The Arctic is one of the planet’s most rapidly warming regions, which will likely alter the biogeochemical processes involved in the bioavailability and transfer of essential (i.e. nutrients such as Cu, Zn, Se) and non-essential (i.e. contaminants such as Pb, Cd, Hg) trace elements. Simultaneously, human presence in the region is increasing due to an expansion of on- and off-shore exploration/extraction of oils & minerals all with a risk of adverse environmental effects. Yet most knowledge about the chemical and biological processes involved in the transfer of trace elements through Arctic food-chains is based on marine and aquatic systems, whereas the Arctic terrestrial ecosystem in general, and Arctic terrestrial food-chains specifically, are vastly unexplored. A better understanding of the cycling of such elements is thus crucial as it may have wide ranging implications on trophic interactions, terrestrial biodiversity and ecosystem functioning as a whole. The ATCAF-project will fill this knowledge gap by targeting the environment – biota – trace element interaction in a high-Arctic terrestrial landscape, while also taking an original approach by directly linking trace elements concentrations in large wildlife to individual and population health. Specifically, the aim is to quantify the above-ground/below-ground linkages of trace elements in a high-Arctic terrestrial ecosystem (i.e. Zackenberg, East Greenland), estimate the transfer efficiency within local food-chains (i.e. in soil, vegetation, prey- & predator-invertebrate insects, herbivores and carnivores), and assess the potential health effects of these elements within large Arctic wildlife (i.e. muskoxen and Arctic fox). To reach this aim, ATCAF will be organized in four interlinked work tasks: Task 1) Ecogeochemistry & the landscape: Mapping of availability and spatial variation in essential and non-essential elements across the soils and vegetation of an Arctic landscape Task 2) Ecogeochemistry & the food-chains: Transfer efficiency of essential- and non-essential elements through Arctic terrestrial food-chains Task 3) Ecogeochemistry & the individual: Trace element distribution within individuals and the potential link between wildlife hair and population health Task 4) Ecogeochemistry and the isotopes: Isotopic composition in blood samples from large Arctic wildlife as diagnostic tool of health By studying the accumulation of contaminants in Arctic terrestrial food-chains in tandem with the uptake of essential elements, ATCAF will provide much needed information on their combined effects on the health and fitness of local biota. Uniting empirical data and predictive modelling approaches, ATCAF will therefore deliver 1) maps showing spatial variation in the trace element composition of different soil and vegetation types in a high-Arctic terrestrial ecosystem, 2) quantitative values of above-ground/below-ground linkages and transfer efficiency of trace elements between trophic levels in the studied system 3) an extensive assessment of within individual trace element distribution and a novel analytical protocol using wildlife hair as a non-invasive long-term bioindicator of individual- and population health, 4) a novel analytical protocol using isotopic composition of blood as a diagnostic tool for wildlife health (e.g. neoplasia, inflammatory status, body condition, pathogen). Combined, the successful outcome of the ATCAF-project will revolutionize science-based monitoring of wildlife health and population trends in systems under pressure. This, in turn, will promote well-informed conservation strategies, wildlife monitoring and ecosystem management with additional socioeconomic and cultural values as it pertains to local populations depending on this wildlife and the ecosystem for their livelihood.

Nanomaterials (NMs) are rapidly entering the market, in fact possibly at the fastest rate seen for any new group of materials. Hence, NMs are bound to end up in the environment where they may be toxic to organisms. Due to their small size NMs have a different molecular packing than equivalent bulk materials, presenting very different physicochemical properties. It is predicted that nanoparticles (NPs) will exhibit a more pronounced biological activity per given mass than larger size materials. Ecotoxicological studies with NPs are nearly all focused on testing one or few NPs. A more systematic approach to compare effects across many NMs with identified different but related characters (e.g. different aspect-ratios or surface activities) is only sparsely reported. Hence, there is an urgent need to provide tool that allow us to fast develop an (eco)toxicological dataset containing comprehensive toxicity information relating to a diverse spectra of NPs characters. For this it is necessary to have the relevant and adequate testing materials, high throughput (HTP) ecotox tools, and data-analysis tools. In bio-CHIP we have a custom made NPs library and a gene based HTP biological impact test system, which is combined with cutting edge expertise in handling such NP-biological data. This is the foundation for making this exploratory project feasible within the short framework. Hence, its success stands with the fact that we have the necessary tools and expertise already advanced and optimized, leaving the room for the actual exploring connections between biological effects and NPs descriptors. This can be implemented in a fast, reliable, and standardised HTP tool. The experimental setup has three main components: (A) Nano Material: NMs custom library – TiO2-Fe (1-10%); (B) Biological Material: Standard test species with HTP transcriptomic library – Enchytraeus crypticus; (C) Network analysis (third generation of pathway tools: the Pathway Topology approach) and QNAR type modelling. The main aims (& tasks) of bio-CHIP can be summarised as follows: 1: To study HTP biological responses to a library of highly characterised NPs; delivering a HTP biological effects database. 2: To use the HTP data obtained in quantitative network models to identify possible relationships (affected pathways, they being metabolic, regulatory, signaling, etc); delivering a pathway model and a QNAR type model relating the biological effects to identified NPs descriptors. 3: To use the derived models to design optimal NM-bio-CHIP; delivering a cost effective tool that assesses biological-NM specific effects. E. crypticus will be exposed to the NMs library and assessment of differential gene expression (microarray). Microarrays are basically glass slides where thousands of genes are printed and can cross hybridize with e.g. biological exposed sample RNA. This means that the whole organism transcriptome response can be assessed for a certain time shot, allowing us to know what gene regulations are occurring when an organism is exposed to a NP. For data analysis we propose a network measure to score each pathway and each gene, by shortest path scanning over the complete genome network. We will apply a comparative functional genomics approach to identify shared transcriptional profiles of NMs. The aim is to search for nanospecific effects (across nanos) by comparative analysis between the different tested NMs for each assessed endpoint, with special focus on the high-information-content next generation sequencing techniques. Finally, a cost-effective tool for NMs – bio-CHIP will be delivered. Based on results a selection of genes to be printed will be made envisaging the most significantly affected pathways (to be NP targeted) and representing all pathways (to ensure coverage of other NPs e.g. all metal oxides). Os nanomateriais (NMs) estão a entrar no mercado rapidamente, provavelmente à taxa mais rápida alguma vez vista. Portanto, os NMs chegarão ao ambiente podendo ser tóxicos para os organismos. Devido ao pequeno tamanho, têm um agrupamento molecular diferente dos materiais macro equivalentes, apresentando propriedades fisico-químicas diferentes. Prevê-se que os NPs têm uma maior actividade biologica por massa do que os materiais macro. Estudos ecotoxicologicos com NPs são quase exclusivamente em 1 ou poucos NPs. Uma abordagem mais sistematica para comparar efeitos entre muitos NMs com características diferentes mas relacionadas (p.e. differente aspeto-racio ou atividade de superfície) é muito limitada. Há uma necessidade urgente de uma base de dados (eco)toxicologicos, que inclua toxicidade relacionada com um espetro vasto de carateres de NPs. Para se desenvolver/implementar são necessárias certas ferramentas tais como os materiais teste relevantes e adequados (espécies and químicos) e análise. No bio-CHIP nós temos uma biblioteca de NPs desenhada e um sistema biológico ‘High-throughput’ (HTP), combinado com a experiência em tratamento de dados NP-bio. Estes são os fundamentos base para a exequibilidade deste projeto exploratório num curto período de tempo. O seu sucesso está associado ao fato de possuirmos as ferramentas necessárias e a especialização desenvolvida e otimizada, deixando espaço para a exploração em si das diferenças e conetividades entre efeitos biológicos e parametros de NPs. O desenho explerimental tem 3 principais componentes: (A) Nano Material: biblioteca desenhada de NM – TiO2-Fe (1-10%); (B) Material Biologico: Espécie teste padronizada com biblioteca transcriptomica HTP – Enchytraeus crypticus; (C) Análise de redes (ferramentas de terceira geração de vias: a abordagem de topologia) e modelação do tipo QNAR. Os principais objectivos ( e tarefas) do bio-CHIP são em suma: 1: estudar as respostas biológicas HTP a uma biblioteca de NPs altamente caracterizada - base de dados HTP de efeitos biológicos; 2: usar os dados HTP obtidos em modelos quantitativos de redes para identificar possíveis relações (vias afectadas, metabólicas, regulatórias, sinalizantes, etc.) - modelo de vias e do tipo QNAR relacionando os efeitos biológicos com os parâmetros de NPs; 3: Usar os modelos obtidos para desenhar o bio-CHIP ideal para NM - aplicação otimizada para avaliação de efeitos biologico-NM específicos. E. crypticus vai ser exposto a uma biblioteca de NMs e avaliar a expressão genica diferencial (microarray). Microarrays são basicamente suportes de vidro onde se imprimem milhares de genes que podem hibridizar com p.e. amostras de RNA de organismos expostos. Isto significa que a resposta da totalidade do transcriptoma do organismo pode ser avaliada num determinado tempo, permitindo conhecer quais os mecanismos de regulação genetica que ocorrem aquando da exposição a NPs. Para a análise de dados propomos avaliação de redes para quantificar cada via metabólica e cada gene, através da via mais curta varrendo a totalidade da rede do genoma. Iremos aplicar uma abordagem comparativa de genomica functional para identificar perfis de transcrição partilhados entre NMs. O objetivo é procurar efeitos nanoespecíficos (entre nanos) através de análise comparative entre diferentes NMs testados para cada parâmetro avaliado, com especial atenção nas técnicas de sequenciação de HTP de próxima geração. Por fim, uma ferramenta económica para NMs – bio-CHIP será produzida. Com base nos resultados, uma série de genes serão selecionados para imprimir num microarray de elevada densidade tendo em vista as vias metabolicas mais afetadas (NP específico) e representando todas as vias (garantindo cobertura de outros NPs que não o TiO2, até possivelmente outros oxidos de metais).

BEAUTY aims to unravel the mechanisms of action of stressors (nanopesticides) identifying adverse outcome pathways (AOPs). This will move risk assessment-RA from the current black-box approach to an informed decision making, enabling to transfer key information between RA of related stressors. For the highly ecologically important invertebrate species, Enchytraeus crypticus, we will use analytical information from internal biological levels (genomics to phenomics) to develop AOPs via pathway network analysis. Since, this ecotoxicology standard species has just become a full genome model (we sequenced its genome last year) we can use the most advanced techniques to support our studies. The techniques will among others include gene knockdown (RNAi) to test identified pathways of response. This will be supported by studies at the functional level, which will add further supporting evidences or close the cycle, e.g. targeted proteomics and immunohistochemistry. All of this will allow to describe, and confirm, the mechanisms that lead to the cascade of events and effects onto an AOP. In addition to this we propose a daring approach to design high precision nanopesticides where the organism is attracted to the pesticide, and we will use the above techniques to confirm the advanced efficacy. This means that increase crop protection and food production can be achieved whilst lowering environmental impacts. This project is possible because the tools to study each level are already developed by the PI and the expertise are included via excellent research groups, both national and international, hence the outreach will be worldwide also. BEAUTY tem como objetivo desvendar os mecanismos de ação de agentes de stress (nanopesticidas) identificando vias metabólicas adversas (VMA). Irá mover a Avaliação de Risco-AR da abordagem atual de caixa negra para uma decisão baseada em informação, permitindo a transferência de informação chave entre AR de stressores relacionados. Para uma das espécies de invertebrados mais relevantes ecologicamente, Enchytraeus crypticus, vamos usar informação analítica dos níveis biológicos internos (genómica a fenómica) para desenvolver VMAs através de analise de redes. Uma vez que esta espécie modelo ecotoxicológica padrão se tornou também um modelo completo de genómica (nós sequenciámos o seu genoma no ano passado) podemos usar as técnicas mais avançadas para apoiar o estudo. A metodologia inclui, entre outras, o silenciamento de genes (RNAi) para testar as vias metabólicas de resposta identificadas. Isto será apoiado por estudos ao nível funcional, que irão providenciar mais evidencias ou fechar o ciclo, p.e. analisando proteómica e imuno-histoquímica alvo. Tudo isto irá permitir-nos descrever, e confirmar, os mecanismos que induzem a cascata de eventos e efeitos nas vias metabólicas afetadas. Adicionalmente, propomos uma abordagem inovadora para desenhar nPs de elevada precisão onde os organismos são atraídos para o pesticida, e utilizaremos as técnicas acima para confirmar o aumento na eficácia. Isto significa que podemos aumentar a proteção das colheitas e produção alimentar e ao mesmo tempo diminuir o impacto ambiental. Este projeto é possível porque as ferramentas para estudar cada um dos níveis se encontram já desenvolvidas pelo PI e a equipa reúne especialidades de grupos de investigação de excelência, tanto nacionais como internacionais, logo ao alcance será também mundial.

There is growing evidence that biodiversity of cold regions, including high latitude insular systems, will be increasingly threatened by climate change and biological invasions. In this project, we aim to combine existing data with new experiments and observations from cold environments in polar and alpine regions to better understand the synergisms between climate change and biological invasions on species redistributions, to predict how these may change into the future, and to develop mitigation measures to deal with impacts. (a) We will focus on cold environments at high latitudes and elevations (Arctic, sub-Arctic, Antarctic, sub-Antarctic and alpine systems from Austria, Czech Republic, France, Greenland [Denmark], Norway, Switzerland, South Africa, and the UK, in addition to the data we will be able to access via collaborators and/or public databases [MIREN, GMBA, SoilTemp, National botanic conservatories, ect? ), as they are warming at an unprecedented rate and are becoming increasingly vulnerable to invasions under a milder climate. Hence, these environments are excellent sentinels for change, and can act as natural laboratories in which to study the synergisms between, and the combined effects of climate change and biological invasions. (b) We will focus on terrestrial, freshwater and coastal ecosystems/communities/habitats/organisms from cold environments. We will use long-term monitoring data for plants and invertebrates, including georeferenced non-native and native species records (e.g. presence/absence, abundance) collected in surveys spanning a wide range of habitat types, (micro)climatic conditions and elevation gradients. These extensive datasets are already available from members of the proposed consortium, and will be further enriched by new experimental research. If funded, this proposal will lead to the development of new analytical tools and scientific insights, using cutting-edge modelling techniques relying on databases with an unprecedented size and scope to improve our understanding of the dynamics of native and non-native species biogeography in cold environments. Our effort will greatly improve the current knowledge of the mechanisms and factors driving the distribution and assemblages of species at local and regional scales. (c) We will focus on plants and invertebrates whose ecology, fitness and distribution are driven by the microclimatic characteristics of their habitats. Plants and insects are often keystone or pioneer species structuring ecological networks, and especially so in cold environments. Several plants are also ?umbrella? species, for which the long-term conservation will allow protection of their associated habitats and species. By linking the distribution of plants and invertebrates, we will be able to consider and reveal the cascading effects of changes of their ecological interactions.