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A box was designed to keep the acorn worm Saccoglossus mereschkowskii in laboratory conditions for 60 days and to monitor its behavior and feeding. Locomotion and construction of burrows in the sediment were found to be due to peristaltic movements of the proboscis, which periodically changes its shape from cylindrical to mushroom-like, and vice versa. Worms built U-shaped burrows connected with burrows of neighbor worms by flank anastomoses, thus producing a branched system of passages in a sediment layer up to 8 cm deep. The system is of importance for aeration of the upper sediment layer. When a worm is feeding, the proboscis sticks out from the anterior opening of the burrow and stretches along the surface of the sediment. Organic particles adhere to mucus secreted by the epidermal epithelium of the proboscis and are transported by ciliary beating to a furrow between the collar and proboscis, where the mouth is located., (© 2024. Pleiades Publishing, Ltd.)

Hemichordate relationships remain contentious due to conflicting molecular results [1-7] and the high degree of morphological disparity between the two hemichordate classes, Enteropneusta and Pterobranchia [8-11]. Additionally, hemichordates have a poor fossil record outside of the Cambrian, with the exception of the collagenous tubes of the pterobranchs (which include graptolites). By the middle Cambrian, tube-dwelling colonial pterobranchs [12, 13] and tube-dwelling enteropneusts coexisted [14, 15], supporting the origin of the hemichordate body plan earlier in the Cambrian without clarifying the morphology of their last common ancestor. Here, we describe a new hemichordate, Gyaltsenglossus senis, based on 33 specimens from the 506-million-year-old Burgess Shale (Odaray Mountain, British Columbia). G. senis has a unique combination of soft anatomical characters found in both extant classes of hemichordates, namely a trimeric-vermiform body plan with an elongate proboscis and six feeding arms with tentacles. The trunk possesses a long through-gut and terminates with a bulbous structure potentially used for locomotion and/or as a temporary anchor. There is no evidence of a secreted tube. Our phylogenetic analyses retrieve this new taxon as a stem-group hemichordate, supporting the hypothesis that a vermiform body plan preceded both tube building and colonial ecologies. This new taxon suggests that a bimodal feeding ecology using tentacles to filter feed and a proboscis to deposit feed may be plesiomorphic in hemichordates. Finally, the presence of a muscular, post-anal attachment structure in all known Cambrian hemichordates supports this feature as an additional hemichordate plesiomorphy critical for understanding early hemichordate evolution., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thyroid hormones (THs) are the only iodine-containing hormones that play fundamental roles in chordates and non-chordates. The chemical nature, mode of action and the synthesis of THs are well established in mammals and other vertebrates. Although thyroid-like hormones have been detected in protostomes and non-chordate deuterostomes, TH signaling is poorly understood as compared to vertebrates, particularly in protostomes. Therefore, the central objective of this article is to review TH system components and TH-induced effects in non-vertebrate chordates, non-chordate deuterostomes and protostomes based on available genomes and functional information. To accomplish this task, we integrate here the available knowledge on the THs signaling across non-vertebrate chordates, non-chordate deuterostomes and protostomes by considering studies encompassing TH system components and physiological actions of THs. We also address the possible interactions of thyroid disrupting chemicals and their effects in protostomes and non-chordate deuterostomes. Finally, the perspectives on current and future challenges are discussed., (Copyright © 2019. Published by Elsevier Inc.)

The trunk is a key feature of the bilaterian body plan. Despite spectacular morphological diversity in bilaterian trunk anatomies, most insights into trunk development are from segmented taxa, namely arthropods and chordates. Mechanisms of posterior axis elongation (PAE) and segmentation are tightly coupled in arthropods and vertebrates, making it challenging to differentiate between the underlying developmental mechanisms specific to each process. Investigating trunk elongation in unsegmented animals facilitates examination of mechanisms specific to PAE and provides a different perspective for testing hypotheses of bilaterian trunk evolution. Here we investigate the developmental roles of canonical Wnt and Notch signaling in the hemichordate Saccoglossus kowalevskii and reveal that both pathways play key roles in PAE immediately following the completion of gastrulation. Furthermore, our functional analysis of the role of Brachyury is supportive of a Wnt-Brachyury feedback loop during PAE in S. kowalevskii , establishing this key regulatory interaction as an ancestral feature of deuterostomes. Together, our results provide valuable data for testing hypotheses of bilaterian trunk evolution., Competing Interests: The authors declare no conflict of interest.

Deuterostomes are a morphologically disparate clade, encompassing the chordates (including vertebrates), the hemichordates (the vermiform enteropneusts and the colonial tube-dwelling pterobranchs) and the echinoderms (including starfish). Although deuterostomes are considered monophyletic, the inter-relationships between the three clades remain highly contentious. Here we report, Yanjiahella biscarpa, a bilaterally symmetrical, solitary metazoan from the early Cambrian (Fortunian) of China with a characteristic echinoderm-like plated theca, a muscular stalk reminiscent of the hemichordates and a pair of feeding appendages. Our phylogenetic analysis indicates that Y. biscarpa is a stem-echinoderm and not only is this species the oldest and most basal echinoderm, but it also predates all known hemichordates, and is among the earliest deuterostomes. This taxon confirms that echinoderms acquired plating before pentaradial symmetry and that their history is rooted in bilateral forms. Yanjiahella biscarpa shares morphological similarities with both enteropneusts and echinoderms, indicating that the enteropneust body plan is ancestral within hemichordates.

Three benthic megafaunal species (i.e. sablefish Anoplopoma fimbria; pacific hagfish Eptatretus stoutii and a group of juvenile crabs) were tested for diel behavioral patterns at the methane hydrates site of Barkley Canyon (890 m depth), off Vancouver Island (BC, Canada). Fluctuations of animal counts in linear video-transects conducted with the Internet Operated Deep-Sea Crawler "Wally" in June, July and December of 2013, were used as proxy of population activity rhythms. Count time series and environmental parameters were analyzed under the hypothesis that the environmental conditioning of activity rhythms depends on the life habits of particular species (i.e. movement type and trophic level). Non-linear least squares modeling of biological time series revealed significant diel periods for sablefish in summer and for hagfish and crabs in December. Combined cross-correlation and redundancy (RDA) analyses showed strong relationships among environmental fluctuations and detected megafauna. In particular, sablefish presence during summer months was related to flow magnitude, while the activity of pacific hagfish and juvenile crabs in December correlated with change in chemical parameters (i.e. chlorophyll and oxygen concentrations, respectively). Waveform analyses of animal counts and environmental variables confirmed the phase delay during the 24 h cycle. The timing of detection of sablefish occurred under low flow velocities, a possible behavioral adaptation to the general hypoxic conditions. The proposed effect of chlorophyll concentrations on hagfish counts highlights the potential role of phytodetritus as an alternative food source for this opportunistic feeder. The juvenile crabs seemed to display a cryptic behavior, possibly to avoid predation, though this was suppressed when oxygen levels were at a minimum. Our results highlight the potential advantages such mobile observation platforms offer in multiparametric deep-sea monitoring in terms of both spatial and temporal resolution and add to the vastly understudied field of diel rhythms of deep-sea megafauna., Competing Interests: Ocean Networks Canada and Neptune Canada (www.oceannetworks.ca/) partially funded the development and deployment of the crawler. The authors declare that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Reports on the abundances and on metal concentrations in intertidal estuarine invertebrates from the Eastern Tropical Pacific are rare. Thus, the objectives of this report are to make accessible data on the abundances (1984-1987, 49 dates; 2013, 12 dates) of sipunculans, brachiopods and hemichordates from a sand-mud flat; and on trace metals (1996, 2000) and abundances (2015, 3 dates) of sipunculans and brachiopods at a sand flat in the Gulf of Nicoya estuary (10o N-85o W). Cores (17.7 cm2) were collected at the sand-mud flat, and quadrats (0.2 m2) at the sand flat. The flats contrasted in their sand (65 % vs 90 %) and silt+clay (31.5 % vs 5.6 %) contents. At the sand-mud flat (1984-87: 1.83 m2) the sipunculans were represented by 13 individuals, the brachiopods by 129 and the acorn worms by 185, with estimated maximum densities of: 5.7, 29, and 40 ind./m2, respectively. Trace metal (Fe, Mn, Ni, Cr, Cd, Zn, and Pb) analysis (Atomic Absorption Spectrometry) were conducted in specimens of Sipunculus nudus (Sipuncula) and Glottidia audebarti (Brachiopoda). Maximum mean concentrations in S. nudus were: For non-depurated worms, Fe (16.0 mg/g dw) > Mn (165 μg/g dw) > Zn (81 μg/g dw) > Cu (26 μg/g dw) > Cr (11 μg/g dw) > Ni (10.4 μg/g dw) > Pb (9.3 μg/g dw) > Cd (1.2 μg/g dw). For 72 hour depurated worms: Fe (5.0 mg/g dw) > Mn (61 μg/g dw) > Zn (39 μg/g dw) > Cu (24 μg/g dw) > Ni (8.4 μg/g dw) > Pb (2.7 μg/g dw) > Cd (0.62 μg/g dw). For G. audebarti: Fe (1.6 mg/g dw-soft parts) > Zn (123.5 μg/g dw-soft parts) > Cu (31.4 μg/g dw-pedicles) > Pb (21.0 μg/g dw-shells) > Cd (5.2 μg/g dw-soft parts) > Cr (4.7 μg/g dw-shells). For sediments; Fe (46 mg/g dw) > Mn (41.3 μg/g dw) > Zn (63 μg/g dw) > Cu (36.2 μg/g dw) > Cr (31.5 μg/g dw) > Pb (21.1 μg/g dw) > Ni (16.1 μg/g dw) > Cd (1.1 μg/g dw). These concentrations were expected for a nonindustrialized estuary. At the sand flat (Area sampled: 10.6 m2 ) 76 individuals of G. audebarti, 112 of G. albida, and 366 of S. nudus were collected in 2015, with estimated maximum densities of: 7.1, 10.5, and 31 ind./m2, respectively. Densities of G. audebarti and G. albida were relatively low, while those of S. nudus were relatively high when compared with other reports. The shell lenght of G. audebarti ranged from 9.0 mm to 38.0 mm and from 6.0 mm to 29.0 mm for G. albida. These ranges were within those found for these lingulides elsewhere. The mean length of S. nudus was 41 mm and the maximum weight was 1.6 g, which are small. No brachiopods were found at the sand-mud flat in 2013, nor enteropneusts at the sand flat in 2015. G. audebarti had a relatively stable presence, while G. albida almost vanished from the samples at the end of 2015. The spatial distributions of the three invertebrates were found aggregated at both intertidal flats. Strong ENSO warming events during 1983 and 2015, and red tides in 1985 may have influenced the abundances.

The indirect-developing enteropneust acorn worm Ptychodera flava has been used as a hemichordate model system for studying the developmental evolution of deuterostome body plans and the origins of chordate characteristics. However, research progress has been hindered by the limited accessibility of its embryonic materials and metamorphosing larvae. In this study, we identified an abundant population of P. flava in Penghu, Taiwan, and examined the feasibility of using this animal for developmental studies. Through histological examination, we established that the reproductive season of this population is between September and December, with a peak breeding period in October and November. In addition, we have developed new procedures that can induce P. flava spawning at any time of the day during the breeding season, with a higher successful rate than that achieved using a previously published method. Moreover, the culturing system we developed enables rearing of P. flava larvae through various planktonic stages and eventual metamorphosis into benthic juveniles, all under laboratory conditions. We anticipate that the animal resources and new technical procedures reported here will further facilitate the use of P. flava as a model organism for evolutionary and developmental biology research., (© 2015 Wiley Periodicals, Inc.)

Hedgehog is a toolkit gene conserved in metazoans. However, its function differs among taxa, and it shows versatile expression patterns in morphogenesis. We analyzed the expression pattern of hedgehog in the indirect development of the hemichordate, Ptychodera flava, during development and regeneration. Pf-Hh showed distinct enteropneust-specific expression at the anterior tip of the larvae, as well as deuterostome-conserved expression in the pharyngeal endoderm. In contrast, the gene is expressed only in the pharyngeal region during anterior regeneration, but not in the anterior tip of the proboscis. These data suggest that anterior regeneration is driven not only by conserved developmental mechanisms, but also by some regeneration-specific mechanism(s).

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.

A single specimen of a previously undescribed acorn worm in the family Torquaratoridae was trawled from a bottom depth of about 350 m in the Kara Sea (Russian Arctic). The new species is the shallowest of the exclusively deep-sea torquaratorids found to date, possibly an example of high-latitude emergence. On the basis of ribosomal DNA sequences and morphology, the worm is described here as the holotype of Coleodesmium karaensis n. gen., n. sp. It is most similar in overall body shape to the previously described enteropneust genus Allapasus, but is uniquely characterized by a tubular component of the proboscis skeleton ensheathing the collar nerve cord. Additionally, within the proboscis, the sparseness of the musculature of C. karaensis clearly distinguishes it from the much more muscular members of Allapasus. The holotype is a female bearing about a dozen embryos on the surface of her pharyngeal region, each recessed within a shallow depression in the dorsal epidermis. The embryos, ranging from late gastrula to an early stage of coelom formation, are a little more than 1 mm in diameter and surrounded by a thin membrane. Each embryo comprises an external ectoderm of monociliated cells (not arranged in obvious ciliated bands) and an internal endo-mesoderm; the blastopore is closed. In the most advanced embryos, the anterior coelom is starting to constrict off from the archenteron. Coleodesmium karaensis is the first enteropneust (and indeed the first hemichordate) found brooding embryos on the surface of the mother's body.

A subset of mammalian retinal ganglion cells expresses an opsin photopigment (melanopsin, Opn4) and is intrinsically photosensitive. The human retina contains melanopsin, but the literature lacks a direct investigation of its spectral sensitivity or G-protein selectivity. Here, we address this deficit by studying physiological responses driven by human melanopsin under heterologous expression in HEK293 cells. Luminescent reporters for common second messenger systems revealed that light induces a high amplitude increase in intracellular calcium and a modest reduction in cAMP in cells expressing human melanopsin, implying that this pigment is able to drive responses via both Gq and Gi/o class G-proteins. Melanopsins from mouse and amphioxus had a similar profile of G-protein coupling in HEK293 cells, but chicken Opn4m and Opn4x pigments exhibited some Gs activity in addition to a strong Gq/11 response. An action spectrum for the calcium response in cells expressing human melanopsin had the predicted form for an opsin : vitamin A1 pigment and peaked at 479 nm. The G-protein selectivity and spectral sensitivity of human melanopsin is similar to that previously described for rodents, supporting the utility of such laboratory animals for developing methods of manipulating this system using light or pharmacological agents.

Acorn worms, or enteropneusts, are vermiform hemichordates that occupy an important position in deuterostome phylogeny. Allied to pterobranch hemichordates, small colonial tube dwellers, modern enteropneusts were thought to be tubeless. However, understanding of hemichordate diversity is poor, as evidenced by absence of reports from some oceanic regions and recent descriptions of large epibenthic deep-water enteropneusts, Torquaratoridae. Here we show, based on expeditions to Antarctica, that some acorn worms produce conspicuous tubes that persist for days. Interestingly, recent fossil descriptions show a Middle Cambrian acorn worm lived in tubes, leading to speculation that these fossils may have been pterobranch forbearers. Our discovery provides the alternative interpretation that these fossils are similar to modern-day torquaratorids and that some behaviours have been conserved for over 500 million years. Moreover, the frequency of Antarctic enteropneusts observed attests to our limited knowledge of Antarctic marine ecosystems, and strengthens hypotheses relating more northern deep-sea fauna to Antarctic shelf fauna.

The origin of the body plan of our own phylum, Chordata, is one of the most fascinating questions in evolutionary biology. Yet, after more than a century of debate, the evolutionary origins of the neural tube and notochord remain unclear. Here we examine the development of the collar nerve cord in the hemichordate Balanoglossus simodensis and find shared gene expression patterns between hemichordate and chordate neurulation. Moreover, we show that the dorsal endoderm of the buccal tube and the stomochord expresses Hedgehog RNA, and it seems likely that collar cord cells can receive the signal. Our data suggest that the endoderm functions as an organizer to pattern the overlying collar cord, similar to the relationship between the notochord and neural tube in chordates. We propose that the origin of the core genetic mechanisms for the development of the notochord and the neural tube date back to the last common deuterostome ancestor.

The movement characteristics of the sperm and their flagella obtained from a lancelet and 35 species from almost all orders of fishes were examined using high-speed video microscopy. The aim was to clarify the relationship between the motility parameters of the spermatozoa having different morphologies and how these motility parameters affect the swimming speed of the spermatozoa. The motility parameters representing the flagellar waveform, the wavelength, and the amplitude were neither very different between the spermatozoa of the different species nor related to the swimming speed. In contrast, the beat frequency was remarkably changed in the different spermatozoa and was proportional to the swimming speed. The maximum shear angle of the flagellar wave, which is directly related to the maximum sliding displacement between the doublet microtubules, remained nearly constant while the beat frequency varied widely; therefore, the spermatozoa beat in the constant sliding displacement mode. An analysis of the relationship between swimming speed and flagellar length revealed that short flagella were at a disadvantage in developing swimming speed; however, so were extra-long flagella. The ratio of the swimming speed to the wave velocity calculated from the wavelength and the beat frequency depended on the distance from the glass surface. The swimming speeds calculated using the original resistive-force theory were greater than the measured values. To rationalize the measured values, the ratio between the normal and tangential drag coefficient in the resistive-force theory was corrected; namely, 1.99 at 1 μm and 1.63 at 3 μm from the glass surface.

The morphological study of extinct taxa allows for analysis of a diverse set of macroevolutionary hypotheses, including testing for change in the magnitude of morphological divergence, extinction selectivity on form, and the ecological context of radiations. Late Ordovician graptoloids experienced a phylogenetic bottleneck at the Hirnantian mass extinction (∼445 Ma), when a major clade of graptoloids was driven to extinction while another clade simultaneously radiated. In this study, we developed a dataset of 49 ecologically relevant characters for 183 species with which we tested two main hypotheses: (i) could the biased survival of one graptoloid clade over another have resulted from morphological selectivity alone and (ii) are the temporal patterns of morphological disparity and innovation during the recovery consistent with an interpretation as an adaptive radiation resulting from ecological release? We find that a general model of morphological selectivity has a low probability of producing the observed pattern of taxonomic selectivity. Contrary to predictions from theory on adaptive radiations and ecological speciation, changes in disparity and species richness are uncoupled. We also find that the early recovery is unexpectedly characterized by relatively low morphological disparity and innovation, despite also being an interval of elevated speciation. Because it is necessary to invoke factors other than ecology to explain the graptoloid recovery, more complex models may be needed to explain recovery dynamics after mass extinctions.

An important question in biology is why some animals are able to regenerate, whereas others are not. The basal chordate amphioxus is uniquely positioned to address the evolution of regeneration. We report here the high regeneration potential of the European amphioxus Branchiostoma lanceolatum. Adults regenerate both anterior and posterior structures, including neural tube, notochord, fin, and muscle. Development of a classifier based on tail regeneration profiles predicts the assignment of young and old adults to their own class with >94% accuracy. The process involves loss of differentiated characteristics, formation of an msx-expressing blastema, and neurogenesis. Moreover, regeneration is linked to the activation of satellite-like Pax3/7 progenitor cells, the extent of which declines with size and age. Our results provide a framework for understanding the evolution and diversity of regeneration mechanisms in vertebrates.

Cephalochordate amphioxus is a promising model animal for studying the evolutionary and developmental mechanisms of vertebrates because its unique phylogenetic position, simple body plan and sequenced genome. However, one major drawback for using amphioxus as a model organism is the restricted supply of living embryos since they are available only during spawning season that varies from a couple of days to several months according to species. Therefore we are aiming to develop methods for obtaining viable amphioxus embryos in non-spawning season. In the current study, we found that Branchiostoma belcheri could develop their gonads and spawn consecutively in the laboratory when cultured in a low density at a high temperature (25-28 °C) supplied with sufficient food and proper cleanness. Among the approximate 150 observed animals, which spawned spontaneously between November and December 2011, 10% have spawned twice, 10% three times, and 80% four times, through April 2012. The quality and quantity of the gametes reproduced in the consecutive spawning have no obvious difference with those spawned once naturally. Spawning intervals varied dramatically both among different animals (from 1 to 5 months) and between intervals of a single individual (from 27 to 74 days for one animal). In summary, we developed a method with which, for the first time, consecutive spawnings of amphioxus in captivity can be achieved. This has practical implications for the cultivation of other amphioxus species, and eventually will greatly promote the utilization of amphioxus as a model system.

Sperm are the most diverse cell type known: varying not only among- and within- species, but also among- and within-ejaculates of a single male. Recently, the causes and consequences of variability in sperm phenotypes have received much attention, but the importance of within-ejaculate variability remains largely unknown. Correlative evidence suggests that reduced within-ejaculate variation in sperm phenotype increases a male's fertilization success in competitive conditions; but the transgenerational consequences of within-ejaculate variation in sperm phenotype remain relatively unexplored. Here we examine the relationship between sperm longevity and offspring performance in a marine invertebrate with external fertilization, Styela plicata. Offspring sired by longer-lived sperm had higher performance compared to offspring sired by freshly-extracted sperm of the same ejaculate, both in the laboratory and the field. This indicates that within-ejaculate differences in sperm longevity can influence offspring fitness - a source of variability in offspring phenotypes that has not previously been considered. Links between sperm phenotype and offspring performance may constrain responses to selection on either sperm or offspring traits, with broad ecological and evolutionary implications.

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in "circadian" light detectors of higher vertebrates. Because in amphioxus melanopsin activates a G(q)/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na(+). In addition to Na(+), Ca(2+) is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at V(m) > E(Na); (b) in Na-free solution a small residual inward photocurrent persists at V(m) near resting level, provided that Ca is present; and (c) V(rev) exhibits a modest shift with [Ca](o) manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K(+), as [K](o) only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La(3+) and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates.

Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. Antithrombin (AT) is a member of the serine protease inhibitor superfamily and a major coagulation inhibitor in all vertebrates, but its evolutionary origin remains elusive. In this study we isolated for the first time a cDNA encoding an antithrombin homolog, BjATl, from the protochordate Branchiostoma japonicum. The deduced protein BjATl consisted of 338 amino acids sharing 36.7% to 41.1% identity to known vertebrate ATs. BjATl contains a potential N-linked glycosylation site, two potential heparin binding sites and the reactive center loop with the absolutely conserved sequence Gly-Arg-Ser; all of these are features characteristic of ATs. All three phylogenetic trees constructed using Neighbor-Joining, Maximum-Likelihood and Bayesian-Inference methods also placed BjATl together with ATs. Moreover, BjATl expressed in yeast cells was able to inhibit bovine thrombin activity by forming a SDS-stable BjATl-thrombin complex. It also displays a concentration-dependent inhibition of thrombin that is accelerated by heparin. Furthermore, BjATl was predominantly expressed in the hepatic caecum and hind-gut, agreeing with the expression pattern of AT in mammalian species. All these data clearly demonstrate that BjATl is an ortholog of vertebrate ATs, suggesting that a primitive coagulation system emerged in the protochordate.

Owing to its phylogenetic position at the base of the chordates, the cephalochordate amphioxus is an emerging model system carrying immense significance for understanding the evolution of vertebrate development. One important shortcoming of amphioxus as a model organism has been the unavailability of animal husbandry protocols to maintain amphioxus adults away from the field. Here, we present the first report of successful maintenance and spawning of Branchiostoma lanceolatum adults in a facility run on artificial seawater. B. lanceolatum has been chosen for this study because it is the only amphioxus species that can be induced to spawn. We provide a step-by-step guide for the assembly of such a facility and discuss the day-to-day operations required for successful animal husbandry of B. lanceolatum adults. This work also includes a detailed description of the B. lanceolatum spawning behavior in captivity. Our analysis shows that the induced spawning efficiency is not sex biased, but increases as the natural spawning season progresses. We find that a minor fraction of the animals undergo phases of spontaneous spawning in the tanks and that this behavior is not affected by the treatment used to induce spawning. Moreover, the induced spawning efficiency is not discernibly correlated with spontaneous spawning in the facility. Last, we describe a protocol for long-term cryopreservation of B. lanceolatum sperm. Taken together, this work represents an important step toward further establishing amphioxus as a laboratory animal making it more amenable to experimental research, and hence assists the coming of age of this emerging model., (Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.)

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.

Epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata) standing at the base of Chordata, has been investigated using electron microscope. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions have been characterized. The data concerning details of the organization of the ciliary apparatus and rootlets system are presented. It is justified the presence of complicated supporting construction of cilia which performs a mechanical stabilizing function and possibly also provide synchronization of ciliary movements. The presence of cilia with two centrioles is considered as an adaptation to high functional load on ciliary apparatus. Well developed bundles of myofilaments are found in the cytoplasm of the basal portions of ciliary cells that characterizes these cells as myoepithelial. The features indicating the role of ciliary cells in absorption are described. The capability of these cells to balloon-like secretion is considered. Data on the accumulation of food reserves in the form of lipid droplets and glycogen in the cell cytoplasm are presented. Ciliated cells are characterized by their function as ciliated secretory-absorptive myoepithelial cells. Based on the location of secretory granules both in the apical and basal portions of granular cells, an exocrine-endocrine function of these cells has been suggested. Typical endocrine cells in the intestinal epithelium of S. mereschkowskii are absent. Several types of granules in the nerve fibers cytoplasm are described. Junctions between the nerve fibers and basal portions of ciliary and granular epithelial cells are found. Nerve regulation of contractile and secretory functions of epithelial cells is supposed. The presence of the regulatory nerve-endocrine system that includes receptor cells of open type, secretory endocrine-like cells and nerve elements of nerve layer is supposed in the intestinal epithelium of enteropneusts.

The invertebrate chordate amphioxus (Branchiostoma), which is the most basal living chordate, has become an accepted model for the vertebrate ancestor in studies of development and evolution. Amphioxus resembles vertebrates in regard to morphology, developmental gene expression, and gene function. In addition, the amphioxus genome has representatives of most vertebrate gene families. Although it has not undergone the two rounds of whole genome duplications that occurred early in the vertebrate lineage, the amphioxus genome has retained considerable synteny with vertebrate genomes. Thus, studies of genes and development in amphioxus embryos can reveal the fundamental genetic basis of the vertebrate body plan, giving insights into the developmental mechanisms of such organs as the somites, pharynx, kidney, and the central nervous system. Moreover, amphioxus is very useful for understanding how these characters evolved. This chapter details methods for microinjection of amphioxus eggs with mRNAs or morpholino antisense oligonucleotides to analyze gene networks operating in early development.

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage., (© The Author(s) 2010. Published by Oxford University Press.)

This symposium focused on the evolution of chordate genomes, in particular, those events that occurred before the appearance of jawed vertebrates. The aim was to highlight insights that have come from the genome sequences of jawless chordates (lampreys, tunicates, and amphioxus) not only into evolution of chordate genomes, but also into the evolution of the organism. To this end, we brought together researchers whose recent work on these organisms spans the gap from genomics to the evolution of body forms and functions as exemplified by endocrine systems and embryonic development., (© The Author 2010. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.)

The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone consisting of α and β subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a synteny suggest that amphioxus' thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and reproductive functions in the neuroendocrine control system of chordates., (© The Author(s) 2010. Published by Oxford University Press.)

The regulation of body axis specification in the common ancestor of bilaterians remains controversial. BMP signaling appears to be an ancient program for patterning the secondary, or dorsoventral, body axis, but any such program for the primary, or anteroposterior, body axis is debated. Recent work in invertebrates indicates that posterior Wnt/beta-catenin signaling is such a mechanism and that it evolutionarily predates the cnidarian-bilaterian split. Here, I argue that a Cartesian coordinate system of positional information set up by gradients of perpendicular Wnt and BMP signaling is conserved in bilaterians, orchestrates body axis patterning and contributes to both the relative invariance and diversity of body forms.

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worm's body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.

Las Perlas Archipelago (LPA) is located off the Pacific Coast of Panama and was designated as a marine protected area (MPA) in 2007. This baseline study of the shallow and deeper sedimentary habitats of the islands partly informed the MPA designation. Ninety-two grab stations and twenty trawl stations were sampled. Sediment grab sample results were interpolated to produce a map that showed the area to be dominated by mud (1246 km2, 40%) and sand/shell sediments (780 km2, 25%). A total of 201 taxa were recorded and over 5800 individual specimens were processed, revealing that the sediments hold varying community compositions, with annelids being the dominant group (73%) followed by crustaceans (14%). Relationships were evident between community, feeding guilds, and sediment types, which give an indication of communities that can be expected in similar sediments in other areas of the Tropical Eastern Pacific. A study of this scale and level of detail is rare for this biogeographic region and provides a valuable, comprehensive appreciation of the LPA's benthos.

The population dynamics of Branchiostoma japonicum, formerly known as B. belcheri, were Investigated from September 2003 to August 2005 in the intertidal zone at Takehara (Hiroshima Prefecture), Seto Inland Sea, Japan. The intertidal population appeared from spring to autumn and disappeared during winter. A laboratory experiment showed that exposure to temperatures below 1 degree C for 2 hours resulted in severe mortality. This low temperature corresponds to the minimum temperature in sediments in the study area. This result suggested either that the intertidal population collapses in winter because of low temperature, or that the lancelets escape from the intertidal to the subtidal zone. Throughout the research period, no lancelets smaller than 10 mm in body length were found, indicating that no larvae settled in the intertidal zone. The intertidal population is probably maintained by the influx of individuals from the neighboring subtidal population. The mean annual density of the lancelets was greatest (10.6 individuals/m(2)) at station 1 nearest the low water mark, and lowest (0.3 Individuals/m(2)) at station 3 furthest from the low water mark. In summer, the water content of the sediments was remarkably lower at station 3 (20.2%) than at station 1 (25.8%). Another laboratory experiment showed that higher mortality occurred from exposure to sediments with a water content less than 25% for 2 hours, comparable to the water content at station 3, suggesting that the spatial distribution of the lancelets upward in the intertidal is restricted by sediment dryness.

In amphioxus embryos, the early development of the type 1 sensory neurons was followed by Tlx gene expression and scanning electron microscopy. At the early neurula stage, AmphiTlx transcription is first detectable in a ventral zone of epidermis. In later embryos, expression is also seen in differentiating type 1 receptor cells of the peripheral nervous system, in a few cells in the central nervous system (CNS), and in anterior notochord cells. The type 1 receptors originate from the ventral epidermis by delaminating individually into the subepidermal space, where they can conveniently be visualized because of their strong expression of AmphiTlx. Within the subepidermal space, the apex of each delaminated receptor loses its cilium and microvilli whereas the base of the cell extends lamellate pseudopodia on the surface of the deeper tissues. The pseudopodia become oriented toward the dorsal side of the embryo, the direction of cell migration. After reaching the flanks of the embryo, the receptor cell stops migrating, loses its pseudopodia, and produces an axon that extends to the CNS. As the receptor finishes differentiating, it downregulates Tlx expression and evidently re-inserts its perikaryon into the epidermis. The discussion considers the possible homology of the type 1 receptor neurons of amphioxus with migrating lateral line primordia and hair cells of vertebrates.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

Ptychodera flava is a hemichordate whose anterior structures regenerate reproducibly from posterior trunk pieces when amputated. We characterized the cellular processes of anterior regeneration with respect to programmed cell death and cell proliferation, after wound healing. We found scattered proliferating cells at day 2 of regeneration using a proliferating cell nuclear antigen antibody. On day 4, most proliferating cells were associated with the nerve tract under the epidermis, and on day 6, a small proboscis derived from proliferated cells was regenerated, and a mouth had broken though the epidermis. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) detected elevated levels of apoptosis in the endoderm that began furthest away from the region of wound healing, then moved anteriorly over 8 days. Posterior to anterior apoptosis is likely to remove digestive endoderm for later differentiation of pharyngeal endoderm. We hypothesize that P. flava regeneration is nerve dependent and that remodeling in the gut endoderm plays an important role in regeneration., (Copyright (c) 2008 Wiley-Liss, Inc.)

Since the discovery of the marine worm Xenoturbella bocki in 1915 by Sixten Bock and its first published description by Einar Westblad (Westblad,1949, Arkiv Zoologi 1:3-29), Xenoturbella was generally allied to the turbellarian flatworms, perhaps most closely to acoelomorphs. In 1997, however, analyses of ribosomal DNA (Norén and Jondelius, 1997, Nature 390:31-32) and developing oocytes (Israelsson, 1997, Nature 390:32) [and, subsequently, embryos (Israelsson, 1999, Proc R Soc Lond B 266:835-841)] recovered from Xenoturbella specimens led to the surprising conclusion that it was in fact a highly degenerate bivalve mollusc. Bourlat et al. showed in 2003 that this result was due to contamination from bivalves in its diet (Bourlat et al.,2003, Nature 424:925-928). Our analyses showed Xenoturbella is a deuterostome, related to the Ambulacraria (echinoderms and hemichordates). Subsequent work has shown that Xenoturbellida is a separate lineage from the Ambulacraria and therefore constitutes the fourth deuterostome phylum (Bourlat et al.,2006, Nature 444:85-88). I consider this phylogenetic position in the light of what is known of its genetics, morphology, and ontogeny. I examine what this phylogenetic position for Xenoturbella can tell us about its own evolution and what light this might shine on the common ancestor of the deuterostomes and hence on the origins of the chordates., (Copyright 2008 Wiley-Liss, Inc.)

A new study shows that iodothyronines induce metamorphosis in the cephalochordate amphioxus by binding to a receptor homologous to vertebrate thyroid hormone receptors. Iodothyronine-induced metamorphosis may be an ancestral feature of the chordates.

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

The authors here present the first anatomical, molecular biological, and ethological data on the organization of the circadian system of a lancelet, Branchiostoma lanceolatum, a close invertebrate relative of vertebrates. B. lanceolatum was found to be a nocturnal animal and, since its rhythmic activity persisted under constant darkness, it also appears to possess an endogenous, circadian oscillator. The authors cloned a homolog of the clock gene Period (Per), which plays a central (inhibitory) role in the biochemical machinery of the circadian oscillators of both vertebrates and protostomians. This gene from B. lanceolatum was designated as amphiPer. Both the sequence of its cDNA and that of the predicted protein are more similar to those of the Per paralogs of vertebrates than to those of the single protostomian Per gene. A strong expression of amphiPer was found in a small cell group in the anterior neural tube. The amphiPer mRNA levels fluctuated in a rhythmic manner, being high early in the day and low late at night. The authors' data suggest a homology of the amphiPer expessing cells to the suprachiasmatic nucleus of vertebrates.

Sex steroids are essential hormones for reproduction in vertebrates. The existence of a sex steroidogenic pathway in invertebrates is controversial because cytochrome P450 (CYP) genes have not been detected in the genomes of an echinoderm and a urochordate. However, cloning and gene expressions of sex steroid-metabolizing enzymes have been reported in a cephalochordate. In this study, in vitro conversion of sex steroids from pregnenolone (P5) to progesterone (P4), from 17alpha-hydroxyprogesterone (17alpha-P4) to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha, 20beta-P) and 17alpha, 20alpha-P, and from androstenedione (AD) to estrone (E1), estradiol-17beta (E2), and testosterone (T) were confirmed by an incubation experiment performed using (14)C-labeled precursors and mature ovarian homogenates of the amphioxus Branchiostoma belcheri. In amphioxus, the ability of immature ovaries to synthesize sex steroids was much lower than that of mature ovaries. Post-spawning, the mRNA of CYP11A significantly decreased in females. Transcripts of the CYP19 gene also declined in one-fourth of the females after spawning, although this trend was not supported statistically. The mRNAs of CYP17 and 17beta-HSD did not change before and after spawning. Our results indicate the presence of Delta(4) and another derivative pathway in the amphioxus ovary and suggest that the synthesis of sex steroids, particularly estrogen synthesis, may be low in females after spawning behavior., ((c) 2008 Wiley-Liss, Inc.)

The morphology and development of a new species of the genus Balanoglossus belonging to the family Ptychoderidae are described in detail. This acorn worm was collected from the sandy seashore in the cove near Shimoda Marine Research Center, University of Tsukuba (Shimoda, Shizuoka Prefecture, Japan). This acorn worm is easily distinguished from other balanoglossids by a characteristic hepatic region. There are two kinds of hepatic saccules: large, dark-colored saccules in the anterior region and small, light-colored saccules in the posterior region. Between the two subregions of the hepatic region, there is a small region that has no or tiny saccules. This species does not form distinct burrows or mounds of casts. The breeding season is in winter. The process of embryogenesis from fertilization to metamorphosis was observed. A shift in seawater temperature from about 13 degrees C to about 9 degrees C induced gamete release. Two days after fertilization, embryos hatched and became typical feeding tornaria larvae. Two months after fertilization these larvae metamorphosed into juveniles and began their benthic life.

Serpins (serine protease inhibitors) constitute a class of proteins with an unusually wide spectrum of different functions at extracellular sites and within the nucleocytoplasmic compartment that extends from protease inhibition to hormone transport and regulation of chromatin organization. Recent investigations reveal a growing number of serpins acting in secretory pathway organelles, indicating that they are not simply cargo destined for export, but fulfill distinct roles within the classical organelle-coupled trafficking system. These findings imply that some serpins are part of a quality control system that monitors the export and possibly import routes of eukaryotic cells. The molecular targets of these serpins are often unknown, opening new avenues for future research.

We are successfully maintaining a laboratory colony of the lancelet Branchiostoma belcheri bred in the laboratory. Based on living individuals in this mass culture, morphological characteristics from the seven-day larval to benthic juvenile stages have been studied. Most striking was that later larval development of B. belcheri showed great individual variation even in a rather stable culture environment. Metamorphosis first occurred on 60 days post fertilization (dpf) and was continuously observed throughout the present study up to 100 dpf. Morphological traits such as the number of primary gill slits and body size at the start of metamorphosis are apparently affected by culture condition. Body size measured in the largest individuals showed nearly linear growth at 0.087 mm/day. The variability found in larval development calls for caution when developmental stages and chronological ages are compared between populations. However, the developmental flexibility of this animal also raises the possibility that growth and sexual maturation could be controlled artificially in captivity.