FMRFamide-like peptides (FLPs) are the largest and most diverse family of neuropeptides known (1, 2). Since the identification of the founder sequence FMRFamide from the clam Macrocallista nimbosa (3), structurally similar peptides have shown to be present in animals of all major phyla (4, 5). Sequence variants of the authentic tetrapeptide have been mainly identified in lophotrochozoans (6). In most phyla and especially in nematodes, however, a diverse repertoire of extended peptides sharing the C-terminal RFamide motif is found (5, 6). Though, they are thought to have a common eumetazoan origin, the relatedness of subfamilies of FLPs remains unclear because of the large sequence diversity (7, 8). Some peptides show high sequence similarity to FMRFamide suggesting homology to the tetrapeptide, and are therefore often referred to as FMRFamide-related peptides (FaRPs). FaRPs are broadly defined as peptides containing the C-terminal sequence X₁ X₂ RFamide, with X₁ generally representing an aromatic amino acid, whereas X₂ denotes a hydrophobic residue (6, 7). As many described RFamides differ from the tetrapeptide core for which evolutionary relationships are difficult to determine, the more general term FLP will be used here to address all peptides with a C-terminal RFamide sequence.

FMRFamide-like peptides are intimately involved in a broad pattern of biological processes as diverse as feeding, cardiovascular function, and water homeostasis (4–6, 9). Despite the large sequence diversity, typified by more than 70 family members in the nematode Caenorhabditis elegans, several functions of FLPs in the control of energy balance, feeding behavior, reproduction, and neuromodulation emerge consistently throughout evolution (10, 11). Biochemical and genetic studies, exploiting mainly C. elegans, have provided insight into the FLP-coordinated regulation of these processes in nematodes. The central role of FLPs in nematode biology including reproduction and locomotory activity has also boosted research on FLP signaling as a target for parasite control in pathogenic nematodes (12–14). However, a lack of data on functional nematode FLP-receptor couples slows down the progress in understanding and exploiting the FLP signaling system. Here, we focus on the evolutionary aspect of FLPs, discussing both the sequence conservation and diversity in the phylum Nematoda, and review our knowledge of conserved FLP signaling functions in nematodes.

Initial attempts to identify FLPs relied on molecular cloning of flp-genes (15, 16), and biochemical characterization of immunoreactive peptide fractions by Edman degradation or gas-phase sequencing [reviewed by Maule et al. (17) and Day and Maule (18)]. The first nematode FLP, named AF1 (KNEFIRFa), was biochemically isolated in this way from the parasite Ascaris suum (19). The completion of the C. elegans genome sequence revealed a large diversity in the nematode FLP system, boosting the prediction of neuropeptides through in silico data-mining (20–23). To date, at least 31 flp precursor genes are predicted in C. elegans that give rise to around 70 distinct FLPs [Table 1; Ref. (24)]. Expression has been confirmed for the majority of these peptides (Table 1), mainly by peptidomic strategies enabling a comprehensive analysis of the whole peptide content of organisms (25–27). In addition, these approaches allow determining the presence of posttranslational modifications and the exact processing into bioactive peptides, which may be difficult to accurately predict when multiple or non-conventional cleavage sites are present (25). Peptidomic techniques have also been successfully adopted for characterizing and localizing FLPs in other nematodes, mostly in A. suum (28–32).

Although the FLP repertoire of nematodes has been best studied in C. elegans, evidence emerges on the distribution of FLPs in parasitic and free-living species across the nematode phylum (Table 1). Complete genome sequences have been determined for few phylum members so far, but transcriptome data is available for over 60 nematode species (66, 67). In 2005, McVeigh and co-workers performed a systematic BLAST analysis of EST databases to investigate FLP sequence diversity within the phylum Nematoda identifying more than 500 FLPs across 46 species (13). The FLP complement of other nematodes seems to be generally similar to that of C. elegans (13, 30, 44). This finding is confirmed by a very recent study of McCoy and colleagues, who re-investigated the available genome and transcriptome resources for 17 pathogenic nematodes (24). Many nematode FLPs display a high degree of inter-species structural conservation that is independent of their parasitic or free-living lifestyle [Table 1; Ref. (13)], supporting a fundamental role of FLPs in nematode biology. Corroborating this, only one nematode flp gene is thought to be parasite-specific; the flp-31 gene is absent from the C. elegans genome, but occurs in several plant parasitic nematodes suggesting a function specific in phytoparasitism (13, 24, 44). Although flp-31 was previously predicted from A. suum (32), this gene is considered to be a sequelog of C. elegans flp-15 (24). Initially, flp-29 and flp-30 were also found to be parasite-specific (13), but a recent investigation of their C-terminal motif and genomic location suggests that these genes should be re-designated to respectively flp-28 and flp-2 (24).

Whereas most FLPs are likely widespread throughout the nematode phylum, variable conservation has been reported for some family members. Highly conserved nematode flp-genes include flp-1, flp-6, flp-11, flp-12, flp-14, flp-16, flp-18, flp-19, flp-21, and flp-22; other genes such as flp-2 and flp-10 have shown to be more restricted and structurally diverse (13, 24). Interestingly, parasitic nematodes appear to possess variable proportions of the C. elegans flp-gene complement and variation is highest among distinct clades (24). Genome-wide analysis of the parasite Meloidogyne incognita showed that its FLP complement is reduced to about 60% compared to that of C. elegans (44). Two other nematodes, Trichuris muris and Trichinella spiralis, were shown to display a dramatically reduced complement of only 13%, whereas A. suum possesses 84% of C. elegans flp-genes (24) The finding that fewer flp-genes are expressed in parasitic nematodes as compared to free-living species has been postulated to be an indication of the more contained repertoire of stimuli these nematodes encounter during their endoparasitic stage (68). Furthermore, more FLPs seem to be present in the animal parasitic datasets compared to plant parasitic nematodes (68). Our view on the diversity of FLPs in nematodes however strongly depends on the available sequence data. In depth analyses of the increasing number of completed genome sequences and transcriptome resources should further expand our understanding of the nematode FLP repertoire in the near future.

Recent studies estimate the presence of 32 distinct flp-genes in nematodes (24). Among them are 15 genes that code for N-terminally extended peptides carrying the classical FaRP motif, whereas most others peptides share the restricted RFamide core (Table 1). Although the relatedness of FLPs across metazoans is often unclear, sequences of the neuropeptide F (NPF) family have been identified in several invertebrate groups and predicted in the nematode phylum as well (69). NPF-like peptides are encoded by the flp-27 precursor that is highly conserved in nematodes, and contains the C-terminal RXRFamide motif characteristic of the invertebrate NPF family (70). The plethora of FLPs in nematodes is high, given the structural simplicity of their nervous system harboring around 300 neurons (2, 5, 6). This diversity of neural messengers is magnified by classical neurotransmitters and a broad range of other neuropeptides of insulin (ins) and neuropeptide-like protein (nlp) families, of which about 200 peptides are predicted in C. elegans (46, 71).

Immunocytochemical localization of FLPs has been performed in various nematode species, mainly using antibodies raised against synthetic FMRFamide or the RFamide motif (18, 37, 55). These studies suggest that FLPs are widely expressed in the nervous system of all nematodes, supporting a general role for FLP signaling in nematode biology. The broad distribution of immunoreactive neurons, including in motor neurons, fueled the research effort to decipher nematode FLP signaling and its role in neuromotor function, which had already proven to be a successful target for parasite control (12). Although the vast patterns of FLP immunoreactivity are generally similar between nematode species, HPLC-ELISA studies have identified qualitative differences between free-living and plant parasitic nematodes, suggesting that the distinct peptides present in plant nematodes are structurally different (72, 73).

Despite immunocytochemistry being immensely useful to study gross patterns of FLP localization, most of the C-terminally directed antibodies used were incapable of reliably discriminating between structurally related FLPs. Gene-specific flp expression has been mainly investigated in C. elegans, using reporter transgenes in which LacZ or a fluorescent protein gene is placed under the control of the endogenous promoter region. Li and co-workers applied this molecular approach to map the specific expression patterns of flp-1 to flp-23 genes (22, 23). Just over 50% of the total number of neurons were found to express flp’s, a wide distribution in stark contrast to earlier immunochemical studies in which only 10% of all C. elegans neurons showed FLP reactivity (74). Expression could be detected in all neuronal cell types, including interneurons, sensory neurons, and motor neurons. Six flp-genes were also expressed in non-neuronal cells, including in head muscle (flp-2 and flp-11), pharyngeal muscle (flp-5 and flp-15), socket and/or sheath cells (flp-11 and flp-15), vulval cells (flp-10), and uterine cells (flp-11 and flp-2). Although the expression of each flp gene can be precisely delineated, there is a considerable overlap with many cells expressing more than one flp gene (23). Most flp-genes are also expressed in multiple neurons suggesting that some FLPs have overlapping functions, unlike others fulfilling unique roles.

In situ hybridization (ISH), which uses nucleotide probes complementary to specific gene transcripts, offers an attractive alternative to delineate flp gene expression in other nematodes that are less amenable to transgenesis than C. elegans (38, 42, 54, 75). Furthermore, as immunochemistry allows the determination of neurite morphology facilitating neuronal identification, several antibodies highly specific to certain A. suum FLPs were recently generated (42, 76). Another approach for FLP localization consists of direct mass spectrometric analysis on dissected neuronal tissue without the need of an extensive extraction process (29, 76). Importantly, this technique can identify previously unknown peptides as no prior sequence information is required. Yew et al. (29) subjected individually dissected nerve ganglia from A. suum to mass spectrometric analysis, producing a peptidomic map of the individual anterior ganglions. FLP distribution appeared to be much less restricted to specific cells as compared to the gene expression studies in C. elegans or G. pallida using reporter constructs and ISH, thus supporting the notion that FLP expression differs among nematode species. The authors however also stress the sampling biases inherent in the use of mass spectrometry. In large nematodes such as the foot-long A. suum, cell-specific FLP content can also be rapidly determined by precisely dissecting individual cells (31, 42).

Taken together, abovementioned studies paint a picture in which FLPs widely occur in all known nematode neuronal subtypes and even in non-neuronal tissues. Whereas the gross patterns of FLP distribution remain consistent across the phylum, more recent studies indicate that the cellular expression of homologous FLPs can substantially differ between nematode species (2, 42). This is remarkable, given that both the general FLP complement and the basic nervous architecture are conserved, with C. elegans even considered as a miniature version of A. suum at the level of neuronal morphology (2, 24, 51, 77, 78). Caution is however warranted as dissimilarities could be attributed to experimental differences, with each technique suffering from inherent caveats. Besides the limited specificity of the commonly used antibodies, transgenic reporter constructs may not contain all regulatory sequences necessary to recapitulate endogenous gene expression. Moreover, variability of cellular expression patterns of different gene products has repeatedly been observed in A. suum, partially due to genetic differences since the worms are not isogenic (31, 42, 75, 76).

Most FLPs are known to act through binding of G protein-coupled receptors (GPCRs) (79–81). Although the early work on nematode FLPs primarily focused on peptide identifications, in vitro and functional studies have started to address the biology of their receptors and mode of action. In C. elegans, sequence similarity or homology to the FLP-receptor family has been postulated for several of the more than 100 peptide GPCR genes predicted in the genome (81, 82). The neuropeptide receptor NPR-1 was previously suggested as a member of the invertebrate NPF receptor (NPFR) family and related neuropeptide Y receptors (NPYRs) in mammals (83). Sequence similarity and phylogenetic clustering suggests additional NPFR/NPYR-like family members are likely to be present in C. elegans, as well as representatives related to vertebrate neuropeptide FF receptors, and Drosophila myosuppressin and FMRFamide receptors (8, 82, 84). Peptides that functionally activate these GPCRs, with exception of NPR-1, unfortunately remain unknown.

In general, few nematode FLPs have been matched to their receptor(s) and the identification of FLP-receptor couples has only been undertaken in C. elegans (2, 81). Activation by FLPs has been reported for 13 C. elegans receptors encoded by 10 genes (Table 1), all of which are members of the rhodopsin family of GPCRs [reviewed in Ref. (81)]. Deorphanization, i.e., the identification of receptor ligand(s), is typically done by expressing GPCRs in a heterologous cellular system such as Xenopus oocytes, mammalian cells or yeast. Receptor activation can then be detected by monitoring downstream steps in the GPCR signaling pathway including levels of secondary messenger molecules or GTP exchange upon G protein activation (85). When heterologous GPCRs are challenged with a peptide library, multiple FLPs are generally found to activate a single receptor. Peptide motifs essential for receptor activation are often shared by FLPs derived from the same precursor protein. For example, Kubiak and co-workers showed that all peptides from the FLP-15 precursor carrying the highly similar GPXGPLRFamide motif, recognize the neuropeptide receptor NPR-3 (60). Likewise, two structurally similar FLPs processed from the FLP-2 precursor were found to activate C. elegans receptors encoded by the frpr-18 locus (45). By monitoring intracellular calcium levels, Mertens et al. showed that both FLP-2 peptides activate two isoforms of the receptor FRPR-18 though with different potencies. Whereas SPREPIRFamide (FLP-2A) was active with nanomolar half-maximal effective concentrations (EC₅₀ values), FRPR-18 receptors were only activated at micromolar concentrations by LRGEPIRFamide (FLP-2B). In contrast, Larsen and co-workers found FLP-2A and FLP-2B to be equipotent on the FRPR-18b isoform using a similar calcium mobilization assay in a different type of cells (14). Receptor pharmacology can thus vary dependent on the heterologous system, which may be due to differences in the available G protein signaling machinery or folding properties that affect the functional expression of a GPCR. Although in vitro expression systems may not fully reflect endogenous settings, most ligand-receptor couples identified in C. elegans are supported by functional studies on FLPs and their receptors (61, 62, 81). Functional evidence on peptide GPCRs and putative FLP ligands is also emerging in other nematodes (86, 87), which may serve as a lead in the search for FLP-receptors in these species.

In C. elegans and likely other nematodes, the FLP signaling network is highly expanded by GPCRs able of binding multiple FLPs that, can even originate from different precursor proteins (Table 1). The neuropeptide receptor NPR-1 was the first FLP-receptor to be deorphanized in C. elegans, and shown to recognize both FLP-18 and FLP-21 peptides (61, 63). Interestingly, this GPCR exists in two variants differing by a single amino acid at position 215, NPR-1.215F or NPR-1.215V that is likely implicated in G protein coupling. Substitution of this residue is sufficient for affecting ligand binding and potency resulting in the differential regulation of feeding behavior (61, 63, 83). Both receptor variants are activated by the FLP-21 peptide that is, however, 10-fold more potent in binding NPR-1.215V than NPR-1.215F (61, 63). In addition, Rogers and co-workers found that the NPR-1.215V variant expressed in Xenopus oocytes can be activated by peptides from the FLP-18 precursor, albeit with lower potencies than FLP-21 (61). A second study reported by Kubiak et al. did not identify FLP-18 peptides as the ligands of NPR-1.215V (63), but the receptor variant was expressed in mammalian cells and differences in expression system may account for the discrepancy in identified ligands in both studies. Both FLP-21 and FLP-18 peptides have been found to activate other C. elegans receptors as well, including NPR-11 and NPR-5 (40, 62, 88). In addition, FLP-18 peptides were also identified as ligands of the receptors NPR-4 and NPR-10 (40, 62). An unusual structure-activity relationship has been suggested for the C. elegans receptor FRPR-3 (47, 50). Ligands identified for this GPCR include a FLP-7 (TPMQRSSMVRFamide) and FLP-11 (AMRNALVRFamide) peptide, whereas structurally similar peptides encoded on the same precursor proteins were ineffective at activating the receptor (50). However, it should be noted that EC₅₀ values for both peptides reside in the micromolar range (50), and other functional ligands might activate FRPR-3 with higher potency. FLP-7 and FLP-11 peptides were also shown to activate another receptor, NPR-22, together with an array of FLPs including FLP-1, FLP-9, FLP-13, and FLP-22 peptides (35). The potencies of receptor activation varied from the nanomolar to the micromolar range (Table 1). Finally, the EGL-6 receptor found to be involved in C. elegans egg-laying has been coupled to its FLP-10 and FLP-17 ligands in two ways, by making use of an in vitro assay but also by screening neuropeptide-encoding transgenes for the ability to inhibit egg-laying (53).

Although knowledge has been gathered on the receptor biology of several C. elegans FLPs, our view of nematode FLP-receptors is far from complete. Deorphanization of GPCRs has been successful in matching some FLP-receptor couples; however, often a sub-set of the predicted peptide repertoire is tested such that the array of ligands acting on a receptor remains incomplete. FLPs are thought to exert most of their effects through the activation of GPCRs, but some family members are capable of eliciting fast responses by gating ion channels (89–91). This mode of action likely also applies for several nematode FLPs (90–94). The coupling of multiple peptides to a single GPCR and vice versa greatly enhances the complexity of FLP signaling in C. elegans. However, the characterization of all functional FLP-receptor couples will be crucial to further expand our understanding of nematode FLP signaling, and will uncover whether promiscuity of FLP-receptors can be generalized in nematodes.

Despite the apparent simplicity of the nematode nervous system, harboring around 300 cells, a surprisingly rich behavioral repertoire has been described (86, 87, 95, 96). The structural and spatiotemporal gene expression diversity of the nematode FLP system is reflected in the range of FLP-induced physiological responses. The role of FLPs has been extensively described in previous reviews (2, 5, 6); here, we focus on FLP signaling functions emerging consistently throughout evolution to illustrate some of the general principles of FLP signaling gleaned from the study of nematode peptides. Although C. elegans has been heavily exploited to investigate the basic biology of FLP signaling, we highlight some pharmacological and behavioral studies performed on related nematodes.

The nematode FLP system comprises an intertwined signaling network with a broad array of neuropeptides operating within an anatomically small nervous system. FLP diversity translates into a central role of this neuropeptide family in various aspects of nematode biology. Functional studies in nematodes support the evolutionary continuity of FLPs as key regulators of energy balance, feeding behavior, reproduction, and sensory modulation. In general, the FLP complement has shown to be widely conserved throughout the phylum though some peptides show a more restricted distribution, with the latter potentially as a consequence of adaptation to a specific lifestyle such as parasitism (2, 24, 42). The particular cellular distribution of FLPs appears not to be fully conserved across nematodes, in contrast to the slow rate at which the nematode nervous system evolves at the cellular level. Rapidly evolving peptide expression could therefore reveal to be an essential factor in the generation of species-specific behavior, furthermore facilitating the radiation of nematodes into a variety of habitats including as parasites of both animals and plants (12).

In C. elegans, the flp-genes have overlapping expression patterns, with at least half of all neurons expressing one or more FLPs (173). This implies that some neurons use a repertoire of FLP peptides in addition to other messengers, which may be deployed in a context-dependent way rendering these cells multifunctional. Such multiplexing could contribute to increase the complexity of information processing in a numerally simple nervous system, hereby supporting the rich behavioral palette of nematodes. Given their broad diversity and expression, neuropeptides are exquisitely suited to actively recruit particular cellular circuits depending on the environmental and internal context. This type of neuromodulation appears to be an irreducible part of circuit flexibility in the nematode nervous system (174).

The considerable amounts of data on nematode FLP function derived from neuronal and neuromuscular bioassays demonstrate an impressive complexity in the FLP signaling system. On the other hand, the knowledge of FLP-receptor interplay remains sparse, and most of our current understanding is derived from C. elegans in which several FLP-receptors have been coupled to their peptide ligands by in vitro assays and in vivo functional studies. A common theme in these studies is that a single receptor can be activated by multiple FLPs encoded by one or more genes. However, this apparent receptor promiscuity will need to be proven physiologically relevant, as a whole layer in the control of FLP signaling may reside in the spatiotemporal expression patterns of both receptor and ligand molecules. With the increasing number of completed genome projects and transcriptome resources, putative FLP-receptors can readily be identified using bioinformatics, and C. elegans data as a scaffold, broadening our view on FLP signaling in other nematodes. In addition, further receptor deorphanization and subsequent localization of these proteins will, together with the extensive data regarding FLP distribution, shed light on specific FLP functioning within the modulation and coordination of nematode behavior and physiology.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.