Development and Testing of Species-Specific Primers for Detecting the Presence of the Northern Pacific Sea Star (Asterias amurensis) from Environmental DNA.

The starfish Asterias amurensis, a well-known predator of molluscan species in intertidal ecosystems, has caused substantial ecological and economic losses in North China such as offshore Qingdao. Effective monitoring and prevention measures are urged to minimize its negative impacts. Compared with traditional biomonitoring methods, environmental DNA technology has emerged as a powerful and cost-efficient tool for inferring species' presence and abundance. In this study, we developed a pair of species-specific primers (i.e., Ast-F and Ast-R) for the A. amurensis mitochondrial COI gene and tested its utility in amplifying and quantifying the DNA fragments from environmental samples under both laboratory and field conditions. The results of controlled water tank experiments demonstrated that the amount of eDNA released by A. amurensis was positively related to its biomass; after the removal of the starfish, the eDNA degraded significantly in 24 h and remained detectable for 8 days. The number of eDNA copies enriched tended to increase with smaller pore size of filter membrane and larger volume of filtered water. For field tests, we confirmed the validation of our approach in six locations in Qingdao by filtering 1000 ml water per sample with a 0.45-µm pore size filtration. All the amplification products generated a single and bright band via gel electrophoresis, and the quantitative PCR results unveiled significant differences in eDNA copies. This study provided an eDNA-based approach for investigating the distribution and biomass of A. amurensis, which may help to formulate early warning and management strategies in coastal Qingdao and other regions.

High-quality chromosome-level genome assembly of the Northern Pacific sea star Asterias amurensis.

Asterias amurensis, a starfish species that is native to countries such as China and Japan, as well as non-native regions like Australia, has raised serious concerns in terms of its impact on ecology and economy. To gain a better understanding of its population genomics and dynamics, we successfully assembled a high-quality chromosome-level genome of A. amurensis using PacBio and Hi-C sequencing technologies. A total of 87 scaffolds assembly with contig N50 length of 10.85 Mb and scaffold N50 length of 23.34 Mb were obtained, with over 98.80% (0.48 Gb) of them anchored to 22 pseudochromosomes. We predicted 16,673 protein-coding genes, 95.19% of which were functionally annotated. Our phylogenetic analysis revealed that A. amurensis and Asterias rubens formed a clade, and their divergence time was estimated ~ 28 million years ago (Mya). The significantly enriched pathways and Gene Ontology terms related to the amplified gene family were mainly associated with immune response and energy metabolism, suggesting that these factors might have contributed to the adaptability of A. amurensis to its environment. This study provides valuable genomic resources for comprehending the genetics, dynamics, and evolution of A. amurensis, especially when population outbreaks or invasions occur.

Neuropeptide expression and action in the reproductive system of the starfish Asterias rubens.

Reproductive processes are regulated by a variety of neuropeptides in vertebrates and invertebrates. In starfish (phylum Echinodermata), relaxin-like gonad-stimulating peptide triggers oocyte maturation and spawning. However, little is known about other neuropeptides as potential regulators of reproduction in starfish. To address this issue, here, we used histology and immunohistochemistry to analyze the reproductive system of the starfish Asterias rubens at four stages of the seasonal reproductive cycle in male and female animals, investigating the expression of eight neuropeptides: the corticotropin-releasing hormone-type neuropeptide ArCRH, the calcitonin-type neuropeptide ArCT, the pedal peptide-type neuropeptides ArPPLN1b and ArPPLN2h, the vasopressin/ocytocin-type neuropeptide asterotocin, the gonadotropin-releasing hormone-type neuropeptide ArGnRH, and the somatostatin/allatostatin-C-type neuropeptides ArSS1 and ArSS2. The expression of five neuropeptides, ArCRH, ArCT, ArPPLN1b, ArPPLN2h, and asterotocin, was detected in the gonoducts and/or gonads. For example, extensive ArPPLN2h expression was revealed in the coelomic epithelial layer of the gonads throughout the seasonal reproductive cycle in both males and females. However, seasonal and/or sexual differences in the patterns of neuropeptide expression were also observed. Informed by these findings, the in vitro pharmacological effects of neuropeptides on gonad preparations from male and female starfish were investigated. This revealed that ArSS1 causes gonadal contraction and that ArPPLN2h causes gonadal relaxation, with both neuropeptides being more effective on ovaries than testes. Collectively, these findings indicate that multiple neuropeptide signaling systems are involved in the regulation of reproductive function in starfish, with some neuropeptides exerting excitatory or inhibitory effects on gonad contractility that may be physiologically relevant when gametes are expelled during spawning.

Chromosome-level genome assembly of the northern Pacific seastar Asterias amurensis.

Asterias amurensis has attracted widespread concern because of its population outbreaks, which has impacted fisheries and aquaculture, as well as disrupting local ecosystems. A high-quality reference genome is necessary to better investigate mechanisms of outbreak and adaptive changes. Combining PacBio HiFi and Hi-C sequencing data, we generated a chromosome-level A. amurensis genome with a size of 491.53 Mb. The contig N50 and scaffold N50 were 8.05 and 23.75 Mb, respectively. The result of BUSCO analysis revealed a completeness score of 98.85%. A total of 16,531 protein-coding genes were predicted in the genome, of which 94.63% were functionally annotated. The high-quality genome assembly resulting from this study will provide a valuable genetic resource for future research on the mechanism of population outbreaks and invasion ecology.

Gonadotropic activity of a second relaxin-type peptide in starfish.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

Cellular uptake of liposome consisting mainly of glucocerebroside from the starfish Asterias amurensis into Caco-2 cells.

Glucocerebroside (GlcCer) is a group of compounds consisting of ß-linked glucose and ceramide with various chain lengths, some of which possess anti-tumor activity and improve skin barrier function for atopic patients when administered orally. The amphiphilic GlcCer molecules are generally easy to aggregate in aqueous solution and result in low absorption in the gut, which can be improved by forming a liposome. With a recognition that a relatively large amount of GlcCer is contained in the starfish and is being discarded, we prepared a liposome consisting mainly of GlcCer (over 95%) with 100 nm in diameter. The adsorption efficiency of the liposome into cultured Caco-2 cells was investigated by live-cell imaging using fluorescently labeled liposomes. We found an immediate internalization of GlcCer-liposome on exposure without significant accumulation on the plasma membrane. The membrane fluidity was transiently affected as evidenced by fluorescence recovery after photobleaching (FRAP) experiments without no significant cellular damage, which indicates a liposome with high content of GlcCer might be useful as the carrier of dietary and/or drug molecules.

Fin systems comparative anatomy in model Batoidea Raja asterias and Torpedo marmorata: Insights and relatioships between musculo-skeletal layout, locomotion and morphology.

The macroscopic and microscopic morphology of the appendicular skeleton was studied in the two species Raja asterias (order Rajiformes) and Torpedo marmorata (Order Torpediniformes), comparing the organization and structural layout of pectoral, pelvic, and tail fin systems. The shape, surface area and portance of the T. marmorata pectoral fin system (hydrodynamic lift) were conditioned by the presence of the two electric organs in the disk central part, which reduced the pectoral fin surface area, suggesting a lower efficiency of the "flapping effectors" than those of R. asterias. Otherwise, radials' rays alignment, morphology and calcification pattern showed in both species the same structural layout characterized in the fin medial zone by stiffly paired columns of calcified tiles in the perpendicular plane to the flat batoid body, then revolving and in the horizontal plane to continue as separate mono-columnar rays in the fin lateral zone with a morphology suggesting fin stiffness variance between medial/lateral zone. Pelvic fins morphology was alike in the two species, however with different calcified tiles patterns of the 1st compound radial and pterygia in respect to the fin-rays articulating perpendicularly to the latter, whose tile rows lay-out was also different from that of the pectoral fins radials. The T. marmorata tail-caudal fin showed a muscular and connective scaffold capable of a significant oscillatory forward thrust. On the contrary, the R. asterias dorsal tail fins were stiffened by a scaffold of radials-like calcified segments. Histomorphology, heat-deproteination technique and morphometry provided new data on the wing-fins structural layout which can be correlated to the mechanics of the Batoid swimming behavior and suggested a cartilage-calcification process combining interstitial cartilage growth (as that of all vertebrates anlagen) and a mineral deposition with accretion of individual centers (the tiles). The resulting layout showed scattered zones of un-mineralized matrix within the calcified mass and a less compact texture of the matrix calcified fibers suggesting a possible way of fluid diffusion throughout the mineralized tissue. These observations could explain the survival of the embedded chondrocytes in absence of a canalicular system as that of the cortical bone.

Localization of relaxin-like gonad-stimulating peptide expression in starfish reveals the gonoducts as a source for its role as a regulator of spawning.

Oocyte maturation and gamete release (spawning) in starfish are triggered by relaxin-like gonad-stimulating peptide (RGP), a neuropeptide that was first isolated from the radial nerve cords of these animals. Hitherto, it has generally been assumed that the radial nerve cords are the source of RGP that triggers spawning physiologically. To investigate other sources of RGP, here we report the first comprehensive anatomical analysis of its expression, using both in situ hybridization and immunohistochemistry to map RGP precursor transcripts and RGP, respectively, in the starfish Asterias rubens. Cells expressing RGP precursor transcripts were revealed in the ectoneural epithelium of the radial nerve cords and circumoral nerve ring, arm tips, tube feet, cardiac stomach, pyloric stomach, and, most notably, gonoducts. Using specific antibodies to A. rubens RGP, immunostaining was revealed in cells and/or fibers in the ectoneural region of the radial nerve cords and circumoral nerve ring, tube feet, terminal tentacle and other arm tip-associated structures, body wall, peristomial membrane, esophagus, cardiac stomach, pyloric stomach, pyloric caeca, and gonoducts. Our discovery that RGP is expressed in the gonoducts of A. rubens proximal to its gonadotropic site of action in the gonads is important because it provides a new perspective on how RGP may act as a gonadotropin in starfish. Thus, we hypothesize that it is the release of RGP from the gonoducts that triggers gamete maturation and spawning in starfish, while RGP produced in other parts of the body may regulate other physiological/behavioral processes.

Extraction, Characterization and Osteogenic Activity of a Type I Collagen from Starfish (Asterias amurensis).

Outbreaks of starfish (Asterias amurensis) pose a major threat to aquaculture and marine ecosystems in Qingdao, China, and no effective methods have been found to control them. A comprehensive study of collagen in starfish could be an alternative to high efficient utilization. Based on this, collagen was firstly extracted from Qingdao A. amurensis. Then, its protein pattern, amino acid composition, secondary structure, microstructure and thermal stability were investigated. The results showed that the A. amurensis collagen (AAC) is a type I collagen composed of α1, α2, and ß chains. Glycine, hydroxyproline, and alanine were the major amino acids. The melting temperature was 57.7 °C. From FTIR, UV spectra and CD chromatography, the AAC had an intact triple helix and secondary structure, and microstructural analysis showed that the AAC had a loose, fibrous porous structure. Next, the osteogenic differentiation effect of AAC on Mouse bone marrow stem cells (BMSCs) was investigated, and the results showed that AAC induced osteogenic differentiation of cells by promoting the proliferation of BMSCs, enhancing alkaline phosphatase (ALP) activity, promoting cell mineralization nodules and upregulating the expression of mRNA of relevant osteogenic genes. These results suggest that AAC might have the potential application to bone health-related functional foods.

Morphological and Physiological Aspects of Mutable Collagenous Tissue at the Autotomy Plane of the Starfish Asterias rubens L. (Echinodermata, Asteroidea): An Echinoderm Paradigm.

The mutable collagenous tissue (MCT) of echinoderms has the capacity to undergo changes in its tensile properties within a timescale of seconds under the control of the nervous system. All echinoderm autotomy (defensive self-detachment) mechanisms depend on the extreme destabilisation of mutable collagenous structures at the plane of separation. This review illustrates the role of MCT in autotomy by bringing together previously published and new information on the basal arm autotomy plane of the starfish Asterias rubens L. It focuses on the MCT components of breakage zones in the dorsolateral and ambulacral regions of the body wall, and details data on their structural organisation and physiology. Information is also provided on the extrinsic stomach retractor apparatus whose involvement in autotomy has not been previously recognised. We show that the arm autotomy plane of A. rubens is a tractable model system for addressing outstanding problems in MCT biology. It is amenable to in vitro pharmacological investigations using isolated preparations and provides an opportunity for the application of comparative proteomic analysis and other "-omics" methods which are aimed at the molecular profiling of different mechanical states and characterising effector cell functions.

Immunohistochemical localisation of vasopressin/oxytocin-type, corazonin-type and luqin-type neuropeptide expression in the starfish Asterias rubens using antibodies to the C-terminal region of precursor proteins.

Neuropeptides derived from larger precursor proteins are secreted as signalling molecules by neurons and regulate diverse physiological and behavioural processes in animals. Recently, we reported the discovery of ArCRZ (HNTFTMGGQNRWKAG-NH2) and ArLQ (EEKTRFPKFMRW-NH2)-novel neuropeptides in the starfish Asterias rubens that are orthologs of arthropod corazonins and molluscan luqins, respectively. However, our efforts to generate antibodies to ArCRZ and ArLQ have been unsuccessful, precluding immunohistochemical analysis of their expression. Here, we investigated an alternative experimental approach for neuropeptide immunohistochemistry by generating antibodies to peptides corresponding to the C-terminal region of the precursor proteins. As proof of principle, we generated antibodies to the C-terminal region of the precursor of the vasopressin/oxytocin-type neuropeptide asterotocin and show that these reveal immunostaining in A. rubens that is very similar to that observed with asterotocin antibodies. Furthermore, antibodies to the C-terminal region of the ArCRZ precursor (ArCRZP) and the ArLQ precursor (ArLQP) produced patterns of immunostaining consistent, respectively, with the distribution of ArCRZP and ArLQP transcripts revealed by mRNA in situ hybridisation. Detailed immunohistochemical analysis revealed widespread expression of ArCRZP and ArLQP in A. rubens, including the central nervous system, digestive system and the body wall and its associated appendages (e.g. tube feet), providing a neuroanatomical framework for investigation and interpretation of the pharmacological actions of ArCRZ and ArLQ in A. rubens. Furthermore, our findings provide a basis for use of antibodies to the C-terminal region of neuropeptide precursor proteins in other species where the production of antibodies to the bioactive neuropeptides is unsuccessful.

Crowding in the middle of marine food webs: A focus on Raja asterias and other mediterranean batoids.

The Mediterranean Sea is among the three biodiversity hotspots of the world where elasmobranchs are severely threatened. Elasmobranchs act as apex or meso-predators within marine food webs and the loss/decline of apex predators determines the mesopredator release, leading in turn to increased predation on smaller prey. However, also several mesopredators (including rays, skates and small sharks) are intensively fished, being of commercial interest, or by-caught, and thus mesopredators increase could not be so evident. We analysed the trophic ecology of an endemic Mediterranean ray, the starry ray Raja asterias, at a seasonal scale from the Adriatic basin, one of the most intensively exploited area of the Mediterranean, by means of stomach contents and stable isotopes analyses. Our results evidenced that starry rays rely on benthic sources including species of local commercial values, such as swimming crabs, small cephalopods, and stomatopods and share the same trophic position with other elasmobranchs (rays, skates, and small sharks) and other mesopredators (e.g., common soles, Norway lobsters and mullets). As all mesopredators are overexploited, as well as their benthic prey are affected by intense trawl-fishing, the whole food webs are disrupted and neither the classical trophic cascade nor the mesopredator release hypothesis could be verified. Conservation measures for these species, such as the release after capture or the application of exclusion grids to the net, should be applied in areas where populations are strongly impacted by trawling.

Asterias forbesi-Inspired SERS Substrates for Wide-Range Detection of Uric Acid.

Uric acid (UA), the final metabolite of purine, is primarily excreted through urine to maintain an appropriate concentration in the bloodstream. However, any malfunction in this process can lead to complications due to either deficiency or excess amount of UA. Hence, the development of a sensor platform with a wide-range detection is crucial. To realize this, we fabricated a surface-enhanced Raman spectroscopy (SERS) substrate inspired by a type of starfish with numerous protrusions, Asterias forbesi. The Asterias forbesi-inspired SERS (AF-SERS) substrate utilized an Au@Ag nanostructure and gold nanoparticles to mimic the leg and protrusion morphology of the starfish. This substrate exhibited excellent Raman performance due to numerous hotspots, demonstrating outstanding stability, reproducibility, and repeatability. In laboratory settings, we successfully detected UA down to a concentration of 1.16 nM (limit of detection) and demonstrated selectivity against various metabolites. In the experiments designed for real-world application, the AF-SERS substrate detected a broad range of UA concentrations, covering deficiencies and excesses, in both serum and urine samples. These results underscore the potential of the developed AF-SERS substrate as a practical detection platform for UA in real-world applications.

Asterias pectinifera-Derived Collagen Peptides Mixed with Halocynthia roretzi Extracts Exhibit Anti-Photoaging Activities during Exposure to UV Irradiation, and Antibacterial Properties.

Asterias pectinifera, a species of starfish and cause of concern in the aquaculture industry, was recently identified as a source of non-toxic and highly water-soluble collagen peptides. In this study, we investigated the antioxidant and anti-photoaging functions of compounds formulated using collagen peptides from extracts of Asterias pectinifera and Halocynthia roretzi (AH). Our results showed that AH compounds have various skin protective functions, including antioxidant effects, determined by measuring the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals, as well as anti-melanogenic effects, determined by measuring tyrosinase inhibition activity. To determine whether ethosome-encapsulated AH compounds (E(AH)) exert ultraviolet (UV)-protective effects, human dermal fibroblasts or keratinocytes were incubated with E(AH) before and after exposure to UVA or UVB. E(AH) treatment led to inhibition of photoaging-induced secretion of matrix metalloproteinase-1 and interleukin-6 and -8, which are associated with inflammatory responses during UV irradiation. Finally, the antibacterial effects of AH and E(AH) were confirmed against both gram-negative and gram-positive bacteria. Our results indicate that E(AH) has the potential for use in the development of cosmetics with a range of skin protective functions.

New Three-Finger Protein from Starfish Asteria rubens Shares Structure and Pharmacology with Human Brain Neuromodulator Lynx2.

Three-finger proteins (TFPs) are small proteins with characteristic three-finger ß-structural fold stabilized by the system of conserved disulfide bonds. These proteins have been found in organisms from different taxonomic groups and perform various important regulatory functions or act as components of snake venoms. Recently, four TFPs (Lystars 1-4) with unknown function were identified in the coelomic fluid proteome of starfish A. rubens. Here we analyzed the genomes of A. rubens and A. planci starfishes and predicted additional five and six proteins containing three-finger domains, respectively. One of them, named Lystar5, is expressed in A. rubens coelomocytes and has sequence homology to the human brain neuromodulator Lynx2. The three-finger structure of Lystar5 close to the structure of Lynx2 was confirmed by NMR. Similar to Lynx2, Lystar5 negatively modulated α4ß2 nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Incubation with Lystar5 decreased the expression of acetylcholine esterase and α4 and α7 nAChR subunits in the hippocampal neurons. In summary, for the first time we reported modulator of the cholinergic system in starfish.

Are myoepithelial cells confined to genital coelomic sinus in the gonads of sea stars?

An ultrastructural study of the gonadal wall in 10 sea star species from the orders Forcipulatida, Paxillosida, Spinulosida, Valvatida and Velatida has shown variations in the presence of myoepithelial cells in the visceral peritoneal epithelium. These cells have only been found in the peritoneal epithelium of the gonads in Aphelasterias japonica (Forcipulatida: Asteriidae), Asterias amurensis (Forcipulatida: Asteriidae), Distolasterias nipon (Forcipulatida: Asteriidae), Diplopteraster multipes (Velatida: Pterasteridae), Luidia quinaria (Paxillosida: Ctenodiscidae), and Pteraster sp. (Velatida: Pterasteridae). Our results may shed light on the evolution of peritoneal epithelium of sea star gonads. It is probable that, initially sea stars had myoepithelial cells in visceral peritoneal epithelium of the gonads. The species from the orders Forcipulatida and Velatida have retained this plesiomorphic state, while many species from the orders Paxillosida, Spinulosida and Valvatida have lost myoepithelial cells from visceral peritoneal epithelium of their gonads.

Potential habitats of an alien species (Asterias rubens Linnaeus, 1758) in the Black Sea: its current and future distribution patterns.

The Atlantic common starfish, Asterias rubens, has arrived firstly at the Marmara Sea in 1996 and to the Black Sea in 2007. In this study, we have exhibited the possible potential distribution of Asterias rubens throughout the Black Sea. For this, we predicted and determined the present and future distributions, and habitat preferences of this starfish in the Black Sea using environmental variables. The ecological niche modeling was used to detect the suitable habitat of A. rubens. In the current model, shallow areas seem to be the suitable habitat for A. rubens. However, this trend may change in the future distribution pattern. For the future projection, two representative concentration pathways (RCPs) that are a greenhouse gas concentration was used: RCP2.6 that is likely to keep global temperature rise below 2 °C by 2100 and RCP8.5 that will happen approximately 5 °C in range of global mean temperature increase in 2100 from pre-industrial baseline. According to RCP2.6 scenarios as well as the RCP8.5 scenario in 2040-2050, the suitable habitats in the Black Sea will probably decrease due to climate change. The most suitable habitats in these scenarios will remain the western and southern coasts of the Black Sea because these areas will be less affected by the change in the climate. In contrast, for the 2090-2100 periods of the RCP8.5, there will likely be a significant unsuitable habitat throughout the Black Sea. Therefore, the suitable habitat for A. rubens will be restricted to the western and southern coasts of the Black Sea.

In Silico Reconstruction of Sperm Chemotaxis.

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca2+ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research.

Ancient role of sulfakinin/cholecystokinin-type signalling in inhibitory regulation of feeding processes revealed in an echinoderm.

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome - the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

A specific and sensitive enzyme-linked immunosorbent assay for measurement of relaxin-like gonad-stimulating peptide in the starfish Asterias rubens.

A relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropic hormone in starfish. In this study, antibodies to Asterias rubens RGP (AruRGP) were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure AruRGP. Biotin-conjugated RGP (biotin-AruRGP) that binds to peroxidase-conjugated streptavidin was synthesized chemically so that it could be specifically detected using 3, 3', 5, 5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate. Similar to AruRGP, biotin-AruRGP bound to AruRGP antibodies. In binding experiments with biotin-AruRGP using wells coated with AruRGP antibodies, a displacement curve was obtained using serial dilutions of AruRGP. Using this ELISA system, AruRGP could be measured in the range 0.01-5.0 pmol per 50 µl test solution. Furthermore, 0.22 ± 0.03 and 0.20 ± 0.04 pmol AruRGP/mg wet weight tissue were detected in the radial nerve cords and circumoral nerve-rings of A. rubens, respectively. Smaller amounts of AruRGP were detected in tube feet, pyloric stomach and cardiac stomach but AruRGP was not detected in pyloric caeca, ovaries and testes. Analysis of the specificity of the AruRGP antibodies revealed that the A- and B-chains of AruRGP, Patiria pectinifera RGP, Aphelasterias japonica RGP, and human relaxin exhibit little or no cross-reactivity in the ELISA. We conclude, therefore, that we have successfully generated an ELISA system that is highly sensitive and specific for detection of AruRGP.