Characterisation of two members of a macroschizont gene family, Tashat1 and Tashat2, from Theileria annulata

Theileria annulata is a protozoan parasite of cattle, that causes the disease tropical theileriosis throughout sub-tropical regions of the Old World. Theileria parasites have the ability to immortalise the host leukocyte they infect causing clonal expansion and dissemination of infected leukocytes throughout the host. This property has allowed the development of an in vitro system for the culture of bovine cells infected by the macroschizont stage of the parasite. In addition, differentiation of the parasite towards the next life cycle stage, the merozoite, can be induced in culture. The signals that cause the macroschizont to differentiate into merozoites are not fully understood, although it is known that this event is associated with a major elevation in merozoite gene expression (Shiels et al., 1994). Recently a small family of parasite genes that are negatively regulated early during differentiation to the merozoite were identified. One member, known as TashAT2 contained predicted AT hook DNA binding motifs and was shown to be localised to the host cell nucleus. It has been postulated that the TashAT2 polypeptide may play a role in the regulation of macroschizont or modulation of host cell gene expression (Swan et al., 1999). The focus of this project was to characterise TashAT1, a second member of the TashAT gene family. To this end, the TashAT1 gene was sub-cloned and sequenced and mapped to a region of the genome containing TashAT2 and a third Task AT gene, TashAT3. The 1.4kb open reading frame of TashAT1 was virtually identical to the five prime end of TashAT3, indicating that TashAT1 or TashAT3 (TashAT1/3) were derived from a recent duplication event. The predicted amino acid sequence of TashAT1/3 contained four AT hook motifs, a nuclear localisation signal and a signal sequence. Northern blot analysis revealed that TashAT1, TashAT2 and TashAT3 mRNA were down regulated early, during differentiation to the merozoite in vitro. However, no down regulation was observed for any of the TashAT transcripts in a cell line that was severely attenuated with respect to parasite differentiation. Sequence analysis of the upstream regions of TashAT1/3 identified a motif element (TashUM) located 43bp upstream of the putative transcription start site of TashAT1/3 that was highly related to a sequence upstream of TashAT1 and another, unrelated macroschizont gene, Tash1. Preliminary electromobility band shift analysis of TashUM revealed that it bound to a factor found in host and parasite enriched nuclear extract, which appeared to decrease in abundance as the parasite differentiated towards merogony. Antisera generated against a region of TashAT1 failed to recognise a TashAT1 polypeptide by Western blot analysis. However, a 180kDa polypeptide that was down regulated with respect to merogony and co-localised to the host nucleus was specifically recognised. The detected polypeptide was identified as TashAT3 on the basis of size, sequence identity and predicted expression profile. Immunofluorescence analysis showed that the anti-TashAT1 antisera reacted against both the host nucleus and parasite. This reactivity was lost as the parasite differentiated to the merozoite. The host reactivity was probably due to recognition of TashAT3, while it could not be concluded that the parasite reactivity was directed against TashAT1. Taken together, the results indicated that TashAT3 and possibly TashAT1 are additional candidates for parasite encoded factors that are translocated to the host nucleus, bind to DNA and alter host cell gene expression. This modulation of gene expression could directly or indirectly alter the phenotype of the host cell and be involved in parasite dependent regulation of leukocyte cell division

Cognitive dysfunction in naturally occurring canine idiopathic epilepsy

Globally, epilepsy is a common serious brain disorder. In addition to seizure activity, epilepsy is associated with cognitive impairments including static cognitive impairments present at onset, progressive seizure-induced impairments and co-morbid dementia. Epilepsy occurs naturally in domestic dogs but its impact on canine cognition has yet to be studied, despite canine cognitive dysfunction (CCD) recognised as a spontaneous model of dementia. Here we use data from a psychometrically validated tool, the canine cognitive dysfunction rating (CCDR) scale, to compare cognitive dysfunction in dogs diagnosed with idiopathic epilepsy (IE) with controls while accounting for age. An online cross-sectional study resulted in a sample of 4051 dogs, of which n = 286 had been diagnosed with IE. Four factors were significantly associated with a diagnosis of CCD (above the diagnostic cut-off of CCDR ≥50): (i) epilepsy diagnosis: dogs with epilepsy were at higher risk; (ii) age: older dogs were at higher risk; (iii) weight: lighter dogs (kg) were at higher risk; (iv) training history: dogs with more exposure to training activities were at lower risk. Impairments in memory were most common in dogs with IE, but progression of impairments was not observed compared to controls. A significant interaction between epilepsy and age was identified, with IE dogs exhibiting a higher risk of CCD at a young age, while control dogs followed the expected pattern of low-risk throughout middle age, with risk increasing exponentially in geriatric years. Within the IE sub-population, dogs with a history of cluster seizures and high seizure frequency had higher CCDR scores. The age of onset, nature and progression of cognitive impairment in the current IE dogs appear divergent from those classically seen in CCD. Longitudinal monitoring of cognitive function from seizure onset is required to further characterise these impairments

Molecular analyses of protists in long-term observation programmes—current status and future perspectives

Protists (microbial eukaryotes) are diverse, major components of marine ecosystems, and are fundamental to ecosystem services. In the last 10 years, molecular studies have highlighted substantial novel diversity in marine systems including sequences with no taxonomic context. At the same time, many known protists remain without a DNA identity. Since the majority of pelagic protists are too small to identify by light microscopy, most are neither comprehensively or regularly taken into account, particularly in Long-term Ecological Research Sites. This potentially undermines the quality of research and the accuracy of predictions about biological species shifts in a changing environment. The ICES Working Group for Phytoplankton and Microbial Ecology conducted a questionnaire survey in 2013–2014 on methods and identification of protists using molecular methods plus a literature review of protist molecular diversity studies. The results revealed an increased use of high-throughput sequencing methods and a recognition that sequence data enhance the overall datasets on protist species composition. However, we found only a few long-term molecular studies and noticed a lack of integration between microscopic and molecular methods. Here, we discuss and put forward recommendations to improve and make molecular methods more accessible to Long-term Ecological Research Site investigators

The Continuous Plankton Recorder survey: how can long-term phytoplankton datasets contribute to the assessment of Good Environmental Status?

Phytoplankton are crucial to marine ecosystem functioning and are important indicators of environmental change. Phytoplankton data are also essential for informing management and policy, particularly in supporting the new generation of marine legislative drivers, which take a holistic ecosystem approach to management. The Marine Strategy Framework Directive (MSFD) seeks to achieve Good Environmental Status (GES) of European seas through the implementation of such a management approach. This is a regional scale directive which recognises the importance of plankton communities in marine ecosystems; plankton data at the appropriate spatial, temporal and taxonomic scales are therefore required for implementation. The Continuous Plankton Recorder (CPR) survey is a multidecadal, North Atlantic–basin scale programme which routinely records approximately 300 phytoplankton taxa. Because of these attributes, the survey plays a key role in the implementation of the MSFD and the assessment of GES in the Northeast Atlantic region. This paper addresses the role of the CPR's phytoplankton time-series in delivering GES through the development and informing of MSFD indicators, the setting of targets against a background of climate change and the provision of supporting information used to interpret change in non-plankton indicators. We also discuss CPR data in the context of other phytoplankton data types that may contribute to GES, as well as explore future possibilities for the use of new and innovative applications of CPR phytoplankton datasets in delivering GES. Efforts must be made to preserve long-term time series, such as the CPR, which supply vital ecological information used to informed evidence-based environmental policy

Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Rowena F. Stern is with University of British Columbia, Ales Horak is with University of British Columbia, Rose L. Andrew is with University of British Columbia, Mary-Alice Coffroth is with State University of New York at Buffalo, Robert A. Andersen is with the Bigelow Laboratory for Ocean Sciences, Frithjof C. Küpper is with the Scottish Marine Institute, Ian Jameson is with CSIRO Marine and Atmospheric Research, Mona Hoppenrath is with the German Center for Marine Biodiversity Research, Benoît Véron is with University of Caen Lower Normandy and the National Institute for Environmental Studies, Fumai Kasai is with the National Institute for Environmental Studies, Jerry Brand is with UT Austin, Erick R. James is with University of British Columbia, Patrick J. Keeling is with University of British Columbia.Background -- Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings -- In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance -- COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.This project was funded by Genome Canada and the Canadian Barcode of Life Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Characterisation of two members of a macroschizont gene family, Tashat1 and Tashat3, from Theileria annulata

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