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.

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.

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.

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.

Gene Regulatory Mechanisms Underlying the Spatial and Temporal Regulation of Target-Dependent Gene Expression in Drosophila Neurons.

Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly activates FMRFa gene expression through an atypical gene regulatory mechanism.

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.

Prediction of Scylla olivacea (Crustacea; Brachyura) peptide hormones using publicly accessible transcriptome shotgun assembly (TSA) sequences.

The aquaculture of crabs from the genus Scylla is of increasing economic importance for many Southeast Asian countries. Expansion of Scylla farming has led to increased efforts to understand the physiology and behavior of these crabs, and as such, there are growing molecular resources for them. Here, publicly accessible Scylla olivacea transcriptomic data were mined for putative peptide-encoding transcripts; the proteins deduced from the identified sequences were then used to predict the structures of mature peptide hormones. Forty-nine pre/preprohormone-encoding transcripts were identified, allowing for the prediction of 187 distinct mature peptides. The identified peptides included isoforms of adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon ß, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, FMRFamide-like peptide, HIGSLYRamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide and tachykinin-related peptide, all well-known neuropeptide families. Surprisingly, the tissue used to generate the transcriptome mined here is reported to be testis. Whether or not the testis samples had neural contamination is unknown. However, if the peptides are truly produced by this reproductive organ, it could have far reaching consequences for the study of crustacean endocrinology, particularly in the area of reproductive control. Regardless, this peptidome is the largest thus far predicted for any brachyuran (true crab) species, and will serve as a foundation for future studies of peptidergic control in members of the commercially important genus Scylla.

flp-32 Ligand/receptor silencing phenocopy faster plant pathogenic nematodes.

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R-silenced worms also display an increase in migration rate. This work demonstrates that Gp-flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR.

FMRFamide signaling promotes stress-induced sleep in Drosophila.

Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

In silico characterization of the neuropeptidome of the Western black widow spider Latrodectus hesperus.

Technological advancements in high-throughput sequencing have resulted in the production/public deposition of an ever-growing number of arthropod transcriptomes. While most sequencing projects have focused on hexapods, transcriptomes have also been generated for members of the Chelicerata. One chelicerate for which a large transcriptome has recently been released is the Western black widow Latrodectus hesperus, a member of the Araneae (true spiders). Here, a neuropeptidome for L. hesperus was predicted using this resource. Thirty-eight peptide-encoding transcripts were mined from the L. hesperus transcriptome, with 216 distinct peptides predicted from the deduced pre/preprohormones. The identified peptides included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon ß, CAPA/periviscerokinin/pyrokinin, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, neuropeptide F (NPF), orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. Of particular note were the identifications of a carboxyl (C)-terminally extended corazonin, FLPs possessing -IMRFamide, -MMYFamide, and -MIHFamide C-termini, a NPF and a sulfakinin each ending in -RYamide rather than -RFamide, a precursor whose orcokinins include C-terminally amidated isoforms, and a collection of TRPs possessing -FXPXLamide rather than the stereotypical -FXGXLamide C-termini. The L. hesperus peptidome is by far the largest thus far published for any member of the Chelicerata. Taken collectively, these data serve as a reference for future neuropeptide discovery in the Araneae and provide a foundation for future studies of peptidergic control in L. hesperus and other spiders.

Myotropic effects of FMRFamide containing peptides on the heart of the mosquito Anopheles gambiae.

FMRFamide-like peptides (FLPs) are produced by invertebrate and vertebrate animals, and regulate diverse physiological processes. In insects, several FLPs modulate heart physiology, with some increasing and others decreasing dorsal vessel contraction dynamics. Here, we describe the FMRFamide gene structure in the mosquito, Anopheles gambiae, quantify the developmental and spatial expression of FMRFamide and its putative receptor (FMRFamideR), and show that the peptides FMRFamide and SALDKNFMRFamide have complex myotropic properties. RACE sequencing showed that the FMRFamide gene encodes eight putative FLPs and is alternatively spliced. Of the eight FLPs, only one is shared by A. gambiae, Aedes aegypti and Culex quinquefasciatus: SALDKNFMRFamide. Quantitative PCR showed that peak expression of FMRFamide and FMRFamideR occurs in second instar larvae and around eclosion. In adults, FMRFamide is primarily transcribed in the head and thorax, and FMRFamideR is primarily transcribed in the thorax. Intravital video imaging of mosquitoes injected FMRFamide and SALDKNFMRFamide revealed that at low doses these peptides increase heart contraction rates. At high doses, however, these peptides decrease heart contraction rates and alter the proportional directionality of heart contractions. Taken altogether, these data describe the FMRFamide gene in A. gambiae, and show that FLPs are complex modulators of mosquito circulatory physiology.

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.

Identification and Characterization of Hdh-FMRF2 Gene in Pacific Abalone and Its Possible Role in Reproduction and Larva Development.

FMRFamide-related peptides are neuropeptides involved in a wide range of biological processes, including reproduction and larval development. To characterize the involvement of FMRFamide in the reproduction and larval development of Pacific abalone Haliotis discus hannai, an FMRFamide cDNA (Hdh-FMRF2) was cloned from the cerebral ganglion (CG). Fluorescence in situ hybridization and qRT-PCR were performed for functional characterization. The Hdh-FMRF2 cDNA encoded 204 deduced amino acids that contained a putative signal peptide and four FaRP domains. The major population of Hdh-FMRF2 neuronal cell bodies was localized in the cortex of CG. Hdh-FMRF2 mRNA expression was significantly upregulated in CG during the mature stage of gonadal development and effective accumulative temperature (EAT) exposed abalone in both sexes. In the induced spawning event, Hdh-FMRF2 expression was significantly upregulated during spawning in males. However, no upregulation was observed in females, suggesting Hdh-FMRF2 might inhibit gamete release in female abalone. These results revealed Hdh-FMRF2 as a reproduction related peptide. Furthermore, mRNA expression in larval development suggested that this peptide was also involved in larval development during development of Pacific abalone. Collectively, this study provides evidence of possible involvement of an FMRFamide neuropeptide in the reproduction and larval development of Pacific abalone.

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.

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.

Neuropeptide evolution: neurohormones and neuropeptides predicted from the genomes of Capitella teleta and Helobdella robusta.

Genes encoding neurohormones and neuropeptide precursors were identified in the genomes of two annelids, the leech Helobdella robusta and the polychaete worm Capitella teleta. Although no neuropeptides have been identified from these two species and relatively few neuropeptides from annelids in general, 43 and 35 such genes were found in Capitella and Helobdella, respectively. The predicted peptidomes of these two species are similar to one another and also similar to those of mollusks, particular in the case of Capitella. Helobdella seems to have less neuropeptide genes than Capitella and it lacks the glycoprotein hormones bursicon and GPA2/GPB5; in both cases the genes coding the two subunits as well as the genes coding their receptors are absent from its genome. In Helobdella several neuropeptide genes are duplicated, thus it has five NPY genes, including one pseudogene, as well as four genes coding Wwamides (allatostatin B). Genes coding achatin, allatotropin, allatostatin C, conopressin, FFamide, FLamide, FMRFamide, GGRFamide, GnRH, myomodulin, NPY, pedal peptides, RGWamide (a likely APGWamide homolog), RXDLamide, VR(F/I)amide, WWamide were found in both species, while genes coding cerebrin, elevenin, GGNG, LFRWamide, LRFYamide, luqin, lymnokinin and tachykinin were only found in Capitella.

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.

FMRFamide gene and peptide expression during central nervous system development of the cephalopod mollusk, Idiosepius notoides.

Mollusks are a showcase of brain evolution represented by several classes with a varying degree of nervous system centralization. Cellular and molecular processes involved in the evolution of the highly complex cephalopod brain from a simple, monoplacophoran-like ancestor are still obscure and homologies on the cellular level are poorly established. FMRFamide (Phe-Ile-Arg-Phe-NH(2))-related peptides (FaRPs) constitute an evolutionarily conserved and diverse group of neuropeptides in the central nervous system (CNS) of many metazoans. Herein, we provide a detailed description of the developing FMRFamide-like immunoreactive (Fa-lir) CNS of the pygmy squid Idiosepius notoides using gene expression analyses and immunocytochemistry. The open reading frame of the I. notoides FMRFamide gene InFMRF predicts one copy each of FIRFamide, FLRFamide (Phe-Leu-Arg-Phe-NH(2)), ALSGDAFLRFamide (Ala-Leu-Ser-Gly-Asp-Ala-Phe-Leu-Arg-Phe-NH(2)), and 11 copies of FMRFamide. Applying matrix-assisted laser desorption/ionization time-of-flight (ToF) mass spectrometry-based peptide profiling, we characterized all predicted FaRPs except ALSGDAFLRFamide. Two cell clusters express InFMRF and show FMRFamide-like-immunoreactivity within the palliovisceral ganglia, that is, the future posterior subesophageal mass, during the lobe differentiation phase. They project neurites via ventral axonal tracts, which form the scaffold of the future subesophageal mass. In the supraesophageal mass, InFMRF is first expressed during mid-embryogenesis in the superior and inferior buccal lobes. A neurite of the peduncle commissure represents the first Fa-lir element. Later, the sub- and supraesophageal mass interconnect via Fa-lir neurites and more brain lobes express InFMRF and FMRFamide-like peptides. InFMRF expression was observed in fewer brain lobes than Fa-lir elements. The early expression of InFMRF and FMRFamide-lir peptides in the visceral system and not the remaining CNS of the cephalopod I. notoides resembles the condition found in the majority of investigated gastropods.

FMRFamide regulates oscillatory activity of the olfactory center in the slug.

In the olfactory center of terrestrial animals, changes in the oscillatory frequency of the local field potential (LFP) are thought to be involved in olfaction-based behavior and olfactory memory. The terrestrial slug Limax has a highly developed olfactory center, the procerebrum, in which the LFP spontaneously oscillates. Although changes in the oscillatory frequency are thought to correspond to the preference for specific odors, our knowledge about the mechanism of this frequency regulation is limited. To clarify the mechanism of the bidirectional frequency changes in the procerebrum, we focused on the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide), which is known to have neuromodulatory functions in invertebrate nervous systems. Application of FMRFamide decreased the oscillatory frequency via G-protein-mediated cascades. Immunohistochemistry showed that FMRFamide-like-immunoreactive neuronal cell bodies are located in the cell mass layer of the procerebrum, projecting their neurites to the neuropile layers. The procerebrum was shown to also receive innervation from other regions of the cerebral ganglion. Furthermore, according to their morphological and projection characteristics, FMRFamide-containing neurons belong to the subpopulations of both bursting and nonbursting neurons in the procerebrum. The mRNA splice variant encoding multiple copies of canonical FMRFamide was specifically expressed in the procerebrum. Taking into account previous results showing that serotonin increases the oscillatory frequency, our results indicate that FMRFamide and serotonin both regulate the LFP frequency but in exactly the opposite direction in the olfactory center of the terrestrial slug.