Neoblast-like stem cells of Fasciola hepatica.

The common liver fluke (Fasciola hepatica) causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of putative novel targets due to their role in development and homeostasis, including at host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, differentiation, and are sensitive to ionising radiation- all attributes of neoblasts in other flatworms. Changes in cell proliferation were also noted during the early stages of in vitro juvenile growth/development (around four to seven days post excystment), which coincided with a marked reduction in the nuclear area of proliferating cells. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologues associated with neoblast-like cells in Schistosoma mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblast-like cells and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics.

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.

Thermoregulatory differences in African mole-rat species from disparate habitats: Responses and limitations.

Individuals and populations possess physiological adaptations to survive local environmental conditions. To occur in different regions where ambient temperature varies, animals must adopt appropriate thermoregulatory mechanisms. Failure to adjust to environmental challenges may result in species distributional range shifts or decreased viability. African mole-rats (Bathyergidae) occupy various habitats in sub-Saharan Africa from deserts to montane regions to mesic coastal areas. We examined thermoregulatory characteristics of three African mole-rat species originating from disparate (montane, savannah, and arid/semi-arid) habitats. Animals were exposed to various ambient temperatures, whilst core body temperature and the surface temperature of different body parts were measured. Oxygen consumption was determined as a measure of heat production. Core body temperatures of Natal (montane) mole-rats (Cryptomys hottentotus natalensis) increased significantly at ambient temperatures >24.5 °C, while those of the highveld (Cryptomys hottentotus pretoriae) (savannah) and Damaraland (Fukomys damarensis) (arid/semi-arid) mole-rats remained within narrower ranges. In terms of surface temperature variation, while pedal surfaces were important in regulating heat loss in Natal and Damaraland mole-rats at high ambient temperatures, the ventral surface was important for heat dissipation in Damaraland and highveld mole-rats. This study provides evidence of the variation and limitations of thermo-physiological mechanisms for three mole-rat species relative to their habitats. Information on physiological adaptations to particular habitats may inform predictive modelling of species movements, declines, and extinctions in response to a changing environment, such as climate change.

Dietary effects on pelage emissivity in mammals: Implications for infrared thermography.

Infrared thermography is becoming popular to measure animal surface temperature non-invasively. However, its application in quantitative mammal research is restricted by a paucity of pelage emissivity measurements, which are necessary to acquire accurate temperature readings. Furthermore, the factors influencing pelage emissivity remain largely unknown. We therefore examined the putative links between diet (fat content), hair length, hair diameter, and pelage emissivity in laboratory mice. Individuals maintained on high-fat diets had higher pelage emissivity values than those on standard diets, which may be due to fur being oily and/or the fact that the fur clumped together, exposing the skin underneath. Alternatively, the chemical composition of the fur of individuals on a high-fat diet may vary from those on a standard diet. We found no significant relationships between various hair metrics and emissivity. This study highlights that aspects of an animal's life history (e.g. age, sex, diet) may contribute to the emissivity of its pelage. As such, a single emissivity value may be inappropriate for use in infrared thermography across all species or individuals; other aspects of an animal's biology, which may affect emissivity, should also be considered. Best practice should involve measuring emissivity for every individual animal used in thermography studies.

No energetic cost of tuberculosis infection in European badgers (Meles meles).

Energy availability and energy use directly influence an organism's life history, fitness and ecological function. In wild animals, abiotic factors such as ambient temperature, season and rainfall, and biotic factors such as body mass, age, social group size and disease status, all potentially influence energy balance. Relatively few studies have examined the effects of disease on the energy expenditure of wild animals. Such studies could further our understanding of factors influencing the transmission of zoonotic diseases. The European badger (Meles meles) is a medium-sized carnivore that occurs in mixed-sex, familial groups across much of its range. In the UK, they are a protected species but are also involved in the epidemiology of bovine tuberculosis (TB) in cattle. We measured the daily energy expenditure (DEE) and resting metabolic rate (RMR) of wild badgers and related this to their TB infection status and a range of other interacting factors including season, group size, disease status, sex, age, body mass and body fat. Individuals were larger and fatter when they were older, and fatter during the winter. Males were also heavier than females during the summer. In addition, individuals from smaller groups that were exposed to TB tended to have lower body mass. There were no direct effects of disease status on DEE or RMR; however, there was a significant interaction whereby DEE increased with body mass in small groups but decreased with body mass in large groups. Results are consistent with the costs of TB infection being met by compensatory mechanisms enabling badgers to survive for extended periods without exhibiting measurable energetic consequences.

flp-32 Ligand/receptor silencing phenocopy faster plant pathogenic nematodes.

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R-silenced worms also display an increase in migration rate. This work demonstrates that Gp-flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR.

Identifying animal behaviours from accelerometers: Improving predictive accuracy of machine learning by refining the variables selected, data frequency, and sample duration.

Observing animals in the wild often poses extreme challenges, but animal-borne accelerometers are increasingly revealing unobservable behaviours. Automated machine learning streamlines behaviour identification from the substantial datasets generated during multi-animal, long-term studies; however, the accuracy of such models depends on the qualities of the training data. We examined how data processing influenced the predictive accuracy of random forest (RF) models, leveraging the easily observed domestic cat (Felis catus) as a model organism for terrestrial mammalian behaviours. Nine indoor domestic cats were equipped with collar-mounted tri-axial accelerometers, and behaviours were recorded alongside video footage. From this calibrated data, eight datasets were derived with (i) additional descriptive variables, (ii) altered frequencies of acceleration data (40 Hz vs. a mean over 1 s) and (iii) standardised durations of different behaviours. These training datasets were used to generate RF models that were validated against calibrated cat behaviours before identifying the behaviours of five free-ranging tag-equipped cats. These predictions were compared to those identified manually to validate the accuracy of the RF models for free-ranging animal behaviours. RF models accurately predicted the behaviours of indoor domestic cats (F-measure up to 0.96) with discernible improvements observed with post-data-collection processing. Additional variables, standardised durations of behaviours and higher recording frequencies improved model accuracy. However, prediction accuracy varied with different behaviours, where high-frequency models excelled in identifying fast-paced behaviours (e.g. locomotion), whereas lower-frequency models (1 Hz) more accurately identified slower, aperiodic behaviours such as grooming and feeding, particularly when examining free-ranging cat behaviours. While RF modelling offered a robust means of behaviour identification from accelerometer data, field validations were important to validate model accuracy for free-ranging individuals. Future studies may benefit from employing similar data processing methods that enhance RF behaviour identification accuracy, with extensive advantages for investigations into ecology, welfare and management of wild animals.

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.

Cheetahs, Acinonyx jubatus, balance turn capacity with pace when chasing prey.

Predator-prey interactions are fundamental in the evolution and structure of ecological communities. Our understanding, however, of the strategies used in pursuit and evasion remains limited. Here, we report on the hunting dynamics of the world's fastest land animal, the cheetah, Acinonyx jubatus. Using miniaturized data loggers, we recorded fine-scale movement, speed and acceleration of free-ranging cheetahs to measure how hunting dynamics relate to chasing different sized prey. Cheetahs attained hunting speeds of up to 18.94 m s(-1) and accelerated up to 7.5 m s(-2) with greatest angular velocities achieved during the terminal phase of the hunt. The interplay between forward and lateral acceleration during chases showed that the total forces involved in speed changes and turning were approximately constant over time but varied with prey type. Thus, rather than a simple maximum speed chase, cheetahs first accelerate to decrease the distance to their prey, before reducing speed 5-8 s from the end of the hunt, so as to facilitate rapid turns to match prey escape tactics, varying the precise strategy according to prey species. Predator and prey thus pit a fine balance of speed against manoeuvring capability in a race for survival.

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.

Considering RNAi experimental design in parasitic helminths.

Almost a decade has passed since the first report of RNA interference (RNAi) in a parasitic helminth. Whilst much progress has been made with RNAi informing gene function studies in disparate nematode and flatworm parasites, substantial and seemingly prohibitive difficulties have been encountered in some species, hindering progress. An appraisal of current practices, trends and ideals of RNAi experimental design in parasitic helminths is both timely and necessary for a number of reasons: firstly, the increasing availability of parasitic helminth genome/transcriptome resources means there is a growing need for gene function tools such as RNAi; secondly, fundamental differences and unique challenges exist for parasite species which do not apply to model organisms; thirdly, the inherent variation in experimental design, and reported difficulties with reproducibility undermine confidence. Ideally, RNAi studies of gene function should adopt standardised experimental design to aid reproducibility, interpretation and comparative analyses. Although the huge variations in parasite biology and experimental endpoints make RNAi experimental design standardization difficult or impractical, we must strive to validate RNAi experimentation in helminth parasites. To aid this process we identify multiple approaches to RNAi experimental validation and highlight those which we deem to be critical for gene function studies in helminth parasites.

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.

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.

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.

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.

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.

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.