CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae.

Extensive use of fossil fuels is leading to increasing CO(2) concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO(2). As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO(2) 41 Pa e.g. 399 µatm) and CO(2) acidified seawater with pH of 7.7 (pCO(2) 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO(2) treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO(2) effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na(+)/K(+)-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

CO2 induced seawater acidification impacts sea urchin larval development I: elevated metabolic rates decrease scope for growth and induce developmental delay.

Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129 Pa, 1271 µatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.

An endogenous peptide stimulates secretory activity in the elasmobranch rectal gland.

Extraction and partial purification of peptide material from the intestine of the elasmobranch Scyliorhinus canicula yielded a fraction that shows potent stimulatory activity in the rectal gland. The extracted material appears to contain an endogenous peptide (or peptides) that represents the natural hormone responsible for the control of rectal gland secretion in vivo.

The isolation and chemical characterization of a novel vasoactive intestinal peptide-related peptide from a teleost fish, the cod, Gadus morhua.

An octacosapeptide that shows sequence homology to porcine vasoactive intestinal peptide (VIP) has been isolated from a teleost fish, the cod, Gadus morhua. The full primary sequence is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Ala- Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Ala. This peptide contains four, or five substitutions, compared with dogfish and porcine VIP, respectively. The residues in positions 13, 26 and 28 are different in all three species. These substitutions seem to have little effect on bioactivity, since cod VIP was virtually equipotent with porcine VIP in stimulating amylase release from guinea-pig pancreatic acini. During the isolation procedure an N-terminally modified form of VIP (Des-His, or 2-28 cod VIP) was also isolated. The available data suggest the sequence of VIP is well conserved in vertebrates which is consistent with an important biological role.

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors.

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.

Pattern of substance P- and cholecystokinin-like immunoreactivity during regeneration of the neural complex in the ascidian Ciona intestinalis.

The neural ganglion of ascidians exhibits a novel and rapid pattern of regeneration whereby within approximately 28-35 days of total ablation an entirely new neural complex is formed. In normal adults, neuronal cell bodies expressing substance P- (SP-Li), neurokinin A-(NKA-Li), CCK/gastrin- (CCK-Li), and insulin-like immunoreactivity exhibit a clearly defined pattern of localization in the cortical rind of the ganglion with characteristic long processes arising from the perikarya running throughout the neuropile. CCK-Li cell bodies are particularly concentrated close to the points of exit of the main nerve trunks. We have used antisera raised against these peptides to monitor the process of regeneration up to postoperative (pa) day 35. Only SP and CCK antisera produced positive staining in the regenerating tissue. Immunoreactive cell bodies first appear following 14 days pa. At this time CCK-Li neurons are more abundant than SP-Li neurons and in contrast to the pattern found in the normal adult ganglion, immunoreactive cell bodies are located both peripherally and centrally in the core of the ganglion and processes were rarely seen. Later stages exhibited an increasing number of SP-Li neurons and at 35 days pa SP-Li cell bodies clearly predominate. CCK-Li neurons typically become clustered close to the points of emergence of the anterior nerve roots. The early expression of CCK-Li and SP-Li molecules during regeneration is considered in terms of their potential role in development and cell proliferation in the newly forming ganglion.

Tissue distribution of the SALMFamide neuropeptides S1 and S2 in the starfish Asterias rubens using novel monoclonal and polyclonal antibodies. I. Nervous and locomotory systems.

The recent isolation and characterization of the SALMFamide neuropeptides (S1 and S2) from the starfish Asterias rubens has initiated a series of single- and double-labelling immunocytochemical studies to ascertain their tissue distribution and cellular localization. Specific novel monoclonal and polyclonal antisera have been raised against these neuropeptides and used in optical immunocytochemistry (ICC). The results of the present study reveal, for the first time, the widespread neuronal distribution of S2 localized to axons and perikarya of the radial nerve cord and tube foot ectoneural nerve plexus. Double labelling revealed a predominantly separate localization for S1 and S2 immunoreactivity. The potential functional roles of S1 and S2 in the radial nerve cord (RNC) and tube feet of Asterias rubens are discussed.

Localization of S1- and S2-like immunoreactivity in the nervous system of the brittle star Amphipholis squamata (Delle Chiaje 1828).

The recent isolation and characterization of the SALMFanide neuropeptides S1 GFNSALMFamide; and S2 (SGPYSFNSGLTFamide) from the sea stars. Asterias rubens and Asterias forbesi have initiated numerous studies on their morphological localization and distribution within the phylum Echinodermata. It has been shown by immunocytochemistry and radioimmunoassay that these peptides are widely distributed in the nervous system of some asteroids, echinoids and ophiuroids. A physiological approach has also shown that S1 and S2 potentiate the luminescence of the small ophiuroid Amphipholis squamata. In the present study. S1- and S2-like immunoreactivity have been localized in A. squamata by immunocytochemistry on both wholemount preparation and histological sections. The results reveal a widespread neuronal distribution of S1-like immunoreactivity in the circumoral ring, radial nerve cord, and tube feet. S1-like immunoreactivity was found to be associated with axons and cell bodies in both the ectoneural and hyponeural components of the nervous. S2-like immunoreactivity was detected only in the ectoneural plenus of the circumoral ring and radial nerve cord.

The SALMFamides: a new family of neuropeptides isolated from an echinoderm.

We have isolated two novel related neuropeptides from the radial nerve cords of the starfishes Asterias rubens and Asterias forbesi. One is an octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2 and the other is a dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2. The peptides were purified using high performance liquid chromatography (HPLC) and a radioimmunoassay for the molluscan FMRFamide-related neuropeptide, pQDPFLRFamide. Both peptides share minimal sequence identity with members of the family of FMRFamide-like peptides so we have designated them as founder members of a new family, the SALMFamides. We refer to the octapeptide as SALMFamide 1 (S1) and the dodecapeptide as SALMFamide 2 (S2). S1 and S2 are the first neuropeptides identified in species belonging to the phylum Echinodermata.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor-beta (TGF-beta) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF-beta-like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF-beta-like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non-neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF-beta-like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone.

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.

Molecular approach to echinoderm regeneration.

Until very recently echinoderm regeneration research and indeed echinoderm research in general has suffered because of the lack of critical mass. In terms of molecular studies of regeneration, echinoderms in particular have lagged behind other groups in this respect. This is in sharp contrast to the major advances achieved with molecular and genetic techniques in the study of embryonic development in echinoderms. The aim of our studies has been to identify genes involved in the process of regeneration and in particular neural regeneration in different echinoderm species. Our survey included the asteroid Asterias rubens and provided evidence for the expression of Hox gene homologues in regenerating radial nerve cords. Present evidence suggests: 1) ArHox1 expression is maintained in intact radial nerve cord and may be upregulated during regeneration. 2) ArHox1 expression may contribute to the dedifferentiation and/or cell proliferation process during epimorphic regeneration. From the crinoid Antedon bifida, we have been successful in cloning a fragment of a BMP2/4 homologue (AnBMP2/4) and analysing its expression during arm regeneration. Here, we discuss the importance of this family of growth factors in several regulatory spheres, including maintaining the identity of pluripotent blastemal cells or as a classic skeletal morphogenic regulator. There is clearly substantial scope for future echinoderm research in the area of molecular biology and certain aspects are discussed in this review.

Purification and characterisation of VIP from two species of dogfish.

Acid extracts of intestine from the dogfish Scyliorhinus canicula and Squalus acanthius were purified by gel filtration, ion exchange chromatography, and reversed-phase HPLC. In radioimmunoassays, VIP-like material from both species of dogfish cross reacted with N-terminal, but not C-terminal antisera. Like porcine VIP, both Scyliorhinus and Squalus VIP were stimulants of exocrine pancreatic secretion in the turkey. The time course of the responses to dogfish VIP were, however, different from that seen with porcine VIP. The present study has developed methods for the isolation of VIP-like peptides from elasmobranchs and has demonstrated that elasmobranch VIP differs from Porcine VIP in the C-terminal region, and that these differences may affect biological activity.

Biochemical and physiological studies on peptides from the elasmobranch gut.

A peptide fraction has been purified from intestinal extracts of the dogfish Scyliorhinus canicula which is a powerful stimulant of rectal gland secretory activity. This peptide is distinct from elasmobranch vasoactive intestinal polypeptide (VIP). The elasmobranch VIP fraction is not active in the rectal gland assay and mammalian VIP is only an effective agonist in Squalus and not in Scyliorhinus or Raja. Preliminary characterisation of the elasmobranch VIP indicate that it has strong N-terminal similarities with mammalian VIP but has limited C-terminal comparability. It is suggested that the rectal gland stimulating peptide, rectin, rather than VIP is responsible for the control of fluid and electrolyte secretion of elasmobranchs.

Isolation of the neuropeptide SALMFamide-1 from starfish using a new antiserum.

We have raised antisera in rabbits to a conjugate of thyroglobulin and Lys-Tyr-Ser-Ala-Leu-Met-Phe-NH2 (KYSALMFamide), a synthetic analog of the starfish neuropeptide S1 (Gly-Phe-Asn-Ser-Ala-Leu-Met- Phe-NH2). The sensitivity and specificity of two antisera (BL and SL) for S1 were established by testing the ability of S1 and structurally related peptides (SALMFamide-2 and various FMRFamide-related peptides) to displace iodinated KYSALMFamide from the serum antibodies in an RIA. Both antisera are sensitive to femtomolar amounts of S1. BL is highly specific for S1 but SL is not, since it is also able to detect femtomolar amounts of the FMRFamide-related peptides. We have used the BL antiserum in the RIA to monitor the purification of S1 immunoreactivity from radial nerve cord extracts of both Asterias rubens and Pycnopodia helianthoides. The partial amino acid sequence GFNSALM was obtained from automated Edman degradation sequencing of pure immunoreactive peaks from both species.

Cholecystokinin (CCK)/gastrin-like immunoreactive neurones in the cerebral ganglion of the protochordate ascidians Styela clava and Ascidiella aspersa.

Antisera raised against the COOH-terminal sequence of mammalian CCK/gastrin were used to ascertain the distribution of CCK/gastrin-like immunoreactive cells in the cerebral ganglion of two ascidian protochordates. Styela clava and Ascidiella aspersa. the cell bodies were found to have a specific regional distribution in Ascidiella, but not in Styela. In addition to central immunoreactive nerve fibres, a number of peripherally located immunoreactive fibres was found. These observations support the idea that some centrally originating protochordate neuronal peptides may have a peripheral role and give weight to the hypothesis that many vertebrate brain-gut peptides had their origin in the neuronal elements of more primitive species.

Isolation and partial sequence of elasmobranch VIP.

We have used a combination of gel filtration, ion exchange chromatography and reversed-phase HPLC to isolate and characterize a VIP-related peptide from the gut of the elasmobranch Scyliorhinus canicula. The N-terminal decapeptide of the Scyliorhinus material was identical with that of porcine VIP. However, Scyliorhinus VIP did not cross react with antisera specific for the C-terminus of porcine VIP. Like porcine VIP, Scyliorhinus VIP was a potent stimulant of exocrine pancreatic secretion in the turkey, but the response to Scyliorhinus VIP had a shorter duration. VIP from a second elasmobranch, Squalus acanthius was partially purified, and had biological and immunochemical properties similar to those of Scyliorhinus VIP. The results indicate that elasmobranch VIP is identical to porcine VIP at its N-terminus, but differs at the C-terminus. These structural differences may influence the rate of metabolism of the peptide.

Isolation of serotonin from the accessory salivary glands of the marine snail Nucella lapillus.

We report here the presence of a bioactive compound in the secretion of the accessory salivary glands (ASGs) of Nucella lapillus. We have purified the compound using HPLC and identified it as serotonin by mass spectrometry, UV spectroscopy, HPLC and capillary electrophoresis. Serotonin was not found in the secretions of the acinous salivary glands or the hypobranchial gland. The amount of serotonin in the secretion of the ASGs does not show seasonal or regional variation.

Present of a toxin in the salivary glands of the marine snail Cymatium intermedius that targets nicotinic acetylcholine receptors.

Presence of a toxin in the salivary glands of the marine snail Cymatium intermedius that targets nicotinic acetylcholine receptors. Toxicon 36, 25-29, 1998.-We present evidence of a neurotoxin from the salivary glands of Cymatium intermedius that displays acetylcholine-like effects on vertebrate (mouse ileum) and invertebrate (molluscan smooth muscle; molluscan heart; leech body wall) tissues. These effects were completely blocked by (+)-tubocurarine (10-100 muM) but not by atropine (up to 200 muM) suggesting that the toxin targets nicotinic-like acetylcholine receptors. This affirms the proposal that this genus may overcome their prey with a paralytic secretion.

Changes in ubiquitin conjugates and Hsp72 levels during arm regeneration in echinoderms.

All organisms show a common defensive mechanism that results in the expression of conserved heat shock proteins (Hsps). These proteins function in a wide range of stressful conditions. We have monitored their levels in species of regenerating echinoderms with different mechanisms of regeneration and from different geographical locations. The effect of an artificial higher temperature on expression of Hsps was also studied. Two stress proteins (Hsp72 and ubiquitin) that are important in processes such as development and protein degradation were investigated. Using Western blot analysis and immunocytochemistry, we found significant changes in the level (Hsp72) and pattern of conjugation (ubiquitin) that corresponded with the repair phase (early regenerative stages) and with the later growth and regeneration of new tissues. Animals from the intertidal environment showed a distinctly sustained expression pattern of Hsp72 compared with benthic animals which suggests a functionally adaptative and dynamic stress response program.