Regulation of forager honey bee appetite independent of the glucose-insulin signaling pathway.

Introduction: To maintain energetic homeostasis the energetic state of the individual needs to communicate with appetite regulatory mechanisms on a regular basis. Although hunger levels indicated by the energetic state and appetite levels, the desire for food intake, tend to be correlated, and on their own are well studied, how the two cross-talk and regulate one another is less known. Insects, in contrast to vertebrates, tend to have trehalose as the primary sugar found in the hemolymph, which could possibly serve as an alternative monitor of the energetic state in comparison to the glucose-insulin signaling pathway, found in vertebrates. Methods: We investigate how manipulating hemolymph sugar levels alter the biogenic amines in the honey bee brain, appetite levels, and insulin like peptide gene expression, across three age classes, to determine how the energetic state of the honey bee might be connected to appetite regulation. Results: We found that only in the forager bees, with a lowering of hemolymph trehalose levels, there was an increase in octopamine and a decrease in tyramine levels in the honey bee brain that corresponded with increased appetite levels, while there was no significant changes in Insulin Like Peptide-1 or 2 gene expression. Discussion: Our findings suggest that hemolymph trehalose levels aid in regulating appetite levels, in forager bees, via octopamine and tyramine, and this regulation appears to be functioning independent of the glucose insulin signaling pathway. Whether this potentially more direct and rapid appetite regulatory pathway can be generalized to other insects, which also undergo energy demanding activities, remains to be investigated.

Inactivation of Nosema spp. with zinc phthalocyanine.

Most honey bee pathogens, such as Vairimorpha (Nosema), cannot be rapidly and definitively diagnosed in a natural setting, consequently there is typically the spread of these diseases through shared and re-use of beekeeping equipment. Furthermore, there are no viable treatment options available for Nosema spores to aid in managing the spread of this bee disease. We therefore aimed to develop a new method using novel Zinc Phthalocyanine (ZnPc) as a photosensitizer for the photodynamic inactivation of Nosema spores that could be used for the decontamination of beekeeping equipment. Nosema spores were propagated for in vitro testing using four caged Apis mellifera honey bees. The ZnPc treatment was characterized, encapsulated with a liposome, and then used as either a 10 or 100 µM treatment for the freshly harvested Nosema spores, for either a 30 and or 60-minute time period, under either light or dark conditions, in-vitro, in 96-well plates. In the dark treatment, after 30-min, the ZnPc 100 µM treatment, caused a 30 % Nosema mortality, while this increased to 80 % at the same concentration after the light treatment. The high rate of anti-spore effects, in a short period of time, supports the notion that this could be an effective treatment for managing honey bee Nosema infections in the future. Our results also suggest that the photo activation of the treatment could be applied in the field setting and this would increase the sterilization of beekeeping equipment against Nosema.

3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface.

Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional (3D) tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, anin situcrosslinked hybrid, multi-material 3D bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow mesenchymal stem cells-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing anin situcrosslink approach with ruthenium (Ru) and sodium persulfate photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

Increased alarm pheromone component is associated with Nosema ceranae infected honeybee colonies.

Use of chemicals, such as alarm pheromones, for rapid communication with conspecifics is widespread throughout evolutionary history. Such chemicals are particularly important for social insects, such as the honeybee (Apis mellifera), because they are used for collective decision-making, coordinating activities and self-organization of the group. What is less understood is how these pheromones change due to an infection and what the implications might be for social communication. We used semiquantitative polymerase chain reaction (sqPCR) to screen for a common microsporidian gut parasite, Nosema ceranae, for 30 hives, across 10 different locations. We then used high-resolution accurate mass gas chromatography-quadrupole time of flight mass spectrometry to generate an exposome profile for each hive. Of the 2352 chemical features identified, chemicals associated with infection were filtered for cosanes or cosenes. A significant association was found between N. ceranae and the presence of (Z)-11-eicosen-1-ol, a known alarm pheromone component. The increase in (Z)-11-eicosen-1-ol could be the recognition mechanism for healthy individuals to care for, kill, or quarantine infected nestmates. Nosema ceranae has contributed to the global decline in bee health. Therefore, altered alarm pheromones might play a role in disrupting social harmony and have potential impacts on colony health.

Gas chromatography - Mass spectrometry as a preferred method for quantification of insect hemolymph sugars.

Insects, due to their small size, have limited energy storage space, but they also have high metabolic rate, so their hemolymph sugars are incredibly dynamic and play a number of important physiological functional roles in maintaining energetic homeostasis. In contrast to vertebrates, trehalose is generally the primary sugar found in insect hemolymph, which is followed by glucose and fructose. Many analytical chemistry methods exist to measure sugars, yet a direct comparison of methods that can measure all three simultaneously, and trehalose in particular, from low sample volumes, are sparse. Using the honey bee as a model, we directly compare the leading current methods of using High Performance Liquid Chromatography (HPLC) with an evaporative light-scattering detector and Gas Chromatography coupled with Mass Spectrometry (GC-MS) to determine which method would be better for measuring trehalose, glucose, and fructose in terms of reproducibility, accuracy, and sensitivity. Furthermore, we injected the enzyme inhibitors trehalozin (a trehalase inhibitor) and sorbose (a trehalase p-synthase inhibitor) to manipulate the trehalose levels in honey bee foragers as a proof of concept that this sugar can be altered independently of hemolymph glucose and fructose levels. Overall the HPLC method was less reproducible for measuring fructose and glucose, and it also had lower sensitivity for measuring trehalose. Consequently, significant differences in trehalose levels within the forager class were only detected with the GC-MS and not the HPLC method. Lastly, using the GC-MS method in the follow up study we found that trehalozin and sorbose causes a significant increase and decrease of trehalose levels respectively, in forager honey bees, independent of the glucose and fructose levels, ten minutes after injection. Taken together, these methods will provide useful tools for future studies exploring the many different physiological functional roles that trehalose can play in maintaining insect energetic homeostasis.

THROUGH THE LOOKING GLASS: Real-Time Imaging in Brachypodium Roots and Osmotic Stress Analysis.

To elucidate dynamic developmental processes in plants, live tissues and organs must be visualised frequently and for extended periods. The development of roots is studied at a cellular resolution not only to comprehend the basic processes fundamental to maintenance and pattern formation but also study stress tolerance adaptation in plants. Despite technological advancements, maintaining continuous access to samples and simultaneously preserving their morphological structures and physiological conditions without causing damage presents hindrances in the measurement, visualisation and analyses of growing organs including plant roots. We propose a preliminary system which integrates the optical real-time visualisation through light microscopy with a liquid culture which enables us to image at the tissue and cellular level horizontally growing Brachypodium roots every few minutes and up to 24 h. We describe a simple setup which can be used to track the growth of the root as it grows including the root tip growth and osmotic stress dynamics. We demonstrate the system's capability to scale down the PEG-mediated osmotic stress analysis and collected data on gene expression under osmotic stress.

The efficacy of Beauveria bassiana, jasmonic acid and chlorantraniliprole on larval populations of Helicoverpa armigera in chickpea crop ecosystems.

BACKGROUND: A robust integrated pest management (IPM) programme is needed to reduce the use of insecticides in controlling Helicoverpa armigera. Therefore, a 2 year field study was conducted to evaluate the use of alternative control measures (biochemical use) for H. armigera relative to exclusively using chemical insecticides. The entomopathogenic fungus Beauveria bassiana, jasmonic acid and the insecticide chlorantraniliprole were each applied twice during the chickpea growing season. RESULTS: All three applied materials (either alone or combined) significantly (P ≤ 0.05) reduced the larval population of H. armigera and pod infestation. Effects increased with time, and the maximum difference was observed 7 days after the second application in each year. The lowest numbers of larvae per plant and pod infestation were in the B. bassiana 3.21 × 106 + chlorantraniliprole treatment in both 2009/2010 and 2010/2011 year. The reduction in the larval population and pod infestation increased chickpea yield and the highest yield in both seasons, and the maximum yield was obtained in the B. bassiana 3.21 × 106 + chlorantraniliprole treatment. The populations of natural enemies were highest in the jasmonic acid treatment. CONCLUSION: The results suggest that B. bassiana, jasmonic acid and chlorantraniliprole may be useful components for the H. armigera IPM strategy. © 2016 Society of Chemical Industry.

From Genetics to Functional Genomics: Improvement in Drought Signaling and Tolerance in Wheat.

Drought being a yield limiting factor has become a major threat to international food security. It is a complex trait and drought tolerance response is carried out by various genes, transcription factors (TFs), microRNAs (miRNAs), hormones, proteins, co-factors, ions, and metabolites. This complexity has limited the development of wheat cultivars for drought tolerance by classical breeding. However, attempts have been made to fill the lost genetic diversity by crossing wheat with wild wheat relatives. In recent years, several molecular markers including single nucleotide polymorphisms (SNPs) and quantitative trait loci (QTLs) associated with genes for drought signaling pathways have been reported. Screening of large wheat collections by marker assisted selection (MAS) and transformation of wheat with different genes/TFs has improved drought signaling pathways and tolerance. Several miRNAs also provide drought tolerance to wheat by regulating various TFs/genes. Emergence of OMICS techniques including transcriptomics, proteomics, metabolomics, and ionomics has helped to identify and characterize the genes, proteins, metabolites, and ions involved in drought signaling pathways. Together, all these efforts helped in understanding the complex drought tolerance mechanism. Here, we have reviewed the advances in wide hybridization, MAS, QTL mapping, miRNAs, transgenic technique, genome editing system, and above mentioned functional genomics tools for identification and utility of signaling molecules for improvement in wheat drought tolerance.

History and current status of wheat miRNAs using next-generation sequencing and their roles in development and stress.

As small molecules that aid in posttranscriptional silencing, microRNA (miRNA) discovery and characterization have vastly benefited from the recent development and widespread application of next-generation sequencing (NGS) technologies. Several miRNAs were identified through sequencing of constructed small RNA libraries, whereas others were predicted by in silico methods using the recently accumulating sequence data. NGS was a major breakthrough in efforts to sequence and dissect the genomes of plants, including bread wheat and its progenitors, which have large, repetitive and complex genomes. Availability of survey sequences of wheat whole genome and its individual chromosomes enabled researchers to predict and assess wheat miRNAs both in the subgenomic and whole genome levels. Moreover, small RNA construction and sequencing-based studies identified several putative development- and stress-related wheat miRNAs, revealing their differential expression patterns in specific developmental stages and/or in response to stress conditions. With the vast amount of wheat miRNAs identified in recent years, we are approaching to an overall knowledge on the wheat miRNA repertoire. In the following years, more comprehensive research in relation to miRNA conservation or divergence across wheat and its close relatives or progenitors should be performed. Results may serve valuable in understanding both the significant roles of species-specific miRNAs and also provide us information in relation to the dynamics between miRNAs and evolution in wheat. Furthermore, putative development- or stress-related miRNAs identified should be subjected to further functional analysis, which may be valuable in efforts to develop wheat with better resistance and/or yield.