Discovery of multiple neuropeptide families in the phylum Platyhelminthes
Introduction
The nervous system occupies a position of pivotal importance in flatworm biology. In addition to carrying sensory and neuromuscular signals, it may be responsible for the systemic transmission of developmental and hormonal cues, because as acoelomates these organisms lack the body cavity and circulatory system which would otherwise contribute to such functions. Amidated neuropeptides display constitutive and widespread immunoreactivity in flatworms and are linked to roles in locomotion, reproduction, feeding and larval host-finding (see Marks and Maule, 2008 for review). Physiology studies demonstrate that neuropeptides have contractile and regulatory effects on flatworm muscle and other tissues (Day et al., 1994, Marks et al., 1997, Hrckova et al., 2004, Humphries et al., 2004, Kreshchenko et al., 2008). The breadth of these insights is remarkable given the paucity of structural data on endogenous flatworm neuropeptides – only eight native sequences have been described, defining two structural families: four FMRFamide-like peptides (FLPs; short bioactive peptides with C-terminal -RF.NH2 motifs) and four neuropeptide Fs (NPFs; 36–39 amino acid peptides with conserved C-terminal Y(−17)Y(−10)GRPRF.NH2 motif) (Curry et al., 1992, Maule et al., 1992, Maule et al., 1993, Maule et al., 1994, Johnston et al., 1995, Johnston et al., 1996). FLPs and NPFs are functionally different, reflecting their distinct structures. Available evidence suggests that FLPs function at the flatworm neuromuscular synapse/junction, with a clear myoexcitatory effect on muscle strips and dispersed/individual muscle fibres in vitro (Day et al., 1994, Marks et al., 1997, Moneypenny et al., 2001). These monospecific effects suggest the presence of a single, muscle-based, FLP receptor (Day et al., 1994, Day et al., 1997). Schistosome NPF displays a potent inhibition of forskolin-stimulated cAMP accumulation in schistosome homogenates (Humphries et al., 2004), indicating conservation of the archetypal neuropeptide Y (NPY) receptor signalling mechanism by NPF receptors. Additionally in regenerating turbellarians, NPF appears to stimulate the mitotic division of neoblasts, the “stem cells” involved in regenerating the head and associated neuromusculature after decapitation (Kreshchenko et al., 2008). This finding suggests that flatworm neuropeptides may have core functions in controlling neoblast proliferation during regeneration.
The importance of peptide signalling in flatworm biology implies that recently characterised neuropeptide receptors (G-protein coupled receptors, GPCRs) and associated signalling mechanisms (Omar et al., 2007) represent attractive targets for novel anthelmintics. Improved data on the primary structures of native neuropeptide ligands will provide a source of molecular templates to guide the development of peptidomimetics with potential as next-generation anthelmintics (Greenwood et al., 2005). Despite successful applications of mass spectrometry (MS) to identification of neuropeptides and neurohormones in several invertebrates (Husson et al., 2005, Behrens et al., 2008, Ma et al., 2008, Weaver and Audsley, 2008), no such studies have yet been performed on flatworms. MS-based neuropeptidomic studies are performed on either dissected neural cells/tissues, or gram-scale homogenisations of whole worms (Hummon et al., 2006). Currently, parasitic flatworms preclude both of these methods: the former because flatworms are acoelomate, compromising the selective recovery of neural tissues from surrounding parenchyma and thereby offering source tissues with a very low neural to non-neural tissue ratio; the latter because parasitic flatworms must be cultured in animal hosts, and are therefore extremely difficult to obtain in large quantities. Coupled to these technical difficulties, flatworm neurobiology has suffered from a scarcity of bioinformatic datasets such that knowledge of flatworm neuropeptide diversity has remained static for ∼15 years. Only recently have exploitable genomic and transcriptomic datasets provided the opportunity to expand this knowledge-base.
The primary aim of this work was to improve our understanding of native flatworm neuropeptides using in silico analyses of neuropeptide-encoding transcripts. The approach to their discovery utilised web-based basic local alignment search tool (BLAST) searches to identify potential neuropeptide precursors in expressed sequence tag (EST) and genomic databases that are available for six many parasitic species (Clonorchis sinensis, Echinococcus granulosus, Opisthorchis viverrini, Schistosoma japonicum, Schistosoma mansoni, Taenia solium), well as a number of free-living forms that serve as model organisms (Dugesia ryukyuensis, Macrostomum lignano and Schmidtea mediterranea). Our initial approach using BLAST searches was followed up by manual curation/inspection and led to the identification of 60 neuropeptide precursors incorporating 96 short peptide transmitters in 10 species of flatworm. Two of the predicted mature peptides were chosen for immunodetection and shown to be expressed in schistosomes.
Section snippets
Bioinformatics
An approach based on using BLAST searches (Altschul et al., 1990) was employed to identify putative neuropeptides from phylum Platyhelminthes. In an initial specific approach, known invertebrate neuropeptides flanked by “KR” di-basic cleavage sites were used as search queries. These included adipokinetic and related hormones, allatostatins, bombesins, corazonins, diuretic and antidiuretic hormones, myosuppressins, pigment dispersing factor peptides, proctolins, prothoracicotropic hormones,
Overview
Using a search strategy based on the BLAST algorithm, we believe this is the first study to describe a global dataset of neuropeptide-encoding transcripts from phylum Platyhelminthes. We identified 60 prepropeptide transcripts (detailed in Supplementary Figs. S2–S26) from 10 species of parasitic and free-living flatworms, which we have grouped by sequence similarity into 25 sequelogs (Table 1). Sequelogs are genes which encode peptides of similar sequence among different species (Varshavsky,
Discussion
We believe that this study reports the first description of new flatworm neuropeptides in ∼15 years and represents a major expansion in our knowledge of flatworm neuropeptide biology. We have described 25 npp sequelogs in phylum Platyhelminthes which encode putative neuropeptide precursor proteins grouped into nine distinct families of neuropeptide (Table 1). While most of the predicted peptides are completely novel, showing little similarity with peptides from other genera, several NPPs appear
Acknowledgements
We acknowledge the laboratories responsible for generating the publicly available genomic and transcriptomic resources for flatworms, without which this study would not have been possible. This work was funded by The National Institutes of Health grant ROI-AI49162. A research studentship (for LA) from the Department of Education and Learning for Northern Ireland and a Wellcome Trust equipment grant (069411) is also acknowledged.
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