Drosophila neuronal Glucose-6-Phosphatase is a modulator of neuropeptide release that regulates muscle glycogen stores via FMRFamide signaling.

Neuropeptides (NPs) and their cognate receptors are critical effectors of diverse physiological processes and behaviors. We recently reported of a noncanonical function of the Drosophila Glucose-6-Phosphatase (G6P) gene in a subset of neurosecretory cells in the central nervous system that governs systemic glucose homeostasis in food-deprived flies. Here, we show that G6P-expressing neurons define six groups of NP-secreting cells, four in the brain and two in the thoracic ganglion. Using the glucose homeostasis phenotype as a screening tool, we find that neurons located in the thoracic ganglion expressing FMRFamide NPs (FMRFaG6P neurons) are necessary and sufficient to maintain systemic glucose homeostasis in starved flies. We further show that G6P is essential in FMRFaG6P neurons for attaining a prominent Golgi apparatus and secreting NPs efficiently. Finally, we establish that G6P-dependent FMRFa signaling is essential for the build-up of glycogen stores in the jump muscle which expresses the receptor for FMRFamides. We propose a general model in which the main role of G6P is to counteract glycolysis in peptidergic neurons for the purpose of optimizing the intracellular environment best suited for the expansion of the Golgi apparatus, boosting release of NPs and enhancing signaling to respective target tissues expressing cognate receptors.

A conserved peptide-binding pocket in HyNaC/ASIC ion channels.

The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.

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.

Efficacy and safety of fractional micro-needling radiofrequency for the treatment of enlarged pores on the cheeks of a chinese cohort: a retrospective study.

To explore the efficacy and safety of fractional micro-needling radiofrequency (FMRF) in the treatment of enlarged pores on the cheek in a Chinese cohort. Patients with enlarged facial pores who underwent FMRF between January 2020 and December 2022 were included in this study. Blinded clinical assessments were performed by two independent dermatologists using a six-grade photographic enlarged pore scale and a quartile grading scale. Patients were asked to rate the degree of pain related to treatment on a visual analog scale (VAS), with scores ranging from 0 (no pain) to 10 (worst pain ever). A paired t-test was used to analyze the six-grade photographic enlarged pore scores. A total of 22 patients received three consecutive sessions of FMRF treatment, with intervals of 1-3 months, and underwent follow-up as scheduled. The mean six-grade photographic enlarged score was 3.55 ± 0.96 at baseline, while the score decreased significantly to 2.59 ± 0.59 after three treatment sessions (P < 0.05). The improvement score of the patients, assessed by two independent dermatologists, was 2.31 ± 0.71, according to the quartile grading scale. The mean VAS score was 6.42 ± 1.44. FMRF is effective and safe for the treatment of enlarged facial pores after three sessions.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

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.

Aromatic amino acids in the finger domain of the FMRFamide-gated Na[Formula: see text] channel are involved in the FMRFamide recognition and the activation.

FMRFamide-gated Na[Formula: see text] channel (FaNaC) is a member of the DEG/ENaC family and activated by a neuropeptide, FMRFamide. Structural information about the FMRFamide-dependent gating is, however, still elusive. Because two phenylalanines of FMRFamide are essential for the activation of FaNaC, we hypothesized that aromatic-aromatic interaction between FaNaC and FMRFamide is critical for FMRFamide recognition and/or the activation gating. Here, we focused on eight conserved aromatic residues in the finger domain of FaNaCs and tested our hypothesis by mutagenic analysis and in silico docking simulations. The mutation of conserved aromatic residues in the finger domain reduced the FMRFamide potency, suggesting that the conserved aromatic residues are involved in the FMRFamide-dependent activation. The kinetics of the FMRFamide-gated currents were also modified substantially in some mutants. Some results of docking simulations were consistent with a hypothesis that the aromatic-aromatic interaction between the aromatic residues in FaNaC and FMRFamide is involved in the FMRFamide recognition. Collectively, our results suggest that the conserved aromatic residues in the finger domain of FaNaC are important determinants of the ligand recognition and/or the activation gating in FaNaC.

An examination of nervous system revealed unexpected immunoreactivity of both secretory apparatus and excretory canals in plerocercoids of two broad tapeworms (Cestoda: Diphyllobothriidea).

Dibothriocephalus ditremus and Dibothriocephalus latus are diphyllobothriidean tapeworms autochthonous to Europe. Their larval stages (plerocercoids) may seriously alter health of their intermediate fish hosts (D. ditremus) or cause intestinal diphyllobothriosis of the final human host (D. latus). Despite numerous data on the internal structure of broad tapeworms, many aspects of the morphology and physiology related to host­parasite co-existence remain unclear for these 2 species. The main objective of this work was to elucidate functional morphology of the frontal part (scolex) of plerocercoids, which is crucial for their establishment in fish tissues and for an early attachment in final hosts. The whole-mount specimens were labelled with different antibodies and examined by confocal microscope to capture their complex 3-dimensional microanatomy. Both species exhibited similar general pattern of immunofluorescent signal, although some differences were observed. In the nervous system, FMRF amide-like immunoreactivity (IR) occurred in the bi-lobed brain, 2 main nerve cords and surrounding nerve plexuses. Differences between the species were found in the structure of the brain commissures and the size of the sensilla. Synapsin IR examined in D. ditremus occurred mainly around FMRF amide-like IR brain lobes and main cords. The unexpected finding was an occurrence of FMRF amide-like IR in terminal reservoirs of secretory gland ducts and excretory canals, which has not been observed previously in any tapeworm species. This may indicate that secretory/excretory products, which play a key role in host­parasite relationships, are likely to contain FMRF amide-related peptide/s.

The FMRFamide-like peptide FLP-1 modulates larval development by regulating the production and secretion of the insulin-like peptide DAF-28 in Caenorhabditis elegans.

The FMRFamide-like peptides (FLPs) are conserved in both free-living and parasitic nematodes. This molecular genetic study verified the relevance of the flp-1 gene, which is conserved in many nematode species, to the larval development of the free-living soil nematode Caenorhabditis elegans. Using C. elegans as a model, we found that: (1) FLP-1 suppressed larval development, resulting in diapause; (2) the secretion of FLP-1, which is produced in AVK head neurons, was suppressed by the presence of food (Escherichia coli) as an environmental factor to continue larval development; (3) the FLP-1 reduced the production and secretion of DAF-28, which is produced in ASI head neurons and is the predominant insulin-like peptide (INS) present. FLP-1 is conserved in many species of plant-parasitic root-knot nematodes that cause severe damage to crops. Therefore, our findings may provide insight into the development of new nematicides that can disturb their infection and development.

The FMRFamide-like peptide FLP-2 is involved in the modulation of larval development and adult lifespan by regulating the secretion of the insulin-like peptide INS-35 in Caenorhabditis elegans.

In the animal kingdom, neuropeptides regulate diverse physiological functions. In invertebrates, FMRFamide and its related peptides, a family of neuropeptides, play an important role as neurotransmitters. The FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families and are conserved in nematodes. Our screen for flp genes of the free-living soil nematode Caenorhabditis elegans revealed that the flp-2 gene is involved in the larval development. The gene is also conserved in plant-parasitic root-knot nematodes. Our molecular genetic analyses of the C. elegans flp-2 gene demonstrated as follows: (1) the production and secretion of FLP-2, produced in the head neurons, are controlled by environmental factors (growth density and food); (2) the FLP-2 is involved in not only larval development but also adult lifespan by regulating the secretion of one of the insulin-like peptides INS-35, produced in the intestine. These findings provide new insight into the development of new nematicides.

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.

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.

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.

Involvement of RFamide neuropeptides in polyp contraction of the adult scleractinian corals Euphyllia ancora and Stylophora pistillata.

The distribution and functions of neurons in scleractinian corals remain largely unknown. This study focused on the Arg-Phe amide family of neuropeptides (RFamides), which have been shown to be involved in a variety of biological processes in animals, and performed molecular identification and characterization in the adult scleractinian coral Euphyllia ancora. The deduced amino acid sequence of the identified RFamide preprohormone was predicted to contain 20 potential neuropeptides, including 1 Pro-Gly-Arg-Phe (PGRF) amide and 15 Gln-Gly-Arg-Phe (QGRF) amide peptides. Tissue distribution analysis showed that the level of transcripts in the tentacles was significantly higher than that in other polyp tissues. Immunohistochemical analysis with the FMRFamide antibody showed that RFamide neurons were mainly distributed in the epidermis of the tentacles and mouth with pharynx. Treatment of E. ancora polyps with synthetic QGRFamide peptides induced polyp contraction. The induction of polyp contraction by QGRFamide peptide treatment was also observed in another scleractinian coral, Stylophora pistillata. These results strongly suggested that RFamides play a role in the regulation of polyp contraction in adult scleractinians.

Specification of Drosophila neuropeptidergic neurons by the splicing component brr2.

During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. In this study, we focus on the role of RNA processing during target-derived signaling. We found that brr2 and Prp8 play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.

FMRFamide-like peptides expand the behavioral repertoire of a densely connected nervous system.

Animals, including humans, can adapt to environmental stress through phenotypic plasticity. The free-living nematode Caenorhabditis elegans can adapt to harsh environments by undergoing a whole-animal change, involving exiting reproductive development and entering the stress-resistant dauer larval stage. The dauer is a dispersal stage with dauer-specific behaviors for finding and stowing onto carrier animals, but how dauers acquire these behaviors, despite having a physically limited nervous system of 302 neurons, is poorly understood. We compared dauer and reproductive development using whole-animal RNA sequencing at fine time points and at sufficient depth to measure transcriptional changes within single cells. We detected 8,042 genes differentially expressed during dauer and reproductive development and observed striking up-regulation of neuropeptide genes during dauer entry. We knocked down neuropeptide processing using sbt-1 mutants and demonstrate that neuropeptide signaling promotes the decision to enter dauer rather than reproductive development. We also demonstrate that during dauer neuropeptides modulate the dauer-specific nictation behavior (carrier animal-hitchhiking) and are necessary for switching from repulsion to CO2 (a carrier animal cue) in nondauers to CO2 attraction in dauers. We tested individual neuropeptides using CRISPR knockouts and existing strains and demonstrate that the combined effects of flp-10 and flp-17 mimic the effects of sbt-1 on nictation and CO2 attraction. Through meta-analysis, we discovered similar up-regulation of neuropeptides in the dauer-like infective juveniles of diverse parasitic nematodes, suggesting the antiparasitic target potential of SBT-1. Our findings reveal that, under stress, increased neuropeptide signaling in C. elegans enhances their decision-making accuracy and expands their behavioral repertoire.

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.

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.

The immunomodulatory function of invertebrate specific neuropeptide FMRFamide in oyster Crassostrea gigas.

As one of the most important neuropeptides identified only in invertebrates of Mollusca, Annelida and Arthropoda, FMRFamide (Phe-Met-Arg-Phe-NH2) involves in multiple physiological processes, such as mediating cardiac frequency and contraction of somatic and visceral muscles. However, its modulatory role in the immune defense has not been well understood. In the present study, an FMRFamide precursor (designed as CgFMRFamide) was identified in oyster Crassostrea gigas, which could be processed into nineteen FMRFamide peptides. Phylogenetic analysis revealed that CgFMRFamide shared high similarity with other identified FMRFamides in mollusks. The mRNA of CgFMRFamide was mainly concentrated in the tissues of visceral ganglia, hepatopancreas and hemocytes, and a consistent distribution of FMRFamide peptide was confirmed by immunohistochemistry and immunocytochemistry assays. The mRNA expression level of CgFMRFamide in hemocytes was significantly up-regulated after immune stimulation with lipopolysaccharide (LPS). After the concentration of FMRFamide was increased by exogenous injection, the in vivo expressions of pro-inflammatory cytokine CgIL17-5, as well as the apoptosis-related CgCaspase-1 and CgCaspase-3 in hemocytes were promptly increased (p < 0.05), but the concentration of signal molecule nitric oxide (NO) was significantly down-regulated (p < 0.05). Meanwhile, an increased phosphorylation of p38 MAP kinase in hemocytes was also detected after the FMRFamide injection. These results collectively demonstrated that the conserved FMRFamide could not only respond to immune stimulation, but also regulate the expression of immune effectors and apoptosis-related genes, which might be mediated by p38 MAP kinase pathway, thereby effectively involved in clearing pathogens and maintaining homeostasis in oysters.