Host movement dominates the predicted effects of climate change on parasite transmission between wild and domestic mountain ungulates.

Climate change is shifting the transmission of parasites, which is determined by host density, ambient temperature and moisture. These shifts can lead to increased pressure from parasites, in wild and domestic animals, and can impact the effectiveness of parasite control strategies. Understanding the interactive effects of climate on host movement and parasite life histories will enable targeted parasite management, to ensure livestock productivity and avoid additional stress on wildlife populations. To assess complex outcomes under climate change, we applied a gastrointestinal nematode transmission model to a montane wildlife-livestock system, based on host movement and changes in abiotic factors due to elevation, comparing projected climate change scenarios with the historic climate. The wildlife host, Alpine ibex (Capra ibex ibex), undergoes seasonal elevational migration, and livestock are grazed during the summer for eight weeks. Total parasite infection pressure was more sensitive to host movement than to the direct effect of climatic conditions on parasite availability. Extended livestock grazing is predicted to increase parasite exposure for wildlife. These results demonstrate that movement of different host species should be considered when predicting the effects of climate change on parasite transmission, and can inform decisions to support wildlife and livestock health.

Discovery of long non-coding RNAs in the liver fluke, Fasciola hepatica.

Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a custom in silico pipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite, Fasciola hepatica, none of which are conserved with those previously described from Schistosoma mansoni. F. hepatica lncRNAs were distinct from F. hepatica mRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2%) and mRNAs (12.8%) were differentially expressed (p<0.001), supporting a functional role for lncRNAs in F. hepatica life stages. While most lncRNAs (81%) were intergenic, we identified some that overlapped protein coding loci in antisense (13%) or intronic (6%) configurations. We found no unequivocal evidence for correlated developmental expression within positionally correlated lncRNA:mRNA pairs, but global co-expression analysis identified five lncRNA that were inversely co-regulated with 89 mRNAs, including a large number of functionally essential proteases. The presence of micro (mi)RNA binding sites in 3135 lncRNAs indicates the potential for miRNA-based post-transcriptional regulation of lncRNA, and/or their function as competing endogenous (ce)RNAs. The same annotation pipeline identified 24,141 putative lncRNAs in F. gigantica. This first description of lncRNAs in F. hepatica provides an avenue to future functional and comparative genomics studies that will provide a new perspective on a poorly understood aspect of parasite biology.

Impact of test, vaccinate or remove protocol on home ranges and nightly movements of badgers a medium density population.

In the British Isles, the European badger (Meles meles) is thought to be the primary wildlife reservoir of bovine tuberculosis (bTB), an endemic disease in cattle. Test, vaccinate or remove ('TVR') of bTB test-positive badgers, has been suggested to be a potentially useful protocol to reduce bTB incidence in cattle. However, the practice of removing or culling badgers is controversial both for ethical reasons and because there is no consistent observed effect on bTB levels in cattle. While removing badgers reduces population density, it may also result in disruption of their social behaviour, increase their ranging, and lead to greater intra- and inter-species bTB transmission. This effect has been recorded in high badger density areas, such as in southwest England. However, little is known about how TVR affects the behaviour and movement of badgers within a medium density population, such as those that occur in Northern Ireland (NI), which the current study aimed to examine. During 2014-2017, badger ranging behaviours were examined prior to and during a TVR protocol in NI. Nightly distances travelled by 38 individuals were determined using Global Positioning System (GPS) measurements of animal tracks and GPS-enhanced dead-reckoned tracks. The latter was calculated using GPS, tri-axial accelerometer and tri-axial magnetometer data loggers attached to animals. Home range and core home range size were measured using 95% and 50% autocorrelated kernel density estimates, respectively, based on location fixes. TVR was not associated with measured increases in either distances travelled per night (mean = 3.31 ± 2.64 km) or home range size (95% mean = 1.56 ± 0.62 km2, 50% mean = 0.39 ± 0.62 km2) over the four years of study. However, following trapping, mean distances travelled per night increased by up to 44% eight days post capture. Findings differ from those observed in higher density badger populations in England, in which badger ranging increased following culling. Whilst we did not assess behaviours of individual badgers, possible reasons why no differences in home range size were observed include higher inherent 'social fluidity' in Irish populations whereby movements are less restricted by habitat saturation and/or that the numbers removed did not reach a threshold that might induce increases in ranging behaviour. Nevertheless, short-term behavioural disruption from trapping was observed, which led to significant increases in the movements of individual animals within their home range. Whether or not TVR may alter badger behaviours remains to be seen, but it would be better to utilise solutions such as oral vaccination of badgers and/or cattle as well as increased biosecurity to limit bTB transmission, which may be less likely to cause interference and thereby reduce the likelihood of bTB transmission.

Transcriptomic analysis supports a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles.

Fasciola spp. liver flukes have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, including fhe-let-7a-5p, fhe-mir-124-3p and miRNAs predicted to target Wnt-signalling, which supports a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control.

The Strongyloides bioassay toolbox: A unique opportunity to accelerate functional biology for nematode parasites.

Caenorhabditis elegans is a uniquely powerful tool to aid understanding of fundamental nematode biology. While C. elegans boasts an unrivalled array of functional genomics tools and phenotype bioassays the inherent differences between free-living and parasitic nematodes underscores the need to develop these approaches in tractable parasite models. Advances in functional genomics approaches, including RNA interference and CRISPR/Cas9 gene editing, in the parasitic nematodes Strongyloides ratti and Strongyloides stercoralis provide a unique and timely opportunity to probe basic parasite biology and reveal novel anthelmintic targets in species that are both experimentally and therapeutically relevant pathogens. While Strongyloides functional genomics tools have progressed rapidly, the complementary range of bioassays required to elucidate phenotypic outcomes post-functional genomics remain more limited in scope. To adequately support the exploitation of functional genomic pipelines for studies of gene function in Strongyloides a comprehensive set of species- and parasite-specific quantitative bioassays are required to assess nematode behaviours post-genetic manipulation. Here we review the scope of the current Strongyloides bioassay toolbox, how established Strongyloides bioassays have advanced knowledge of parasite biology, opportunities for Strongyloides bioassay development and, the need for investment in tractable model parasite platforms such as Strongyloides to drive the discovery of novel targets for parasite control.

Pan-phylum In Silico Analyses of Nematode Endocannabinoid Signalling Systems Highlight Novel Opportunities for Parasite Drug Target Discovery.

The endocannabinoid signalling (ECS) system is a complex lipid signalling pathway that modulates diverse physiological processes in both vertebrate and invertebrate systems. In nematodes, knowledge of endocannabinoid (EC) biology is derived primarily from the free-living model species Caenorhabditis elegans, where ECS has been linked to key aspects of nematode biology. The conservation and complexity of nematode ECS beyond C. elegans is largely uncharacterised, undermining the understanding of ECS biology in nematodes including species with key importance to human, veterinary and plant health. In this study we exploited publicly available omics datasets, in silico bioinformatics and phylogenetic analyses to examine the presence, conservation and life stage expression profiles of EC-effectors across phylum Nematoda. Our data demonstrate that: (i) ECS is broadly conserved across phylum Nematoda, including in therapeutically and agriculturally relevant species; (ii) EC-effectors appear to display clade and lifestyle-specific conservation patterns; (iii) filarial species possess a reduced EC-effector complement; (iv) there are key differences between nematode and vertebrate EC-effectors; (v) life stage-, tissue- and sex-specific EC-effector expression profiles suggest a role for ECS in therapeutically relevant parasitic nematodes. To our knowledge, this study represents the most comprehensive characterisation of ECS pathways in phylum Nematoda and inform our understanding of nematode ECS complexity. Fundamental knowledge of nematode ECS systems will seed follow-on functional studies in key nematode parasites to underpin novel drug target discovery efforts.

Developmental Regulation and Functional Prediction of microRNAs in an Expanded Fasciola hepatica miRNome.

The liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analyzed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.

Categorising cheetah behaviour using tri-axial accelerometer data loggers: a comparison of model resolution and data logger performance.

BACKGROUND: Extinction is one of the greatest threats to the living world, endangering organisms globally, advancing conservation to the forefront of species research. To maximise the efficacy of conservation efforts, understanding the ecological, physiological, and behavioural requirements of vulnerable species is vital. Technological advances, particularly in remote sensing, enable researchers to continuously monitor movement and behaviours of multiple individuals simultaneously with minimal human intervention. Cheetahs, Acinonyx jubatus, constitute a "vulnerable" species for which only coarse behaviours have been elucidated. The aims of this study were to use animal-attached accelerometers to (1) determine fine-scale behaviours in cheetahs, (2) compare the performances of different devices in behaviour categorisation, and (3) provide a behavioural categorisation framework. METHODS: Two different accelerometer devices (CEFAS, frequency: 30 Hz, maximum capacity: ~ 2 g; GCDC, frequency: 50 Hz, maximum capacity: ~ 8 g) were mounted onto collars, fitted to five individual captive cheetahs. The cheetahs chased a lure around a track, during which time their behaviours were videoed. Accelerometer data were temporally aligned with corresponding video footage and labelled with one of 17 behaviours. Six separate random forest models were run (three per device type) to determine the categorisation accuracy for behaviours at a fine, medium, and coarse resolution. RESULTS: Fine- and medium-scale models had an overall categorisation accuracy of 83-86% and 84-88% respectively. Non-locomotory behaviours were best categorised on both loggers with GCDC outperforming CEFAS devices overall. On a coarse scale, both devices performed well when categorising activity (86.9% (CEFAS) vs. 89.3% (GCDC) accuracy) and inactivity (95.5% (CEFAS) vs. 95.0% (GCDC) accuracy). This study defined cheetah behaviour beyond three categories and accurately determined stalking behaviours by remote sensing. We also show that device specification and configuration may affect categorisation accuracy, so we recommend deploying several different loggers simultaneously on the same individual. CONCLUSION: The results of this study will be useful in determining wild cheetah behaviour. The methods used here allowed broad-scale (active/inactive) as well as fine-scale (e.g. stalking) behaviours to be categorised remotely. These findings and methodological approaches will be useful in monitoring the behaviour of wild cheetahs and other species of conservation interest.

Decision rules for determining terrestrial movement and the consequences for filtering high-resolution global positioning system tracks: a case study using the African lion (Panthera leo).

The combined use of global positioning system (GPS) technology and motion sensors within the discipline of movement ecology has increased over recent years. This is particularly the case for instrumented wildlife, with many studies now opting to record parameters at high (infra-second) sampling frequencies. However, the detail with which GPS loggers can elucidate fine-scale movement depends on the precision and accuracy of fixes, with accuracy being affected by signal reception. We hypothesized that animal behaviour was the main factor affecting fix inaccuracy, with inherent GPS positional noise (jitter) being most apparent during GPS fixes for non-moving locations, thereby producing disproportionate error during rest periods. A movement-verified filtering (MVF) protocol was constructed to compare GPS-derived speed data with dynamic body acceleration, to provide a computationally quick method for identifying genuine travelling movement. This method was tested on 11 free-ranging lions (Panthera leo) fitted with collar-mounted GPS units and tri-axial motion sensors recording at 1 and 40 Hz, respectively. The findings support the hypothesis and show that distance moved estimates were, on average, overestimated by greater than 80% prior to GPS screening. We present the conceptual and mathematical protocols for screening fix inaccuracy within high-resolution GPS datasets and demonstrate the importance that MVF has for avoiding inaccurate and biased estimates of movement.

Soil δ13C and δ15N baselines clarify biogeographic heterogeneity in isotopic discrimination of European badgers (Meles meles).

Isotopic techniques have been used to study phenomena in the geological, environmental, and ecological sciences. For example, isotopic values of multiple elements elucidate the pathways energy and nutrients take in the environment. Isoscapes interpolate isotopic values across a geographical surface and are used to study environmental processes in space and time. Thus, isoscapes can reveal ecological shifts at local scales, and show distribution thresholds in the wider environment at the macro-scale. This study demonstrates a further application of isoscapes, using soil isoscapes of 13C/12C and 15N/14N as an environmental baseline, to understand variation in trophic ecology across a population of Eurasian badgers (Meles meles) at a regional scale. The use of soil isoscapes reduced error, and elevated the statistical signal, where aggregated badger hairs were used, and where individuals were identified using genetic microarray analysis. Stable isotope values were affected by land-use type, elevation, and meteorology. Badgers in lowland habitats had diets richer in protein and were adversely affected by poor weather conditions in all land classes. It is concluded that soil isoscapes are an effective way of reducing confounding biases in macroscale, isotopic studies. The method elucidated variation in the trophic and spatial ecology of economically important taxa at a landscape level. These results have implications for the management of badgers and other carnivores with omnivorous tendencies in heterogeneous landscapes.

In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes.

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target.

How often should dead-reckoned animal movement paths be corrected for drift?

BACKGROUND: Understanding what animals do in time and space is important for a range of ecological questions, however accurate estimates of how animals use space is challenging. Within the use of animal-attached tags, radio telemetry (including the Global Positioning System, 'GPS') is typically used to verify an animal's location periodically. Straight lines are typically drawn between these 'Verified Positions' ('VPs') so the interpolation of space-use is limited by the temporal and spatial resolution of the system's measurement. As such, parameters such as route-taken and distance travelled can be poorly represented when using VP systems alone. Dead-reckoning has been suggested as a technique to improve the accuracy and resolution of reconstructed movement paths, whilst maximising battery life of VP systems. This typically involves deriving travel vectors from motion sensor systems and periodically correcting path dimensions for drift with simultaneously deployed VP systems. How often paths should be corrected for drift, however, has remained unclear. METHODS AND RESULTS: Here, we review the utility of dead-reckoning across four contrasting model species using different forms of locomotion (the African lion Panthera leo, the red-tailed tropicbird Phaethon rubricauda, the Magellanic penguin Spheniscus magellanicus, and the imperial cormorant Leucocarbo atriceps). Simulations were performed to examine the extent of dead-reckoning error, relative to VPs, as a function of Verified Position correction (VP correction) rate and the effect of this on estimates of distance moved. Dead-reckoning error was greatest for animals travelling within air and water. We demonstrate how sources of measurement error can arise within VP-corrected dead-reckoned tracks and propose advancements to this procedure to maximise dead-reckoning accuracy. CONCLUSIONS: We review the utility of VP-corrected dead-reckoning according to movement type and consider a range of ecological questions that would benefit from dead-reckoning, primarily concerning animal-barrier interactions and foraging strategies.

Phylum-Spanning Neuropeptide GPCR Identification and Prioritization: Shaping Drug Target Discovery Pipelines for Nematode Parasite Control.

Nematode parasites undermine human health and global food security. The frontline anthelmintic portfolio used to treat parasitic nematodes is threatened by the escalation of anthelmintic resistance, resulting in a demand for new drug targets for parasite control. Nematode neuropeptide signalling pathways represent an attractive source of novel drug targets which currently remain unexploited. The complexity of the nematode neuropeptidergic system challenges the discovery of new targets for parasite control, however recent advances in parasite 'omics' offers an opportunity for the in silico identification and prioritization of targets to seed anthelmintic discovery pipelines. In this study we employed Hidden Markov Model-based searches to identify ~1059 Caenorhabditis elegans neuropeptide G-protein coupled receptor (Ce-NP-GPCR) encoding gene homologs in the predicted protein datasets of 10 key parasitic nematodes that span several phylogenetic clades and lifestyles. We show that, whilst parasitic nematodes possess a reduced complement of Ce-NP-GPCRs, several receptors are broadly conserved across nematode species. To prioritize the most appealing parasitic nematode NP-GPCR anthelmintic targets, we developed a novel in silico nematode parasite drug target prioritization pipeline that incorporates pan-phylum NP-GPCR conservation, C. elegans-derived reverse genetics phenotype, and parasite life-stage specific expression datasets. Several NP-GPCRs emerge as the most attractive anthelmintic targets for broad spectrum nematode parasite control. Our analyses have also identified the most appropriate targets for species- and life stage- directed chemotherapies; in this context we have identified several NP-GPCRs with macrofilaricidal potential. These data focus functional validation efforts towards the most appealing NP-GPCR targets and, in addition, the prioritization strategy employed here provides a blueprint for parasitic nematode target selection beyond NP-GPCRs.

Animal lifestyle affects acceptable mass limits for attached tags.

Animal-attached devices have transformed our understanding of vertebrate ecology. To minimize any associated harm, researchers have long advocated that tag masses should not exceed 3% of carrier body mass. However, this ignores tag forces resulting from animal movement. Using data from collar-attached accelerometers on 10 diverse free-ranging terrestrial species from koalas to cheetahs, we detail a tag-based acceleration method to clarify acceptable tag mass limits. We quantify animal athleticism in terms of fractions of animal movement time devoted to different collar-recorded accelerations and convert those accelerations to forces (acceleration × tag mass) to allow derivation of any defined force limits for specified fractions of any animal's active time. Specifying that tags should exert forces that are less than 3% of the gravitational force exerted on the animal's body for 95% of the time led to corrected tag masses that should constitute between 1.6% and 2.98% of carrier mass, depending on athleticism. Strikingly, in four carnivore species encompassing two orders of magnitude in mass (ca 2-200 kg), forces exerted by '3%' tags were equivalent to 4-19% of carrier body mass during moving, with a maximum of 54% in a hunting cheetah. This fundamentally changes how acceptable tag mass limits should be determined by ethics bodies, irrespective of the force and time limits specified.

Ascaris suum Informs Extrasynaptic Volume Transmission in Nematodes.

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other nonsynaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum, FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we investigate nonsynaptic neuropeptide signaling in nematodes mediated by the body cavity fluid. Our data show that (i) A. suum PCF (As-PCF) contains a catalog of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins, (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome, (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides, (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5, (v) As-PCF alters C. elegans behavior, and (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. This study provides the first direct experimental evidence to support an extrasynaptic volume route for neuropeptide transmission in nematodes. These data indicate nonsynaptic signaling within the nematode functional connectome and are particularly pertinent to receptor deorphanization approaches underpinning drug discovery programs for nematode pathogens.

Molecular characterisation and vaccine efficacy of two novel developmentally regulated surface tegument proteins of Fasciola hepatica.

The surface tegument of Fasciola hepatica is a crucial tissue due to its key role at the host-parasite interface. We characterised three novel proteins, termed Fhteg1, Fhteg5 and Fhteg8, that are found in the tegument membrane fraction of adult F. hepatica. Bioinformatic analysis of proteomic datasets identified Fhteg5 and Fhteg8 as tegumental glycoproteins and revealed that Fhteg1, Fhteg5 and Fhteg8 are associated with exosomes of adult F. hepatica. Fhteg1, Fhteg5 and Fhteg8 appear to be related to uncharacterised sequences in F. gigantica, Fasciolopsis buski, Echinostoma caproni, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum and S. mansoni, although F. hepatica appears to have expanded this family. Fhteg1 and Fhteg5 were characterised in detail. The Fhteg1 and Fhteg5 gene transcripts each demonstrate significant upregulation in juvenile fluke 2-4 days post-excystment, with transcript levels maintained during development over 3 weeks in vitro. RNAseq data showed that both Fhtegs are expressed in the adult life stage, although the transcript levels were about 8 fold lower than those in juveniles (3 week post infection). Using immunocytochemistry, Fhteg1 and Fhteg5 were each shown to be expressed in cells adjacent to the muscle layer as well as on the surface of 1 week old juveniles, whilst Fhteg5 was also present in cells at the base of the pharynx. RNAi mediated knockdown of Fhteg1 and Fhteg5 transcripts in 4-10 day old juveniles had no effect on parasite survival, movement or growth in vitro. Although no IgG responses were observed for Fhteg1 or Fhteg5 during infection in sheep and cattle, both proteins elicited a low IgG response in a proportion of infected rats. Rats vaccinated with Fhteg1 and Fhteg5 showed good IgG responses to both proteins and a mean 48.2 % reduction in worm burden following parasite challenge. Although vaccination of cattle with both proteins induced a range of IgG responses, no protection was observed against parasite challenge. This is the first study to provide insights into the molecular properties of two novel, developmentally regulated surface tegument proteins in F. hepatica.

RNA interference dynamics in juvenile Fasciola hepatica are altered during in vitro growth and development.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

Surviving in steep terrain: a lab-to-field assessment of locomotor costs for wild mountain lions (Puma concolor).

BACKGROUND: Under current scenarios of climate change and habitat loss, many wild animals, especially large predators, are moving into novel energetically challenging environments. Consequently, changes in terrain associated with such moves may heighten energetic costs and effect the decline of populations in new localities. METHODS: To examine locomotor costs of a large carnivorous mammal moving in mountainous habitats, the oxygen consumption of captive pumas (Puma concolor) was measured during treadmill locomotion on level and incline (6.8°) surfaces. These data were used to predict energetic costs of locomotor behaviours of free-ranging pumas equipped with GPS/accelerometer collars in California's Santa Cruz Mountains. RESULTS: Incline walking resulted in a 42.0% ± 7.2 SEM increase in the costs of transport compared to level performance. Pumas negotiated steep terrain by traversing across hillsides (mean hill incline 17.2° ± 0.3 SEM; mean path incline 7.3° ± 0.1 SEM). Pumas also walked more slowly up steeper paths, thereby minimizing the energetic impact of vertical terrains. Estimated daily energy expenditure (DEE) based on GPS-derived speeds of free-ranging pumas was 18.3 MJ day- 1 ± 0.2 SEM. Calculations show that a 20 degree increase in mean steepness of the terrain would increase puma DEE by less than 1% as they only spend a small proportion (10%) of their day travelling. They also avoided elevated costs by utilizing slower speeds and shallower path angles. CONCLUSIONS: While many factors influence survival in novel habitats, we illustrate the importance of behaviours which reduce locomotor costs when traversing new, energetically challenging environments, and demonstrate that these behaviours are utilised by pumas in the wild.