FMRFamide-related Peptides Research Articles

Family of FLP Peptides in Caenorhabditis elegans and Related Nematodes.

Neuropeptides regulate all aspects of behavior in multicellular organisms. Because of their ability to act at long distances, neuropeptides can exert their effects beyond the conventional synaptic connections, thereby adding an intricate layer of complexity to the activity of neural networks. In the nematode Caenorhabditis elegans, a large number of neuropeptide genes that are expressed throughout the nervous system have been identified. The actions of these peptides supplement the synaptic connections of the 302 neurons, allowing for fine tuning of neural networks and increasing the ways in which behaviors can be regulated. In this review, we focus on a large family of genes encoding FMRFamide-related peptides (FaRPs). These genes, the flp genes, have been used as a starting point to identifying flp genes throughout Nematoda. Nematodes have the largest family of FaRPs described thus far. The challenges in the future are the elucidation of their functions and the identification of the receptors and signaling pathways through which they function.

Neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti

For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti. J. Comp. Neurol. 522:592–608, 2014.

Distribution of neuropeptides in the antennal lobes of male Spodoptera littoralis

Olfaction is an important sensory modality that regulates a plethora of behavioural expressions in insects. Processing of olfactory information takes place in the primary olfactory centres of the brain, namely the antennal lobes (ALs). Neuropeptides have been shown to be present in the olfactory system of various insect species. In the present study, we analyse the distribution of tachykinin, FMRFamide-related peptides, allatotropin, allatostatin, myoinhibitory peptides and SIFamide in the AL of the male Egyptian cotton leafworm, Spodoptera littoralis. Immunocytochemical analyses revealed that most neuropeptides were expressed in different subpopulations of AL neurons. Their arborisation patterns within the AL suggest a significant role of neuropeptide signalling in the modulation of AL processing. In addition to local interneurons, our analysis also revealed a diversity of extrinsic peptidergic neurons that connected the antennal lobe with other brain centres. Their distributions suggest that extrinsic neurons perform various types of context-related modulation.

Presence and distribution of FMRFamide-like immunoreactivity in the sea cucumber Holothuria scabra (Jaeger, 1833)

Since the discovery of FMRFamide, a cardioactive neuropeptide in the mollusk Macrocallista nimbosa (Lightfoot, 1786) by Price and Greenberg (Science 197:670–671, 1977), there has been a steady increase in our understanding of the distribution and diversity of FMRFamide and FMRFamide-related peptides (FaRPs) across phyla. The sea cucumber Holothuria scabra (Jaeger, 1833) (Echinodermata: Holothuroidea) is a high-premium tropical economic species that is overexploited globally and may be in imminent danger of extinction in some areas. Conservation of this species and its amenability to culture is however hampered by the limited knowledge about its biology, including the neurohormonal system. Using indirect immunofluorescence technique with antisera raised against the tetrapeptide FMRFamide as well as reverse-phase HPLC, we have demonstrated the widespread distribution of FMRFamide-like immunoreactivity in the tissues of H. scabra, including the coelomic fluid, suggesting that FaRPs might have neurohormonal activities in this species.

Invertebrate FMRFamide Related Peptides

In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

Expression of the SOFaRP2 gene in the central nervous system of the adult cuttlefish Sepia officinalis

FMRFamide-related Peptides (FaRPs) are involved in a variety of physiological processes, including reproduction, feeding, development, body patterning and osmoregulation in vertebrates and invertebrates. Here we investigate the expression pattern of cuttlefish Sepia officinalis FaRP2 gene in the brain by in situ hybridization. The SOFaRP2 gene was found to be expressed most intensively in the posterior chromatophore lobe, vasomotor lobe and subvertical lobe. In addition, positive staining was also found in the fin lobe, brachial lobe, anterior chromatophore lobe, anterior, dorsal and lateral basal lobes, inferior and superior frontal lobes, and optic lobe. The expression pattern of SOFaRP2 suggests its involvement in chromatophore regulation, feeding behavior, and learning and memory.

The FMRFamide-related neuropeptide FLP-20 is required in the mechanosensory neurons during memory for massed training in C. elegans

Lasting memories are likely to result from a lasting change in neurotransmission. In the nematode Caenorhabditis elegans, spaced training with a tap stimulus induces habituation to the tap that lasts for >24 h and is dependent on glutamate transmission, postsynaptic AMPA receptors, and CREB. Here we describe a distinct, presynaptic mechanism for a shorter lasting memory for tap habituation induced by massed training. We report that a FMRFamide-related peptide (FMRF = Phe-Met-Arg-Phe-NH(2)), FLP-20, is critical for memory lasting 12 h following massed training, but is not required for other forms of memory. Massed training correlated with a flp-20-dependent increase in synaptobrevin tagged with green fluorescent protein in the presynaptic terminals of the PLM mechanosensory neurons that followed the timeline of the memory trace. We also demonstrated that flp-20 is required specifically in the mechanosensory neurons for memory 12 h after massed training. These findings show that within the same species and form of learning, memory is induced by distinct mechanisms to create a lasting alteration in neurotransmission that is dependent upon the temporal pattern of training: memory of spaced training results from postsynaptic changes in the interneurons of the neural circuit, whereas memory of massed training results from presynaptic changes in the mechanosensory neurons of the neural circuit.

Peptidergic control of food intake and digestion in insects1This review is part of a virtual symposium on recent advances in understanding a variety of complex regulatory processes in insect physiology and endocrinology, including development, metabolism, cold hardiness, food intake and digestion, and diuresis, through the use of omics technologies in the postgenomic era.

Like all heterotrophic organisms, insects require a strict control of food intake and efficient digestion of food into nutrients to maintain homeostasis and to fulfill physiological tasks. Feeding and digestion are steered by both external and internal signals that are transduced by a multitude of regulatory factors, delivered either by neurons innervating the gut or mouthparts, or by midgut endocrine cells. The present review gives an overview of peptide regulators known to control feeding and digestion in insects. We describe the discovery and functional role in these processes for insect allatoregulatory peptides, diuretic hormones, FMRFamide-related peptides, (short) neuropeptide F, proctolin, saliva production stimulating peptides, kinins, and tachykinins. These peptides control either gut myoactivity, food intake, and (or) release of digestive enzymes. Some peptides exert their action at multiple levels, possibly having a biological function that depends on their site of delivery. Many regulatory peptides have been physically extracted from different insect species. However, multiple peptidomics, proteomics, transcriptomics, and genome sequencing projects have led to increased discovery and prediction of peptide (precursor) and receptor sequences. In combination with physiological experiments, these large-scale projects have already led to important steps forward in unraveling the physiology of feeding and digestion in insects.

Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform

Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio (m/z) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m/z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin β-4-like protein 3, and tight junction-associated protein 1. Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment.

Toward a single‐cell‐based analysis of neuropeptide expression in Periplaneta americana antennal lobe neurons

A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiological recordings combined with immunocytochemical studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiologically distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, we systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochemistry and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-containing cells. Finally, we demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiology with peptide profiling at the single-cell level.

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP2) and its expression pattern in the brain of the European cuttlefish Sepia officinalis

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP2). The complete 835-base pair cDNA sequence of the SOFaRP2 gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP2 gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP2 gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP2 gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP2 might be involved in controlling chromatophore activity in cuttlefish.

Oral activity of FMRFamide-related peptides on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1–0.5μg peptide/μl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5μg Acypi-MS/μl diet whereas 40% of aphids feeding on 0.5μg LMS/μl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t½=54min). No toxicity was observed when the analogues δR9 LMS and citrolline9 Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.

Molecular analysis of two FMRFamide‐encoding transcripts expressed during the development of the tropical abalone haliotis asinina

FMRFamide-related peptides (FaRPs) are involved in numerous neural functions across the animal kingdom and serve as important models for understanding the evolution of neuropeptides. Gastropod molluscs have proved to be particularly useful foci for such studies, but the developmental expression of FaRPs and the evolution of specific transcripts for different peptides are unclear within the molluscs. Here we show that FaRPs are encoded by two transcripts that appear to be splice variants of a single gene in the abalone, Haliotis asinina, which represents the basal vetigastropods. Has-FMRF1 comprises 1,438 nucleotides and encodes a precursor protein of 329 amino acids that can potentially produce two copies of FLRFamide, one copy each of TLAGDSFLRFamide, QFYRIamide, SDPDLDDVIRASLLAYSLDDSPNN, and SVATAPVEAKAVEAGNKDIE, and 13 copies of FMRFamide. The second 1,241-nucleotide transcript, Has-FMRF2, encodes a 206-amino acid precursor protein with single copies of FLRFamide and FMRFamide along with such extended forms as NFGEPFLRFamide, FDSYEDKALRFamide, and NGWLHFamide, in addition to SDPGEDMLKSILLRGAPSNNGLQY and DTUDETTUNDNAHSRQ. Both transcripts are present early in life and are expressed in different but overlapping patterns within the developing larval nervous system. Mass spectrometry and immunocytochemistry demonstrate that FaRPs are cleaved from larger precursors and localize to the developing nervous system. Our results confirm previous evidence that FaRPs are expressed early and potentially play many roles during molluscan development and suggest that the last common ancestor to living gastropods used alternative splicing of an FMRFamide gene to generate a diversity of FaRPs in spatially restricted patterns in the nervous system.

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda)

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the ‘receptive areas’ via the CNS to the chromatophore effectors.

Neuropeptide action in insects and crustaceans.

Physiological processes are regulated by a diverse array of neuropeptides that coordinate organ systems. The neuropeptides, many of which act through G protein-coupled receptors, affect the levels of cyclic nucleotides (cAMP and cGMP) and Ca(2+) in target tissues. In this perspective, their roles in molting, osmoregulation, metabolite utilization, and cardiovascular function are highlighted. In decapod crustaceans, inhibitory neuropeptides (molt-inhibiting hormone and crustacean hyperglycemic hormone) suppress the molting gland through cAMP- and cGMP-mediated signaling. In insects, the complex movements during ecdysis are controlled by ecdysis-triggering hormone and a cascade of downstream neuropeptides. Adipokinetic/hypertrehalosemic/hyperprolinemic hormones mobilize energy stores in response to increased locomotory activity. Crustacean cardioacceleratory (cardioactive) peptide, proctolin, and FMRFamide-related peptides act on the heart, accessory pulsatile organs, and excurrent ostia to control hemolymph distribution to tissues. The osmoregulatory challenge of blood gorging in Rhodnius prolixus requires the coordinated release of serotonin and diuretic and antidiuretic hormones acting on the midgut and Malpighian tubules. These studies illustrate how multiple neuropeptides allow for flexibility in response to physiological challenges.

Human RFamide-related peptide-1 diminishes cellular and integrated cardiac contractile performance

Peptides influence cardiac dysfunction; however, peptidergic modulation of contractile performance remains relatively uncharacterized. We identified a novel human peptide that modulates mammalian contractile performance. Members of the FMRFamide-related peptide (FaRP) family contain a C-terminal RFamide but structurally variant N-terminal extension. We report human RFamide-related peptide-1 (hRFRP-1) and rat RFRP-1 rapidly and reversibly decreased shortening and relaxation in isolated mammalian cardiac myocytes in a dose dependent manner. The mammalian FaRP, 26RFa, structurally related to RFRP-1 by only an RFamide did not influence myocyte contractile function. The protein kinase C (PKC) inhibitor bisindolylmaleimide-1 blocked hRFRP-1 activity. Pretreatment with pertussis toxin (PTX) did not diminish hRFRP-1 influence on contractile function. In addition, intravenous injection of hRFRP-1 in mice decreased heart rate, stroke volume, ejection fraction, and cardiac output. Collectively these findings are consistent with the conclusion RFRP-1 is an endogenous signaling molecule that activates PKC and acts through a PTX-insensitive pathway to modulate cardiac contractile function. Taken together these negative chronotropic, inotropic, and lusitropic effects of hRFRP-1 are significant; they suggest direct acute cellular and organ-level responses in mammalian heart. This is the first known study to identify a mammalian FaRP with cardio-depressant effects, opening a new area of research on peptidergic modulation of contractile performance. The high degree of RFRP structure conservation from amphibians to mammals, and similarity to invertebrate cardioinhibitory peptides suggests RFRP-1 is involved in important physiological functions. Elucidation of mechanisms involved in hRFRP-1 synthesis, release, and signaling may aid the development of strategies to prevent or attenuate cardiac dysfunction.

An immunohistochemical study of Carassius RF amide in the stomach, intestine, and pancreas of the Japanese butterfly ray, Gymnura japonica

Carassius RFamide (C-RFa) is a brain-gut peptide, isolated originally from the brain of a teleost fish, Japanese crucian carp (Carassius auratus langsdorfii). It is a novel FMRFamide-related peptide sharing homologies with mammalian prolactin-related peptide. We observed the localization of C-RFa-like immunoreactivity (C-RFa-IR) in the stomach, intestine, and pancreas of Japanese butterfly ray using an immunohistochemical technique. In the whole gastrointestinal tract, C-RFa-IR was observed in smooth muscle cells, and in the Meissner’s and Auerbach’s plexus ganglia. Furthermore, in the stomach C-RFa-IR was also observed at the bottom of the fundic glands in both the body and pyloric region. In the intestine C-RFa-IR was observed in epithelial cells at the tip of mucosal folds. In the pancreas, C-RFa-IR was observed in islet cells. Thus, we suggest that ray C-RFa may have functional roles in both peripheral neurotransmission and endocrine function of the gastroenteropancreatic system.

The association of the FMRFamide-related peptide family with the heart of the stick insect, Baculum extradentatum

The association of the FMRFamide-related peptide family with the heart of the stick insect, Baculum extradentatum

Bioinformatic analyses of the publicly accessible crustacean expressed sequence tags (ESTs) reveal numerous novel neuropeptide-encoding precursor proteins, including ones from members of several little studied taxa

ESTs have been generated for many crustacean species, providing an invaluable resource for peptide discovery in members of this arthropod subphylum. Here, these data were mined for novel peptide-encoding transcripts, with the mature peptides encoded by them predicted using a combination of online peptide prediction programs and homology to known arthropod sequences. In total, 70 mature full-length/partial peptides representing members of 16 families/subfamilies were predicted, the vast majority being novel; the species from which the peptides were identified included members of the Branchiopoda ( Daphnia carinata and Triops cancriformis), Maxillopoda ( Caligus clemensi, Caligus rogercresseyi, Lepeophtheirus salmonis and Lernaeocera branchialis) and Malacostraca ( Euphausia superba, Marsupenaeus japonicus, Penaeus monodon, Homarus americanus, Petrolisthes cinctipes, Callinectes sapidus and Portunus trituberculatus). Of particular note were the identifications of an intermediate between the insect adipokinetic hormones and crustacean red pigment concentrating hormone and a modified crustacean cardioactive peptide from the daphnid D. carinata; Arg 7-corazonin was also deduced from this species, the first identification of a corazonin from a non-decapod crustacean. Our data also include the first reports of members of the calcitonin-like diuretic hormone, FMRFamide-related peptide (neuropeptide F subfamily) and orcokinin families from members of the Copepoda. Moreover, the prediction of a bursicon α from the euphausid E. superba represents the first peptide identified from any member of the basal eucaridean order Euphausiacea. In addition, large collections of insect eclosion hormone- and neuroparsin-like peptides were identified from a variety of species, greatly expanding the number of known members of these families in crustaceans.

FMRFamide elicits chromatophore expansion and retraction depending on its type and development in the squid, Sepioteuthis lessoniana

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development.