Structure and mechanism of a neuropeptide-activated channel in the ENaC/DEG superfamily.

Phe-Met-Arg-Phe-amide (FMRFamide)-activated sodium channels (FaNaCs) are a family of channels activated by the neuropeptide FMRFamide, and, to date, the underlying ligand gating mechanism remains unknown. Here we present the high-resolution cryo-electron microscopy structures of Aplysia californica FaNaC in both apo and FMRFamide-bound states. AcFaNaC forms a chalice-shaped trimer and possesses several notable features, including two FaNaC-specific insertion regions, a distinct finger domain and non-domain-swapped transmembrane helix 2 in the transmembrane domain (TMD). One FMRFamide binds to each subunit in a cleft located in the top-most region of the extracellular domain, with participation of residues from the neighboring subunit. Bound FMRFamide adopts an extended conformation. FMRFamide binds tightly to A. californica FaNaC in an N terminus-in manner, which causes collapse of the binding cleft and induces large local conformational rearrangements. Such conformational changes are propagated downward toward the TMD via the palm domain, possibly resulting in outward movement of the TMD and dilation of the ion conduction pore.

The head mesodermal cell couples FMRFamide neuropeptide signaling with rhythmic muscle contraction in C. elegans.

FMRFamides are evolutionarily conserved neuropeptides that play critical roles in behavior, energy balance, and reproduction. Here, we show that FMRFamide signaling from the nervous system is critical for the rhythmic activation of a single cell of previously unknown function, the head mesodermal cell (hmc) in C. elegans. Behavioral, calcium imaging, and genetic studies reveal that release of the FLP-22 neuropeptide from the AVL neuron in response to pacemaker signaling activates hmc every 50 s through an frpr-17 G protein-coupled receptor (GPCR) and a protein kinase A signaling cascade in hmc. hmc activation results in muscle contraction through coupling by gap junctions composed of UNC-9/Innexin. hmc activation is inhibited by the neuronal release of a second FMRFamide-like neuropeptide, FLP-9, which functions through its GPCR, frpr-21, in hmc. This study reveals a function for two opposing FMRFamide signaling pathways in controlling the rhythmic activation of a target cell through volume transmission.

Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse.

Obesity imposes a global health threat and calls for safe and effective therapeutic options. Here, we found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. We also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without behavioral abnormalities. Therefore, our study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases.

Inhibitory Effect of FMRFamide on NO Production During Immune Defense in Sepiella japonica.

Neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), specifically existing in invertebrates, plays pivotal roles in various physiological processes. The involvement in neuroendocrine-immune regulation was explored in recent years, and it could modulate nitric oxide (NO) production under immune stress. However, detailed knowledge is still little known. In this study, we identified FMRFamide as an inhibitory factor on NO production in the immune reaction of Sepiella japonica. Firstly, Vibrio harveyi incubation caused significantly upregulated expression of FMRFamide precursor and NO synthase (NOS) in just hatched cuttlefish with quantitative Real-time PCR (qRT-PCR), which indicated that both were likely to be involved in the immune defense. The whole-mount in situ hybridization (ISH) detected FMRFamide precursor and NOS-positive signals appeared colocalization, suggesting that at histological and anatomical levels FMRFamide might interact with NOS. Next, NOS mRNA was highly significantly upregulated at 72 h when FMRFamide precursor mRNA was knocked down effectively with the RNA interference (RNAi) method; the results hinted that FMRFamide was likely to regulate NO production. Continuously, the inflammatory model was constructed in RAW 264.7 cells induced by lipopolysaccharide (LPS), FMRFamide administration resulted in a highly significant reduction of the NO level in dose- and time-response manners. Although the addition of the selected inducible NOS (iNOS) inhibitor had inhibited the NO production induced by LPS, the additional FMRFamide could still furtherly sharpen the process. Collectively, it was concluded that neuropeptide FMRFamide could indeed inhibit NO production to serve as feedback regulation at the late stage of immune response to protect hosts from excessive immune cytotoxicity. The inhibitory effect on NO production could not only be mediated by the NOS pathway but also be implemented through other pathways that needed to be furtherly explored. The results will provide data for comparing the structure and immune function of neuroendocrine-immune system (NEIS) between "advanced" cephalopods and other invertebrates and will provide new information for understanding the NEIS of cephalopods.

Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity.

FMRFamide (Phe-Met-Arg-Phe-amide, FMRFa) and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFa activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFa potency against one of its native targets, the excitatory FMRFa-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of neuropeptide activity, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFa, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching degenerin (DEG)/epithelial sodium channel (ENaC) superfamily, incorporating mutagenesis and experimental dissection of channel function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands but enhanced by FMRFa, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFa neuropeptide activity at FaNaC receptor channels and illuminates the evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

Effect of Air Exposure-Induced Hypoxia on Neurotransmitters and Neurotransmission Enzymes in Ganglia of the Scallop Azumapecten farreri.

The nervous system expresses neuromolecules that play a crucial role in regulating physiological processes. Neuromolecule synthesis can be regulated by oxygen-dependent enzymes. Bivalves are a convenient model for studying air exposure-induced hypoxia. Here, we studied the effects of hypoxia on the expression and dynamics of neurotransmitters, and on neurotransmitter enzyme distribution, in the central nervous system (CNS) of the scallop Azumapecten farreri. We analyzed the expression of the neurotransmitters FMRFamide and serotonin (5-HT) and the choline acetyltransferase (CHAT) and universal NO-synthase (uNOS) enzymes during air exposure-induced hypoxia. We found that, in early-stage hypoxia, total serotonin content decreased in some CNS regions but increased in others. CHAT-lir cell numbers increased in all ganglia after hypoxia; CHAT probably appears de novo in accessory ganglia. Short-term hypoxia caused increased uNOS-lir cell numbers, while long-term exposure led to a reduction in their number. Thus, hypoxia weakly influences the number of FMRFamide-lir neurons in the visceral ganglion and does not affect peptide expression in the pedal ganglion. Ultimately, we found that the localization and level of synthesis of neuromolecules, and the numbers of cells expressing these molecules, vary in the scallop CNS during hypoxia exposure. This indicates their possible involvement in hypoxia resistance mechanisms.

The FMRFamide Neuropeptide FLP-20 Acts as a Systemic Signal for Starvation Responses in Caenorhabditis elegans.

Most animals face frequent periods of starvation throughout their entire life and thus need to appropriately adjust their behavior and metabolism during starvation for their survival. Such adaptive responses are regulated by a complex set of systemic signals, including hormones and neuropeptides. While much progress has been made in identifying pathways that regulate nutrient-excessive states, it is still incompletely understood how animals systemically signal their nutrient-deficient states. Here, we showed that the FMRFamide neuropeptide FLP-20 modulates a systemic starvation response in Caenorhabditis elegans. We found that mutation of flp-20 rescued the starvation hypersensitivity of the G protein ß-subunit gpb-2 mutants by suppressing excessive autophagy. FLP-20 acted in AIB neurons, where the metabotropic glutamate receptor MGL-2 also functions to modulate a systemic starvation response. Furthermore, FLP-20 modulated starvation-induced fat degradation in a manner dependent on the receptor-type guanylate cyclase GCY-28. Collectively, our results reveal a circuit that senses and signals nutrient-deficient states to modulate a systemic starvation response in multicellular organisms.

FMRF-related peptides in Aedes aegypti midgut: neuromuscular connections and enteric nervous system.

FMRFamide-related peptides (FaRPs) are a class of neuropeptides that participate in a variety of physiological processes in invertebrates. They occur in nerves of stomatogastric ganglia and enteroendocrine cells of the insect digestive tract, where they may control muscle functions. However, their direct involvement in muscle function has never been shown in situ. We studied the relationship between FaRPs and midgut muscle during larval-pupal transition of the mosquito Aedes aegypti. In late L4, FaRP-positive neuronal extensions attach to the bundles of the external circular muscle layer, and muscle stem cells start to undergo mitosis in the internal circular layer. Thereafter, the external muscle layer degenerates, disappearing during early pupal development, and is completely absent in the adult mosquito. Our results indicate that FaRP-based neural signals are involved in the reorganization of the muscle fibers of the mosquito midgut during the larval-pupal transition. In addition to confirming FaRP involvement in muscle function, we show that the mosquito midgut muscles are largely innervated, and that circular and longitudinal muscle have specific neuron bodies associated with them.

FMRF-NH2 -related neuropeptides in Biomphalaria spp., intermediate hosts for schistosomiasis: Precursor organization and immunohistochemical localization.

Freshwater snails of the genus Biomphalaria serve as intermediate hosts for the digenetic trematode Schistosoma mansoni, the etiological agent for the most widespread form of intestinal schistosomiasis. As neuropeptide signaling in host snails can be altered by trematode infection, a neural transcriptomics approach was undertaken to identify peptide precursors in Biomphalaria glabrata, the major intermediate host for S. mansoni in the Western Hemisphere. Three transcripts that encode peptides belonging to the FMRF-NH2 -related peptide (FaRP) family were identified in B. glabrata. One transcript encoded a precursor polypeptide (Bgl-FaRP1; 292 amino acids) that included eight copies of the tetrapeptide FMRF-NH2 and single copies of FIRF-NH2 , FLRF-NH2 , and pQFYRI-NH2 . The second transcript encoded a precursor (Bgl-FaRP2; 347 amino acids) that comprised 14 copies of the heptapeptide GDPFLRF-NH2 and 1 copy of SKPYMRF-NH2 . The precursor encoded by the third transcript (Bgl-FaRP3; 287 amino acids) recapitulated Bgl-FaRP2 but lacked the full SKPYMRF-NH2 peptide. The three precursors shared a common signal peptide, suggesting a genomic organization described previously in gastropods. Immunohistochemical studies were performed on the nervous systems of B. glabrata and B. alexandrina, a major intermediate host for S. mansoni in Egypt. FMRF-NH2 -like immunoreactive (FMRF-NH2 -li) neurons were located in regions of the central nervous system associated with reproduction, feeding, and cardiorespiration. Antisera raised against non-FMRF-NH2 peptides present in the tetrapeptide and heptapeptide precursors labeled independent subsets of the FMRF-NH2 -li neurons. This study supports the participation of FMRF-NH2 -related neuropeptides in the regulation of vital physiological and behavioral systems that are altered by parasitism in Biomphalaria.

Localization of neurons expressing choline acetyltransferase, serotonin and/or FMRFamide in the central nervous system of the decapod shore crab Hemigrapsus sanguineus.

Although it is now established that neurons in crustacea contain multiple transmitter substances, little is know about patterns of expression and co-expression or about the functional effects of such co-transmission. The present study was designed to characterize the distributions and potential colocalization of choline acetyltransferase (ChAT), serotonin (5-HT) and neuropeptide H-Phe-Met-Arg-Phe-NH2 (FMRFamide) in the central nervous system (CNS) of the Asian shore crab, Hemigrapsus sanguineus using immunohistochemical analyses in combination with laser scanning confocal microscopy. ChAT was found to be expressed by small, medium-sized, and large neurons in all regions of the brain and ventral nerve cord (VNC). For the most part, ChAT, FMRFamide, and 5-HT are expressed in different neurons, although some colocalization of ChAT- with FMRFamide- or 5-HT-LIR is observed in small and medium-sized cells, mostly neurons that immunostain only weakly. In the brain, such double immunolabeling is observed primarily in neurons of the protocerebrum and, to a particularly great extent, in local olfactory interneurons of the deutocerebrum. The clusters of neurons in the VNC that stain most intensely for ChAT, FMRFamide, and 5-HT, with colocalization in some cases, are located in the subesophageal ganglia. This colocalization appears to be related to function, since it is present in regions of the CNS characterized by multiple afferent projections and outputs to a variety of functionally related centers involved in various physiological and behavioral processes. Further elucidation of the functional significance of these neurons and of the widespread process of co-transmission in the crustaceans should provide fascinating new insights.

Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis).

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC-MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.

The neuroactive substances and associated muscle system in Rhipidocotyle campanula (Digenea, Bucephalidae) from the intestine of the pike Esox lucius.

We report about the muscular system and the serotonergic and FMRFamidergic components of the nervous system of the Bucephalidae trematode, Rhipidocotyle campanula, an intestinal parasite of the pike. We use immunocytochemical methods and confocal scanning laser microscopy (CLSM). The musculature is identified by histochemical staining with fluorescently labeled phalloidin. The body wall musculature of R. campanula contains three layers of muscle fibres - the outer thin circular, intermediate longitudinal and inner diagonal muscle fibres running in two opposite directions. The digestive system of R. campanula possess of a well-developed musculature: radial, longitudinal and circular muscle elements are detected in the pharynx, circular and longitudinal muscle filaments seen in the oesophagus, and longitudinal and the circular muscle fibres were found in the intestinal wall. Specific staining indicating the presence of actin muscle filaments occurs in the cirrus sac localized in the posterior body region. The frontal region of anterior attachment organ, the rhynchus, in R. campanula is represented by radial muscle fibres. The posterior part of the rhynchus comprise of radial muscles forming the organ's wall, and several strong longitudinal muscle bundles. Serotonergic and FMRFamidergic structures are detected in the central and peripheral compartments of the nervous system of R. campanula, that is, in the paired brain ganglia, the brain commissure, the longitudinal nerve cords, and connective nerve commissures. The innervations of the rhynchus, pharynx, oesophagus and distal regions of the reproductive system by the serotonergic and FMRFamidergic nervous elements are revealed. We compare our findings obtained on R. campanula with related data for other trematodes.

An FMRFamide Neuropeptide in Cuttlefish Sepia pharaonis: Identification, Characterization, and Potential Function.

Neuropeptides are released by neurons that are involved in a wide range of brain functions, such as food intake, metabolism, reproduction, and learning and memory. A full-length cDNA sequence of an FMRFamide gene isolated from the cuttlefish Sepia pharaonis (designated as SpFMRFamide) was cloned. The predicted precursor protein contains one putative signal peptide and four FMRFamide-related peptides. Multiple amino acid and nucleotide sequence alignments showed that it shares 97% similarity with the precursor FMRFamides of Sepiella japonica and Sepia officinalis and shares 93% and 92% similarity with the SpFMRFamide gene of the two cuttlefish species, respectively. Moreover, the phylogenetic analysis also suggested that SpFMRFamide and FMRFamides from S. japonica and S. officinalis belong to the same sub-branch. Tissue expression analysis confirmed that SpFMRFamide was widely distributed among tissues and predominantly expressed in the brain at the three development stages. The combined effects of SpFMRFamide+SpGnRH and SpFLRFamide+SpGnRH showed a marked decrease in the level of the total proteins released in the CHO-K1 cells. This is the first report of SpFMRFamide in S. pharaonis and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

Neuroanatomy of mud dragons: a comprehensive view of the nervous system in Echinoderes (Kinorhyncha) by confocal laser scanning microscopy.

BACKGROUND: The Scalidophora (Kinorhyncha, Loricifera and Priapulida) have an important phylogenetic position as early branching ecdysozoans, yet the architecture of their nervous organ systems is notably underinvestigated. Without such information, and in the absence of a stable phylogenetic context, we are inhibited from producing adequate hypotheses about the evolution and diversification of ecdysozoan nervous systems. Here, we utilize confocal laser scanning microscopy to characterize serotonergic, tubulinergic and FMRFamidergic immunoreactivity patterns in a comparative neuroanatomical study with three species of Echinoderes, the most speciose, abundant and diverse genus within Kinorhyncha. RESULTS: Neuroanatomy in Echinoderes as revealed by acetylated α-tubulin immunoreactivity includes a circumpharyngeal brain and ten neurite bundles in the head region that converge into five longitudinal nerves within the trunk. The ventral nerve cord is ganglionated, emerging from the brain with two connectives that converge in trunk segments 2-3, and diverge again within segment 8. The longitudinal nerves and ventral nerve cord are connected by two transverse neurites in segments 2-9. Differences among species correlate with the number, position and innervation of cuticular structures along the body. Patterns of serotoninergic and FMRFamidergic immunoreactivity correlate with the position of the brain neuropil and the ventral nerve cord. Distinct serotonergic and FMRFamidergic somata are associated with the brain neuropil and specific trunk segments along the ventral nerve cord. CONCLUSIONS: Neural architecture is highly conserved across all three species, suggesting that our results reveal a pattern that is common to more than 40% of the species within Kinorhyncha. The nervous system of Echinoderes is segmented along most of the trunk; however, posterior trunk segments exhibit modifications that are likely associated with sensorial, motor or reproductive functions. Although all kinorhynchs show some evidence of an externally segmented trunk, it is unclear whether external segmentation matches internal segmentation of nervous and muscular organ systems across Kinorhyncha, as we observed in Echinoderes. The neuroanatomical data provided in this study not only expand the limited knowledge on kinorhynch nervous systems but also establish a comparative morphological framework within Scalidophora that will support broader inferences about the evolution of neural architecture among the deepest branching lineages of the Ecdysozoa.

Architecture of the nervous system in metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 (Digenea).

The development of metacercariae of Diplostomum pseudospathaceum Niewiadomska, 1984 is accompanied by profound morphological transformations often characterized as metamorphosis, which makes these metacercariae an interesting case for studying the morphogenesis of the digenean nervous system. Although the nervous system of D. pseudospathaceum is one of the most extensively studied among digeneans, there are still gaps in our knowledge regarding the distribution patterns of some neuroactive substances, most notably neuropeptides. The present study addresses these gaps by studying pre-infective metacercariae of D. pseudospathaceum using immunochemical staining and confocal microscopy to characterize the distribution patterns of serotonin (5-HT) and two major groups of flatworm neuropeptides, FMRFamide-related (FaRPs) and substance P-related (SP) peptides. The general morphology of the nervous system was examined with antibodies to alpha-tubulin. The nervous system of the metacercariae was shown to conform to the most common morphology of the nervous system in the hermaphroditic generation, with three pairs of posterior nerve cords and four pairs of anterior nerves. The patterns of FaRP- and 5-HT immunoreactivity (IR) were similar to those revealed in earlier studies by cholinesterase activity, which is in accordance with the known role of these neurotransmitters in controlling muscle activity in flatworms. The SP-IR nervous system was significantly different and consisted of mostly bipolar cells presumably acting as mechanoreceptors. The architecture of the nervous system in D. pseudospathaceum metacercariae is discussed in comparison to that in cercariae of D. pseudospathaceum and metacercariae of related digenean species.

Distribution of FMRFamide-related peptides and co-localization with glutamate in Cupiennius salei, an invertebrate model system.

FMRFamide-related proteins have been described in both vertebrate and invertebrate nervous systems and have been suggested to play important roles in a variety of physiological processes. One proposed function is the modulation of signal transduction in mechanosensory neurons and their associated behavioral pathways in the Central American wandering spider Cupiennius salei; however, little is known about the distribution and abundance of FMRFamide-related proteins (FaRPs) within this invertebrate system. We employ immunohistochemistry, Hoechst nuclear stain and confocal microscopy of serial sections to detect, characterize and quantify FMRFamide-like immunoreactive neurons throughout all ganglia of the spider brain and along leg muscle. Within the different ganglia, between 3.4 and 12.6% of neurons showed immunolabeling. Among the immunoreactive cells, weakly and strongly labeled neurons could be distinguished. Between 71.4 and 81.7% of labeled neurons showed weak labeling, with 18.3 to 28.6% displaying strong labeling intensity. Among the weakly labeled neurons were characteristic motor neurons that have previously been shown to express ɣ-aminobutyric acid or glutamate. Ultrastructural investigations of neuromuscular junctions revealed mixed presynaptic vesicle populations including large electron-dense vesicles characteristic of neuropeptides. Double labeling for glutamate and FaRPs indicated that a subpopulation of neurons may co-express both neuroactive compounds. Our findings suggest that FaRPs are expressed throughout all ganglia and that different neurons have different expression levels. We conclude that FaRPs are likely utilized as neuromodulators in roughly 8% of neurons in the spider nervous system and that the main transmitter in a subpopulation of these neurons is likely glutamate.

Subcellular Peptide Localization in Single Identified Neurons by Capillary Microsampling Mass Spectrometry.

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS.

Identification, Characterization, and Expression Analysis of a FMRFamide-Like Peptide Gene in the Common Chinese Cuttlefish (Sepiella japonica).

The peptide FMRFamide is one of the well-known peptides involved in multiple physiological processes in the phylum Mollusca. In this study, a FMRFamide gene (GenBank accession No. KJ933411) was identified in a cuttlefish species called Sepiella japonica and was designated as SjFMRFamide. The total length of the SjFMRFamide sequence was found to be 1880 bp while the open reading frame contained 996 bp encoding a protein of 331 amino acid residues with a predicted isoelectric point (pI) and molecular weight (MW) of 9.18 and 38.8 kDa along with a 333 bp 5'-untranslated region (UTR) and 551 bp 3'-UTR. The deduced SjFMRFamide precursor protein contains one signal peptide and expresses four kinds FMRFamide-related peptides including a single copy of FLRFamide, ALSGDAFLRFamide, and FIRFamide and multiple copies of FMRFamide. Results of phylogenetic relation analysis strongly indicated that the sequence of this gene shares high identity with the genes of known FMRFamides. Spatial expression analysis indicated the highest mRNA expression of SjFMRFamide in the brain of male and female cuttlefishes among the eight tissues analyzed. An in situ hybridization assay of the brain indicated that SjFMRFamide was transcribed in several functional lobes, which suggests that it might be related to many physiological regulatory mechanisms. This is the first study describing FMRFamide in S. japonica and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide.

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.

An ancient FMRFamide-related peptide-receptor pair induces defence behaviour in a brachiopod larva.

Animal behaviour often comprises spatially separated sub-reactions and even ciliated larvae are able to coordinate sub-reactions of complex behaviours (metamorphosis, feeding). How these sub-reactions are coordinated is currently not well understood. Neuropeptides are potential candidates for triggering larval behaviour. However, although their immunoreactivity has been widely analysed, their function in trochozoan larvae has only been studied for a few cases. Here, we investigate the role of neuropeptides in the defence behaviour of brachiopod larvae. When mechanically disturbed, the planktonic larvae of Terebratalia transversa protrude their stiff chaetae and sink down slowly. We identified endogenous FLRFamide-type neuropeptides (AFLRFamide and DFLRFamide) in T. transversa larvae and show that the protrusion of the chaetae as well as the sinking reaction can both be induced by each of these peptides. This also correlates with the presence of FLRFamidergic neurons in the apical lobe and adjacent to the trunk musculature. We deorphanized the AFLRFamide/DFLRFamide receptor and detected its expression in the same tissues. Furthermore, the ability of native and modified FLRFamide-type peptides to activate this receptor was found to correspond with their ability to trigger behavioural responses. Our results show how FLRFamide-type neuropeptides can induce two coherent sub-reactions in a larva with a simple nervous system.