality of caged bees exposed to Apistan® compared to oxalic acid or untreated control bees. However, this mortality is not believed to negatively impact the colony. We therefore recommend combining OA applications with artificial broodless colony conditions achieved either by brood removal or queen caging as an effective management strategy for varroa mites.