The development and metamorphosis of the indirect developing acorn worm Schizocardium californicum (Enteropneusta: Spengelidae)

Abstract

Background: Enteropneusts are benthic marine invertebrates that belong to the deuterostome phylum Hemichordata. The two main clades of enteropneusts are defined by differences in early life history strategies. In the Spengelidae and Ptychoderidae, development is indirect via a planktotrophic tornaria larva. In contrast, development in the Harrimanidae is direct without an intervening larval life history stage. Most molecular studies in the development and evolution of the enteropneust adult body plan have been carried out in the harrimanid Saccoglossus kowalevskii. In order to compare these two developmental strategies, we have selected the spengelid enteropneust Schizocardium californicum as a suitable indirect developing species for molecular developmental studies. Here we describe the methods for adult collecting, spawning and larval rearing in Schizocardium californicum, and describe embryogenesis, larval development, and metamorphosis, using light microscopy, immunocytochemistry and confocal microscopy.

Results: Adult reproductive individuals can be collected intertidally and almost year-round. Spawning can be triggered by heat shock and large numbers of larvae can be reared through metamorphosis under laboratory conditions. Gastrulation begins at 17 h post-fertilization (hpf) and embryos hatch at 26 hpf as ciliated gastrulae. At 3 days post-fertilization (dpf), the tornaria has a circumoral ciliary band, mouth, tripartite digestive tract, protocoel, larval muscles and a simple serotonergic nervous system. The telotroch develops at 5 dpf. In the course of 60 days, the serotonergic nervous system becomes more elaborate, the posterior coeloms develop, and the length of the circumoral ciliary band increases. At the end of the larval stage, larval muscles disappear, gill slits form, and adult muscles develop. Metamorphosis occurs spontaneously when the larva reaches its maximal size (ca. 3 mm), and involves loss and reorganization of larval structures (muscles, nervous system, digestive tract), as well as development of adult structures (adult muscles, tripartite body organization).

Conclusions: This study will enable future research in S. californicum to address long standing questions related to the evolution of axial patterning mechanisms, germ layer induction, neurogenesis and neural patterning, the mechanisms of metamorphosis, the relationships between larval and adult body plans, and the evolution of metazoan larval forms.

Keywords: Enteropneusta; Hemichordata; Indirect development; Metamorphosis; Planktotrophy; Schizocardium californicum; Tornaria.

Fig. 1

Adult Schizocardium californicum. Light micrographs. a Female. b Male. Dashed lines mark the boundaries between the main body regions. bg, branchiogenital region; c, collar; g, genital region; h, hepatic region; hs, hepatic sacs; p, proboscis; t, trunk. Scale bars: 10 mm

Fig. 2

Cleavage and gastrulation in Schizocardium californicum. Light micrographs. Animal pole is up. a Zygote. b 2-cell stage. c 4-cell stage. d 8-cell stage. e 16-cell stage. f 32-cell stage. g-i Formation of the coeloblastula. j Flat plate gastrula. k Mid-gastrula. l Pre-hatching gastrula. m Gastrula at time of hatching. n Post-hatching gastrula. a, archenteron; bc, blastocoel; bp, blastopore. Scale bars: 50 μm

Fig. 3

Early larval development in Schizocardium californicum. a-d, f-g Light DIC micrographs of live specimens. Optical sections through the middle of the embryo (a, c-d, f) or surface views (b, g). e, h-o Maximum intensity projections of confocal stacks. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. All panels are anterior to the top. Lateral views are ventral to the left. a, b Hatched gastrula stage embryos at the time of pore canal formation, 33 hpf. c-e Developing tornaria larva at the time of protocoel formation, 2 dpf. f-o Early tornaria after mouth formation, 3 dpf. i Close-up lateral view of the apical organ_. j Close-up view of the squamous epithelium of the dorsal ectoderm. o Close-up dorsal view of the apical organ. a, archenteron; ao, apical organ; at, apical tuft; cb, serotonergic cell body; cc, cells of the presumptive circumoral ciliary band; dv, dorsal view; lv, lateral view; m, mouth; of, oral field; p, protocoel; pd, protocoel duct (pore canal); pof, postoral field; post, postoral loop of the circumoral ciliary band; pp, protocoel pore (hydropore); pre, preoral loop of the circumoral ciliary band; prf, preoral field; st, stomodeum; vv, ventral view. Scale bars: 50 μm

Fig. 4

Morphology of the tornaria larva of Schizocardium californicum at 5 dpf and 10 dpf. a-c, p-r Light DIC micrographs of live specimens. Optical sections through the middle of the embryo (a-p) or surface views (b-c, q-r). d-o, s-y Maximum intensity projections of confocal stacks. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. All panels are anterior to the top, and lateral views are ventral to the left. a-o Tornaria larva at 5 dpf. f, m Close-up views of the circular pharyngeal muscles and apical strand. i-j, o Close-up views of the apical organ and dorsally projecting neurites. p-y Tornaria larva at 10 dpf. w-y Close-up views of the apical organ and dorsal nerve tract. af, anal field; am, anchoring muscles; ao, apical organ; as, apical strand; av, anterior view; dc, dorsal cluster of the apical organ; dn, dorsal nerve tract; dv, dorsal view; e, eyespots; i, intestine; lv, lateral view; m, mouth; mc, mesenchymal cell; n, neuropil; nt, neurotroch; of, oral field; p, protocoel; ph, pharynx; pm, pharyngeal muscles; pof, post-oral field; post, postoral loop of the circumoral ciliary band; pp, protocoel pore (hydropore); pre, preoral loop of the circumoral ciliary band; prf, preoral field; s, stomach; t, telotroch; vc, ventral cluster of the apical organ; vv, ventral view. Scale bars: 100 μm (a-e, g-l, p-v), 50 μm (f, m, o, w-y)

Fig. 5

Morphology of the tornaria larva of Schizocardium californicum at 18 and 22 dpf. a-j Light DIC micrographs of live larvae. Optical sections through the middle of the embryo (a, c, f, h) or surface views (b, d-e, g, i-j). k-v Maximum intensity projections of confocal stacks. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. All panels are anterior to the top, and lateral views are ventral to the left. a-e Tornaria larva at 18 dpf. f-v Tornaria larva at 22 dpf. l Close-up lateral view of pharyngeal circular and longitudinal muscles. n Close-up lateral view of apical organ (box in panel m). v Close-up dorsal view of apical organ. a, anus; aa, autofluorescence caused by algal cells in digestive tract; af, anal field; am, anchoring muscles; ao, apical organ; as, apical strand; at, apical tuft; cm, circular pharyngeal muscles; dc, dorsal cluster of the apical organ; dn, dorsal nerve tract; dv, dorsal view; i, intestine; lg, lateral groove; lm, longitudinal pharyngeal muscles; ln, lateral nerve tract; lv, lateral view; m, mouth; mc, mesenchymal cell; n, neuropil; nt, neurotroch; of, oral field; p, protocoel; pdl, primary dorsal lobe; pds, primary dorsal saddle; ph, pharynx; pof, postoral field; post, postoral loop of the circumoral ciliary band; pp, protocoel pore (hydropore); pre, preoral loop of the circumoral ciliary band; prf, preoral field; s, stomach; t, telotroch; tn, telotroch nerve tract; vc, ventral cluster of the apical organ; vs, ventral saddle; vv, ventral view. Scale bars: 200 μm (a-k, m, o-u), 100 μm (l, n, v)

Fig. 6

Morphology of the tornaria larva of Schizocardium californicum at 26, 31 and 36 dpf. Light DIC micrographs of live larvae. All panels are anterior to the top and lateral views are ventral to the left. Optical sections through the middle of the larva (d, g-h, l) or surface views (a-c, e-f, i-k). a-d Tornaria larva at 26 dpf. d Close-up ventral view of the mesocoels and metacoels around the posterior stomach. e-h Tornaria larva at 31 dpf. Inset is a close-up of the telotroch showing pigment spots. h Close-up view of area boxed in panel g showing formation of the proboscis vesicle. i-l Tornaria larva at 36 dpf. Inset is a close-up view of the region highlighted in l, showing movement of the proboscis vesicle closer to the protocoel. as, apical strand; dv, dorsal view; i, intestine; ldl, lower dorsal lobe; ll, lateral lobe; ls, lateral saddle; lv, lateral view; m, mouth; mc, mesenchymal cell; of, oral field; pd, protocoel duct (pore canal); pdl, primary dorsal lobe; pds, primary dorsal saddle; ph, pharynx; pof, postoral field; post, postoral loop of the circumoral ciliary band; pp, protocoel pore (hydropore); pre, preoral loop of the circumoral ciliary band; ps, pigment spots; pvl, primary ventral lobe; pvs, primary ventral saddle; s, stomach; t, telotroch; v, proboscis vesicle; vs, ventral saddle; vv, ventral view. Scale bars: 400 μm (a-c, e-g, i-l), 100 μm (d, l inset), 200 μm (h)

Fig. 7

Morphology of the tornaria larva of Schizocardium californicum at 36 dpf. Maximum intensity projections of confocal stacks. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. All panels are anterior to the top and lateral views are ventral to the left. a-c lateral views. b Tubulin channel only, showing organization of the ciliary bands. c DAPI and serotonin channels only, showing the lateral nerve tract passing through the lateral groove and the telotroch nerve tract. d Ventral view, acetylated tubulin and serotonin channels only showing the nerve tract underlying the telotroch. e Close-up ventral view of the pharynx, phalloidin channel only, showing the circular and longitudinal muscles in the anterior and posterior pharynx, respectively. f-i Dorsal views. g DAPI and serotonin channels only, showing the dorsal nerve tract descending along the dorsal midline, and the telotroch nerve tract. h Acetylated tubulin channel only, showing organization of the ciliary bands. i Close-up view of the telotroch, showing the shorter cilia of the secondary telotroch. aa, autofluorescence caused by algal cells in digestive tract; ao, apical organ; as, apical strand; cm, circular pharyngeal muscles; dc, dorsal cluster of the apical organ; ldl, lower dorsal lobe; ll, lateral lobe; lm, longitudinal pharyngeal muscles; ln, lateral nerve tract; ls, lateral saddle; pdl, primary dorsal lobe; pds, primary dorsal saddle; pm, pharyngeal muscles; pof, postoral field; pvl, primary ventral lobe; pvs, primary ventral saddle; st, secondary telotroch; t, telotroch; tn, telotroch nerve tract. vc, ventral cluster of the apical organ. Scale bar: 500 μm (a-d, f-h), 200 μm (e, i)

Fig. 8

Morphology of the tornaria larva of Schizocardium californicum between 50 dpf and the end of the larval stage. Light DIC micrographs of live larvae. All panels are anterior to the top and lateral views are ventral to the left. a-k Tornaria larva at 50 dpf. b, d-e Surface views showing external morphology and organization of the ciliary bands. a, c Optical sections through the middle of the larva, showing internal anatomy. f-g Close-up view of the anterior end of the larva, showing regression of the apical strand. Arrows indicate where cells of the apical strand are no longer present. h-i Dorsal view of the protocoel region, showing the gradual movement of the proboscis vesicle towards the protocoel. j-k Growth and shape change of the posterior coeloms between 50 dpf (j) and 65 dpf (k). l-p Tornaria larva at 65 dpf. l-n Optical sections through the middle of the larva, showing internal anatomy. m, o-p Surface views showing external morphology and organization of the ciliary bands. q-t Morphological changes occurring between 65 dpf and metamorphosis. q-r Close-up view of the pharynx, showing development of gill bars. s Close-up view of the protocoel in its final form before metamorphosis. t Close-up view of the metacoel in its final form before metamorphosis. as, apical strand; gb, gill bar; i, intestine; ldl, lower dorsal lobe; lg, lateral groove; ll, lateral lobe; m, mouth; mes, mesocoel; lv, lateral view; met, metacoel; of, oral field; og, oral groove; p, protocoel; pd, protocoel duct (pore canal); pdl, primary dorsal lobe; pds, primary dorsal saddle; ph, pharynx; pof, postoral field; post, postoral loop of the circumoral ciliary band; pre, preoral loop of the circumoral ciliary band; prf, preoral field; pvl, primary ventral lobe; pvs, primary ventral saddle; s, stomach; t, telotroch; v, proboscis vesicle; vs, ventral saddle. Scale bars: 400 μm (a), 200 μm (f-k, q-t), 800 μm (l)

Fig. 9

Morphology of the late tornaria larva prior to metamorphosis. Maximum intensity projections of confocal stacks. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. Anterior to the top and ventral to the left. a-e Lateral view. b Tubulin channel only, showing organization of ciliary bands. c Phalloidin channel only, showing highly muscularized coeloms and reduced pharyngeal musculature. Note that the apical strand is no longer present. d Close-up side view of the protocoel, showing circular and longitudinal muscle fibers. e Close-up side view of the left mesocoel and metacoel. f-h Dorsal view. g Serotonin channel only, showing dense epidermal neural net. h Phalloidin channel only, showing highly muscularized coeloms. i-k Ventral view. j Tubulin channel only, showing organization of ciliary bands. k Serotonin channel only, showing dense epidermal neural net. l Close-up anterior view of the apical organ, serotonin channel only. ao, apical organ; av, anterior view; cm, circular muscle fibers; dc, dorsal cluster of the apical organ; dn, dorsal nerve tract; dv, dorsal view; ll, lateral lobe; lm, longitudinal muscle fibers; ls, lateral saddle; mes, mesocoel; met, metacoel; n, neurotroch; np, neuropil; p, protocoel; pdl, primary dorsal lobe; pds, primary dorsal saddle; pl, primary lobe; pm, pharyngeal muscles; post, post-oral loop of the circumoral ciliary band; pre, pre-oral loop of the circumoral ciliary band; prf, pre-oral field; pvl, primary ventral lobe; pvs, primary ventral saddle; sv, side view; t, telotroch; tn, telotroch nerve tract; vc, ventral cluster of the apical organ. Scale bar: 600 μm (a-c, f-k), 300 μm (d-e, l)

Fig. 10

Metamorphosis in Schizocardium californicum. Light DIC micrographs of live individuals. Anterior to the top and ventral to the left. a Larva at onset of metamorphosis, showing enlarged coelomic cavities. b-c Larvae between 3 h and 5 h after onset of metamorphosis, showing thickening of the ectoderm, posterior retraction of the pharynx, and further enlargement of coelomic cavities. d Larva around 10–12 h after onset of metamorphosis, showing almost fully formed proboscis, thickening collar, and folding of the gut. e Larva around 24 h after onset of metamorphosis, showing fully formed proboscis and collar, further folding of the gut, enlargement of pharynx and gill slits, and formation of the trunk. f Post-metamorphic juvenile, 48 h after onset of metamorphosis, showing fully formed proboscis and collar, and elongating trunk. g Close-up ventral view of the forming proboscis. Same individual as c. The anterior ciliary grooves are disappearing as the loops of the circumoral ciliary band begin to fuse. h-j Changes in coelomic organization during metamorphosis. h Onset of metamorphosis. Same individual as a. i 5 h after onset of metamorphosis. j 12 h after onset of metamorphosis. The metacoels are forming the longitudinal muscles of the trunk. k-m Changes in pharyngeal morphology during metamorphosis. k Same individual as a, showing 6 gill slits at onset of metamorphosis, and the presence of tongue bars. l pharynx at 12 h after onset of metamorphosis, showing relative position with the mesocoels. m Pharynx of larva around 24 h after onset of metamorphosis, showing addition of a seventh pair of gill slits. n-o Ectodermal gill pore formation. n Same individual as c. o Same individual as e. ao, apical organ; c, collar; gb, gill bars; gp, gill pores; i, intestine; lm, longitudinal trunk muscle; m, mouth; mes, mesocoel; met, metacoel; of, oral field; p, protocoel; ph, pharynx; post, postoral loop of the circumoral ciliary band; pr, proboscis; pre, preoral loop of the circumoral ciliary band; prf, preoral field; pvl, primary ventral lobe; s, stomach; t, telotroch; tb, tongue bar; tr, trunk. White arrows mark the posterior boundary of forming collar. Scale bars: 500 μm (a-j, l-n), 250 μm (k, o)

Fig. 11

Early metamorphosis in Schizocardium californicum. Maximum intensity projections of confocal stacks. a-g Larvae labeled with phalloidin (green), anti-acetylated tubulin antibody (red) and DAPI (blue). h-j Larvae labeled with anti-serotonin (yellow) antibodies, and DAPI (blue). All panel are anterior to the top and lateral views are ventral to the left. a Onset of metamorphosis, same stage as Fig. 10a. b Larva during early metamorphosis, same stage as Fig. 10b. The primary lobes are disappearing as the loops of the circumoral ciliary band begin to move toward each other. c Larva during mid-metamorphosis, similar stage as Fig. 10c. d-g Close-up views of coeloms and associated musculature, same individuals as panel a (f-g) and panel c (d-e). h-i Serotonergic nervous system during early metamorphosis (similar stage as panel b), showing neurites and cell bodies scattered throughout the future proboscis, collar and anterior trunk down to the telotroch. Neurite bundles are still present in the lobes and lateral grooves of the forming proboscis. j Serotonergic nervous system during early metamorphosis (similar stage as panel c). Neurite bundles are no longer present in the lobes and lateral grooves. Neurites of the anterior trunk are less abundant. A dorsal nerve tract runs along the trunk midline. cm, circular muscle fibers; dn, dorsal nerve tract; dv, dorsal view; gp, gill pore; lm, longitudinal muscle fibers; ln, lateral nerve tract; lv, lateral view; mes, mesocoel; met, metacoel; pdl, primary dorsal lobe; pds, primary dorsal saddle; pof, postoral field; prf, preoral field; pvl, primary ventral lobe; pvs, primary ventral saddle; t, telotroch; tn, telotroch nerve tract; vv, ventral view. Scale bars: 500 μm

Fig. 12

Late metamorphosis and early juvenile development in Schizocardium californicum. Larvae are labeled with four markers: phalloidin (green), anti-acetylated tubulin (red) and anti-serotonin (yellow) antibodies, and DAPI (blue) unless otherwise indicated. a-e 24 h after onset of metamorphosis. Anterior to the top. b Serotonin labeling only. Inset shows a close-up on the larval apical organ, still visible. c Phalloidin labeling only, showing longitudinal muscles in the trunk. d Close-up of the anterior proboscis, serotonin and DAPI labeling only. Neurons of the larval apical organ are still visible, but become less distinct from the broadly distributed neurons of the juvenile proboscis as time progresses. e Serotonin labeling only. Insets show optical slices through the anterior tip and ventral side of the proboscis epidermis. f Early juvenile, 48 h after onset of metamorphosis. Surface optical slices are excluded from the stack, showing muscles inside the proboscis and trunk. g Close-up view of the trunk epidermis, showing basiepidermal serotonergic nerve net and muscle fibers underlying the ciliated epithelium. h Juvenile, 15 days post-metamorphosis. ao, apical organ; c, collar; dn, dorsal neurite bundle; dv, dorsal view; gb, gill bars; gp, gill pore; lv, lateral view; m, mouth; pm, proboscis muscles; pr, proboscis; t, telotroch; tm, longitudinal trunk muscles; tr, trunk; vn, ventral neurite bundle. Scale bars: 500 μm (a-c, e-f, h), 100 μm (d), 200 μm (g)