Kinome Analysis of Honeybee (Apis mellifera L.) Dark-Eyed Pupae Identifies Biomarkers and Mechanisms of Tolerance to Varroa Mite Infestation.

The mite Varroa destructor is a serious threat to honeybee populations. Selective breeding for Varroa mite tolerance could be accelerated by biomarkers within individual bees that could be applied to evaluate a colony phenotype. Previously, we demonstrated differences in kinase-mediated signaling between bees from colonies of extreme phenotypes of mite susceptibility. We expand these findings by defining a panel of 19 phosphorylation events that differ significantly between individual pupae from multiple colonies with distinct Varroa mite tolerant phenotypes. The predictive capacity of these biomarkers was evaluated by analyzing uninfested pupae from eight colonies representing a spectrum of mite tolerance. The pool of biomarkers effectively discriminated individual pupae on the basis of colony susceptibility to mite infestation. Kinome analysis of uninfested pupae from mite tolerant colonies highlighted an increased innate immune response capacity. The implication that differences in innate immunity contribute to mite susceptibility is supported by the observation that induction of innate immune signaling responses to infestation is compromised in pupae of the susceptible colonies. Collectively, biomarkers within individual pupae that are predictive of the susceptibility of colonies to mite infestation could provide a molecular tool for selective breeding of tolerant colonies.

Neonicotinoid insecticides in pollen, honey and adult bees in colonies of the European honey bee (Apis mellifera L.) in Egypt.

Honeybee losses have been attributed to multiple stressors and factors including the neonicotinoid insecticides (NIs). Much of the study of hive contamination has been focused upon temperate regions such as Europe, Canada and the United States. This study looks for the first time at honey, pollen and bees collected from across the Nile Delta in Egypt in both the spring and summer planting season of 2013. There is limited information upon the frequency of use of NIs in Egypt but the ratio of positive identification and concentrations of NIs are comparable to other regions. Metabolites of NIs were also monitored but given the low detection frequency, no link between matrices was possible in the study. Using a simple hazard assessment based upon published LD50 values for individual neonicotinoids upon the foraging and brood workers it was found that there was a potential risk to brood workers if the lowest reported LD50 was compared to the sum of the maximum NI concentrations. For non-lethal exposure there was significant risk at the worst case to brood bees but actual exposure effects are dependant upon the genetics and conditions of the Egyptian honeybee subspecies that remain to be determined.

Concentrations of neonicotinoid insecticides in honey, pollen and honey bees (Apis mellifera L.) in central Saskatchewan, Canada.

Neonicotinoid insecticides (NIs) and their transformation products were detected in honey, pollen and honey bees, (Apis mellifera) from hives located within 30 km of the City of Saskatoon, Saskatchewan, Canada. Clothianidin and thiamethoxam were the most frequently detected NIs, found in 68 and 75% of honey samples at mean concentrations of 8.2 and 17.2 ng g(-1) wet mass, (wm), respectively. Clothianidin was also found in >50% of samples of bees and pollen. Concentrations of clothianidin in bees exceed the LD50 in 2 of 28 samples, while for other NIs concentrations were typically 10-100-fold less than the oral LD50. Imidaclorpid was detected in ∼30% of samples of honey, but only 5% of pollen and concentrations were

Identification of developmentally-specific kinotypes and mechanisms of Varroa mite resistance through whole-organism, kinome analysis of honeybee.

Recent investigations associate Varroa destructor (Mesostigmata: Varroidae) parasitism and its associated pathogens and agricultural pesticides with negative effects on colony health, resulting in sporadic global declines in domestic honeybee (Apis mellifera) populations. These events have motivated efforts to develop research tools that can offer insight into the causes of declining bee health as well as identify biomarkers to guide breeding programs. Here we report the development of a bee-specific peptide array for characterizing global cellular kinase activity in whole bee extracts. The arrays reveal distinct, developmentally-specific signaling profiles between bees with differential susceptibility to infestation by Varroa mites. Gene ontology analysis of the differentially phosphorylated peptides indicates that the differential susceptibility to Varroa mite infestation does not reflect compromised immunity; rather, there is evidence for mite-mediated immune suppression within the susceptible phenotype that may reduce the ability of these bees to counter secondary viral infections. This hypothesis is supported by the demonstration of more diverse viral infections in mite-infested, susceptible adult bees. The bee-specific peptide arrays are an effective tool for understanding the molecular basis of this complex phenotype as well as for the discovery and utilization of phosphorylation biomarkers for breeding programs.

A fatty acyl-CoA reductase highly expressed in the head of honey bee (Apis mellifera) involves biosynthesis of a wide range of aliphatic fatty alcohols.

Honey bees (Apis mellifera) are social insects which have remarkable complexity in communication pheromones. These chemical signals comprise a mixture of hydrocarbons, wax esters, fatty acids, aldehydes and alcohols. In this study, we detected several long chain aliphatic alcohols ranging from C18-C32 in honey bees and the level of these alcohols varied in each body segment. C18:0Alc and C20:0Alc are more pronounced in the head, whereas C22:0Alc to C32Alc are abundant in the abdomen. One of the cDNAs coding for a fatty acyl-CoA reductase (AmFAR1) involved in the synthesis of fatty alcohols was isolated and characterized. AmFAR1 was ubiquitously expressed in all body segments with the predominance in the head of honey bees. Heterologous expression of AmFAR1 in yeast revealed that AmFAR1 could convert a wide range of fatty acids (14:0-22:0) to their corresponding alcohols, with stearic acid 18:0 as the most preferred substrate. The substrate preference and the expression pattern of AmFAR1 were correlated with the level of total fatty alcohols in bees. Reconstitution of the wax biosynthetic pathway by heterologous expression of AmFAR1, together with Euglena wax synthase led to the high level production of medium to long chain wax monoesters in yeast.

Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses.

BACKGROUND AND AIMS: Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. METHODS: Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. KEY RESULTS: Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. CONCLUSIONS: The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.

Identification and immunogold localization of a novel bromegrass (Bromus inermis Leyss) peroxisome channel protein induced by ABA, cold and drought stresses, and late embryogenesis.

A cDNA (BG-15) was isolated through differential screening of a cDNA library made from an ABA-treated bromegrass (Bromus inermis Leyss) suspension cell culture. The 819 bp pair cDNA encoded a 174 amino acid polypeptide with a calculated molecular mass of 18.08 kD and isolectric point of 7.50. The deduced amino acid sequences for the cDNA were 29.5% and 32.6% homologous to the known amino acid-selective channel proteins of the chloroplastic outer membrane in pea and barley, but were highly homologous (55.6% to 83.2%) to the putative membrane channel proteins from rice and Arabidopsis. Immunogold localization demonstrated that the channel protein encoded by this cDNA was present on the peroxisome membrane. High stringency southern analysis revealed that 1 to 2 copies of the peroxisomal channel protein (PCP) genes were present in the bromegrass genome. Northern and Western blots revealed that the PCP gene was responsive to both cold and drought stresses, and was rapidly induced by ABA (75 microM). The transcript of the PCP gene also accumulated during late embryogenesis, but declined rapidly during germination. Data taken together, responsiveness of the PCP to cold and drought stresses, and accumulation during late embryogenesis suggest this novel peroxisomal channel protein is associated with sugar and fatty acid metabolism through fatty acid import or succinate export from peroxisome during desiccation tolerance and energy metabolism.

A lipid transfer protein gene BG-14 is differentially regulated by abiotic stress, ABA, anisomycin, and sphingosine in bromegrass (Bromus inermis).

The objective was to investigate the expression of a lipid transfer protein gene (LTP) both in bromegrass (Bromus inermis) cells and seedlings after exposure to abiotic stresses, abscisic acid (ABA), anisomycin, and sphingosine. A full-length cDNA clone BG-14 isolated from bromegrass suspension cell culture encodes a polypeptide of 124 amino acids with typical LTP characteristics, such as a conserved arrangement of cysteine residues. During active stages of cold acclimation LTP expression was up-regulated, whereas at the final stage of cold acclimation LTP transcript level declined to pre-acclimation level. A severe drought stress induced the LTP gene; yet, LTP expression doubled 3 d after re-hydration. Both temperature and heat shock duration influence LTP induction; however temperature is the primary factor. Treatment with NaCl stimulated accumulation of LTP mRNA within 15 min and the transcripts remained at elevated levels for the duration of the salinity stress. Most interestingly, Northern blots showed LTP was rapidly induced not only by ABA, but also by anisomycin and sphingosine in suspension cell cultures. Of the three chemicals, ABA induced the most rapid and highest response in LTP expression as well as highest freezing tolerance, whereas sphingosine was the least active for both LTP expression and freezing tolerance.