Final Report Summary - PARAFROGS (Emerging Protist Parasites of Frogs: Global prevalence and host/parasite interaction) It is widely recognized that amphibians (e.g. frogs) are among the most threatened animal groups. For example, in 2008, 32% of amphibian species were listed as ‘threatened or extinct’ and 42% were listed as in decline. The causes of this decline have been identified as: habitat loss, environmental change, and the introduction of non-native species. Emerging infectious diseases have also been shown to play a key role in many amphibian declines, for example the chytrid fungal pathogen Batrachochytrium dendrobatidis has caused mass mortality events (MMEs) in Australia, Europe and across the Americas. Infection by Ranavirus has also been documented in, for example, the United Kingdom (UK), United States of America (USA), and Canada. Recent work has linked local MMEs in the USA with the infection of larval frogs (tadpoles) of the genera Lithobates and Acris by a protist. In 2006, histological examinations of tadpole tissues revealed the presence of thousands of small spherical cells infecting preferentially the liver of Southern Leopard Frog tadpoles (Lithobates sphenocephalus, formerly Rana sphenocephala) sampled from a MME in Georgia (USA) (Davis et al. Ecohealth 2007 4: 310-317). Phylogenetic analysis of the SSU rDNA showed that this infectious protist closely related to Perkinsus, a parasite of marine bivalves (Azevedo C. Int. J. Parasitol. 1989 75:627–635). Phylogenetic analysis has shown that Perkinsea are a deeply divergent sister-group of dinoflagellate alveolates. Actually, only three representative groups of Perkinsea were previously described: Perkinsus spp., parasites of marine bivalves (e.g. oysters and clams), Parvilucifera spp. parasites of dinoflagellates and Rastrimonas subtilis parasites of cryptophyte algae. However, environmental sequencing of the SSU rDNA gene diversity targeting the small eukaryotic microbes (cells of 0.2 to 5µm diameter) in freshwater environments has demonstrated that the Rana pathogen is part of a highly diverse clade present in numerous freshwater ecosystems suggesting the pathogen has a free-living phase (e.g. Richards T.A. et al., Environ. Microbiol., 2005 7(9): 1413-1425, Brate J., et al., ISME J. 2010 4:1144-1153.)During this project, I developed new molecular methodologies to investigate the diversity of Perkinsea microbes in natural environments and animal or plant tissues. This methodology was first tested on freshwater environments where we identified a wide diversity of Perkinsea lineages. Using the same-targeted PCR protocol we also tested for Perkinsea-like lineages in livers sampled from 182 tadpoles across multiple families of frogs sampled from three continents and six global localities. Our results provide the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments. A manuscript describing this work has been submitted to a leading peer-review journal. As part of my work, I also collaborated with international-peers in the authorship of a review article describing the range of disease threats to natural amphibian populations (Gleason et al. Fungal Ecology 2014 11:181-192.). Taken together these results provide foundation of numerous projects for my future research career and will provide information to inform sustainable conservation planning and disease detection for amphibian populations.During our work with tadpoles, we identified a different pathogen of interest in the liver tissue samples. Although emerging diseases affecting adult life stage are becoming well-documented, infectious agents targeting tadpoles life stage are still poorly described. Indeed, tadpoles are more susceptible to infectious disease because they have only an innate immune system function (e.g. macrophages), while their adaptive immune system does not function until they reach maturity. However, the innate immunity macrophage cells are also the targets of different parasites (e.g. Goussia sp., Apicomplexa) that can affect host immune system and consequently host fitness. Through a collaborative network with Dr M. Jirků and Dr A. Valigurová (Biology Centre, Academy of Sciences of the Czech Republic, Czech Republic) in parallel to the Perkinsea work, we also studied a new association between amphibian macrophages and a putative gregarine parasite named Nematopsis temporaria. Further understanding of how biotic factors influence the capacity of tadpoles to face emerging disease are necessary to develop new strategy for conservation plans. We plan to submit this paper for publication shortly.In parallel, I was involved in the European FP6 Biodiversa Project BioMarKs, which uses high throughput sequencing methods to explore the genetic diversity of the whole protist community in marine waters. This work gave me the opportunity to analyse large sequence datasets. As part of this collaboration I led a subproject within this consortium aiming to understand the molecular diversity of Perkinsea. Although Perkinsea described organisms are mainly from marine ecosystems (e.g. Perkinsus sp. and Parvilucifera sp. parasite of molluscs and dinoflagellates respectively), the molecular diversity of this lineage is infrequently detected in the marine environmental clone libraries. Using the high-throughput sequencing methods based on RNA SSU as template, I demonstrated a hitherto undetected diversity of ribosomally “active” Perkinsea lineages in the sediment environments, suggesting that Perkinsea might play a significant but unrecognized role as parasites in marine sediments or as part of the ‘seedbank’ microbial community (Chambouvet et al. BMC Microbiol. 2014 14:110.). These first results will build the foundation for further project, to identify the putative parasitic organisms identified by their genetic signature and understand their role in the marine food web.Finally, as part of my fellowship I took part in a field-trip project collaboration with Dr A. Worden (MBARI, California, USA) where we developed a new sampling process for deep-sea sediment (up to 4,000m depth) with the aim of sequencing targeted meta-transcriptomes. Our goal was to access the transcriptome of putative parasites from the Kinetoplastida phylum related to well-known human parasites (e.g. Trypanosoma sp. or Leishmania sp.) present in deep-sea sediments. I have developed a new targeted transcriptome sequencing approach to access the transcriptome of the whole community of the Kinetoplastida in the sediment using the two unique-conserved regions on the 5’ end of mRNA, the splice leader (SL) (Chambouvet et al. in prep). This new protocol is then applicable to any protist group which have splice-leader modified mRNA (this includes Perkinsea) and can be applied to any environment type including host tissues. I believe I have developed an approach to link the above-mentioned tools to the studying microbial function in their natural environments, making this new research avenue very powerful and something that will become a primary focus of my work.This fellowship was essential for me to establish my own sustainable high profile research because I have now acquired a strong background in state of the art molecular biology tools and bioinformatic analysis (e.g. phylogenetic analysis, diversity tag sequencing, targeted meta-transcriptomics). These methodologies will allow my research to integrate the analysis of large ‘omics’ datasets, which are of increasing importance in microbial ecology. Dr Thomas Richards acted as strong mentor during the whole duration of the fellowship and lead me to enhance my research capabilities, communication skills, scientific experience and maturity. Finally with the support and advice of Dr Richards, I have established a strong scientific collaborative network, making me well placed to develop and lead both my own research team and future collaborations with the broader scientific community.This project has resulted in the following scientific publications: four publications as first authors, one as last author and at least three publications as a co-author. I was also the lead author in a book chapter for the Cambridge University Press published in 2015. During the project the fellowship I was also sponsored by EMBO to presents the results of this project at an international meeting “Integrated microbial biology” in Czech Republic 2014. Finally, I have also attended to EMBO laboratory management courses in 2015 to enhance my managing skills to develop and lead my own research group. Hence this project allows me to develop strong skills and establish my own sustainable research and apply for further funding.