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Larval development and metamorphosis of the deep-sea Cidaroid urchin Cidaris blakei.

Introduction

Extant echinoids are divided into two major sister clades, the Cidaroida and the Euechinoida. Euechinoids are diverse, comprising more than 720 living species, including both regular and irregular echinoids (Smith, 1984; Kroh and Smith, 2010). Abundant literature is available on the embryonic and larval development of euechinoids (reviewed by Guidice, 1986; Emlet et at., 1987; Pearse and Cameron, 1991). Cidaroida, the order of "pencil urchins," is much less diverse, comprising 29 genera and 129 living species (Kroh and Mooi, 2010). Since they first appeared in the Permian (definitively in the lower Zechstein, 255 mya) cidaroids have undergone few morphological changes and are often regarded as "living fossils" (Kier, 1977; Smith and Holling-worth, 1990; Smith, 2005; Smith et al., 2006). Relatively few studies have detailed cidaroid development. The only planktotrophic cidaroid reared through metamorphosis and fully described is Eucidaris thouarsi, a shallow-water urchin collected in the Bay of Panama (Emlet, 1988). Other species for which some portion of development has been described include Eucidaris metularia and Eucidaris tribu-thides (Mortensen, 1937; Schroeder, 1981), Cidaris cidaris (Prouho, 1887), and Prionocidaris baculosa (Mortensen, 1938). Development in two lecithotrophic species, Phyllacanthus parvispinus and Phyllacanthus imperialis (Parks et al., 1989; Olson et al., 1993) has also been described.

The reproduction, spawning, and early larval development of Cidaris blakei were studied more than 20 years ago by C. M. Young, J. L. Cameron, and P. A. Tyler, but none of the cultures survived to metamorphosis (C. M. Young, unpubl. obs.). Only the reproductive periodicity, breeding behavior, and egg size have been reported (Young, 1994a, 2003). In the present study, we extend and complete this preliminary work by describing the complete embryonic and larval development through metamorphosis and into the juvenile stage. This is the first description of a deep-sea echinoid reared through metamorphosis. Previous studies of the morphology, larval duration, physiological tolerances, and developmental timetables of deep-sea larvae from the bathyal zone have been reviewed by Young (2003). The long-held belief that brooding is the dominant mechanism of reproduction in the deep sea (Thorson, 1950) has been summarily disproved (Pearse, 1994; Young, 1994b). It is now known that deep-sea organisms employ a wide range of developmental modes (reviewed by Young, 2003). Although planktotrophy is not uncommon in the deep sea (e.g., Bouchet and Waren, 1994; Young et at, 1998; Van Gaest, 2006), there is still the question of what planktotrophic larvae might eat as they begin to develop far below the productive waters of the euphotic zone. Evidence for ontogenetic vertical migration by deep-sea gastropods includes isotopic analyses of the protoconchs of bathyal and abyssal gastropods (Rex and Waren, 1982), and capture and identification of the veligers of deep-sea molluscs in surface plankton tows (reviewed by Bouchet and Waren, 1994; Van Gaest, 2006; Arellano and Young, 2009). Other studies have shown that physiological tolerances might present a barrier to migration for some species but not for others (Young and Cameron, 1989; Young and Tyler, 1993; Young et at, 1996, 1998).

In this paper we compare development of C. blakei with that of other cidaroids and also other deep-sea echinoids. We discuss which characteristics are common to cidaroids, and potentially attributable to phylogenetic constraints, and which adaptations may be specific to the deep-sea environment. Additionally, we describe morphological features not previously observed in a planktotrophic echinoid and provide preliminary evidence for physiological barriers to ontogenetic vertical migration.

Materials and Methods

Collection and husbandry

Adult specimens of the deep-water echinoid Cidaris blakei (A. Agassiz, 1878) were collected from 12 sites in the northern Bahamas, using the manned submersible Johnson Sea-Link II (Harbor Branch Oceanographic Research Institute, Fort Pierce, FL). C. blakei was collected at depths ranging from 540 m to 685 m on 14 and 22 May 2008. In situ temperatures at the collection sites ranged from 11 to 13 [degrees] C. Organisms were brought from depth to the ship in closed, seawater-filled containers and immediately transferred into aquaria in a 13 [degrees] C cold room. At the end of the cruise, on 25 May, adult specimens were transported to the Oregon Institute of Marine Biology in Charleston, Oregon, and placed in seawater tables with recirculating filtered seawater maintained at 11 [degrees] C and a salinity of 32. Adult C. blakei were fed algae (e.g., Ulva sp. and Sargassum muticum) as well as encrusting organisms including the bryozoan Membranipom membranacea, the sponge Halichondria panacea, and the gorgonian Leptogorgia sp., all collected from the intertidal and subtidal zones of Oregon.

Spawning and culturing

On 12 June 2008, we induced spawning of C. blakei with an intracoelomic injection of 1.0-5.0 ml of 0.55 mol [1.sup.-1] KCL. Adults of C. blakei were spawned again on 23 and 25 June 2008, using intracoelomic injections of 0.5-1 ml of 10 mmol [1.sup.-1] acetylcholine in 0.45 [micro]m of filtered seawater (FSW). Sperm from seven different males (one on 12 June, four on 23 June, and two on 25 June) were used to fertilize eggs from three different females. Within hours of fertilization, embryos were transferred to 1-1 beakers of Millipore-filtered (0.45 pm) seawater. Cultures were stirred constantly using swinging paddles (about 12 cycles per min) that hung from a rack similar to that described by Strathmann (1987). The embryos and larvae resulting from the 12 June spawn-ing were kept in a 16 [degrees] C incubator. These did not survive, so subsequent cultures were maintained between 11 and 13 [degrees] C by partial submersion in flowing seawater tables. Larval cultures were cleaned every 3 days by reverse filtration through a 100-[micro]m-mesh sieve. Once larvae developed complete digestive tracts, they were fed a combination of Chaetoceros gracility, Dunaliella tertiolecta, and Rhodomonas lens at a total concentration of 2000 cell/ml. Algae were cultured in f/2 medium (Guillard, 1975). Algal cells were removed from the culture medium by centrifugation and decanting and were then re-suspended in FSW prior to feeding to the larvae.

About 90 days after spawning, larvae with well-developed rudiments were moved from the larval cultures into small dishes with spines and test plates from dead adults. Once they had settled and metamorphosed, juveniles were kept in culture dishes maintained at 11 [degrees] C and fed the alga Ulva sp. that had been macerated with a razor blade. Juve-niles were also fed macerated and whole colonies of the arborescent bryozoan Bugula pacifica. Individuals consumed some Ulva and were seen on the bryozoan and with bryozoan fragments in their guts.

Microscopy

A Nikon Coolpix 4500 camera mounted on either an Olympus BH-2 compound microscope or an Olympus SZH10 dissecting microscope was used to photograph embryos and larvae. Absolute measurements were determined with calibrated ocular micrometers or by photographing a stage micrometer at the same magnification as that of a larva or embryo of interest.

For scanning electron microscopy (SEM), samples were fixed in 10% buffered formalin, then washed and stored in Millonig's phosphate buffer. Samples were post-fixed in 2% osmium tetroxide, washed in phosphate buffer, and dehydrated through an alcohol series before critical-point-drying, mounting, and sputter-coating with gold/palladium. All SEM micrographs were taken on a Tescan VEGA TC microscope.

Temperature tolerances

We tested the ability of larvae of C. blakei to survive a range of temperatures that they might experience during an ontogenetic vertical migration to the upper water column by exposing them to temperatures representing the thermal profile of the Bahamas during their spawning season (Young et at, 1998). Fifty days after fertilization, we placed ten 8-arm plutei in each of six 20-ml scintillation vials. These vials were then placed in an aluminum temperature-gradient block (Young and Sewell, 1999) with one recirculating water bath at each end that maintained a thermal gradient from 11 to 22 [degrees] C. Three replicate vials, each containing 10 plutei, were moved up through the thermal gradient with a 12-h acclimation period for every 2-degree temperature increase. As a control, three additional vials containing 10 larvae each were held in the thermal block at 11 [degrees] C. At every temperature transfer we checked the larvae for survivorship and noted morphological changes.

Results

Development of Cidaris blakei from fertilization to metamorphosis took 120 days (Table 1). After injection on 12, 23, and 25 June 2008, a single female on each date shed eggs that were large and opaque relative to eggs of other echinoids with planktotrophic development. Eggs of the female spawned on 12 June 2008 were 160.6 [micro]m ([+ or -] 3.3 [micro]m SD, n = 20) in diameter. Eggs of the females spawned on 23 and 25 June had mean diameters of 153.1 [micro]m ([+ or -] 5.0 [micro]m SD, n = 20) and 158.1 ([+ or -] 4.9 [micro]m SD, n = 20), respectively (Fig. 1A).

Table 1
Developmental timetable of Cidaris blakei, cultured at 11-13
[degrees] C, from fertilization to metamorphosis

Time since     Developmental stage
fertilization

2.5 h          Two-cell embryos
6h             Four-cell embryos
7.5 h          Eight-cell embryos
9 h            16-cell embryos
23 h           Unhatched blastulae; wrinkled with a clear
               blastocoel and irregular blastoderm
47 h *         Hatched blastulae; apical tuft visible
73 h           Initiation of gastrulation
4 days         Mid-stage gastrulae; archenteron about 1/2 way into
               blastocoel
7 days         Gastrulae that are compressed; first spicules
               visible with cross-polarized light
8 days         Prisms
14 days        Two-arm plutei with open mouths
33 days        Four and six-arm plutei
48 days        Formation of lobes begins
79 days        Juvenile rudiments with podial buds and
               pedicellariae
120 days       Metamorphosis; juvenile spine count ranges from 5
               to 23

* Blastulae were unhatched at 23 and 33 h. We saw hatching begin
between 46 and 48 h in three cultures.


The blastomeres of early embryos were widely spaced (Fig. 1 B, C, D). Attempts to visualize the hyaline layer on the embryos of C blakei by using light microscopy were unsuccessful, suggesting that the hyaline layer was either very reduced or absent. The blastomeres appeared to be constrained only by the perimeter of the fertilization envelope. Multicellular and morula stages were disorganized (Fig. 1C, D), and unhatched blastulae were irregularly spherical (Fig. 1E). In many but not all cases after the fourth cleavage, there were smaller cells present at one end (presumed vegetal) of the embryo, numbering anywhere from zero to five (Fig. 1C). Primary mesenchyme was not specifically noted in the gastrulae of C. blakei, although we observed a mass of cells in the middle of the embryo (Fig. 1F). Gastrulae were yellow-white and very opaque, with numerous red pigment cells in the ectoderm. Because of the opacity of the embryos, we were unable to observe and describe coelom formation. Seven days after fertilization, during the gastrula stage, a pair of calcified spicules was evident when viewed with cross-polarized light (Fig. 1G). The prism stage formed by day 8 as the pair of skeletal elements grew into triradiate spicules (Fig. 1H, I). They formed fenestrated postoral rods characteristic of all cidaroid larvae that have skeletons. The rods were fenestrated from base to tip (Fig. 1I).

Later embryos of C. blakei were different from those of other cidaroids with feeding larvae in several ways. The ectoderm of 4-day-old gastrulae had "pits," or invaginations (Fig. 2A, B). Though absent in some other cidaroid embyros, an apical tuft (83-gm long on a 165-[micro]m-long embryo) first became evident in recently hatched blastulae and persisted through the early two-arm pluteus stage (Figs. 1F, 3A). The mouth of C. blakei did not open until well into the two-arm pluteus stage, 14 days after fertilization (Fig. 3A, B). Fifteen days after fertilization, the anterolateral arms began to elongate from the preoral hood and the postoral arms were 484 [micro]m (Fig. 3B). After this stage, the paired, fenestrated posterodorsal spicules also began to form and lengthen (Fig. 3C). The postoral arms of 25-day-old plutei were 680-[micro]m long. Subsequently a pair of lobes, the posterior lobes, formed at the posterior end of the larva between the postoral and posterodorsal arms. A second pair of epi-dermal lobes, the ventro-posterior lobes, were growing ven-tro-medially at the base of the posterodorsal arms 51 days after fertilization (Table 1, Fig. 3C); a third pair, the antero-ventral lobes, are also seen at this stage (Fig. 3C). Larvae eventually formed five pairs of lobes: the posterior pair, two pairs on the dorsal surface, and two more pairs on the ventral surface. The oral region of the juvenile did not develop within a vestibule as has been shown repeatedly and without exception for euechinoids. Juvenile structures first became evident 77 days after fertilization (Table 1) in the form of podial buds exposed on the left side of the larva and a single pedicellaria at the base of the posterodorsal arms (Fig. 4A, B).

Juvenile structures continued to develop for 43 more days before the first larva metamorphosed (Table 1). Unfortunately, most cultures became infected with bacteria at about 80 days after fertilization, and only nine larvae survived to metamorphosis. Of these, all metamorphosed with a set of five primary podia growing from the left hydrocoel and between one and three pedicellariae, one on the dorsal surface associated with the base of the dorsal arch spicule, and two posterior on the left side--one at the base of the posterodorsal arm rod and one at the base of the pastoral arm rod (Fig. 4A, B). In all competent larvae, there was always a pedicellaria associated with the dorsal arch spicule. All spines present at metamorphosis were of the same type, and they varied widely in number between 5 and 23 (Fig. 5A, B). We saw competent larvae using their podia to adhere to the bottoms of dishes. Some larvae metamorphosed (lost larval traits) in less than a day, whereas others metamorphosed more gradually over several days. Competent larvae would adhere to the substratum with their podia; subsequently they would reorient their larval arms toward the posterior of the larval body and resorb the tissue from the arms so that only larval rods remained. All during this time, 'juvenile spines were becoming more apparent and the body was becoming round when viewed from the aboral side.

Juveniles

One day after metamorphosis, juvenile test diameter was 471 [micro]m ([+ or -] 49 pm SD, n = 4). Juvenile spines after metamorphosis were all identical in morphology. They were straight, gradually tapered to a simple point, and lacked any outwardly directed points at their distal ends (Fig. 5B, C). Adult spines developed 14 days after metamorphosis. These spines were more stout at their bases than juvenile spines, grew long, and had spinelets that projected distolaterally at regular intervals along their lengths. The first set of secondary podia appeared 20 days after metamorphosis. At 35 days after metamorphosis, the two remaining juveniles had test diameters of 769 gm and 762 gm, and they had buccal plates and calcified teeth (40-day-old juveniles are shown in Fig. SC, D).

Temperature

We were unable to culture C. blakei larvae at temperatures higher than 12 [degrees] C. Cultures placed in incubators and maintained at 16 [degrees] C did not survive. Attempts to acclimate larvae to increased temperatures using the thermal block were also unsuccessful. After 24 h and a temperature increase to 15 [degrees] C, 100% of plutei in all vials became morphologically abnormal. At 22 [degrees] C, all larvae had resorbed arms, deformed mouths, and reduced or lost lobes compared to control plutei held at 11 [degrees] C (Fig. 6).

Discussion

Characteristic embryology and larval form of cidaroids

In many respects, the development of Cidaris blakei resembles that described for other cidaroids. In early development there is an apparent lack of a hyaline layer, irregularly arranged blastomeres, variable numbers of micromeres, and an absence of primary mesenchyme (Schroeder, 1981). Later in development there are five pairs of lobes arising from the ciliated band (Prouho, 1887; Mortensen, 1937, 1938; Emlet, 1988). As in both the planktotrOphic cidaroid Eucidaris thouarsi and the lecithotrophic cidaroid Phyllacanthus imperialis, a vestibule for juvenile rudiment formation is lacking (Emlet, 1988; Olson et al., 1993).

For all species of cidaroids in which early development has been described, the blastomeres are widely spaced. In Prionocidaris baculosa, Mortensen (1938, p. 14) observed that the cells were so separate after first cleavage that they formed twins from a single egg. Mortensen also noted that a large percentage of embryos were of an unusual shape that ultimately went on to form a normal, spherical blastula. Schroeder (1981, p. 145) noted the same phenomenon in Eucidaris tribuloides and cited a "virtual absence" of the hyaline layer. Though we did not notice any instances in which two embryos developed within the same fertilization envelope, our observations of relatively large distances between the cells of developing embryos, our inability to see a hyaline layer, and our finding that embryos that were irregularly shaped after fourth cleavage later formed regular blastulae certainly concur with those of Mortensen (1938) and Schroeder (1981).

In euechinoids, there are two types of mesenchyme cells. The first mesenchyme cells to ingress are called primary mesenchyme and are derived from the small micromeres that arise at the vegetal pole of the embryo during an unequal fourth cleavage. These primary mesenchyme cells produce the larval skeleton. The secondary mesenchyme cells ingress later and are associated with pigment cells and musculature (Horstadius, 1973; Okazaki, 1975). Although cidaroids appear to lack primary mesenchyme, the skeleto-genic cells in E. tribuloides derive from the 16-cell micromeres just as they do in euechinoids, and these cells ingress earlier than other mesenchyme cells but not as early as in euechinoids (Wray and McClay, 1988). In experiments on cell lineage in E. tribuloides (Wray and McClay, 1988), blastomeres were size-fractionated using a sucrose gradient, then micromeres from donor embryos were combined with host blastomeres to form chimeras. The small donor cells went on to form the larval skeleton. In a much earlier study, Horstadius (1939) showed that when different numbers of micromeres were added to host embryos from which micromeres had been removed, the outcomes were vastly different. For instance, addition of a single micromere to isolated rings of animal or vegetal cells resulted in a ciliated blob, whereas the addition of four micromeres resulted in a normal pluteus. It would be interesting to know whether cidaroids, which already have variable numbers of micrometes, would also show a difference in development if these same experiments were performed.

Some embryos of C. blakei were observed without any micromeres, or at least no size distinction was evident between cells. Euechinoids embryos from which micromeres have been excised still produce a larval skeleton, but the skeleton is derived from the veg2 cells (Horstadius, 1973). It is possible that the same is true for those embryos of C. blakei that lack micromeres. Embryos from which micromeres have been removed have delayed initiation of gastrulation and a slower rate of archenteron elongation compared to embryos with micromeres (Ishizuka et al., 2001). Alternatively, the size distinction may be volumetric, and cells with a skeletogenic fate may not always be smaller than other cells. It is possible that the extended time before spicules first appear in C. blakei (e.g., 8 days compared to 72 h in Strongylocentrotus droebachiensis, which has similar egg size and was reared at a lower temperature [Stephens, 1972]), is due to many of the embryos having few or no micromeres.

The only cidaroid for which primary mesenchyme has been noted is Cidaris cidaris (Prouho, 1887). Wray and McClay (1988, p. 313) suggested that Prouho's drawing of primary mesenchyme on C. cidaris "may actually represent the very early tip of the invaginating archenteron, which in Eucidaris is loosely organized and could be mistaken for ingressing cells." We observed C. Nakei at a similar stage and noted a cluster of cells very similar to that figured by Prouho. This stage was soon followed by elongation of the archenteron, and further investigation is warranted to determine the origin of these cells.

Juvenile structures developed on C. hlakei larvae in the absence of a vestibule, as has been described for the planktotrophic cidaroid E. thouarsi and the direct-developing cidaroids Phyllacanthus parvispinus and P. imperious (Emlet, 1988; Parks et al., 1989; Olson et al., 1993). In euechinoids, the vestibule is formed from an invagination of the larval epidermis that comes into contact with the left hydrocoel. In those species without an invagination, the structures developed on the larval epidermis, resulting in an apparently simpler metamorphosis compared to the eversion of the vestibule in euechinoids. This type of avestibular rudiment has now been described for two lecithotrophic and two planktotrophic cidaroids in three genera and from both shallow and deep-water habitats; it seems likely, therefore, that this character is conserved in the entire order.

Differences among cidaroids

The larvae of C. blakei differ from other planktotrophic cidaroids in several ways that distinguish them from other species or genera. These include egg size, the length of larval life, the presence of an apical tuft (present only in the congener C. cidaris), pits on the ectoderm of gastrulae, a mouth that opens late in the two-arm pluteus stage, and juvenile spine morphology.

The eggs of C. blakei are among the largest reported for planktotrophic cidaroids (Table 2). They represent an almost 5-fold increase in volume over the eggs of E. thouarsi, E. tribuloides, and E. metularict (Table 2). In general, echinoids with larger eggs (and therefore more maternal investment in materials and energy) have shorter developmental times to metamorphosis than those with smaller eggs (Emlet et al., 1987; Emlet, 1995; Levitan, 2000). Mortensen (1938, p. 15) considered the differences in time to skeletal formation and time to two-armed pluteus in P. baculosa and E. metularia (reared in consecutive summers along the Red Sea) as "an enormous difference in the rate of the developmental processes of these two species." Development to these stages was essentially four times faster for P. baculosa than for E. metularia, and Mortensen postulated that the difference was due to the smaller egg of E. metularia. Though the extended developmental period of C. blakei (a 4-fold time increase over the smaller-egged Eucidaris species) seems somewhat anomalous in this regard, there was a large difference between the rearing temperature of C. blakei and those of other cidaroid larvae that have been studied (Table 2).

Table 2
Comparison of known cidaroid larvae with respect to egg size,
developmental time, and morphology

Species            Depth       Egg     Egg  Apical        Time to
               collected  diameter  volume  tuft *  metamorphosis
                     (m)     ([mu]    (nl)                 (days)
                                m)

Cidaris               ND       170    2.57  P       (reared for 3
cidaris                                                   months)

Cidaris          450-685       157    2.03  P                 120
blakei

Eucidaris             ND        90    0.38  A                  ND
metularia

Eucidaris            2-3        90    0.38  A                  30
thouarsi

Eucidaris            3-5        95    0.45  A                  ND
tribuloides

Prionocidaris         73       150    1.77  A                  30
baculosa

Species        Temperature  Juvenile  Source
                ([degrees]      test
                        C)  diameter
                             ([mu]m)

Cidaris         11[dagger]        ND  Prouho (1887)
cidaris

Cidaris                 11       471  This study
blakei

Eucidaris       23 [double        ND  Mortensen
metularia          dagger]            (1937)

Eucidaris               28       510  Emlet(1988)
thouarsi

Eucidaris               26        ND  Schroeder(1981)
tribuloides

Prionocidaris   23 [double       375  Mortensen
baculosa           dagger]            (1938)

ND = no data available.
* P = present: A = absent.
[dagger] Prouho did not note the temperature at which larvae of C.
cidaris were reared. This value is the temperature of surface water
at the location and during
the season where the study was completed.
[double dagger] Mortensen did not regulate the exact temperatures
at which he reared E. metidaria and P. baculosa. These values are
a rough estimate of culture temperature based on notes in the text.


Temperature affects the rate at which larvae develop, with increased temperatures shortening time to metamorphosis (e.g., Emlet et at, 1987; Pearse and Cameron, 1991; Emlet, 1995). E. thouarsi metamorphosed after only 30 days at 28 [degrees] C (Emlet, 1987). P. baculosa also metamorphosed 30 days after fertilization, at temperatures above 23 [degrees] C (Mortensen, 1937). The only cidaroid with a developmental timetable similar to that of C. blakei is the congeneric C. cidaris (Prouho, 1887). Although C. cidaris was not reared to metamorphosis (and Prouho does not note the appearance of any juvenile structures), his final observations were of a larva cultured for 3 months. In his study of C. cidaris, Prouho (1887) does not include the temperature at which cultures were reared, but he does note (somewhat surprisingly) that the urchins spawned without chemical stimulation in the month of February at the Arago Laboratory, which is situated on the Mediterranean Sea on the southeastern coast of France, where surface water temperatures in February have been recently recorded as 11.5 [degrees] C (Charles et at, 2005).0n the basis of this evidence, the congeners C. cidaris and C. blakei have a similar developmental timetable at similar temperatures. We cautiously suggest that prolonged development at relatively cool temperatures is characteristic of this genus, assuming adequate nutrition and culture conditions.

To examine whether the extended larval duration of C. blakei is a functional adaptation to life in the deep sea or a result of phylogenetic relationships, we must first try to normalize for the wide variation in temperatures at which cidaroid larvae have been raised. Previous studies have used [Q.sub.10], the factor by which the rate of a process changes with a 10 [degrees] C increase in temperature, to compensate for differences in temperature when comparing larvae (Emlet, 1995). Previous studies on echinoid [Q.sub.10] values have shown a range of 3.0-3.6 for the tropical urchins Lytechinus variegatus and Echinometra lucunter, and for the temperate sand dollar Dendraster excentricus (Cameron et al., 1985; McEdward, 1985). If we apply this range of [Q.sub.10] values to E. thouarsi and C. blakei and normalize the temperature to 20 [degrees] C, there is still a wide difference in developmental times, but in this case C. blakei would reach metamorphosis twice as fast as E. thouarsi (Table 3). This supports previous evidence that smaller eggs have a longer pre-metamorphic stage. Other factors, such as depth of distribution, resource availability, and culture conditions have also been shown to affect developmental duration. Low food concentrations and increased depth can increase developmental times (Strath-mann, 1987; Emlet, 1995), and we might expect to see even longer developmental times for C. blakei in situ.

Table 3
Days to metamorphosis of Cidaris blakei and Eucidaris
thouarsi when temperature is adjusted to 20 [degrees] C
using the equation for
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

                    Days to metamorphosis

[Q.sub.10] values  C. blakei  E. thouarsi

3.0                       45           72
3.2                       42           76
3.4                       40           80
3.6                       38           84

Original data were 120 days to metamorphosis at 11 [degrees] C
for C. blakei (this study) and 30 days to metamorphosis at
28 [degrees] C for E. thouarsi (Emlet, 1988)


Extended developmental periods are not unknown for deep-sea larvae. The bathyal echinoid Aspidodiaclema jacobyi was reared in culture for 5 months (Young and George, 2000). The cold-seep gastropod Bathynerita naticoidea may spend up to one year in the water column before settlement (Van Gaest, 2006), and the deep-sea mussel "Bathymodiolus" childressi has been estimated to spend between 9 and 13 months in the plankton (Arellano and Young, 2009). This extended larval period may result in long-range dispersal and a wider geographic range (Arellano and Young, 2009). C. blakei has a relatively small geographic range, being found in the North Atlantic only between the Bahamas and Barbados (Mortensen, 1938). In an analysis of 33 species of regular echinoids, including four species of cidaroids, larval duration was not related to geographic range (Emlet, 1995). The bathyal congeners C. cidaris and C. blakei were reared at similar temperatures and had similar developmental timetables. A notable difference, however, is in the completion of the gut, which occurs in C. cidaris at 8 days (it may have opened before this, but Prouho [1887] first notes an open mouth in a figure of an 8 day-old gastrula), but not until 14 days in C. blakei (this study). It is difficult to discuss this difference in the context of habitat, because Prouho does not note the depth at which he collected his specimens. C. cidaris occurs at a broad range of depths, extending from 50 to 1000 in (Mortensen, 1938). There is, however, an example of a deep-sea euechinoid with a prolonged non-feeding stage. The deep-sea diadematid Aspidodiadema jacobvi, which has eggs only 98 p.m in diameter, was shown to have an extended lecithotrophic stage prior to development of a -complete gut. The. mouth of A. jacobvi did not open until late in the two-arm pluteus stage, 11 to 21 days after fertilization (Young et al., 1989; Young and George. 2000). Young et al. (1989) hypothesized that the extended pre-feeding period of A. jacobvi could be an adaptation to deal with patchy food resources in the deep-sea. This hypothesis could apply to C. blakei as well. Alternatively, the opaque eggs and mass of cellular material demonstrated to occur in blastulae of A. jacobvi (Young et al., 1989) and seen in the gastrulae of C. blakei (Fig. 1F) may impede fusion of the tip of the archenteron with the ectoderm and delay formation of the mouth in each species. Ectodermal pits have been described previously only on the direct-developing, lecithotrophic echinoids Asthenosoma ijimai (an echinothurioid) (Amemiya and Emlet, 1992) and Phyllacanthus! parvispinus. (a cidaroid) (Parks et al., 1989). Amemiya and Emlet (1992) postulated that the ectodermal pits could be associated with the deep wrinkles in lecithotrophic blastulae, but Parks et al. (1989) noted that position and number of pits did not coincide with location of the wrinkles of P. parvispinus. C. blakei did form a wrinkled blastula, probably because blastomeres of the early embryo are widely spaced. We do not know the function or derivation of these invaginations in C. blakei, but their presence on three different species of echinoids warrants further investigation.

When we compare the descriptions of the six planktotrophic cidaroids for which a portion of development has been described, two distinct morphological forms emerge. The three species of Eucidaris have eggs that are 90-95 Am in diameter (Table 2). The prism form of the Eucidaris congeners is triangular, transparent, and has numerous pigment spots. There is no apical tuft present at any stage. Eggs of the two species of Cidaris are 150 [micro]m in diameter or larger. Larvae have an apical tuft, and the prism is elongate and very opaque. Drawings by Mortensen (1938) of the early stages of Prionocidaris bactdosa show that it closely resembles the two species of Cidaris, although it does not have an apical tuft. Additionally, Mortensen (1938) notes that the postoral rods of P. baculosa are fenestrated from base to tip, which is the same as the postoral rods of C. blakei, but differs from E. metularia, in which the fenestration begins farther from the base. In phylogenies constructed using adult morphological characteristics, C. blakei is in the subtribe Cidarina, but Prionocidaris. EucidariS, and Phyllacanthus are all in the sister subtribe Phyllacanthina (Smith and Wright, 1989). Within the Phyllacanthina, Prionocidaris is a sister taxa of the clade containing both Eucidaris and Phyllacanthus. If this interpretation is correct, then among the planktotrophic species, large egg size, apical tuft, and opaque, elongate gastrulae may represent the more primitive condition (Fig. 7).

Juveniles

The juveniles of C. Merkel differ from other cidaroid juveniles in the shape of their juvenile and adult spines. The 471-pm test diameter of C. biakei juveniles falls within the expected range for echinoids (Emlet et al., 1987) and between the reported 350-[micro]m test of newly settled P. baculosa and the 510-[micro]m test of E. thouarsi (Mortensen, 1938; Emlet, 1988). The morphology of the juvenile spines of C. blakei is unique among cidaroids. All other known juvenile cidaroids have spines with a triradiate tip (Fig. 8) (Mortensen, 1938; Emlet, 1988; Parks et al., 1989).

Thermal tolerance and ontogenetic vertical migration

Some larvae from the deep sea may migrate to the euphotic zone. Direct evidence for ontogenetic vertical migration of deep-sea organisms comes from capture of larvae in surface-water plankton tows (reviewed by Bouchet. and Waren, 1994; Van Gaest, 2006; Arellano, 2008). In the laboratory, potential for vertical migration can be explored indirectly by testing the physiological tolerances of embryos and larvae (Van Gaest et al., 2007). If larvae migrate up through the water column, then they must be able to withstand a wide range of temperatures and salinities,. Water temperatures for the northern Bahamas during the spawning season of C. blakei range from 10 [degrees] C at the deepest location at which C. blakei occurs to 25 [degrees] C at the water's surface (Young et at, 1998). Our preliminary results suggest that larvae of C. blakei are unable to withstand temperatures above 15 [degrees] C (Fig. 6). In the Bahamas, this temperature is reached at depths of 300 to 500 m, depending on the season (Young and Cameron, 1989), and therefore larvae are unlikely to successfully migrate into the photic zone. While the larvae of C. blakei ate phytoplankton in laboratory culture, it is unclear what they may feed on below the photic zone. Larvae of some Antarctic asteroids apparently can consume bacteria (Rivkin et at, 1986), and there is limited evidence that some deep-sea echinoderm larvae may also have this ability (Young, Bosch, and Cameron, unpubl. data). However, preliminary studies in our laboratories using cyanobacteria (Synechococcus sp.) as a food source indicate that these larvae do not concentrate cells the size of bacteria (<2 um) (Smart, Bennett, Emlet, and Young, unpubl. data). Given our observation that the larvae of C. blakei can develop all the way to metamorphosis with conventional phytoplanktonic food, it seems plausible that the larvae simply feed on larger phytoplankton cells or seston particles sinking to the depths where larvae reside.

This report on C. blakei is the first published description of a deep-sea echinoid reared through metamorphosis. The development of this species differs in several ways from that of shallow-water cidaroids. Characteristics such as embryonic and larval morphology, ectodermal pits at the gastrula stage, presence of an apical tuft, and juvenile spine morphology may represent the ancestral form of more-derived echinoids, including other cidaroids and euechinoids. Other characteristics, such as an extended larval period, may he adaptations to the deep-sea habitat.

Acknowledgments

This study was supported by NSF grant OCE 0527139 to C. M. Young, R. B. Emlet, and A. M. Wood. K. C. Bennett was supported by the NSF's Graduate Teaching Fellows in GK-12 grants OCE 0338153 and 0638731. Thanks to T. Smart and M. Wolf for their assistance in the laboratory and for editing and commenting on this manuscript.

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KATHLEEN C. BENNETT *, CRAIG M. YOUNG, AND RICHARD B. EMLET

Oregon Institute of Marine Biology and the Department of Biology, University of Oregon, P.O. Box 5389, Charleston, Oregon 97420

Received 22 September 2011; accepted l March 2012.

* To whom correspondence should be addressed. E-mail: k8Obennett@gmail.com
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