Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis

The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

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 Gp30 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. © 2013 Atkinson et al.

The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999-2009

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions. (C) 2012 Elsevier B.V. All rights reserved.

Neuropeptides in Helminths: Occurrence and Distribution

Nematode neuropeptide systems comprise an exceptionally complex array of similar to 250 peptidic signaling molecules that operate within a structurally simple nervous system of similar to 300 neurons. A relatively complete picture of the neuropeptide complement is available for Caenorhabditis elegans, with 30 flp, 38 ins and 43 nlp genes having been documented; accumulating evidence indicates similar complexity in parasitic nematodes from clades I, III, IV and V. In contrast, the picture for parasitic platyhelminths is less clear, with the limited peptide sequence data available providing concrete evidence for only FMRFamide-like peptide (FLP) and neuropeptide F (NPF) signaling systems, each of which only comprises one or two peptides. With the completion of the Schmidtea meditteranea and Schistosoma mansoni genome projects and expressed sequence tag datasets for other flatworm parasites becoming available, the time is ripe for a detailed reanalysis of neuropeptide signaling in flatworms. Although the actual neuropeptides provide limited obvious value as targets for chemotherapeutic-based control strategies, they do highlight the signaling systems present in these helminths and provide tools for the discovery of more amenable targets such as neuropeptide receptors or neuropeptide processing enzymes. Also, they offer opportunities to evaluate the potential of their associated signaling pathways as targets through RNA interference (RNAi)-based, target validation strategies. Currently, within both helminth phyla, the flp signaling systems appear to merit further investigation as they are intrinsically linked with motor function, a proven target for successful anti-parasitics; it is clear that some nematode NLPs also play a role in motor function and could have similar appeal. At this time, it is unclear if flatworm NPF and nematode INS peptides operate in pathways that have utility for parasite control. Clearly, RNAi-based validation could be a starting point for scoring potential target pathways within neuropeptide signaling for parasiticide discovery programs. Also, recent successes in the application of in planta-based RNAi control strategies for plant parasitic nematodes reveal a strategy whereby neuropeptide encoding genes could become targets for parasite control. The possibility of developing these approaches for the control of animal and human parasites is intriguing, but will require significant advances in the delivery of RNAi-triggers.

A First Report of Anguina pacificae in Ireland

Anguina pacificae is a significant pest of Poa annua golf course greens in northern California. This study presents the first confirmed case of an A. pacificae infestation outside of North America, where the nematode's distribution is further restricted to a relatively limited coastal region. Species confirmation was made by morphometric and molecular methods and comparisons to closely related species including the European species, Anguina agropyri. The A. pacificae population detected on an Irish golf course was monitored over a 2-yr period and the life cycle compared with Californian population dynamics. A. pacificae was assessed for the potential risk of spreading to the local agricultural sector, in addition, the biosecurity risks from A. pacificae and plant parasitic nematodes in general were reviewed for northwest Europe.

Effects of Parasitism and Morphology on Squirrelpox Virus Seroprevalence in Grey Squirrels (Sciurus carolinensis)

Invasive species have been cited as major causes of population extinctions in several animal and plant classes worldwide. The North American grey squirrel (Sciurus carolinensis) has a major detrimental effect on native red squirrel (Sciurus vulgaris) populations across Britain and Ireland, in part because it can be a reservoir host for the deadly squirrelpox virus (SQPV). Whilst various researchers have investigated the epizootiology of SQPV disease in grey squirrels and have modelled the consequent effects on red squirrel populations, less work has examined morphological and physiological characteristics that might make individual grey squirrels more susceptible to contracting SQPV. The current study investigated the putative relationships between morphology, parasitism, and SQPV exposure in grey squirrels. We found geographical, sex, and morphological differences in SQPV seroprevalence. In particular, larger animals, those with wide zygomatic arch widths (ZAW), males with large testes, and individuals with concurrent nematode and/or coccidial infections had an increased seroprevalence of SQPV. In addition, males with larger spleens, particularly those with narrow ZAW, were more likely to be exposed to SQPV. Overall these results show that there is variation in SQPV seroprevalence in grey squirrels and that, consequently, certain individual, or populations of, grey squirrels might be more responsible for transmitting SQPV to native red squirrel populations.

Biosecurity and Emerging Plant Health Problems in Turf Production and Maintenance

Recent research has supported the view that the distributions of many pests and diseases have extended towards higher latitudes over the last 50 years. Probably driven by a combination of climate change and trade, this extension to the ranges of hundreds of plant pathogens may have serious implications not only for agriculture, horticulture and forestry, but also for turf production &maintenance. Here we review our data relating to the current status of three emerging pest and disease problems across North West Europe (rapid blight, Labyrinthula sp. , the root knot nematode, Meloidogyne minor and the pacific stem gall nematode, Anguina pacificae ) and discuss the factors which may be involved in their spread and increasing impact on the turf industry. With turf production and maintenance becoming an increasingly international business, we ask if biosecurity and the promotion of plant health in turf production fields and associated sport facilities should be a greater priority for the industry. We also examine if a lack of effective biosecurity measures in the materials supply chain has led to increased plant health problems.

New insights into the FLPergic complements of parasitic nematodes: Informing deorphanisation approaches

FMRFamide-like peptide (FLP) receptors are appealing as putative anthelmintic targets. To date, 31 flp-encoding genes have been identified in Caenorhabditis elegans and thirteen FLP-activated G-protein coupled receptors (FLP-GPCRs) have been reported. The lack of knowledge on FLPs and FLP-GPCRs in parasites impedes their functional characterisation and chemotherapeutic exploitation. Using homology-based BLAST searches and phylogenetic analyses this study describes the identification of putative flp and flp-GPCR gene homologues in 17 nematode parasites providing the first pan-phylum genome-based overview of the FLPergic complement. These data will facilitate FLP-receptor deorphanisation efforts in the quest for novel control targets for nematode parasites.

Unraveling flp-11/flp-32 dichotomy in nematodes

FMRFamide-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.