Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

Genome‐wide analysis of genes related to ovary activation in worker honey bees

A defining characteristic of eusocial animals is their division of labour into reproductive and nonreproductive specialists. Here, we used a microarray study to identify genes associated with functional sterility in the worker honey bee Apis mellifera. We contrasted gene expression in workers from a functionally sterile wild‐type strain with that in a mutant (anarchist) strain selected for high rates of ovary activation. We identified a small set of genes from the brain (n = 7) and from the abdomen (n = 5) that are correlated in their expression with early stages of ovary activation. Sterile wild‐type workers up‐regulated two unknown genes and a homologue of Drosophila CG6004. By contrast, reproductive anarchist workers up‐regulated genes for the yolk protein vitellogenin, venom peptides and a member of the AdoHycase superfamily, among others. The differentially expressed genes identified are likely to be involved in early differentiation into sterile and reproductive worker phenotypes and may therefore form part of the gene networks associated with the regulation of honey bee worker sterility. Our study may have lacked sufficient power to detect all but a minority of biologically relevant changes taking place; however, the differential expression of vitellogenin and a putative AdoHycase suggests that our screen has captured core reproductive genes and that ovary activation may involve an epigenetic mechanism.     

Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6‐year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long‐chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa‐tolerant honey bees.     

The honey bee gut microbiota: strategies for study and characterization

Gut microbiota research is an emerging field that improves our understanding of the ecological and functional dynamics of gut environments. The honey bee gut microbiota is a highly rewarding community to study, as honey bees are critical pollinators of many crops for human consumption and produce valuable commodities such as honey and wax. Most significantly, unique characteristics of the Apis mellifera gut habitat make it a valuable model system. This review discusses methods and pipelines used in the study of the gut microbiota of Ap. mellifera and closely related species for four main purposes: identifying microbiota taxonomy, characterizing microbiota genomes (microbiome), characterizing microbiota–microbiota interactions and identifying functions of the microbial community in the gut. The purpose of this contribution is to increase understanding of honey bee gut microbiota, to facilitate bee microbiota and microbiome research in general and to aid design of future experiments in this growing field.     

Expression of genes related to reproduction and pollen foraging in honey bees (Apis mellifera) narcotized with carbon dioxide

It has been proposed that a honey bee (Apis mellifera) worker's preference for foraging for pollen or nectar is modulated by a gene network that was originally involved in regulating the reproductive cycles of an ancestral solitary species. We used carbon dioxide to induce narcosis in queens and workers. This treatment is known to initiate oogenesis in queens, reduce oogenesis in queenless workers and to change worker foraging preference. We then assessed changes in gene expression of genes suspected to be involved in either foraging behaviour or reproduction. We show that some genes change expression in the opposite direction between castes in response to treatment. Our results therefore support the hypothesis that reproductive and foraging traits are causally related in the honey bee.     

Division of labour in honey bees: age‐ and task‐related changes in the expression of octopamine receptor genes

The honey bee (Apis mellifera L.) has developed into an important ethological model organism for social behaviour and behavioural plasticity. Bees perform a complex age‐dependent division of labour with the most pronounced behavioural differences occurring between in‐hive bees and foragers. Whereas nurse bees, for example, stay inside the hive and provide the larvae with food, foragers leave the hive to collect pollen and nectar for the entire colony. The biogenic amine octopamine appears to play a major role in division of labour but the molecular mechanisms involved are unknown. We here investigated the role of two characterized octopamine receptors in honey bee division of labour. AmOctαR1 codes for a Ca²⁺‐linked octopamine receptor. AmOctβR3/4 codes for a cyclic adenosine monophosphate‐coupled octopamine receptor. Messenger RNA expression of AmOctαR1 in different brain neuropils correlates with social task, whereas expression of AmOctβR3/4 changes with age rather than with social role per se. Our results for the first time link the regulatory role of octopamine in division of labour to specific receptors and brain regions. They are an important step forward in our understanding of complex behavioural organization in social groups.     

Decreased detoxification genes and genome size make the human body louse an efficient model to study xenobiotic metabolism

The human body louse, Pediculus humanus humanus, has one of the smallest insect genomes, containing ～10 775 annotated genes. Annotation of detoxification [cytochrome P450 monooxygenase (P450), glutathione‐S‐transferase (GST), esterase (Est) and ATP‐binding cassette transporter (ABC transporter)] genes revealed that they are dramatically reduced in P. h. humanus compared to other insects except for Apis mellifera. There are 37 P450, 13 GST and 17 Est genes present in P. h. humanus, approximately half the number found in Drosophila melanogaster and Anopheles gambiae. The number of putatively functional ABC transporter genes in P. h. humanus and Ap. mellifera are the same (36) but both have fewer than An. gambiae (44) or Dr. melanogaster (65). The reduction of detoxification genes in P. h. humanus may be a result of this louse's simple life history, in which it does not encounter a wide variety of xenobiotics. Neuronal component genes are highly conserved across different insect species as expected because of their critical function. Although reduced in number, P. h. humanus still retains at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to xenobiotics (eg Cyp3 clade P450s, Delta GSTs, B clade Ests and B/C subfamily ABC transporters), suggestive of its high potential for resistance development.     

Silencing of Apis mellifera dorsal genes reveals their role in expression of the antimicrobial peptide defensin‐1

Like all other insects, two key signalling pathways [Toll and immune deficiency (Imd)] regulate the induction of honey bee immune effectors that target microbial pathogens. Amongst these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the nuclear factors kappa B (NF‐κB) Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin‐1 was regulated by the Toll pathway, whereas hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin‐1. Honey bees have two dorsal genes (dorsal‐1 and dorsal‐2) and two splicing isoforms of dorsal‐1 (dorsal‐1A and dorsal‐1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down‐regulation of defensin‐1 was observed for dorsal‐1A and dorsal‐2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin‐1 is regulated by Dorsal (Toll pathway).     

Four serine proteinases expressed in Manduca sexta haemocytes

Several putative serine proteinases were detected in Manduca sexta larval plasma by labelling with radio-active diisopropyl fluorophosphate. To begin to identify and characterize such enzymes, a polymerase chain reaction was carried out using haemocyte cDNA as template and primers designed to amplify conserved sequences from serine proteinases. Four serine proteinase cDNA fragments were cloned. These were used as probes to screen an M. sexta larval haemocyte cDNA library to obtain full-length clones encoding haemocyte proteinases 1–4 (HP1, HP2, HP3 and HP4). HP1 and HP2 contain an aminoterminal ‘clip’ domain similar to those found in horse-shoe crab clotting enzyme and clotting factor B and also in the Drosophila melanogaster proteinases snake and easter. HP3 and HP4 are most similar to proteinases from mammalian leucocytes. HP1 and HP2 are both present in plasma. HP1 is expressed in haemocytes (granular cells and oenocytoids) and not in fat body. HP2 is expressed in fat body and in granular haemocytes, plasmatocytes and oenocytoids. After injection of larvae with bacteria, the level of HP2 mRNA decreased in haemocytes and increased in fat body.     

A honey bee can threat ear: Sudden sensorineural hearing loss

Sudden sensorineural hearing loss is an otologic emergency. Many etiological factors can lead to this pathology. Honey bee (Apis mellifera) sting may lead to local and systemic reactions due to sensitization of the patient. In this paper we described a sudden sensorineural hearing loss occurred after honey bee sting.     

Initial evaluation of vascular ingrowth into superporous hydrogels

There is a need for new materials and architectures for tissue engineering and regenerative medicine. Based upon our recent results developing novel scaffold architecture, we hypothesized that this new architecture would foster vascularization, a particular need for tissue engineering. We report on the potential of superporous hydrogel (SPH) scaffolds for in vivo cellular infiltration and vascularization. Poly(ethylene glycol) diacrylate (PEGDA) SPH scaffolds were implanted in the dorsum of severe combined immunodeficient (SCID) mice and harvested after 4 weeks of in vivo implantation. The SPHs were visibly red and vascularized, as apparent when compared to the non‐porous hydrogel controls, which were macroscopically avascular. Host cell infiltration was observed throughout the SPHs. Blood cells and vascular structures, confirmed through staining for CD34 and smooth muscle α‐actin, were observed throughout the scaffolds. This novel soft material may be utilized for cell transplantation, tissue engineering and in combination with cell therapies. The neovasularization and limited fibrotic response suggest that the architecture may be conducive to cell survival and rapid vessel development. Copyright © 2009 John Wiley & Sons, Ltd.     

The Emotional and Behavioral Impact of Delivering Bad News to Virtual versus Real Standardized Patients: A Pilot Study

Abstract

Phenomenon: Virtual standardized patients (vSPs) are becoming increasingly common in medical education, though one limitation of vSPs is the artificiality of computer-based simulators. Past research on the use of vSPs has not clearly established whether learners have different emotional responses to real SPs (rSPs) compared with vSPs; however, understanding learners’ emotional responses to vSPs is important in providing realistic learning experiences and establishing the validity of this teaching and assessment tool. This study compared the emotional experiences of individuals who interacted with rSPs and vSPs. Approach: Sixty medical students at a medical school in the southeastern United States participated in the study. Participants were randomly assigned to deliver bad news to an rSP or vSP. The vSP for this study used a hybrid intelligence model that allowed a person to “inhabit” the vSP. Salivary cortisol and a self-report measure of mood—the Profile of Mood States, Second Edition (POMS 2)—were gathered before and after delivering the bad news. The SP and 2 independent evaluators rated the behavioral performance of each participant in real and virtual conditions. Participants also rated the performance of the SP. Findings: Participants in both conditions reported increased negative emotionality on the POMS 2 following the SP interaction. There were no significant between-group differences on the POMS 2 or salivary cortisol concentration following the SP interaction. Ratings by the SP and independent evaluators indicated that participants performed similarly on most interpersonal dimensions, except tone of voice. Participants perceived the vSP as less realistic than the rSP. Insights: These results suggest that medical students may have similar emotional and behavioral responses when delivering bad news to a vSP when compared to an rSP. These findings provide support for the continued use of vSPs in training learners to deliver bad news and other communication-based skills and to assess their performance on these tasks.     

Advances in superporous hydrogels.

Superporous hydrogels (SPHs) are different from superabsorbent polymers (SAPs) in that SPHs swell fast, within minutes, to the equilibrium swollen state regardless of their size. The fast swelling property is based on water absorption through open porous structure by capillary force. The poor mechanical strength of SPHs was overcome by developing the second-generation SPH composites (SPHCs) and the third-generation SPH hybrids (SPHHs). This review examines the differences between SAPs and SPHs and describes three different generations of SPHs.     

Proteome profile and lentiviral transduction of cultured honey bee (Apis mellifera L.) cells

Honey bees (Apis mellifera L.) play a vital role in agriculture as pollinators, and serve as model organisms of social behaviour and immunity. The lack of both immortalized cell lines and methods to introduce recombinant DNA reliably into primary cells hinders cellular and molecular studies in this organism. We hereby demonstrate the expression of a GFP gene delivered by lentivirus transduction to cultured embryonic cells. The success of this approach indicates that viral transduction could be used to deliver constitutively active oncogenes in order to immortalize honey bee cells. We were able to revive cells successfully after several months of cryogenic storage and we show how the proteome varies between freshly collected and cultured embryonic cells.