Michael C. Thorndyke

Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

Impact of near-future ocean acidification on echinoderms

As a consequence of increasing atmospheric CO2, the world’s oceans are warming and slowly becoming more acidic (ocean acidification, OA) and profound changes in marine ecosystems are certain. Calcification is one of the primary targets for studies of the impact of CO2-driven climate change in the oceans and one of the key marine groups most likely to be impacted by predicted climate change events are the echinoderms. Echinoderms are a vital component of the marine environment with representatives in virtually every ecosystem, where they are often keystone ecosystem engineers. This paper reviews and analyses what is known about the impact of near-future ocean acidification on echinoderms. A global analysis of the literature reveals that echinoderms are surprisingly robust to OA and that important differences in sensitivity to OA are observed between populations and species. However, this is modulated by parameters such as (1) exposure time with rare longer term experiments revealing negative impacts that are hidden in short or midterm ones; (2) bottlenecks in physiological processes and life-cycle such as stage-specific developmental phenomena that may drive the whole species responses; (3) ecological feedback transforming small scale sub lethal effects into important negative effects on fitness. We hypothesize that populations/species naturally exposed to variable environmental pH conditions may be pre-adapted to future OA highlighting the importance to understand and monitor environmental variations in order to be able to to predict sensitivity to future climate changes. More stress ecology research is needed at the frontier between ecotoxicology and ecology, going beyond standardized tests using model species in order to address multiple water quality factors (e.g. pH, temperature, toxicants) and organism health. However, available data allow us to conclude that near-future OA will have negative impact on echinoderm taxa with likely significant consequences at the ecosystem level.

Near-future levels of ocean acidification reduce fertilization success in a sea urchin

Summary Although it is widely believed that seawater is chemically well-buffered, CO 2 -induced acidification of the worlds oceans threatens the viability of many species [1–3]. Research to date has focused on the responses of adult stages of calcifying taxa to gross pH changes relevant for the years 2200–2400 [3,4]. We investigated the consequences of exposure of gametes and larvae of the sea urchin Heliocidaris erythrogramma to CO 2 -induced acidification by −0.4 pH units (the upper limit of predictions for the year 2100 [5]), and found statistically significant reductions in sperm swimming speed and percent sperm motility. We predicted the effects of these changes using an established model [6], and tested fertilization success experimentally in assays using the same gametes and pH treatments. Observed reductions in fertilization success corresponded closely to model predictions (24% reduction). If general, these findings have important implications for the reproductive and population viability of broadcast spawning marine species in the future acidified ocean.

Formation and evolution of the chordate neurotrophin and Trk receptor genes.

Neurotrophins are structurally related neurotrophic polypeptide factors that regulate neuronal differentiation and are essential for neuronal survival, neurite growth and plasticity. It has until very recently been thought that the neurotrophin system appeared with the vertebrate species, but identification of a cephalochordate neurotrophin receptor (Trk), and more recently neurotrophin sequences in several genomes of deuterostome invertebrates, show that the system already existed at the stem of the deuterostome group. Comparative genomics supports the hypothesis that two whole genome duplications produced many of the vertebrate gene families, among those the neurotrophin and Trk families. It remains to be proven to what extent the whole genome duplications have driven macroevolutionary change, but it appears certain that the formation of the multi-gene copy neurotrophin and Trk receptor families at the stem of vertebrates has provided a foundation from which the various functions and pleiotropic effects produced by each of the four extant neurotrophins have evolved.

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.

FMRFamide-like Immunoreactivity in the Nervous System of the Starfish Asterias rubens

The nervous system of the starfish Asterias rubens was subjected to immunocytochemical investigation using antisera raised against the molluscan neuropeptide FMRFamide. Immunoreactivity was detected in the radial nerve cords and the circumoral nerve ring, as well as in the sub-epithelial nerve plexus of the tube foot system. The hyponeural part of the radial cords contained numerous immunoreactive cell bodies. In the ectoneural tissue, immunoreactive cells were present in the epithelium, with cell bodies especially abundant in the lateral parts of the nerve, close to the site of emergence of the innervation to the tube feet. The sub-epithelial nerve plexus of the tube feet contained immunoreactive fibers that were continuous with an extensive system of ectoneural immunoreactive fibers in the radial nerve cords. Immunoreactive fibers were particularly evident in the regenerating radial nerves of previously sectioned arms.

Afuni, a novel transforming growth factor-beta gene is involved in arm regeneration by the brittle star Amphiura filiformis

The bone morphogenetic proteins (BMPs) are a family of the transforming growth factor-β (TGF-β) superfamily that perform multiple roles during vertebrate and invertebrate development. Here, we report the molecular cloning of a novel BMP from regenerating arms of the ophiuroid Amphiura filiformis. The theoretically translated amino acid sequence of this novel BMP has high similarity to that of the sea urchin BMP univin. This novel BMP has been named afuni. Whole-mount in situ hybridisation implicates afuni in arm regeneration. Expression occurs in distinct proximal and distal regions of late regenerates (3- and 5-week postablation). These sites are at different stages of regeneration, suggesting multiple roles for this gene in adult arm development. Cellular expression of this gene occurs in migratory cells within the radial water canal (RWC) of regenerating and nonregenerating arms. These migrating coelomocytes suggest a key role for the coelomic RWC as a source of the cellular material for use in arm regeneration by A. filiformis.

Evolution of echinoderms may not have required modification of the ancestral deuterostome HOX gene cluster: first report of PG4 and PG5 Hox orthologues in echinoderms

Is the extreme derivation of the echinoderm body plan reflected in a derived echinoderm Hox genotype? Building on previous work, we exploited the sequence conservation of the homeobox to isolate putative orthologues of several Hox genes from two asteroid echinoderms. The 5-peptide motif (LPNTK) diagnostic of PG4 Hox genes was identified immediately downstream of one of the partial homeodomains from Patiriella exigua. This constitutes the first unequivocal report of a PG4 Hox gene orthologue from an echinoderm. Subsequent screenings identified genes of both PG4 and PG4/5 in Asterias rubens. Although in echinoids only a single gene (PG4/5) occupies these two contiguous cluster positions, we conclude that the ancestral echinoderm must have had the complete deuterostome suite of medial Hox genes, including orthologues of both PG4 and PG4/5 (= PG5). The reported absence of PG4 in the HOX cluster of echinoids is therefore a derived state, and the ancestral echinoderm probably had a HOX cluster not dissimilar to that of other deuterostomes. Modification of the ancestral deuterostome Hox genotype may not have been required for evolution of the highly derived echinoderm body plan.

Molecular characterisation of SALMFamide neuropeptides in sea urchins

SUMMARY The SALMFamides are a family of neuropeptides found in species belonging to the phylum Echinodermata. Members of this family have been identified in starfish (class Asteroidea) and in sea cucumbers (class Holothuroidea) but not in other echinoderms. Our aim here was to characterise SALMFamide neuropeptides in sea urchins (class Echinoidea). Radioimmunoassays for the starfish SALMFamides S1 and S2 were used to test for related peptides in whole-body acetone extracts of the sea urchin Echinus esculentus. Fractionation of extracts using high performance liquid chromatography (HPLC) revealed several peaks of SALMFamide-like immunoreactivity, with two S2-like immunoreactive peaks (3 and 4) being the most prominent. However, peak 4 could not be purified to homogeneity and although peak 3 was purified, only a partial sequence (MRYH) could be obtained. An alternative strategy for identification of echinoid SALMFamides was provided by sequencing the genome of the sea urchin Strongylocentrotus purpuratus. Analysis of whole-genome shotgun sequence data using the Basic Local Alignment Search Tool (BLAST) identified a contig (347664) that contains a coding region for seven putative SALMFamide neuropeptides (PPVTTRSKFTFamide, DAYSAFSFamide, GMSAFSFamide, AQPSFAFamide, GLMPSFAFamide, PHGGSAFVFamide and GDLAFAFamide), which we have named SpurS1-SpurS7, respectively. Three of these peptides (SpurS1-3) have the C-terminal sequences TFamide or SFamide, which are identical or similar to the C-terminal region of the starfish SALMFamide S2. This may explain the occurrence of several S2-like immunoreactive peptides in extracts of Echinus esculentus. Detailed analysis of the sequence of contig 347664 indicated that the SALMFamide gene in Strongylocentrotus purpuratus comprises two exons, with the first exon encoding a signal peptide sequence and the second exon encoding SpurS1-SpurS7. Characterisation of this gene is important because it is the first echinoderm neuropeptide precursor sequence to be identified and, more specifically, it provides our first insight into the structure and organisation of a SALMFamide gene in an echinoderm. In particular, it has revealed a hitherto unknown complexity in the diversity of SALMFamide neuropeptides that may occur in an echinoderm species because all previous studies, which relied on peptide purification and sequencing, revealed only two SALMFamide neuropeptides in each species examined. It now remains to be established whether or not the occurrence of more than two SALMFamides in Strongylocentrotus purpuratus is a feature that is peculiar to this species and to echinoids in general or is more widespread across the phylum Echinodermata. Identification of SpurS1-SpurS7 provides the basis for comparative analysis of the physiological actions of these peptides in sea urchins and for exploitation of the sea urchin genome sequence to identify the receptor(s) that mediate effects of SALMFamides in echinoderms.

Localisation of gamma aminobutyric acid (GABA)-like immunoreactivity in the echinoderm Asterias rubens

Gamma amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. We have used a specific antiserum raised against GABA to demonstrate GABA-like immunoreactivity in the radial nerve cord (RNC), tube feet and the digestive system of the asteroid Asterias rubens. In the RNC, immunoreactivity was restricted to ectoneural fibres and cell bodies while in the tube feet fibres were revealed in the basal nerve ring and longitudinal nerve. In the gut, extensive labelling was apparent in the basi-epithelial plexus as well as in mucosal perikarya.