Pigment Dispersing Factors and Their Cognate Receptors in a Crustacean Model, With New Insights Into Distinct Neurons and Their Functions

Pigment dispersing factors (PDFs, or PDHs in crustaceans) form a structurally related group of neuropeptides found throughout the Ecdysozoa and were first discovered as pigmentary effector hormones in crustaceans. In insects PDFs fulfill crucial neuromodulatory roles, most notably as output regulators of the circadian system, underscoring their central position in physiological and behavioral organization of arthropods. Intriguingly, decapod crustaceans express multiple isoforms of PDH originating from separate genes, yet their differential functions are still to be determined. Here, we functionally define two PDH receptors in the crab Carcinus maenas and show them to be selectively activated by four PDH isoforms: PDHR 43673 was activated by PDH-1 and PDH-2 at low nanomolar doses whilst PDHR 41189 was activated by PDH-3 and an extended 20 residue e-PDH. Detailed examination of the anatomical distribution of all four peptides and their cognate receptors indicate that they likely perform different functions as secreted hormones and/or neuromodulators, with PDH-1 and its receptor 43,673 implicated in an authentic hormonal axis. PDH-2, PDH-3, and e-PDH were limited to non-neurohemal interneuronal sites in the CNS; PDHR 41189 was largely restricted to the nervous system suggesting a neuromodulatory function. Notably PDH-3 and e-PDH were without chromatophore dispersing activity. This is the first report which functionally defines a PDHR in an endocrine system in a crustacean and to indicate this and other putative roles of this physiologically pivotal peptide group in these organisms. Thus, our findings present opportunities to further examine the endocrine and circadian machinery in this important arthropod phylum

Metabolic Stress Responses in Drosophila Are Modulated by Brain Neurosecretory Cells That Produce Multiple Neuropeptides

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas

Abstract Background Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. Results Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. Conclusions The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans

Neuroarchitecture of Peptidergic Systems in the Larval Ventral Ganglion of Drosophila melanogaster

Recent studies on Drosophila melanogaster and other insects have revealed important insights into the functions and evolution of neuropeptide signaling. In contrast, in- and output connections of insect peptidergic circuits are largely unexplored. Existing morphological descriptions typically do not determine the exact spatial location of peptidergic axonal pathways and arborizations within the neuropil, and do not identify peptidergic in- and output compartments. Such information is however fundamental to screen for possible peptidergic network connections, a prerequisite to understand how the CNS controls the activity of peptidergic neurons at the synaptic level. We provide a precise 3D morphological description of peptidergic neurons in the thoracic and abdominal neuromeres of the Drosophila larva based on fasciclin-2 (Fas2) immunopositive tracts as landmarks. Comparing the Fas2 “coordinates” of projections of sensory or other neurons with those of peptidergic neurons, it is possible to identify candidate in- and output connections of specific peptidergic systems. These connections can subsequently be more rigorously tested. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the projections and compartmentalization of neurons expressing 12 different peptide genes, encoding approximately 75% of the neuropeptides chemically identified within the Drosophila CNS. Results are assembled into standardized plates which provide a guide to identify candidate afferent or target neurons with overlapping projections. In general, we found that putative dendritic compartments of peptidergic neurons are concentrated around the median Fas2 tracts and the terminal plexus. Putative peptide release sites in the ventral nerve cord were also more laterally situated. Our results suggest that i) peptidergic neurons in the Drosophila ventral nerve cord have separated in- and output compartments in specific areas, and ii) volume transmission is a prevailing way of peptidergic communication within the CNS. The data can further be useful to identify colocalized transmitters and receptors, and develop peptidergic neurons as new landmarks

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn13-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn13-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day

A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions

<p>Abstract</p> <p>Background</p> <p>Insect neuropeptides are distributed in stereotypic sets of neurons that commonly constitute a small fraction of the total number of neurons. However, some neuropeptide genes are expressed in larger numbers of neurons of diverse types suggesting that they are involved in a greater diversity of functions. One of these widely expressed genes, <it>snpf</it>, encodes the precursor of short neuropeptide F (sNPF). To unravel possible functional diversity we have mapped the distribution of transcript of the <it>snpf </it>gene and its peptide products in the central nervous system (CNS) of <it>Drosophila </it>in relation to other neuronal markers.</p> <p>Results</p> <p>There are several hundreds of neurons in the larval CNS and several thousands in the adult <it>Drosophila </it>brain expressing <it>snpf </it>transcript and sNPF peptide. Most of these neurons are intrinsic interneurons of the mushroom bodies. Additionally, sNPF is expressed in numerous small interneurons of the CNS, olfactory receptor neurons (ORNs) of the antennae, and in a small set of possibly neurosecretory cells innervating the corpora cardiaca and aorta. A sNPF-Gal4 line confirms most of the expression pattern. None of the sNPF immunoreactive neurons co-express a marker for the transcription factor DIMMED, suggesting that the majority are not neurosecretory cells or large interneurons involved in episodic bulk transmission. Instead a portion of the sNPF producing neurons co-express markers for classical neurotransmitters such as acetylcholine, GABA and glutamate, suggesting that sNPF is a co-transmitter or local neuromodulator in ORNs and many interneurons. Interestingly, sNPF is coexpressed both with presumed excitatory and inhibitory neurotransmitters. A few sNPF expressing neurons in the brain colocalize the peptide corazonin and a pair of dorsal neurons in the first abdominal neuromere coexpresses sNPF and insulin-like peptide 7 (ILP7).</p> <p>Conclusion</p> <p>It is likely that sNPF has multiple functions as neurohormone as well as local neuromodulator/co-transmitter in various CNS circuits, including olfactory circuits both at the level of the first synapse and at the mushroom body output level. Some of the sNPF immunoreactive axons terminate in close proximity to neurosecretory cells producing ILPs and adipokinetic hormone, indicating that sNPF also might regulate hormone production or release.</p

Unravelling the evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor gene families in Bilaterians: insights from Anopheles Mosquitoes

Allatostatin type A receptors (AST-ARs) are a group of G-protein coupled receptors activated by members of the FGL-amide (AST-A) peptide family that inhibit food intake and development in arthropods. Despite their physiological importance the evolution of the AST-A system is poorly described and relatively few receptors have been isolated and functionally characterised in insects. The present study provides a comprehensive analysis of the origin and comparative evolution of the AST-A system. To determine how evolution and feeding modified the function of AST-AR the duplicate receptors in Anopheles mosquitoes, were characterised. Phylogeny and gene synteny suggested that invertebrate AST-A receptors and peptide genes shared a common evolutionary origin with KISS/GAL receptors and ligands. AST-ARs and KISSR emerged from a common gene ancestor after the divergence of GALRs in the bilaterian genome. In arthropods, the AST-A system evolved through lineage-specific events and the maintenance of two receptors in the flies and mosquitoes (Diptera) was the result of a gene duplication event. Speciation of Anophelesmosquitoes affected receptor gene organisation and characterisation of AST-AR duplicates (GPRALS1 and 2) revealed that in common with other insects, the mosquito receptors were activated by insect AST-A peptides and the iCa(2+)-signalling pathway was stimulated. GPRALS1 and 2 were expressed mainly in mosquito midgut and ovaries and transcript abundance of both receptors was modified by feeding. A blood meal strongly up-regulated expression of both GPRALS in the midgut (p < 0.05) compared to glucose fed females. Based on the results we hypothesise that the AST-A system in insects shared a common origin with the vertebrate KISS system and may also share a common function as an integrator of metabolism and reproduction. Highlights: AST-A and KISS/GAL receptors and ligands shared common ancestry prior to the protostome-deuterostome divergence. Phylogeny and gene synteny revealed that AST-AR and KISSR emerged after GALR gene divergence. AST-AR genes were present in the hemichordates but were lost from the chordates. In protostomes, AST-ARs persisted and evolved through lineage-specific events and duplicated in the arthropod radiation. Diptera acquired and maintained functionally divergent duplicate AST-AR genes.Foundation for Science and Technology, Portugal (FCT) [PTDC/BIA-BCM/114395/2009]; European Regional Development Fund (ERDF) COMPETE - Operational Competitiveness Programme; Portuguese funds through FCT Foundation for Science and Technology [PEst-C/MAR/LA0015/2013, UID/Multi/04326/2013, PEst-OE/SAU/LA0018/2013]; FCT [SFRH/BPD/89811/2012, SFRH/BPD/80447/2011, SFRH/BPD/66742/2009]; auxiliary research contract FCT Pluriannual funds [PEst-C/MAR/LA0015/2013, UID/Multi/04326/2013]info:eu-repo/semantics/publishedVersio

Brain architecture in the terrestrial hermit crab Coenobita clypeatus (Anomura, Coenobitidae), a crustacean with a good aerial sense of smell

<p>Abstract</p> <p>Background</p> <p>During the evolutionary radiation of Crustacea, several lineages in this taxon convergently succeeded in meeting the physiological challenges connected to establishing a fully terrestrial life style. These physiological adaptations include the need for sensory organs of terrestrial species to function in air rather than in water. Previous behavioral and neuroethological studies have provided solid evidence that the land hermit crabs (Coenobitidae, Anomura) are a group of crustaceans that have evolved a good sense of aerial olfaction during the conquest of land. We wanted to study the central olfactory processing areas in the brains of these organisms and to that end analyzed the brain of <it>Coenobita clypeatus </it>(Herbst, 1791; Anomura, Coenobitidae), a fully terrestrial tropical hermit crab, by immunohistochemistry against synaptic proteins, serotonin, FMRFamide-related peptides, and glutamine synthetase.</p> <p>Results</p> <p>The primary olfactory centers in this species dominate the brain and are composed of many elongate olfactory glomeruli. The secondary olfactory centers that receive an input from olfactory projection neurons are almost equally large as the olfactory lobes and are organized into parallel neuropil lamellae. The architecture of the optic neuropils and those areas associated with antenna two suggest that <it>C. clypeatus </it>has visual and mechanosensory skills that are comparable to those of marine Crustacea.</p> <p>Conclusion</p> <p>In parallel to previous behavioral findings of a good sense of aerial olfaction in C. clypeatus, our results indicate that in fact their central olfactory pathway is most prominent, indicating that olfaction is a major sensory modality that these brains process. Interestingly, the secondary olfactory neuropils of insects, the mushroom bodies, also display a layered structure (vertical and medial lobes), superficially similar to the lamellae in the secondary olfactory centers of <it>C. clypeatus</it>. More detailed analyses with additional markers will be necessary to explore the question if these similarities have evolved convergently with the establishment of superb aerial olfactory abilities or if this design goes back to a shared principle in the common ancestor of Crustacea and Hexapoda.</p