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Abbreviated Development of the Brooding Brittle Star Ophioplocus esmarki.

Introduction

Echinoderms with non-feeding developmental stages have evolved numerous times from species with feeding larvae (Mortensen, 1921; Strathmann, 1974, 1975, 1978; Hendler, 1982; Mladenov, 1985; Wray and Raff, 1991; Wray, 1996; Smith, 1997; McEdward and Miner, 2001). The change from the ancestral condition of indirect development to abbreviated development includes an increase in maternally derived nutrients contributed to the egg and a corresponding decrease in larval feeding. Evolutionarily convergent features in the development of these abbreviated forms include large eggs, bullet-shaped larval bodies, loss of feeding structures and a larval skeleton, the presence of uniform ciliation or transverse ciliary bands used for swimming, and decreased time to the juvenile stage (Emlet et al., 1987; Hendler, 1991; Emlet, 1994; Wray, 1996).

Brittle stars (class Ophiuroidea) with ancestral indirect development go through the swimming, feeding ophiopluteus larva stage (MacBride, 1907; Mortensen, 1921, 1931, 1937, 1938; Narasimhamurti, 1933; Olsen, 1942; Hendler, 1975; Rumrill, 1984; Mladenov, 1985; Yamashita, 1985). The larva develops four pairs of long arms with a single, continuous ciliary band that is used for swimming and feeding (Strathmann, 1971, 1975, 1978); and pentamerous juvenile structures develop within the bilateral ophiopluteus (Hyman, 1955; Burke, 1989; Hendler, 1991). The five-lobed hydrocoel grows around the larval esophagus and fuses with itself to form the ring canal and radial canals of the water vascular system. Pentamerous patterning of other juvenile structures (other coelomic compartments, skeleton, nervous system, etc.) begins after the formation of the early water vascular system. There is evidence from sea urchins that the coelomic mesoderm induces the juvenile skeletal and nervous systems (Minsuk and Raff, 2002). At the time of metamorphosis, the brittle star resorbs the original larval structures (Type I or Type II metamorphosis), settles to the benthos, and enters the juvenile phase of life (Mladenov, 1985; Selvakumaraswamy and Byrne, 2006).

The bilaterally symmetrical nervous system of the ophiopluteus first forms at the anterior end (apical organ) of the late gastrula stage embryo; and later it is associated with the ciliary band, oral epidermis, and digestive system of the larva (Cisternas and Byrne, 2003; Byrne et al., 2008; Hirokawa et al., 2008; Dupont et al., 2009; Gliznutsa and Dautov, 2011). Within the larva, a new nervous system forms from the ventral ectoderm in close proximity to the water vascular system (MacBride, 1907; Narasimhamurti, 1933; Olsen, 1942). This juvenile nervous system includes a ring nerve, radial nerves, and podial nerves associated with the tube feet (Hirokawa et al., 2008; Dupont et al., 2009).

One pattern of abbreviated development in brittle stars includes a non-feeding vitellaria larva that swims in the plankton for only a few days before metamorphosing into a juvenile (Brooks and Grave, 1899; Mortensen, 1921, 1938; Stancyk, 1973; Hendler, 1982, 1991; Komatsu and Shoshaku, 1993; Selvakumaraswamy and Byrne, 2004; Cisternas and Byrne, 2005; Fourgon et al., 2005). After cleavage and gastrulation, a large lobe (the preoral lobe) forms at the anterior end, and the embryo undergoes extensive morphogenetic movements to set up the vitellaria body plan. Juvenile structures develop at the mid-ventral surface. Unlike the continuous ophiopluteus larval ciliary band, the vitellaria larva develops three to five transverse ciliary bands that are only used for swimming. The ciliary bands are positioned on ectodermal ridges, the preoral lobe, and a posterior lobe.

Because Ophioplocus esmarki broods its embryos, vitellaria larvae, and juveniles inside the bursal cavities, this species provides an opportunity for learning about how brooding might be associated with changes in vitellaria structure, compared to the pelagic vitellaria. Also, this species can be used to examine the development of the muscles and nervous system, which have not been described for a vitellaria larva. The nervous system is especially interesting because it is closely associated with the continuous ciliary band in feeding echinoderm larvae. In this study, the development of O. esmarki is described from cleavage stages through the beginning of pentamerous symmetry. The external morphology, ciliary bands, nerves, and muscles of the vitellaria larva and juvenile are examined. This brooded vitellaria shares many features with pelagic vitellaria larvae, except for a difference in buoyancy. The vitellaria larval nervous system has similarities with the ophiopluteus, but it also has significant differences. There are greater similarities in the development of the juvenile nervous system when comparing the vitellaria and late stage ophiopluteus larva.

Materials and Methods

Early embryos of Ophioplocus esmarki Lyman, 1874 are brooded from mid-January to April along the southern California coast and from April to June along the central California coast (Rumrill, 1984). For this study, adult brittle stars were obtained from Marinus Scientific (Long Beach, CA). Adults were anesthetized with 1:1 artificial seawater: 7.5% Mg[Cl.sub.2] for 15 minutes. Embryos, vitellaria larvae, and juveniles were flushed out of the bursal cavities using a stream of seawater from a micropipettor, and they were raised at 12-15 [degrees]C in glass dishes. In one batch, early cleavage stage embryos were collected. These embryos were followed for several days to document external development (Table 1; Fig. 1). Ages given for this batch refer to the time after they were removed from the bursal cavities. Specimens were fixed at different stages in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated in a series of ethanol concentrations (30%, 50%, 70%), and imaged with a Zeiss Axiovert 40 CFL inverted microscope (Oberkochen, Germany).

In other batches, the development of the ciliary bands, neurons, and muscles was examined using markers for acetylated alpha tubulin (clone 6-11B-1, Sigma-Aldrich, St. Louis, MO), synaptotagmin (1e 11, Developmental Studies Hybridoma Bank, Iowa City, IA; Nakajima et al., 2004), and BODIPY-FL phallacidin (filamentous actin, Molecular Probes, Eugene, OR), respectively. Specimens were fixed for 15 minutes in 3.7% formaldehyde in seawater, washed 3 times in PBS and 1 time in PBS with Tween (PBST), blocked in 5% goat serum in PBS, and incubated overnight at 4 [degrees]C in 1:1000 anti-acetylated alpha tubulin, or a 1 : 2 dilution of anti-synaptotagmin to PBS. They were washed 5 times in PBS for 30 minutes to 2 hours at room temperature, incubated in 1 : 50 secondary antibody (fluorescein isothiocyanate or Cy3 dye conjugated to goat antimouse immunoglobulin G; Jackson ImmunoResearch, West Grove, PA) in PBS for 2 hours, and then washed 5 times in PBS. PBS was used to wash because young stages become fragile in PBST. Muscles were stained with BODIPY-FL phallacidin by using the protocol from Strickland et al. (2004). Ethanol and methanol were not used in the procedure because they interfere with phallacidin staining. In summary, BODIPYFL phallacidin in methanol was dried on the inside of a 1.5-mL tube and was then resuspended in PBS. This was used to stain the muscle during the secondary antibody step for anti-synaptotagmin. Specimens stained for cilia were mounted in 1:1 PBS : glycerol and were examined with a laser scanning confocal microscope with inverted stage (Leica TCS SP5 II; Wetzlar, Germany). Neural- and muscle-stained specimens were decalcified in 0.05 mol [L.sup.-1] ethylenediaminetetraacetic acid in PBS, dehydrated in an isopropanol series (30%, 50%, 70%, 100% x 3), cleared in 2:1 benzyl benzoate:benzyl alcohol (Strickland et al., 2004), and imaged with confocal microscopy.

The larval stages and structures were labeled according to previous studies. Ectodermal structures and ciliary bands (tracts a-i) were labeled according to Fourgon et al. (2005). Nerves and muscles were labeled according to Hyman (1955). To correct left-right positioning captured by imaging with an inverted microscope, images were transformed 180[degrees] to match the orientation of images in published literature. Fiji (Schindelin et al., 2012) and GIMP (2019) were used to generate images, adjust brightness and contrast, and color code different features in the images (Schindelin et al., 2012).

Results

External morphology

When removed from the brood chambers, ciliated embryo and larval stages swim along the bottom surface of the dish but not in the water column. They are negatively buoyant at all stages. They swim in a circular pattern or a directed pattern, or they rotate in place. They do not swim upward into the water column or hover above the bottom surface of the dish. When they develop functional tube feet, mostly they adhere to the dish and no longer swim.

The external anatomy remains bilaterally symmetrical for the first six days of development (Table 1; Fig. 1A-D). A fertilization envelope encloses the cleavage stages (Fig. 1 A, B), and an elongated gastrula stage develops by four days (Fig. 1C). A pre-vitellaria forms by six days and has four lateral outgrowths (left dorsal bulge, right dorsal bulge, left ventral bulge, and right ventral bulge) and a prominent preoral lobe at the anterior end (Fig. 1D).

The early vitellaria larva forms by 11 days (Table 1; Fig. 1E, F) and has a shift in bilateral symmetry. The preoral lobe changes shape (explained below for the 12-day mid-vitellaria). The ectodermal bulges become raised to form ridges. The posterior end (posterior lobe, right ventral bulge, and left ventral bulge) shifts to the right, or clockwise around the mouth when viewed from the ventral side. A circular oral field is prominent on the larval ventral side with the juvenile mouth developing in the center. The rudiments of the five arms are present, but the terminal tentacle and tube feet are not yet visible in the region around the developing juvenile mouth.

The mid-vitellaria forms by 12 days (Table 1; Fig. 1G, H), and the twisting pattern of the preoral lobe is more obvious than in the 11-day early vitellaria. The arc of the anterior region in Figure ID is transformed into a W-shaped structure. Because region b stays most anterior, region a forms a U-shaped ridge on the dorsal surface of the preoral lobe. At the same time, region c forms a U-shaped ridge on the ventral surface. The overall pattern forms a W shape when viewed from the anterior pole. In the middle region of the embryo, the right dorsal bulge moves to a central position on the dorsal side (Fig. 1G). The developing juvenile structures on the ventral side of the larva include the arm rudiments, each with three outgrowths, which form the terminal tentacle at the arm tip, and a pair of tube feet (Fig. 1H).

The late vitellaria forms by 13 days (Table 1; Fig. 1I, J). The preoral lobe has a cylindrical shape, and the W-shaped ectodermal ridges form two transverse ridges when viewed from the ventral side. In the developing oral field of the juvenile body, five pairs of small buccal podia form close to the mouth. Interradial ridges of ectoderm (associated with the left dorsal bulge, left ventral bulge, posterior lobe, right ventral bulge, and preoral lobe) are positioned near the developing juvenile mouth. These will contribute to the epineural folds that will fuse on the oral side of each arm and to the jaws that will form adjacent to the mouth.

The early juvenile forms by 19 days (Table 1; Fig. 1K, L), and pentamerous symmetry is the predominant pattern. The preoral and posterior lobes are mostly resorbed, and the ectodermal ridges are smoothed out. The larval dorsal surface becomes the juvenile aboral surface, and the larval ventral surface becomes the oral surface. On the oral surface, the epineural folds fuse in the radial positions to enclose the developing epineural canals along each arm. The central plate and radial plates of the juvenile skeleton form on the aboral side. The arms extend outward, and the juvenile resembles a miniature star.

Ciliary band development

During gastrulation, the embryo is uniformly ciliated except in the blastopore region and the site of the future mouth (not shown). Denser bands of cilia, tracts a-i, form in specific regions of ectoderm in the pre-vitellaria larva (Fig. 2A, A'). Dense cilia form across the anterior ridge of the preoral lobe (tracts a, b, c in Fig. 2), along the initial ectodermal bulges of the embryo (right dorsal bulge with tract d; left dorsal bulge with tract e; right ventral bulge with tract h; left ventral bulge with tract i), and along the posterior lobe (tracts f and g). This stage is the end of bilateral symmetry at the ectodermal surface.

By the early vitellaria stage, the ectoderm is still largely ciliated but now has distinct ciliary bands (Fig. 2B, B'). The anterior and posterior regions undergo morphogenetic movements that distort the bilateral symmetry and transform the patterning of the ciliary bands. The preoral lobe changes shape and transforms the anterior strip of cilia (a, b, c) into a looping band shaped like a W when viewed from the anterior pole (described in External morphology). The posterior lobe and its associated C-shaped ciliary bands f and g, the right ventral bulge with ciliary band h, and the left ventral bulge with ciliary band i all shift to the right, or clockwise when viewed from the ventral side. On the dorsal side, large gaps separate ciliary tracts d and e, as well as f and i (Fig. 2B). The developing juvenile with the five arm rudiments displaces the ciliary bands on the ventral side (Fig. 2B').

At the late vitellaria stage, most of the uniform ciliation is lost, and the tracts of cilia lengthen and become narrower (Fig. 2C, C). On the resorbing preoral lobe, the anterior ciliary band, which originated as a single tract (a, b, c) along the anterior surface, is now transformed into two ciliary bands that span the preoral lobe. Tract a is not as distinct as tract b. The tracts spanning the dorsal surface of the middle of the larval body come together to form transverse bands that are nearly continuous (tracts e, d, and h in Fig. 2C). Ciliary tracts i and f form a discontinuous band in a more posterior position. Cilia associated with the hydropore are present on the dorsal surface (Fig. 2C). On the ventral side, tracts h, e, and i extend toward the developing juvenile mouth between the developing arms (Fig. 2C').

As the preoral lobe is resorbed, the associated ciliary bands (a, b, c) are resorbed (not shown), and the remaining bands are resorbed after this. The early juvenile has no remnants of ciliary bands on either surface (Fig. 2D, D'). However, the aboral surface of the developing juvenile disk has a pattern of uniform, sparse ciliation. A small area of denser cilia marks the position of the hydropore on the aboral side in the region posterior to the resorbing preoral lobe (Fig. 2C, D). The oral surface has cilia associated with the terminal tentacles and tube feet.

The nervous and muscular systems

The first neurons are located under the surface of the ectoderm at the pre-vitellaria stage after the formation of the coelomic cavities (Fig. 3A, B). The neurons form in a bilaterally symmetrical, crescent-shaped cluster that is centered on the dorsal side in the region where the preoral lobe connects with the rest of the body (Fig. 3C). Overall, embryos have 10-20 neurons at this stage. At the early vitellaria stage, some neurons are in close proximity to the ciliary bands associated with the ectodermal ridges, but there are no tracts of neuronal processes in parallel with the ciliary bands (Fig. 3D).

In the mid-vitellaria stage, neurons are located at the base of the preoral lobe on both the ventral and dorsal sides (Fig. 3E, H). Neural processes extend around the developing juvenile oral field (Fig. 3F). Some neurons are associated with the developing axial complex (Fig. 3G). There are no neurons in the developing juvenile structures within the oral field (Fig. 3E). Muscle development has not yet begun.

At the late vitellaria stage (Fig. 4A-F), the number of neurons at the base of the preoral lobe decreases to about four per vitellaria. Overall, the larval neurons are less conspicuous than at earlier stages. However, the neurons associated with the axial complex are strongly stained with the synaptotagmin antibody (Fig. 4A, C). At this time, the juvenile nerves and muscles have faint staining with pentamerous symmetry. The early nerves include the ring nerve surrounding the future mouth, the radial nerves extending from the ring nerve to the tips of the arms, and the podial nerves extending from the radial nerves to encircle each tube foot (Fig. 4A, D). The first juvenile muscles include the external interradial muscles between each arm, podial muscles within the tube feet, and terminal tentacle muscles at the tips of the arms (Fig. 4B, E). The water vascular system also has faint labeling in the stone canal in the axial complex, the ring canal, and the radial canals (Fig. 4B, E). Muscle fibers extend dorsally from the base of the podial muscles into the central disk (Fig. 4B, E, arrowheads and inset).

By the early juvenile stage (before the preoral lobe has been fully absorbed), the larval nervous system continues to decrease as the juvenile nervous system develops (Fig. 4G-L). Very few of the juveniles still have neurons associated with the base of the preoral lobe. Most juveniles have synaptotagmin-positive cells associated with the developing axial complex (Fig. 4G, J). The ring, radial, terminal tentacle, and podial nerves of the developing juvenile system are more strongly stained at this stage (Fig. 4G, I). The podial nerves have ring ganglia located at the proximal and distal ends (Fig. 4G). At the distal tips of the radial nerves, the synaptotagmin staining is strongest at the base of the terminal tentacles (Fig. 4G). The early juvenile muscular system is also more strongly stained and distinct at this stage (Fig. 4H-L). As in the late vitellaria stage, the terminal tentacle muscles, external interradial muscles, and podial muscles have the strongest staining (Fig. 4H). The axial complex has filamentous actin staining in the pore canal and the stone canal and synaptotagmin staining in a structure next to them (Fig. 4J). Buccal podial nerves have formed with ring ganglia at the proximal and distal ends (Fig. 4K). The jaws also have both neural and muscle staining with the earliest development of the interior interradial muscles (Fig. 4L).

At the later juvenile stages, the nervous system has a similar structure, but with more muscle groups (Fig. 5). With the exception of synaptotagmin staining within the axial complex (Fig. 5A, C, D), the early neurons associated with the preoral lobe are not apparent. The ring nerve and radial nerves form ganglia, or thickenings (Fig. 5A, D). Radial muscles have formed at the base of each arm, and muscle fibers are associated with the polian vesicles and peristomial membrane (Fig. 5B, D). Additional muscles have developed in a later juvenile stage with two arm segments (Fig. 5E-H). Two pairs of intervertebral muscles form at the proximal end of each arm, distal to the radial muscles. The larger aboral intervertebral muscles obscure the oral intervertebral muscles in Figure 5G. In addition to the buccal podia, tube feet, and terminal tentacle, other components of the water vascular system stain for muscle, including the ring canal, radial canals, polian vesicles, pore canal, and stone canal (Fig. 5E-G). The polian vesicles are adjacent to the exterior interradial muscles in a more aboral position. Some muscle fibers associated with the radial canals have a longitudinal orientation extending down the length of the arm, and other fibers are oriented perpendicular to the arm axis (Fig. 5G). A network of muscle fibers forms under the aboral surface of the central disk (Fig. 5E-H). These muscle fibers originate at the base of the tube feet (Fig. 4E) and contribute to two networks. An aboral muscle fiber network lines the body wall, and a more internal muscle fiber network lines the stomach (Fig. 5H).

Discussion

The ancestral larval form of brittle stars is the feeding, swimming, bilaterally symmetrical ophiopluteus larva. Many species, however, have a short-lived, non-feeding vitellaria larva with transverse ciliary bands, a more streamlined body shape with unique symmetry, and faster development of juvenile structures. The current study adds important knowledge on the development of the vitellaria larva, which is part of the life history of brittle stars within five families (Ophiocomidae, Ophiodermatidae, Ophiopezidae, Ophiolepididae, and Ophionereididae). In summary, we show that (1) the external anatomy of the Ophioplocus esmarki vitellaria larva is similar to pelagic vitellaria larvae, (2) the change in life history from a pelagic to brooded vitellaria is correlated with a change in buoyancy, (3) the vitellaria larval nervous system has similarities to, and differences from, the ophiopluteus larval nervous system, (4) the development of the juvenile nervous system is similar in the vitellaria and late ophiopluteus, and (5) the vitellaria larva develops only juvenile muscles (i.e., there are no muscles associated with larval function).

The vitellaria of O. esmarki share many general morphological features with other vitellaria larvae as described by Selvakumaraswamy and Byrne (2004), Cisternas and Byrne (2005), and Fourgon et al. (2005). Shared features include the unique ectodermal patterning along the anterior-posterior, left-right, and dorsal-ventral axes. In general, the vitellaria larva is divided into three parts: anterior, middle, and posterior. The anterior section forms the preoral lobe, the middle section is where the juvenile rudiment forms, and the posterior section forms the posterior lobe and the right and left ventral bulges. In most vitellaria larvae described, the anterior end undergoes extensive morphogenetic movements in the establishment of the transverse ciliary bands on the preoral lobe, and posterior structures shift to the right (clockwise in a ventral view) (Cisternas and Byrne, 2005; Fourgon et al., 2005; this study). Important ectodermal patterning events also occur along the vitellaria dorsal-ventral axis, which is transformed into the aboral-oral axis of the juvenile. The ectoderm on the central, ventral surface takes on the pentamerous symmetry of the developing juvenile as the terminal tentacle, tube feet, and buccal podia form. The transverse ciliary band-associated ridges of ectoderm mostly span the dorsal side and are in interradial positions relative to developing juvenile structures on the ventral side. Overall, the pattern of the O. esmarki vitellaria is similar to other vitellaria larvae, suggesting that the morphogenetic events in establishing the body plan are the same, even though O. esmarki is a brooder.

Like the general ectoderm, the ciliary band pattern of O. esmarki is similar to the ciliary bands of other species with a vitellaria larva. However, there are subtle differences in the early patterning and in the length of time that the ciliary bands are retained. In Ophionereis schayeri and Ophiomastix venosa, the ciliary bands of the vitellaria larva originate as a single continuous band, suggesting homology with the continuous ciliary band of the ancestral ophiopluteus (Selvakumaraswamy and Byrne, 2004; Fourgon et al., 2005). In O. esmarki, the transverse ciliary bands do not originate as a distinct continuous band, perhaps suggesting a small change in patterning events. The ciliary bands form on raised ectodermal ridges as the uniform ciliation of the gastrula stage embryo becomes sparse. The ciliary bands and associated ectoderm then undergo shape changes as in other vitellaria larva, suggesting that these are homologous structures. The early juveniles of O. esmarki do not have ciliary bands, which is similar to O. schayeri (Selvakumaraswamy and Byrne, 2004), but unlike O. venosa, in which juveniles maintain ciliary bands on the aboral surface of the disk (Fourgon et al., 2005). Overall, the ciliation pattern in O. esmarki appears to be similar to the general transverse banding pattern found in most vitellaria larvae (Mortensen, 1921; Stancyk, 1973; Hendler, 1982; Komatsu and Shosaku, 1993; Selvakumaraswamy and Byrne, 2004; Cisternas and Byrne, 2005; Fourgon et al., 2005; Byrne et al., 2008). The ciliary band similarities in brooded and pelagic vitellaria larvae suggest that the same morphogenetic movements are at work and that, in O. esmarki, brooding is not correlated with loss of ciliary bands.

An important difference found in the brooded vitellaria of O. esmarki in comparison to other species with pelagic vitellaria larvae is the inability to swim in the water column. Even though the vitellaria larvae of O. esmarki have a morphology similar to pelagic vitellaria larvae, including the transverse ciliary bands, they are not able to swim above the bottom surface. With the change from pelagic to brooded vitellaria, one would expect less selective pressure to maintain the vitellaria swimming structures (McEdward and Janies, 1997). In support of this idea is the example of the brooded embryos of Ophionereis olivacea, which develop into reduced vitellaria larvae that lack transverse ciliary bands (Byrne, 1991). However, at least three species, Ophiopeza spinosa, Ophioderma longicauda, and O. esmarki, brood larvae with typical vitellaria transverse ciliary banding patterns, suggesting that it is not uncommon to maintain these swimming structures and that the change from pelagic to brooded vitellaria may have occurred recently. Byrne et al. (2008) suggest that O. spinosa switched from pelagic to brooding relatively recently because the embryos have vitellaria morphology and can swim in the water column when removed from the bursae. Ophioderma longicauda, which had been classified as having a pelagic vitellaria larva (Fenaux, 1969), includes brooding and non-brooding populations (Stohr et al., 2009; Boissin et al., 2011; Weber et al., 2014), also suggesting that brooding has evolved recently. Within the genus Ophioplocus, Ophioplocus incipiens and Ophioplocus bispinosus brood their young (Mortensen, 1936; Byrne and O'Hara, 2017), and Ophioplocus januarii may have a pelagic vitellaria (Brogger et al., 2013). The O. esmarki vitellaria is very similar to the pelagic vitellaria of Ophioplocus japonicus (Komatsu and Shoshaku, 1993). The only apparent difference is the inability to swim in the water column. The O. esmarki vitellaria swimming patterns (circular, directional, and rotational on the bottom) are similar to those described by Webb (1989), with the exception of vertical movements and hovering in the water column. Perhaps the ciliary bands play an important role in the brooded vitellaria (e.g., swimming ability following an accidental early release from the bursae). Perhaps O. esmarki has been brooding long enough for a change in buoyancy to occur, but for too short a time to result in the loss of the ciliary bands used for swimming.

Previous studies show that much of the ophiopluteus nervous and muscular systems is associated with feeding and digestive structures. The ophiopluteus nervous system is associated with the apical organ, continuous ciliary band, and digestive system; and it is lost at metamorphosis (Cisternas and Byrne, 2003; Byrne et al., 2007; Hirokawa et al., 2008; Dupont et al., 2009; Gliznutsa and Dautov, 2011). The first neurons in the apical region are positioned in the anteriormost portion of the developing larva. These cells become displaced to the anterior dorsolateral region and are likely homologous to cells within the neurogenic apical organ in other echinoderms (Byrne et al., 2007; Dupont et al., 2009). Synaptotagmin-expressing cells form extensive neuronal tracts adjacent to the ciliary bands and are also associated with the lower lip, esophagus, pyloric sphincter of the stomach, and anus. Peptidergic neurons (S1-positive) form at the base of the anterolateral arms during larval arm resorption and may be involved in metamorphosis (Cisternas and Byrne, 2003). The muscles of the ophiopluteus are also associated with feeding. These include muscles associated with the esophagus for swallowing and with upper dilators for expanding the mouth (Gliznutsa and Dautov, 2011). Myoepithelial cells form sphincters associated with the different compartments of the digestive system.

How are the larval nervous and muscular systems different in the vitellaria with transverse ciliary bands, no digestive system, and faster onset of metamorphosis? In the vitellaria, we found larval neurons but no larval muscles. Some vitellaria larval neurons may be homologous with specific neural populations in the ophiopluteus larva. In the vitellaria, neurons at the dorsal base of the preoral lobe may be homologous to the apical organ neurons (Byrne et al., 2007; Hirokawa et al., 2008; Dupont et al., 2009) because they are located in a similar position but are farther posterior than they are in the ophiopluteus. The neurons at the ventral base of the preoral lobe in the vitellaria larva (Fig. 4D) may be homologous to the ophiopluteus peptidergic neurons that form at the base of the anterolateral arms around the time of metamorphosis (Cisternas and Byrne, 2003). The scarce vitellaria neurons associated with the ectodermal ridges may have homology with some of the neurons associated with the ciliary band in the ophiopluteus (Hirokawa et al., 2008; Dupont et al., 2009). Staining for additional neural markers, including serotonin and S1, may help to determine the identity of the early neurons in the vitellaria larva. If the populations of vitellaria larval neurons are indeed homologous to ophiopluteus larval neurons, then this would suggest that in both larval forms some neurons have important functions not related to feeding, such as control over cilia for swimming and sensory functions.

Important differences in vitellaria neural development include the lack of tracts of neurons along the ciliary bands and the lack of neurons associated with the larval digestive system. The lack of neurons associated with the larval mouth, esophagus, stomach, and intestine is not surprising because the vitellaria larva does not have a functional digestive system. However, the lack of tracts of neurons along the ciliary bands is unexpected because neurons in these areas are considered to be ancestral features of echinoderm development (Burke, 2011). If neural tracts along the ciliary bands are important for feeding, then one might expect them to be lacking in a non-feeding larva. In the evolution of non-feeding forms, there would be less selective pressure to maintain feeding behaviors and structures; and the neurons associated with feeding would be lost through evolutionary time.

As the ophiopluteus swims and feeds, the pentamerous nervous and muscular systems of the juvenile form within the larva. The juvenile nervous system includes the ring nerve, five radial nerves, paired podial nerves, and neurons associated with the developing jaws (Hirokawa et al., 2008; Dupont et al., 2009). Some muscles of the juvenile system also develop within the late stage ophiopluteus, but details are not given (MacBride, 1907; Olsen, 1942). The adult muscle groups are summarized by Hyman (1955) into three main groups to control the movement of the (1) jaws (external interradial muscles, radial muscles, and internal interradial muscles); (2) arms (two pairs of intervertebral muscles between each arm segment); and (3) components of the water vascular system (terminal tentacle, tube feet, polian vesicles, and buccal podia).

We provide evidence that the development of the juvenile nervous system within the vitellaria is similar to the development of the juvenile nervous system within the late ophiopluteus, with the exception of the neurons in the axial complex. The juvenile nerves follow the same pattern of development, with the formation of the ring nerve, radial nerves, and nerves associated with the tube feet and buccal podia (Hirokawa et al., 2008; Dupont et al., 2009; this study) after the development of the water vascular system (MacBride, 1907; Narasimhamurti, 1933; Olsen, 1942). This suggests that the different larval types, with their different morphologies and modes of development, have the same or similar developmental pathways that lead to the same outcome in building the juvenile nervous system.

Another important finding in the vitellaria larva is the development of neurons in the axial complex. These cells express synaptotagmin shortly after the formation of vitellaria larval neurons but before the development of the pentamerous juvenile nervous system. Unlike the other larval neurons, these axial complex neurons persist into the juvenile stage. Similar synaptotagmin staining occurs in the axial complex of the juvenile ophiuroid Amphiura filiformis (fig. 4 in Dupont et al., 2009) and the developing juvenile asteroid Patiriella regularis (figs. 5 and 6 in Elia et al., 2009), which is consistent with the possibility of neural development in the axial complex of other echinoderms. The function of the axial complex is unknown but may involve circulation and/or excretion; it does include a neural component (Ezhova et al., 2014, 2015). Thus, even though it has not been described before, synaptotagmin staining in this structure is not surprising. However, the finding that the axial complex neurons form early in the vitellaria larva before other juvenile structures suggests that the axial complex is a juvenile system that has evolved accelerated development in this abbreviated form.

Overall, the development of the larval nervous system in echinoderms with abbreviated development, including echinoids (Bisgrove and Raff, 1989; Katow et al., 2009), asteroids (Elia et al., 2009), crinoids (Nakano et al., 2009), and ophiuroids (this study), has changed independently of the juvenile system. These findings support the idea that the larval and juvenile nervous systems are independent in location and developmental timing and that they are subject to different evolutionary pressures (Burke, 2011). Changes to the larval nervous system in a non-feeding larva can include the position of the apical nervous system relative to the anterior end, loss of neuronal tracts associated with ciliary bands used for feeding, loss of neurons associated with the larval digestive system, and the timing of neural development relative to other structures. Neural systems in non-feeding larval forms may be associated with sensory systems and control over cilia for swimming.

The development of the brittle star musculature has been mentioned briefly (MacBride, 1907; Olsen, 1942), but the present study may be the first to describe the development of the juvenile muscles in any brittle star larva. The first muscles to develop in the vitellaria larva control the movement of the tube feet, buccal podia, polian vesicles, terminal tentacle, jaws, and mouth (Hyman, 1955). Muscles that develop later control the movement of the arms. In addition, there are muscle fibers associated with the epigastric coelom lining the body wall and stomach (Fig. 5H) and with additional parts of the water vascular system (ring canal, radial canals, stone canal, and pore canal). This additional staining supports the findings that these structures are made partly of a myoepithelium (Hyman, 1955; Byrne, 1994). The sequence of muscle development in the vitellaria is logical. A pelagic vitellaria larva, which these brooded vitellaria larvae resemble, would settle after a few days and move around and sense the substrate by using the tube feet and terminal tentacles. They would use their buccal podia and jaws to bring food to the stomach. Only later do the arms develop multiple segments, and thus the muscles controlling the arms develop later.

Within the entire class of ophiuroids, there are diverse developmental modes and larval types leading to the juvenile brittle star body plan (Strathmann and Rumrill, 1987; Hendler, 1991). At least eight different developmental patterns occur and include (1) ophiopluteus larvae, (2) reduced ophiopluteus larvae, (3) vitellaria larvae, (4) reduced vitellaria larvae, (5) abbreviated or direct developing internally brooded larvae (viviparous or ovoviviparous), (6) abbreviated or direct developing externally brooded larvae, (7) abbreviated or direct developing larvae that develop within a capsule, and (8) planktotrophic-like larvae that are brooded. With growing capabilities to resolve phylogenetic trees (Hugall et al., 2016; O'Hara et al., 2017) by using comparisons of molecular sequences, the evolution of development within the ophiuroids, including the evolution of the vitellaria larva, will be better understood. Ophiuroid development can be challenging to study because of the difficulties in obtaining gametes and embryos. However, there is so little known about these organisms that, even with the challenges, it would be extremely informative to develop comparative and molecular studies in order to learn more about the evolutionary relationships, development, and evolution of development within the ophiuroids and how they compare with evolution and development within the entire echinoderm phylum.

Acknowledgments

The authors wish to thank the College of Science and the Thomas H. Gosnell School of Life Sciences at Rochester Institute of Technology (RIT) for undergraduate research support, Maureen Ferran and Anne Houtman for critically reviewing the manuscript, and the reviewers for especially helpful feedback. The authors also acknowledge Cheryl A. Hanzlik, the Confocal Microscopy Lab at RIT, and National Science Foundation (NSF) Major Research Instrumentation award 1126629 for assistance with laser scanning confocal microscopy. BJS was supported in part by NSF Undergraduate Research and Mentoring in the Biological Sciences award 0829259.

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HYLA C. SWEET (*), MEGAN C. DOOLIN, CHELSEA N. YANOWIAK, ASHLEY D. COOTS, ALEC W. FREYN, JANE M. ARMSTRONG, AND BARBARA J. SPIECKER

Thomas H. Gosnell School of Life Sciences, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, New York 14623

Received 25 August 2017; Accepted 29 November 2018; Published online 12 March 2019.

(*) To whom correspondence should be addressed. Email: hxssbi@rit.edu.

Abbreviations: PBS. phosphate-buffered saline: PBST. PBS with Tween.

DOl: 10.1086/701916
Table 1
General observations on the development of Ophioplocus esmarki

Stage              Observations         Timing    Figures

16 cells           Surrounded by a      7 hours   Fig. 1B
                   fertilization
                   envelope
Gastrula           Elongated,           4 days    Fig. 1C
                   bilaterally
                   symmetrical
Pre-vitellaria     Bilaterally          6 days    Fig. 1D, 2A, 2A',
                   symmetrical                    3A, B
                   ectodermal
                   ridges form,
                   including a
                   preoral lobe;
                   embryo is
                   uniformly
                   ciliated with
                   denser cilia
                   associated with
                   the ectodermal
                   ridges; larval
                   neurons are
                   prominent at
                   the base of
                   the preoral
                   lobe on the
                   dorsal side
Early vitellaria   Preoral lobe         11 days   Fig. 1E, F. 2B, 2B',
                   has started to                 3C, D
                   twist; posterior
                   structures have
                   started to rotate
                   to the right;
                   oral field has
                   formed; cilia
                   form thick
                   tracts on the
                   ectoderm
                   ridges; some
                   larval neurons
                   are associated
                   with ectodermal
                   ridges
Mid-vitellaria     Preoral lobe         12 days   Fig. 1G, H, 3E-H
                   forms a
                   W-shaped
                   structure;
                   terminal
                   tentacle and
                   tube feet
                   can be seen
                   at the surface
                   of the oral
                   field; larval
                   neurons are
                   on ventral
                   and dorsal
                   sides of
                   preoral lobe
                   base and form
                   projections
                   to ectodermal
                   ridges; neurons
                   are associated
                   with the axial
                   complex; larval
                   neurons are
                   not extensively
                   associated with
                   the ciliary
                   bands; larval
                   neurons are
                   not in close
                   proximity to
                   where the
                   juvenile
                   neurons
                   will form
Late vitellaria    Preoral lobe         13 days   Fig. 11, J, 2C. 2C',
                   is cylindrical                 4A-F
                   with two
                   raised bands
                   of ectoderm;
                   buccal podia
                   can be seen
                   in the oral
                   field; epineural
                   folds have
                   grown
                   interradially
                   toward the
                   mouth to
                   separate the
                   developing
                   arms; tracts
                   of cilia are
                   thinner and
                   more connected
                   to form bands;
                   cilia are
                   associated with
                   the hydropore;
                   larval neurons
                   and neurons
                   in the axial
                   region are
                   still present;
                   juvenile
                   nerves and
                   muscles begin
                   to form;
                   muscles
                   staining
                   occurs in
                   the water
                   vascular
                   system;
                   muscle fibers
                   extend
                   dorsally from
                   the base of
                   the tube
                   feet into
                   the body
                   disk
Juvenile           Juvenile has         19 days   Fig. 1K, L, 2D, 2D',
                   five-fold                      4G-L, 5A-H
                   symmetry;
                   the original
                   anterior end
                   includes
                   remnants of
                   the preoral
                   lobe, hydropore,
                   and axial
                   complex;
                   cilia are
                   sparse but
                   are associated
                   with some
                   juvenile
                   structures
                   including the
                   hydropore;
                   larval neurons
                   are not apparent;
                   axial neurons
                   are positioned
                   to the right
                   of the stone
                   canal: juvenile
                   nervous system
                   is associated
                   with the ring
                   nerve, radial
                   nerves, tube
                   feet, terminal
                   tentacles, buccal
                   podia, and
                   jaws; juvenile
                   muscles are
                   associated with
                   feeding structures
                   (mouth, buccal
                   podia, jaws),
                   movement
                   (terminal
                   tentacle, tube
                   feet), and the
                   water vascular
                   system (stone
                   canal, pore
                   canal, polian
                   vesicles, radial
                   canals); a
                   network of
                   muscle fibers
                   has formed
                   on the dorsal
                   side of the
                   body disk

Stage              Other names

16 cells
Gastrula
Pre-vitellaria     Stage D (Brooks and Grave, 1899); early vitellaria
                   (Cisternas and Byrne, 2005); early premetamorphic
                   stage (Fourgon et al., 2005)
Early vitellaria   Mid-vitellaria (Cisternas and Byrne, 2005); early
                   metamorphic stage (Fourgon et al., 2005)
Mid-vitellaria     Stage E (Brooks and Grave, 1899); advanced vitellaria
                   (Cisternas and Byrne, 2005); late metamorphic stage
                   (Fourgon et al., 2005)
Late vitellaria    Stage F (Brooks and Grave, 1899); advanced vitellaria
                   (Cisternas and Byrne, 2005); post larva (Fourgon et
                   al. 2005)
Juvenile

A summary of observations of development, timing of the stages at 12-15
[degrees]C, figures in which the stages are shown, and other names of
stages used in previous studies.
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Author:Sweet, Hyla C.; Doolin, Megan C.; Yanowiak, Chelsea N.; Coots, Ashley D.; Freyn, Alec W.; Armstrong,
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Date:Apr 1, 2019
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