Cambrian Tentaculate Worms and the Origin of the Hemichordate Body Plan.

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.

The evolutionary road to invertebrate thyroid hormone signaling: Perspectives for endocrine disruption processes.

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.

Untangling posterior growth and segmentation by analyzing mechanisms of axis elongation in hemichordates.

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.

A stem group echinoderm from the basal Cambrian of China and the origins of Ambulacraria.

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.

High-Frequency Patterns in the Abundance of Benthic Species near a Cold-Seep - An Internet Operated Vehicle Application.

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.

Brachiopods, sipunculans, enteropneusts and metals from two estuarine tidal flats, Pacific, Costa Rica / Braquiópodos, sipuncúlidos, enteropneustos y metales en dos planicies estuarinas de marea, Pacífico, Costa Rica

Abstract: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. Rev. Biol. Trop. 64 (3): 1311-1331. Epub 2016 September 01.

ResumenSon raros los reportes sobre las abundancias y concentraciones de metales en invertebrados estuarinos de la zona de entre-mareas del Pacífico Este Tropical. Los objetivos de este informe son el hacer accesibles datos sobre las abundancias (1984-1987, 49 fechas; 2013, 12 fechas) de sipuncúlidos, braquiópodos y hemicordados en una planicie arenoso-fangosa y sobre metales traza (1996, 2000) y abundancias (2015, 3 fechas) de sipuncúlidos y braquiópodos en una planicie arenosa en el estuario del Golfo de Nicoya (10o N-85o W). Barrenos (17.7 cm2) fueron recolectados en la planicie arenoso-fangosa y cuadrantes (0.2 m2) en la arenosa. Las planicies contrastaron en sus contenidos de arena (65 % vs 90 %) y de limo + arcilla (31.5 % vs 5.6 %). En la planicie arenoso-fangosa (1984-87: 1.83 m2) los sipuncúlidos estuvieron representados por 13 individuos, los braquiópodos por 129 y los hemicordados enteropneustos por 185, con densidades estimadas de: 5.7, 29, y 40 ind. /m2, respectivamente. Análisis de metales traza (Fe, Mn, Ni, Cr, Cd, Zn, y Pb) por Espectrometría de Absorción Atómica (AAS) fueron hechos en especímenes de Sipunculus nudus (Sipuncula) y Glottidia audebarti (Brachiopoda). Concentraciones máximas promedio en S. nudus fueron: para gusanos no-depurados, 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). Para gusanos depurados por 72 horas: 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). Para G. audebarti: Fe (1.6 mg/g dw-partes suaves) > Zn (123.5 µg/g dw-partes suaves) > Cu (31.4 µg/g dwpedículos) > Pb (21.0 µg/g dw-conchas) > Cd (5.2 µg/g dw-partes suaves) > Cr (4.7 µg/g dw-conchas). Para sedimentos; 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). Estas concentraciones fueron esperables para un estuario no industrializado. En la planicie arenosa (Area muestreada: 10.6 m2) 76 individuos de G. audebarti, 112 de G. albida y 366 de S. nudus fueron recolectados en el 2015, con densidades estimadas de: 7.1, 10.5, y 31 ind. /m2, respectivamente. Densidades de G. audebarti y G. albida fueron relativamente bajas, mientras que las de S. nudus fueron relativamente altas cuando se les comparó con otros reportes. La longitud de la concha de G. audebarti varió entre 9.0 mm y 38.0 mm y entre 6.0 mm a 29.0 mm la de G. albida. Estos ámbitos estuvieron dentro de los encontrados para estos lingúlidos en otros sitios. La longitud promedio de S. nudus fue 41 mm y el peso máximo fue de 1.6 g que son pequeños. En la planicie arenoso-fangosa no se encontró braquiópodos en el 2013, ni enteropneustos en la planicie arenosa en el 2015. G. audebarti tuvo una presencia relativamente estable, mientras que G. albida casi desapareció de las muestras al final del 2015. La distribución espacial de las tres especies fue de tipo agregado en ambas planicies. Fuertes eventos ENSO durante 1983 y 2015, así como mareas rojas en 1985, pueden haber influenciado las abundancias.

Brachiopods, sipunculans, enteropneusts and metals from two estuarine tidal flats, Pacific, Costa Rica.

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.

Reproductive periodicity, spawning induction, and larval metamorphosis of the hemichordate acorn worm Ptychodera flava.

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.

Hedgehog Expression During Development and Regeneration in the Hemichordate, Ptychodera flava.

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).

A cDNA resource for gene expression studies of a hemichordate, Ptychodera flava.

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.

Modern Antarctic acorn worms form tubes.

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.

Hemichordate neurulation and the origin of the neural tube.

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.

An externally brooding acorn worm (Hemichordata, Enteropneusta, Torquaratoridae) from the Russian arctic.

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.

Human melanopsin forms a pigment maximally sensitive to blue light (λmax ≈ 479 nm) supporting activation of G(q/11) and G(i/o) signalling cascades.

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.

Consecutive spawnings of Chinese amphioxus, Branchiostoma belcheri, in captivity.

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.

Fertilization is not a new beginning: the relationship between sperm longevity and offspring performance.

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.

Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies.

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.

A novel serpin with antithrombin-like activity in Branchiostoma japonicum: implications for the presence of a primitive coagulation system.

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.

Graptoloid diversity and disparity became decoupled during the Ordovician mass extinction.

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.