Novel integrated computational AMP discovery approaches highlight diversity in the helminth AMP repertoire.

Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described. This study highlights limitations in the homology-based approaches, used to identify putative nematode AMPs, for the characterisation of flatworm AMPs, and reveals that innovative algorithmic AMP prediction approaches provide an alternative strategy for novel helminth AMP discovery. The data presented here: (i) reveal that flatworms do not encode traditional lophotrochozoan AMP groups (Big Defensin, CSαß peptides and Myticalin); (ii) describe a unique integrated computational pipeline for the discovery of novel helminth AMPs; (iii) reveal >16,000 putative AMP-like peptides across 127 helminth species; (iv) highlight that cysteine-rich peptides dominate helminth AMP-like peptide profiles; (v) uncover eight novel helminth AMP-like peptides with diverse antibacterial activities, and (vi) demonstrate the detection of AMP-like peptides from Ascaris suum biofluid. These data represent a significant advance in our understanding of the putative helminth AMP repertoire and underscore a potential untapped source of antimicrobial diversity which may provide opportunities for the discovery of novel antimicrobials. Further, unravelling the role of endogenous worm-derived antimicrobials and their potential to influence host-worm-microbiome interactions may be exploited for the development of unique helminth control approaches.

New techniques, applications and perspectives in neuropeptide research.

Neuropeptides are one of the most diverse classes of signaling molecules and have attracted great interest over the years owing to their roles in regulation of a wide range of physiological processes. However, there are unique challenges associated with neuropeptide studies stemming from the highly variable molecular sizes of the peptides, low in vivo concentrations, high degree of structural diversity and large number of isoforms. As a result, much effort has been focused on developing new techniques for studying neuropeptides, as well as novel applications directed towards learning more about these endogenous peptides. The areas of importance for neuropeptide studies include structure, localization within tissues, interaction with their receptors, including ion channels, and physiological function. Here, we discuss these aspects and the associated techniques, focusing on technologies that have demonstrated potential in advancing the field in recent years. Most identification and structural information has been gained by mass spectrometry, either alone or with confirmations from other techniques, such as nuclear magnetic resonance spectroscopy and other spectroscopic tools. While mass spectrometry and bioinformatic tools have proven to be the most powerful for large-scale analyses, they still rely heavily on complementary methods for confirmation. Localization within tissues, for example, can be probed by mass spectrometry imaging, immunohistochemistry and radioimmunoassays. Functional information has been gained primarily from behavioral studies coupled with tissue-specific assays, electrophysiology, mass spectrometry and optogenetic tools. Concerning the receptors for neuropeptides, the discovery of ion channels that are directly gated by neuropeptides opens up the possibility of developing a new generation of tools for neuroscience, which could be used to monitor neuropeptide release or to specifically change the membrane potential of neurons. It is expected that future neuropeptide research will involve the integration of complementary bioanalytical technologies and functional assays.

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.

Advancing Strongyloides omics data: bridging the gap with Caenorhabditis elegans.

Among nematodes, the free-living model organism Caenorhabditis elegans boasts the most advanced portfolio of high-quality omics data. The resources available for parasitic nematodes, including Strongyloides spp., however, are lagging behind. While C. elegans remains the most tractable nematode and has significantly advanced our understanding of many facets of nematode biology, C. elegans is not suitable as a surrogate system for the study of parasitism and it is important that we improve the omics resources available for parasitic nematode species. Here, we review the omics data available for Strongyloides spp. and compare the available resources to those for C. elegans and other parasitic nematodes. The advancements in C. elegans omics offer a blueprint for improving omics-led research in Strongyloides. We suggest areas of priority for future research that will pave the way for expansions in omics resources and technologies. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

Strongyloides questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'.

Exploring the antimicrobial peptidome of nematodes through phylum-spanning in silico analyses highlights novel opportunities for pathogen control.

Antimicrobial Peptides (AMPs) are key constituents of the invertebrate innate immune system and provide critical protection against microbial threat. Nematodes display diverse life strategies where they are exposed to heterogenous, microbe rich, environments highlighting their need for an innate immune system. Within the Ecdysozoa, arthropod AMPs have been well characterised, however nematode-derived AMP knowledge is limited. In this study the distribution and abundance of putative AMP-encoding genes was examined in 134 nematode genomes providing the most comprehensive profile of AMP candidates within phylum Nematoda. Through genome and transcriptome analyses we reveal that phylum Nematoda is a rich source of putative AMP diversity and demonstrate (i) putative AMP group profiles that are influenced by nematode lifestyle where free-living nematodes appear to display enriched putative AMP profiles relative to parasitic species; (ii) major differences in the putative AMP profiles between nematode clades where Clade 9/V and 10/IV species possess expanded putative AMP repertoires; (iii) AMP groups with highly restricted profiles (e.g. Cecropins and Diapausins) and others [e.g. Nemapores and Glycine Rich Secreted Peptides (GRSPs)] which are more widely distributed; (iv) complexity in the distribution and abundance of CSαß subgroup members; and (v) that putative AMPs are expressed in host-facing life stages and biofluids of key nematode parasites. These data indicate that phylum Nematoda displays diversity in putative AMPs and underscores the need for functional characterisation to reveal their role and importance to nematode biology and host-nematode-microbiome interactions.

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.

Purchase rates and energy content of nutritionally promoted and traditional fast foods purchased at lunchtime in Australia - a pilot study.

OBJECTIVE: Nutritionally promoted foods are now available at fast-food establishments. Little is known about their popularity, who is purchasing them, or their impact on dietary intake. Our study aimed to determine: how often nutritionally promoted fast foods were purchased; the demographic characteristics of people purchasing these foods; and if purchasing these foods resulted in reduced energy, and increased vegetable, content of lunches compared with those who purchased traditional fast foods. DESIGN: A survey collecting lunchtime fast-food purchases and demographic details was administered over two months. Nutritionally promoted products included the McDonalds' 'Heart Foundation Tick Approved' range and Subway's 'Six grams of fat or less' range. Energy and vegetable contents were estimated using information from fast-food companies' websites. Differences in demographics, energy and vegetable contents between individuals purchasing nutritionally promoted and traditional lunches were assessed using χ2 and t tests. SETTING: Queensland, Australia. SUBJECTS: Lunchtime diners aged over 16 years at Subway and McDonalds. RESULTS: Surveys were collected from 927 respondents (58 % male, median age 25 (range 16-84) years; 73 % response rate). Only 3 % (n 24/910) of respondents who ordered a main option had purchased a nutritionally promoted item. Purchasers of nutritionally promoted foods were ∼13 years older, predominantly female (79 %), and more often reported involvement in a health-related profession (29 % v. 11 %) than purchasers of traditional foods (P < 0·05). Purchasers of nutritionally promoted foods ordered 1·5 fewer megajoules and 0·6 more vegetable servings than purchasers of traditional foods (P < 0·05). CONCLUSIONS: Nutritionally promoted fast foods may reduce lunchtime energy content, however these foods were infrequently chosen.

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.

Clinical helminth infections alter host gut and saliva microbiota.

BACKGROUND: Previous reports show altered gut bacterial profiles are associated with helminth infected individuals. Our recently published molecular survey of clinical helminthiases in Thailand border regions demonstrated a more comprehensive picture of infection prevalence when Kato Katz microscopy and copro-qPCR diagnostics were combined. We revealed that Opisthorchis viverrini, hookworm, Ascaris lumbricoides and Trichuris trichiura were the most predominant helminth infections in these regions. In the current study, we have profiled the faecal and saliva microbiota of a subset of these helminth infected participants, in order to determine if microbial changes are associated with parasite infection. METHODS: A subset of 66 faecal samples from Adisakwattana et al., (2020) were characterised for bacterial diversity using 16S rRNA gene profiling. Of these samples a subset of 24 participant matched saliva samples were also profiled for microbiota diversity. Sequence data were compiled, OTUs assigned, and diversity and abundance analysed using the statistical software Calypso. RESULTS: The data reported here indicate that helminth infections impact on both the host gut and oral microbiota. The profiles of faecal and saliva samples, irrespective of the infection status, were considerably different from each other, with more alpha diversity associated with saliva (p-value≤ 0.0015). Helminth infection influenced the faecal microbiota with respect to specific taxa, but not overall microbial alpha diversity. Conversely, helminth infection was associated with increased saliva microbiota alpha diversity (Chao 1 diversity indices) at both the genus (p-value = 0.042) and phylum (p-value = 0.026) taxa levels, compared to uninfected individuals. Elevated individual taxa in infected individuals saliva were noted at the genus and family levels. Since Opisthorchis viverrini infections as a prominent health concern to Thailand, this pathogen was examined separately to other helminths infections present. Individuals with an O. viverrini mono-infection displayed both increases and decreases in genera present in their faecal microbiota, while increases in three families and one order were also observed in these samples. DISCUSSION: In this study, helminth infections appear to alter the abundance of specific faecal bacterial taxa, but do not impact on overall bacterial alpha or beta diversity. In addition, the faecal microbiota of O. viverrini only infected individuals differed from that of other helminth single and dual infections. Saliva microbiota analyses of individuals harbouring active helminth infections presented increased levels of both bacterial alpha diversity and abundance of individual taxa. Our data demonstrate that microbial change is associated with helminthiases in endemic regions of Thailand, and that this is reflected in both faecal and saliva microbiota. To our knowledge, this is the first report of an altered saliva microbiota in helminth infected individuals. This work may provide new avenues for improved diagnostics; and an enhanced understanding of both helminth infection pathology and the interplay between helminths, bacteria and their host.

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.

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.

Analysis of Schistosoma mansoni Extracellular Vesicles Surface Glycans Reveals Potential Immune Evasion Mechanism and New Insights on Their Origins of Biogenesis.

Parasitic helminths are master manipulators of host immunity. Their strategy is complex and involves the release of excreted/secreted products, including extracellular vesicles (EVs). The protein and miRNA contents of EVs have been characterised for many parasitic helminths but, despite reports suggesting the importance of EV surface carbohydrate structures (glycans) in the interactions with target cells and thus subsequent effector functions, little is known about parasite EV glycomics. Using lectin microarrays, we identified several lectins that exhibit strong adhesion to Schistosoma mansoni EVs, suggesting the presence of multiple glycan structures on these vesicles. Interestingly, SNA-I, a lectin that recognises structures with terminal sialic acid, displayed strong affinity for S. mansoni EVs, which was completely abolished by neuraminidase treatment, suggesting sialylation in the EV sample. This finding is of interest, as sialic acids play important roles in the context of infection by aiding immune evasion, affecting target recognition, cell entry, etc., but are not thought to be synthesised by helminths. These data were validated by quantitative analysis of free sialic acid released from EVs following treatment with neuraminidase. Lectin histochemistry and fluorescence in situ hybridisation analyses on whole adult worms suggest the involvement of sub-tegumental cell bodies, as well as the digestive and excretory systems, in the release of EVs. These results support previous reports of EV biogenesis diversity in trematodes and potentially highlight new means of immune modulation and evasion employed by schistosomes.

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.

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.

Clinical helminthiases in Thailand border regions show elevated prevalence levels using qPCR diagnostics combined with traditional microscopic methods.

BACKGROUND: Under-regulated national borders in Southeast Asia represent potential regions for enhanced parasitic helminth transmission and present barriers to helminthiasis disease control. METHODS: Three Thailand border regions close to Myanmar, Laos and Cambodia were surveyed for clinical parasitic helminth disease. In-field microscopy was performed on stools from 567 individuals. Sub-samples were transported to Bangkok for molecular analysis comprising three multiplex qPCR assays. RESULTS: The overall helminth infection prevalence was 17.99% as assessed by Kato-Katz and 24.51% by qPCR. The combined prevalence of the two methods was 28.57%; the most predominant species detected were Opisthorchis viverrini (18.34%), hookworm (6.88%; Ancylostoma spp. and Necator americanus), Ascaris lumbricoides (2.29%) and Trichuris trichiura (1.76%). CONCLUSIONS: These data demonstrate the value of molecular diagnostics for determining more precise prevalence levels of helminthiases in Southeast Asia. Availability of such accurate prevalence information will help guide future public health initiatives and highlights the need for more rigorous surveillance and timely intervention in these regions.

Tool-Driven Advances in Neuropeptide Research from a Nematode Parasite Perspective.

Expanding 'omics' datasets for parasitic nematodes have accelerated the identification of putative drug targets derived from the nematode nervous system. However, novel drug target validation is hampered by the absence of adequate localisation, functional characterisation, and receptor deorphanisation tools in key nematode pathogens. Reverse genetics techniques have advanced to encompass transgenic, targeted mutagenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9) approaches in Caenorhabditis elegans. Unfortunately the translation to nematode pathogens has been slow, such that parasite-focused toolbox development and optimisation is critical. Here we review the discovery, localisation, and functional characterisation toolkit available for parasitic nematode neuropeptide research, and assess the scope and limitations of the tools and techniques for novel nematicide discovery.

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.

RNA interference in adult Ascaris suum--an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite.

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.