Correction: The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

[This corrects the article DOI: 10.1371/journal.pgen.1011011.].

The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

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

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.

Roles of crustacean hyperglycaemic hormone in ionic and metabolic homeostasis in the Christmas Island blue crab, Discoplax celeste.

There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.

The adaptive significance of crustacean hyperglycaemic hormone (CHH) in daily and seasonal migratory activities of the Christmas Island red crab Gecarcoidea natalis.

The Christmas Island red crab Gecarcoidea natalis undergoes extreme changes in metabolic status, ranging from inactivity during the dry season, to a spectacular annual breeding migration at the start of the wet season. The dramatic change in metabolic physiology that this polarisation entails should be reflected in changes in endocrine physiology, particularly that of the crustacean hyperglycaemic hormone (CHH), of which we know relatively little. CHH levels were measured using a novel ultrasensitive time-resolved fluoroimmunoassay (TR-FIA), together with metabolites (glucose, lactate), in the field at several scales of temporal resolution, during migratory activities (wet season) and during the inactive fossorial phase (dry season). Release patterns of CHH were measured during extreme (forced) exercise, showing for the first time an unexpectedly rapid pulsatile release of this hormone. A seasonally dependent glucose-sensitive negative-feedback loop was identified that might be important in energy mobilisation during migration. Haemolymph lactate levels were strongly correlated with CHH levels in both field and experimental animals. During migration, CHH levels were lower than during the dry season and, during migration, daytime CHH levels (when most locomotor activity occurred) increased. However, the intense dawn activity in both dry and wet seasons was not always associated with repeatable hyperglycaemia or CHH release. The results obtained are discussed in relation to the life history and behaviour of G. natalis.

Identification and expression of mRNAs encoding bursicon in the plesiomorphic central nervous system of Homarus gammarus.

Ecdysis in arthropods is a complex process, regulated by many neurohormones, which must be released in a precisely coordinated manner. In insects, the ultimate hormone involved in this process is the cuticle tanning hormone, bursicon. Recently, this hormone has been identified in crustaceans. To further define the distribution of bursicon in crustacean nervous systems, and to compare hormone structures within the sub-phylum, cDNAs encoding both bursicon subunits were cloned and sequenced from the nervous system of the European lobster, Homarus gammarus, and expression patterns including those for CCAP determined using in-situ hybridisation, quantitative RT-PCR and immunohistochemistry. Full-length cDNAs encoded bursicon subunits of 121 amino acids (Average M(r): 13365.48) for Burs α, 115 amino acids (Average M(r): 12928.54) for Burs ß. Amino acid sequences were most closely related to those of crabs, and for Burs ß the sequence was identical to that of the American lobster, Homarus americanus. Complete co-localisation with CCAP in the VNC was seen. Copy numbers burs α, burs ß and CCAP mRNAs were between 0.5 and 1.5 × 10(5) for both bursicon subunits, 0.5-6 × 10(5) per cdn neurone for CCAP. The terminal abdominal ganglia (AG 6-8) contained about 52 cdn-type neurons, making it the largest bursicon producing region in the CNS. Double labelling IHC using recombinant Carcinus Burs α and CCAP antisera demonstrated complete co-localisation in the VNC. On the basis of the results obtained, it is proposed that CCAP and bursicon release occur simultaneously during ecdysis in crustaceans.

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.

Functional Identification and Characterization of the Diuretic Hormone 31 (DH31) Signaling System in the Green Shore Crab, Carcinus maenas.

The functional characterization of crustacean neuropeptides and their cognate receptors has not kept pace with the recent advances in sequence determination and, therefore, our understanding of the physiological roles of neuropeptides in this important arthropod sub-phylum is rather limited. We identified a candidate receptor-ligand pairing for diuretic hormone 31 (DH31) in a neural transcriptome of the crab, Carcinus maenas. In insects, DH31 plays species -specific but central roles in many facets of physiology, including fluid secretion, myoactivity, and gut peristalsis but little is known concerning its functions in crustaceans. The C. maenas DH31 transcript codes for a 147 amino acid prepropeptide, and a single receptor transcript translates to a secretin-like (Class B1) G protein-coupled receptor (GPCR). We used an in vitro aequorin luminescence Ca2+ mobilization assay to demonstrate that this candidate DH31R is activated byCarcinus and insect DH31s in a dose-dependent manner (EC50 15-30 nM). Whole mount immunohistochemical and in situ hybridization localization revealed extensive DH31 expressing neurons throughout the central nervous system, most notably in the abdominal ganglion where large, unpaired cells give rise to medial nerves, which terminate in extensive DH31 immunopositive dendritic fields intimately associated with oesophageal musculature. This system constitutes a large and hitherto undescribed neurohemal area adjacent to key muscle groups associated with the gastric system. DH31 expressing neurons were also seen in the cardiac, commissural, oesophageal, and stomatogastric ganglia and intense labeling was seen in dendrites innervating fore- and hindgut musculature but not with limb muscles. These labeling patterns, together with measurement of DH31R mRNA in the heart and hindgut, prompted us test the effects of DH31 on semi-isolated heart preparations. Cardiac superfusion with peptide evoked increased heart rates (10-100 nM). The neuroanatomical distribution of DH31 and its receptor transcripts, particularly that associated with gastric and cardiac musculature, coupled with the cardio- acceleratory effects of the peptide implicate this peptide in key myoactive roles, likely related to rhythmic coordination.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.

Cloning, characterization, and developmental expression of a putative farnesoic acid O-methyl transferase in the female edible crab Cancer pagurus.

Farnesoic acid methyl transferase (FAMTase) catalyzes methylation of farnesoic acid to yield the crustacean juvenoid, methyl farnesoate (MF). A full-length cDNA encoding a 275 amino acid putative FAMTase has been isolated from the mandibular organ of the female edible crab (Cancer pagurus) by reverse transcriptase-polymerase chain reaction in conjunction with cDNA library screening. A high degree of sequence identity was found between this and other putative crustacean FAMTases. Conceptual translation and protein sequence analysis suggested that phosphorylation could occur at multiple sites in the FAMTase. This finding is consistent with the recent observation that endogenous FAMTase activity in mandibular organ extracts can be regulated by phosphorylation in vitro. We demonstrated that the recombinant FAMTase could be expressed as a LacZ-fusion protein in Escherichia coli and have undertaken its partial purification from inclusion bodies. In an established assay system, the recombinant FAMTase lacked activity. Northern blotting demonstrated widespread expression of an approximately 1250-nucleotide FAMTase transcript in female C. pagurus tissues. Levels of FAMTase transcripts in mandibular organs of female C. pagurus were found to fluctuate during vitellogenesis and embryonic development. Throughout the spring of 2002, an HPLC-based method was used to measure hemolymph MF titers in more than 70 female specimens of C. pagurus, which segregated into "high MF" and "low MF" groups. The high MF titers, which occurred before or during early vitellogenesis, coincided with, or were preceded by, elevated levels of putative FAMTase mRNA in the mandibular organs.

Dissociation of circadian and circatidal timekeeping in the marine crustacean Eurydice pulchra.

BACKGROUND: Tidal (12.4 hr) cycles of behavior and physiology adapt intertidal organisms to temporally complex coastal environments, yet their underlying mechanism is unknown. However, the very existence of an independent "circatidal" clock has been disputed, and it has been argued that tidal rhythms arise as a submultiple of a circadian clock, operating in dual oscillators whose outputs are held in antiphase i.e., ~12.4 hr apart. RESULTS: We demonstrate that the intertidal crustacean Eurydice pulchra (Leach) exhibits robust tidal cycles of swimming in parallel to circadian (24 hr) rhythms in behavioral, physiological and molecular phenotypes. Importantly, ~12.4 hr cycles of swimming are sustained in constant conditions, they can be entrained by suitable stimuli, and they are temperature compensated, thereby meeting the three criteria that define a biological clock. Unexpectedly, tidal rhythms (like circadian rhythms) are sensitive to pharmacological inhibition of Casein kinase 1, suggesting the possibility of shared clock substrates. However, cloning the canonical circadian genes of E. pulchra to provide molecular markers of circadian timing and also reagents to disrupt it by RNAi revealed that environmental and molecular manipulations that confound circadian timing do not affect tidal timing. Thus, competent circadian timing is neither an inevitable nor necessary element of tidal timekeeping. CONCLUSIONS: We demonstrate that tidal rhythms are driven by a dedicated circatidal pacemaker that is distinct from the circadian system of E. pulchra, thereby resolving a long-standing debate regarding the nature of the circatidal mechanism.

A novel form of pigment-dispersing hormone in the central nervous system of the intertidal marine isopod, Eurydice pulchra (leach).

Pigment-dispersing factor (PDF) is well known as a circadian clock output factor, which drives daily activity rhythms in many insects. The role of its homologue, pigment-dispersing hormone (PDH), in the regulation of circadian and/or circatidal rhythmicity in crustaceans is, however, poorly understood. The intertidal isopod crustacean, Eurydice pulchra has well-defined circatidal (12.4-hour) activity rhythms. In this study we show that this runs parallel to a circadian (24-hour) cycle of chromatophore dispersion. As a first step in determining the potential role of PDH in these rhythms, we have identified a novel form of PDH expressed in this species. Because conventional homology cloning was unsuccessful, we employed immuno-identification and Edman microsequencing to determine the primary structure of this peptide. From this, cDNA cloning identified the nucleotide encoding sequence and thus facilitated description of PDH neurons by in situ hybridization and immunohistochemistry. We show them to be morphologically similar to those that co-ordinate circadian activity rhythms in insects. In animals expressing both tidal (activity) and circadian (chromatophore) rhythms, however, there was no evidence for a corresponding periodicity in the expression of pdh transcript, as determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in Eurydice heads. It is therefore suggested that any role for PDH in daily/tidal timing in Eurydice is not mediated at the transcriptional level, rather rhythms in neurohemal release may be important in such co-ordination.

Efficient identification of proteins from ovaries and hepatopancreas of the unsequenced edible crab, Cancer pagurus, by mass spectrometry and homology-based, cross-species searching.

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.

Identification and developmental expression of mRNAs encoding putative insect cuticle hardening hormone, bursicon in the green shore crab Carcinus maenas.

Bursicon is the ultimate hormone in insect ecdysis, which is involved in cuticle hardening. Here we show that mRNAs encoding the heterodimeric cystine knot protein bursicon (Burs alpha, beta), are present in crustaceans, suggesting ubiquity of this hormone in arthropods. We firstly report the cloning, sequencing of mRNAs encoding subunits from the water flea, Daphnia arenata and the CNS of the crab, Carcinus maenas, in comparison with insect bursicon subunits. Expression patterns of alpha and beta burs mRNAs were examined by in-situ hybridisation (ISH) and quantitative RT-PCR. In the thoracic ganglion, burs alpha and beta mRNAs were completely colocalised in neurones expressing crustacean cardioactive peptide (CCAP). However, in the brain and eyestalk, bursicon transcripts were never observed, despite a complex expression pattern of CCAP interneurones. Patterns of expression of burs alpha and beta mRNAs were constitutive during the moult cycle of adult crabs, in stark contrast to the situation in insects. Whilst copy numbers of burs beta transcripts closely matched those of CCAP, those of burs alpha mRNA were around 3-fold higher than burs beta. This pattern was apparent during embryogenesis, where bursicon transcripts were first observed at around 50% development-the same time as first expression of CCAP mRNA. Transcript ratios (burs alpha: beta) increased during development. Our studies have shown, for the first time, that bursicon mRNAs are expressed in identified neurones in the nervous system of crustaceans. These findings will now promote further investigation into the functions of bursicon during the moult cycle and development of crustaceans.

Members of the crustacean hyperglycemic hormone (CHH) peptide family are differentially distributed both between and within the neuroendocrine organs of Cancer crabs: implications for differential release and pleiotropic function.

The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH, moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancer suggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles).

Mosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti, together with 8-bromo-cyclic AMP. The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent. Anoga-DH4) and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44 initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31. Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+, whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport. We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.

Expression and release patterns of neuropeptides during embryonic development and hatching of the green shore crab, Carcinus maenas.

Crustacean ecdysis is controlled by at least three neuropeptides: moult-inhibiting hormone (MIH), which represses ecdysteroid synthesis; crustacean hyperglycaemic hormone (CHH), which not only influences ecdysteroid synthesis but also water uptake during moulting; and crustacean cardioactive peptide (CCAP), which is involved in stereotyped ecdysis behaviour. During embryonic development, moulting takes place in the egg, but there is little information regarding developmental expression of these neuropeptides during this period or during hatching--an event that is analogous to eclosion in insects. To address this problem, we determined expression profiles of MIH and CHH mRNA by quantitative RT-PCR, together with developmental peptide expression studies [confocal immunocytochemistry (ICC) and radioimmunoassay (RIA)]. Likely homologous events relating to neuropeptide surges of both CHH and CCAP were seen during larval hatching, when compared to the adult moult, and cell-specific copy concentration of both MIH and CHH mRNAs was identical to that of the adult during late embryonic development. We measured parallel mRNA and peptide expression of two neuropeptides (red pigment-concentrating hormone RPCH) and pigment-dispersing hormone (PDH) during development, as these have roles as neuromodulators and as classical neurohormonal roles. For MIH and CHH, gene expression was in accordance with peptide expression, but novel sites of CHH expression were found (abdominal peripheral neurones), the expression and release patterns of which may be related to larval eclosion and water uptake necessary for eggshell rupture and hatching. For RPCH and PDH, gene transcription and peptide expression were not in accordance. A significant contribution of maternally derived (non-translated) PDH mRNA to the embryo was seen, and for RPCH, high-level mRNA and peptide expression during late embryogenesis is related to a long ignored, but potentially important release site--the enigmatic post-commissural organs--which are the most prominent structures expressing RPCH during late embryogenesis.

Moult cycle-related changes in biological activity of moult-inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) in the crab, Carcinus maenas. From target to transcript.

The currently accepted model of moult control in crustaceans relies entirely on the hypothesis that moult-inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) repress ecdysteroid synthesis of the target tissue (Y-organ) only during intermoult, and that changes in synthesis and/or release of these neurohormones are central to moult control. To further refine this model, we investigated the biological activities of these neuropeptides in the crab Carcinus maenas, at the target tissue, receptor and cellular level by bioassay (inhibition of ecdysteroid synthesis), radioligand (receptor) binding assays, and second messenger (cGMP) assays, at defined stages of the moult cycle. To investigate possible moult cycle-related changes in neuropeptide biosynthesis, steady-state transcript levels of both neuropeptide mRNAs were measured by quantitative RT-PCR, and stored neuropeptide levels in the sinus gland were quantified during intermoult and premoult. The results show that the most important level of moult control lies within the signalling machinery of the target tissue, that expression and biosynthesis of both neuropeptides is constant during the moult cycle, and are not central to the currently accepted model of moult control.

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.