Grooming and hygienic behaviours of the honey bee (Apis mellifera) toward the parasitic mite Varroa destructor

Despite a multitude of environmental stressors, the Varroa mite is still regarded as the greatest cause of honey bee mortality in its invaded range. Breeding honey bees that are resistant to the mite is an important area of research. This thesis aimed to gain a better understanding of the grooming and hygienic behaviours of Russian honey bees (RHB). The effect of a break in the synchrony of a mite’s life cycle on reproductive success was tested through brood inoculation experiments. Mites released by hygienic behaviour and forced to enter a new cell are less likely to lay male offspring. Through laboratory cage assays it was found that daughter mites are more susceptible to grooming behaviour. A new method of marking Varroa mites was developed which would enable a single cohort of mites to be followed after inoculation. A strong brood removal trait was noticed in RHB colonies, therefore they were tested for Varroa sensitive hygienic (VSH) behaviour. RHB demonstrated levels of VSH as high as the USDA line bred specifically for this behaviour. In addition the same QTL found to be responsible for the trait in VSH bees, was associated with VSH in RHB stock. Previous work showed that the ratio of older mites to total trapped mites (O/T) in the debris of honey bee colonies demonstrated the strongest association with colony infestation. This research showed that O/T is associated with VSH and brood removal behaviour. In addition, bees that displayed high levels of VSH in this study were also more likely to spend a longer amount of time grooming in laboratory assays. This indicates that both grooming and hygienic behaviours play important roles in the resistance of RHB stock. Their likelihood to be expressed by other stocks is discussed and recommendations for further research are provided.

Transitions: From novice participation to expertise, pre-school 'matters' in an Irish pre-school community

This in depth, qualitative, participant observer study tracks children's transition experiences from novice to experienced membership of their pre-school community. It also considers adult roles in mediating this process in the context of the recent introduction of a universal free-pre-school year for children growing up in Ireland. Participation and the space to negotiate a participatory identity is understood in this study as a key element of positive experiences of early years transitions, within pre-school and beyond. The underlying theoretical framework is socio-cultural. This approach shifts from a scientific positivist view of thinking and learning as an individual inside the head process and asserts the historical, social, cultural as well as the situated context of learning and meaning making All participants, including myself as researcher, are recognised, explored and valued as embedded in the cultural context studied. In a sense, this approach tilts the worlds being observed through participation in them and reflects them in new light. The aim is to interpret and reflect the multiple realities constructed in this context rather than seek a truth out there waiting to be found. Special efforts are made to be invited in to and acknowledge children's expertise in the cultural worlds they negotiate with peers and adults in pre-school. The aim is to better understand what children may find motivating, interesting or problematic as they interpret reproduce and transform meaning within their play and learning worlds. My aim is for an honest rendering of the voices of stakeholders in pre-school communities from teachers, parents, and policy makers to children themselves. It makes visible constraints; potentials and possibilities within everyday Irish pre-school practices in the situated context studied as well as the broader societal, legislative and macro policy influences it reflects. Casting light on the taken for granted opens the possibility of adaptation or transformation. Transition itself can act as a tool to meet the changing needs of children on their developmental pathways across the life cycle

Micro and nanostructured impedance sensors for biological and biomedical applications

This thesis work covered the fabrication and characterisation of impedance sensors for biological applications aiming in particular to the cytotoxicity monitoring of cultured cells exposed to different kind of chemical compounds and drugs and to the identification of different types of biological tissue (fat, muscles, nerves) using a sensor fabricated on the tip of a commercially available needle during peripheral nerve block procedures. Gold impedance electrodes have been successfully fabricated for impedance measurement on cells cultured on the electrode surface which was modified with the fabrication of gold nanopillars. These nanostructures have a height of 60nm or 100nm and they have highly ordered layout as they are fabricated through the e-beam technique. The fabrication of the threedimensional structures on the interdigitated electrodes was supposed to improve the sensitivity of the ECIS (electric cell-substrate impedance sensing) measurement while monitoring the cytotoxicity effects of two different drugs (Antrodia Camphorata extract and Nicotine) on three different cell lines (HeLa, A549 and BALBc 3T3) cultured on the impedance devices and change the morphology of the cells growing on the nanostructured electrodes. The fabrication of the nanostructures was achieved combining techniques like UV lithography, metal lift-off, evaporation and e-beam lithography techniques. The electrodes were packaged using a pressure sensitive, medical grade adhesive double-sided tape. The electrodes were then characterised with the aid of AFM and SEM imaging which confirmed the success of the fabrication processes showing the nanopillars fabricated with the right layout and dimensions figures. The introduction of nanopillars on the impedance electrodes, however, did not improve much the sensitivity of the assay with the exception of tests carried out with Nicotine. HeLa and A549 cells appeared to grow in a different way on the two surfaces, while no differences where noticed on the BALBc 3T3 cells. Impedance measurements obtained with the dead cells on the negative control electrodes or the test electrodes with the drugs can be compared to those done on the electrodes containing just media in the tested volume (as no cells are attached and cover the electrode surface). The impedance figures recorded using these electrodes were between 1.5kΩ and 2.5 kΩ, while the figures recorded on confluent cell layers range between 4kΩ and 5.5kΩ with peaks of almost 7 kΩ if there was more than one layer of cells growing on each other. There was then a very clear separation between the values of living cell compared to the dead ones which was almost 2.5 - 3kΩ. In this way it was very easy to determine whether the drugs affected the cells normal life cycle on not. However, little or no differences were noticed in the impedance analysis carried out on the two different kinds of electrodes using cultured cells. An increase of sensitivity was noticed only in a couple of experiments carried out on A549 cells growing on the nanostructured electrodes and exposed to different concentration of a solution containing Nicotine. More experiments to achieve a higher number of statistical evidences will be needed to prove these findings with an absolute confidence. The smart needle project aimed to reduce the limitations of the Electrical Nerve Stimulation (ENS) and the Ultra Sound Guided peripheral nerve block techniques giving the clinicians an additional tool for performing correctly the peripheral nerve block. Bioimpedance, as measured at the needle tip, provides additional information on needle tip location, thereby facilitating detection of intraneural needle placement. Using the needle as a precision instrument and guidance tool may provide additional information as to needle tip location and enhance safety in regional anaesthesia. In the time analysis, with the frequency fixed at 10kHz and the samples kept at 12°C, the approximate range for muscle bioimpedance was 203 – 616 Ω, the approximate bioimpedance range for fat was 5.02 - 17.8 kΩ and the approximate range for connective tissue was 790 Ω – 1.55 kΩ. While when the samples were heated at 37°C and measured again at 10kHz, the approximate bioimpedance range for muscle was 100-175Ω. The approximate bioimpedance range of fat was 627 Ω - 3.2 kΩ and the range for connective tissue was 221-540Ω. In the experiments done on the fresh slaughtered lamb carcass, replicating a scenario close to the real application, the impedance values recorded for fat were around 17 kΩ, for muscle and lean tissue around 1.3 kΩ while the nervous structures had an impedance value of 2.9 kΩ. With the data collected during this research, it was possible to conclude that measurements of bioimpedance at the needle tip location can give valuable information to the clinicians performing a peripheral nerve block procedure as the separation (in terms of impedance figures) was very marked between the different type of tissues. It is then feasible to use an impedance electrode fabricated on the needle tip to differentiate several tissues from the nerve tissue. Currently, several different methods are being studied to fabricate an impedance electrode on the surface of a commercially available needle used for the peripheral nerve block procedure.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

The distribution of sales revenues from pharmaceutical innovation.

OBJECTIVE: This report updates our earlier work on the returns to pharmaceutical research and development (R&D) in the US (1980 to 1984), which showed that the returns distributions are highly skewed. It evaluates a more recent cohort of new drug introductions in the US (1988 to 1992) and examines how the returns distribution is emerging for drugs with life cycles concentrated in the 1990s versus the 1980s. DESIGN AND SETTING: Methods were described in detail in our earlier reports. The current sample included 110 new drug entities (including 28 orphan drugs), and sales data were obtained for the period 1988 to 1998, which represented between 7 and 11 years of sales for the drugs included. 20 years was chosen as the expected market life for this cohort, and a 2-step procedure was used to project future sales for the drugs--during the period until patent expiry and then beyond patent expiry until the 20-year time-horizon was completed. Thus, the values in the first half of the life cycle are essentially based on realised sales, while those in the second half are projected using information on patent expiry and other inputs. MAIN OUTCOME MEASURES AND RESULTS: Peak annual sales for the top decile of drugs introduced between 1988 and 1992 in the US amounted to almost $US1.1 billion compared with peak sales of less than $US175 million (1992 values) for the mean compound. In particular, the top decile accounted for 56% of overall sales revenue. Although the sales distributions were skewed in both our earlier and current analysis, the top decile in the later time-period exhibited more rapid rates of growth after launch, a peak that was more than 50% greater in real terms than for the 1980 to 1984 cohort, and a faster rate of expected decline in sales after patent expiry. One factor contributing to the distribution of sales revenues becoming more skewed over time is the orphan drug phenomenon (i.e. most of the orphan drugs are concentrated at the bottom of the distribution). CONCLUSION: The distribution of sales revenues for new drug compounds is highly skewed in nature. In this regard, the top decile of new drugs accounts for more than half of the total sales generated by the 1988 to 1992 cohort analysed. Furthermore, the distribution of sales revenues for this cohort is more skewed than that of the 1980 to 1984 cohort we analysed in previous research.

Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

Sea Urchin Body Plan Development and Evolution: An Integrative Transcriptomic Approach

My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins.

In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning.

In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases.

Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

A study of user requirements for COTS: computer off the shelf software solutions

[This abstract is based on the authors' abstract.]Three new standards to be applied when adopting commercial computer off-the-shelf (COTS) software solutions are discussed. The first standard is for a COTS software life cycle, the second for a software solution user requirements life cycle, and the third is a checklist to help in completing the requirements. The standards are based on recent major COTS software solution implementations.

Towards policy-based self-configuration of embedded systems

This paper describes work towards the deployment of flexible self-management into real-time embedded systems. A challenging project which focuses specifically on the development of a dynamic, adaptive automotive middleware is described, and the specific self-management requirements of this project are discussed. These requirements have been identified through the refinement of a wide-ranging set of use cases requiring context-sensitive behaviours. A sample of these use-cases is presented to illustrate the extent of the demands for self-management. The strategy that has been adopted to achieve self-management, based on the use of policies is presented. The embedded and real-time nature of the target system brings the constraints that dynamic adaptation capabilities must not require changes to the run-time code (except during hot update of complete binary modules), adaptation decisions must have low latency, and because the target platforms are resource-constrained the self-management mechanism have low resource requirements (especially in terms of processing and memory). Policy-based computing is thus and ideal candidate for achieving the self-management because the policy itself is loaded at run-time and can be replaced or changed in the future in the same way that a data file is loaded. Policies represent a relatively low complexity and low risk means of achieving self-management, with low run-time costs. Policies can be stored internally in ROM (such as default policies) as well as externally to the system. The architecture of a designed-for-purpose powerful yet lightweight policy library is described. A suitable evaluation platform, supporting the whole life-cycle of feasibility analysis, concept evaluation, development, rigorous testing and behavioural validation has been devised and is described.

Impact of climate change on marine pelagic phenology and trophic mismatch

Phenology, the study of annually recurring life cycle events such as the timing of migrations and flowering, can provide particularly sensitive indicators of climate change. Changes in phenology may be important to ecosystem function because the level of response to climate change may vary across functional groups and multiple trophic levels. The decoupling of phenological relationships will have important ramifications for trophic interactions, altering food-web structures and leading to eventual ecosystem-level changes. Temperate marine environments may be particularly vulnerable to these changes because the recruitment success of higher trophic levels is highly dependent on synchronization with pulsed planktonic production. Using long-term data of 66 plankton taxa during the period from 1958 to 2002, we investigated whether climate warming signals are emergent across all trophic levels and functional groups within an ecological community. Here we show that not only is the marine pelagic community responding to climate changes, but also that the level of response differs throughout the community and the seasonal cycle, leading to a mismatch between trophic levels and functional groups.

An overview of Calanus helgolandicus ecology in European waters

We review current knowledge and understanding of the biology and ecology of the calanoid copepod Calanus helgolandicus in European waters, as well as provide a collaborative synthesis of data from 18 laboratories and 26 sampling stations in areas distributed from the northern North Sea to the Aegean and Levantine Seas. This network of zooplankton time-series stations has enabled us to collect and synthesise seasonal and multi-annual data on abundance, body size, fecundity, hatching success and vertical distribution of C. helgolandicus. An aim was to enable comparison with its congener Calanus finmarchicus, which has been studied intensively as a key component of European and north east Atlantic marine ecosystems. C. finmarchicus is known to over-winter at depth, whereas the life-cycle of C. helgolandicus is less well understood. Overwintering populations of C. helgolandicus have been observed off the Atlantic coast between 400 and 800 m, while in the Mediterranean there is evidence of significant deep-water populations at depths as great as 4200 m. The biogeographical distribution of C. helgolandicus in European coastal waters covers a wide range of habitats, from open ocean to coastal environments, and its contribution to mesozooplankton biomass ranges from 6% to 93%. Highest abundances were recorded in the Adriatic and off the west coast of Spain. C. helgolandicus is generally found in 9-20 C water, with maximum abundances from 13-17 C. In contrast, C. finmarchicus is found in cooler water between 0 and 15 C, with peak abundances from 0 to 9 C. As water has warmed in the North Atlantic over recent decades, the range of C. helgolandicus and its abundance on the fringes of its expanding range have increased. This review will facilitate development of population models of C. helgolandicus. This will not only help answer remaining questions but will improve our ability to forecast future changes, in response to a warming climate, in the abundance and distribution of this important species.

Impact of climate change on Antarctic krill

Antarctic krill Euphausia superba (hereafter ‘krill’) occur in regions undergoing rapid environmental change, particularly loss of winter sea ice. During recent years, harvesting of krill has increased, possibly enhancing stress on krill and Antarctic ecosystems. Here we review the overall impact of climate change on krill and Antarctic ecosystems, discuss implications for an ecosystem-based fisheries management approach and identify critical knowledge gaps. Sea ice decline, ocean warming and other environmental stressors act in concert to modify the abundance, distribution and life cycle of krill. Although some of these changes can have positive effects on krill, their cumulative impact is most likely negative. Recruitment, driven largely by the winter survival of larval krill, is probably the population parameter most susceptible to climate change. Predicting changes to krill populations is urgent, because they will seriously impact Antarctic ecosystems. Such predictions, however, are complicated by an intense inter-annual variability in recruitment success and krill abundance. To improve the responsiveness of the ecosystem-based management approach adopted by the Commission for the Conservation of Antarctic Marine Living Resources (CCAMLR), critical knowledge gaps need to be filled. In addition to a better understanding of the factors influencing recruitment, management will require a better understanding of the resilience and the genetic plasticity of krill life stages, and a quantitative understanding of under-ice and benthic habitat use. Current precautionary management measures of CCAMLR should be maintained until a better understanding of these processes has been achieved.

Rapid biogeographical plankton shifts in the North Atlantic Ocean

Large-scale biogeographical changes in the biodiversity of a key zooplankton group (calanoid copepods) were detected in the north-eastern part of the North Atlantic Ocean and its adjacent seas over the period 1960–1999. These findings provided key empirical evidence for climate change impacts on marine ecosystems at the regional to oceanic scale. Since 1999, global temperatures have continued to rise in the region. Here, we extend the analysis to the period 1958–2005 using all calanoid copepod species assemblages (nine species assemblages based on an analysis including a total of 108 calanoid species or taxa) and show that this phenomenon has been reinforced in all regions. Our study reveals that the biodiversity of calanoid copepods are responding quickly to sea surface temperature (SST) rise by moving geographically northward at a rapid rate up to about 23.16 km yr−1. Our analysis suggests that nearly half of the increase in sea temperature in the northeast Atlantic and adjacent seas is related to global temperature rises (46.35% of the total variance of temperature) while changes in both natural modes of atmospheric and oceanic circulation explain 26.45% of the total variance of temperature. Although some SST isotherms have moved northwards by an average rate of up to 21.75 km yr−1 (e.g. the North Sea), their movement cannot fully quantify all species assemblage shifts. Furthermore, the observed rates of biogeographical movements are far greater than those observed in the terrestrial realm. Here, we discuss the processes that may explain such a discrepancy and suggest that the differences are mainly explained by the fluid nature of the pelagic domain, the life cycle of the zooplankton and the lesser anthropogenic influence (e.g. exploitation, habitat fragmentation) on these organisms. We also hypothesize that despite changes in the path and intensity of the oceanic currents that may modify quickly and greatly pelagic zooplankton species, these organisms may reflect better the current impact of climate warming on ecosystems as terrestrial organisms are likely to significantly lag the current impact of climate change.

Macroecological study of Centropages typicus in the North Atlantic Ocean

Centropages typicus is a temperate neritic-coastal species of the North Atlantic Oceans, generally found between the latitudes of the Mediterranean and the Norwegian Sea. Therefore, the species experiences a large number of environments and adjusts its life cycle in response to changes in key abiotic parameters such as temperature. Using data from the Continuous Plankton Recorder (CPR) Survey, we review the macroecology of C. typicus and factors that influence its spatial distribution, phenology and year-to-year to decadal variability. The ecological preferences are identified and quantified. Mechanisms that allow the species to occur in such different environments are discussed and hypotheses are proposed as to how the species adapts to its environment. We show that temperature and both quantity and quality of phytoplankton are important factors explaining the space and time variability of C. typicus. These results show that C. typicus will not respond only to temperature increase in the region but also to changes in phytoplankton abundance, structure and composition and timing of occurrence. Methods such as a decision tree can help to forecast expected changes in the distribution of this species with hydro-climatic forcing. (C) 2007 Elsevier Ltd. All rights reserved.