Unraveling flp-11/flp-32 dichotomy in nematodes

AbstractFMRFamide-like peptide (FLP) signalling systems are core to nematode neuromuscular function. Novel drug discovery efforts associated with nematode FLP/FLP receptor biology are advanced through the accumulation of basic biological data that can reveal subtle complexities within the neuropeptidergic system. This study reports the characterisation of FMRFamide-like peptide encoding gene-11 (flp-11) and FMRFamide-like peptide encoding gene-32 (flp-32), two distinct flp genes which encode the analogous peptide, AMRN(A/S)LVRFamide, in multiple nematode species – the only known example of this phenomenon within the FLPergic system of nematodes. Using bioinformatics, in situ hybridisation, immunocytochemistry and behavioural assays we show that: (i) flp-11 and -32 are distinct flp genes expressed individually or in tandem across multiple nematode species, where they encode a highly similar peptide; (ii) flp-11 does not appear to be the most widely expressed flp in Caenorhabditis elegans; (iii) in species expressing both flp-11 and flp-32, flp-11 displays a conserved, restricted expression pattern across nematode clades and lifestyles; (iv) in species expressing both flp-11 and flp-32, flp-32 expression is more widespread and less conserved than flp-11; (v) in species expressing only flp-11, the flp-11 expression profile is more similar to the flp-32 profile observed in species expressing both; and (vi) FLP-11 peptides inhibit motor function in multiple nematode species. The biological significance and evolutionary origin of flp-11 and -32 peptide duplication remains unclear despite attempts to identify a common ancestor; this may become clearer as the availability of genomic data improves. This work provides insight into the complexity of the neuropeptidergic system in nematodes, and begins to examine how nematodes may compensate for structural neuronal simplicity. From a parasite control standpoint, this work underscores the importance of basic biological data, and has wider implications for the utility of C. elegans as a model for parasite neurobiology

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.<br/

The ability of magnetic field sensors to monitor feeding in three domestic herbivores

The rate at which animals ingest food is a fundamental part of animal ecology although it is rarely quantified, with recently-developed animal-attached tags providing a potentially viable approach. However, to date, these methods lack clarity in differentiating various eating behaviours, such as ‘chewing’ from ‘biting’. The aims of this study were to examine the use of inter-mandibular angle sensors (IMASENs), to quantify grazing behaviour in herbivores including cattle (Bos taurus), sheep (Ovis aries) and pygmy goats (Capra aegagrus hircus) eating different foodstuffs. Specifically, we aimed to: (1) quantify jaw movements of each species and determine differences between biting and chewing; (2) assess whether different food types can be discerned from jaw movements; and (3) determine whether species-specific differences in jaw movements can be detected. Subjects were filmed while consuming concentrate, hay, grass and browse to allow comparison of observed and IMASEN-recorded jaw movements. This study shows that IMASENs can accurately detect jaw movements of feeding herbivores, and, based on the rate of jaw movements, can classify biting (taking new material into the mouth) from chewing (masticating material already in the mouth). The biting behaviours associated with concentrate pellets could be identified easily as these occurred at the fastest rate for all species. However, the rates of chewing different food items were more difficult to discern from one another. Comparison of chew:bite ratios of the various food types eaten by each species showed no differences. Species differences could be identified using bite and chew rates. Cattle consistently displayed slower bite and chew rates to sheep and pygmy goats when feeding, while sheep and pygmy goats showed similar bite and chew rates when feeding on concentrate pellets. Species-specific differences in chew:bite ratios were not identified. Magnetometry has the potential to record quantitative aspects of foraging such as the feeding duration, food handling time and food type. This is of major importance for researchers interested in both captive (e.g., agricultural productivity) and wild animal foraging dynamics as it can provide quantitative data with minimal observer interference

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro

Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke

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 o

RNAi dynamics in juvenile Fasciola spp. liver flukes reveals the persistence of gene silencing in vitro

Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection

Squirrelpox virus: assessing prevalence, transmission and environmental degradation

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species

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

Dead-reckoning animal movements in R: a reappraisal using Gundog.Tracks

BackgroundFine-scale data on animal position are increasingly enabling us to understand the details of animal movement ecology and dead-reckoning, a technique integrating motion sensor-derived information on heading and speed, can be used to reconstruct fine-scale movement paths at sub-second resolution, irrespective of the environment. On its own however, the dead-reckoning process is prone to cumulative errors, so that position estimates quickly become uncoupled from true location. Periodic ground-truthing with aligned location data (e.g., from global positioning technology) can correct for this drift between Verified Positions (VPs). We present step-by-step instructions for implementing Verified Position Correction (VPC) dead-reckoning in R using the tilt-compensated compass method, accompanied by the mathematical protocols underlying the code and improvements and extensions of this technique to reduce the trade-off between VPC rate and dead-reckoning accuracy. These protocols are all built into a user-friendly, fully annotated VPC dead-reckoning R function; Gundog.Tracks, with multi-functionality to reconstruct animal movement paths across terrestrial, aquatic, and aerial systems, provided within the Additional file 4 as well as online (GitHub).ResultsThe Gundog.Tracks function is demonstrated on three contrasting model species (the African lion Panthera leo, the Magellanic penguin Spheniscus magellanicus, and the Imperial cormorant Leucocarbo atriceps) moving on land, in water and in air. We show the effect of uncorrected errors in speed estimations, heading inaccuracies and infrequent VPC rate and demonstrate how these issues can be addressed.ConclusionsThe function provided will allow anyone familiar with R to dead-reckon animal tracks readily and accurately, as the key complex issues are dealt with by Gundog.Tracks. This will help the community to consider and implement a valuable, but often overlooked method of reconstructing high-resolution animal movement paths across diverse species and systems without requiring a bespoke application