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Notice: ©1987 University of Washington Press. This manuscript is an author version with the final publication available and may be cited as: McEuen, F. S. (1987). Phylum Echinodermata, class Holothuroidea. In M. F. Strathmann (Ed.), Reproduction and development of marine invertebrates of the northern Pacific coast: data and methods for the study of eggs, embryos, and larvae (pp. 574-596). Seattle, WA: University of Washington Press.

... McEuen, F. S. 1987. Pp . 574-596, in: Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. Data and Methods f~r the Study of E~~ Embryos, and Larvae. M. Strath­ mann, ed. Univ. of Washing~n Press, Seattle. 670 pp.

PHYLUM ECHINODERMATA, CLASS HOLOTHUROIDEA

F. S. McEuen

Chapter advisors: Richard R. Strathrnann, Scott T. Smiley. and Lane Cameron

INTRODUCTION High primary production, constant tidal flushing, and habitat diversity all help to support a large number of sea cucumber species in the San Juan Archipelago. At least twelve species representing four of the six orders of holothuroids (Dendrochirotida, Aspidochirotida, Apodida, and Molpadiida) occur in relatively high densities within diving range (30 m) in this region. The majority of local sea cucumbers have separate sexes , broadcast their gametes, and produce a non-feeding, planktonic larva. Most free-spawning species reproduce from late winter to late spring and brooding species spawn in late fall and early winter. The more recent comprehensive reviews of holothuroid reproduction and development are by Hyman (1955), Inaba (1968), Boolootian (1966), and Smiley et al. (in press). Studies of larval forms and behavior of northeast Pacific holothuroids have been made by Mortensen (1921) , Johnson (1931), Johnson & Johnson (1950), Strathmann (1971), Young & Chia (1982), McEuen & Chia (1985 and in press), Cameron (1985), McEuen (1986) and Smiley (1986a,b).

REPRODUCTION AND DEVELOPMENT Hermaphroditism is relatively common among small apodids but is rare in other holothuroid groups where separate sexes are the rule. Sexes can be externally distinguished in species which have (1) a thin body wall through which the color of ripe gonads shows, (2) females with incubatory behavior or brood pouches, or (3) morphological differences of the genital papilla. Sea cucumbers possess a single gonad composed of one or two tufts of long, slen­ der tubules, sometimes branched, radiating from a gonad basis. Gonadal walls have an inner epithelium of parietal and follicular epithelial cells, a central connective tissue layer and genital hemal sinus, and an outer peritoneum of epithelial cells, axons, and muscle cells (Smiley & Cloney 1985). Oocytes, surrounded by follicle cells, are connected by a follicular stalk to the inner epithelium. Full-grown oocytes in meiotic prophase I are ovulated into the gonadal lumen where they undergo germinal vesicle breakdown (GVBD) before or during spawning. The specific environmental factors entraining holothuroid gametogenic cycles still remain to be determined. Periods of no feeding and autumn or

574 4

HOWTIlUROIDEA 575

winter torpor are probably connected with gonad and oocyte nutrition and development. Kanatani (1979) summarized work on endocrine substances affecting oocyte maturation and gamete shedding. Follicle cells are stimulated by a peptide in radial nerve factor to produce a secondary factor that induces resumption of meiosis (maturation of the oocyte through GVBD) (Maruyama 1985). Hormonal substances regulating holothuroid reproductive behavior and gamete release remain unknown. To aid mixing and dispersal of gametes, sea cucumbers may lift the anterior end, wave their tentacles, sway from side to side, or forcefully expel the eggs and sperm (McEuen 1986 and in prep .). Free-spawning species have one or more gonopores on a genital papilla positioned dorsally in the tentacle crown or as far as 2-3 cm behind the tentacles on the dorsal surface. Male dendrochirotids typically possess a number of gonopores. Sperm of the primitive type are characteristic of almost all species of holothuroids (Chia et al. 1975), including local species (Everingham 1961; Brooks 1973 ; Atwood 1974a,b, 1975a,b; Chia et al. 1975; Chia & Bickell 1983; Fontaine & Lambert 1976; McEuen & Chia 1985 and in press ; McEuen 1986), with the exception of Cucumaria Iubrica and C. pseudocurata. The spermatozoon of C. Iubrica is fusiform (elongate and cylindrical) (Atwood & Chi a 1974; Atwood 1975a) and that of C. pseudocurata is tabloid in shape (elongate and dorsoventrally compressed) (Atwood 1975a,b). The acrosome of the latter is situated medially on the ventral surface. Species-specific chemoattraction of sperm to egg extracts is evident for some sea cucumbers, but is generally lacking among the Dendrochirotida (Miller 1981, 1985). Eggs of holothuroids are probably spawned at first meiotic metaphase (Holland 1981) and are enclosed in a thick jelly coat. Fertilization is assumed to occur after GVBD and before polar body extrusion. Sperm contact with the egg jelly layer sets off an acrosome reaction, sending an acrosomal tubule through the envelope of jelly to fuse with a fertilization cone (Colwin & Col­ win 1955; Colwin & Colwin 1955, 1956; Colwin et al. 1975). Fusion of sperm and egg membranes begins the cortical reaction, and a fertilization envelope is subsequently formed. No hyaline layer is produced within the perivitelline space (Holland 1981); the low fertilization envelope is consequently difficult to see with light microscopy. Polar bodies are then extruded through the oocyte protuberance (formerly "micropyle appendage", Smiley & Cloney 1985) and may be quickly lost or may be present through the blastula stage. Unfertilized naturally spawned eggs of Psolus chitonoides can form polar bodies (McEuen unpubl.).

Pelagic planktotrophic development. Relatively small (185-210 urn in diameter) eggs are broadcast during mass spawnings in late spring and early summer. A feeding larva develops in the plankton. The only local species with this type of development is Parastichopus californicus. In this type of development, spawned eggs are negatively buoyant and develop into a coeloblastula through radial, equal holoblastic cleavage. The wall of the coeloblastula at the vegetal pole thickens, then flattens into a gastral

.- ... 576 F. S. McEUEN plate. Gastrulation begins by invagination of the gastral plate and subsequent­ ly produces an archenteron one-half to two-thirds the length of the embryo. Within the blastocoel, mesenchyme cells are proliferated from the tip of the archenteron. As the transparent gastrula elongates, a mid-ventral indentation appears; the archenteron bends and fuses with the indentation to form the mouth. The early auricularia is formed with the appearance of a looped band of cilia. Further growth produces arms and lobes over which the single, continu­ ous ciliary band is looped. This band of cilia is employed for feeding and locomotion (see Strathmann 1971, 1974, 1975). The width of the band varies; there are 3-7 cilia across the band, one cilium per cell. The band separates the body surface into aboral and circumoral fields. A transverse groove, along which food is conducted to the mouth, lies between the preoral and postoral transverse portions of the ciliary band. Encircling the mouth is an adoral band of cilia that is continuous with a ciliary band at the top of the esophagus. Calcareous ossicles are present in the posterolateral lobes of the auricu­ laria. The larva is transparent with occasional tinting of the ciliary band. Cells proliferate in the larval body cavity close to where the archenteron bends and form a hydroporic canal that opens on the dorsal surface as the hydropore. Other cells form a pouch, the axohydrocoel, near the base of the esophagus. Metamorphosis commences with diminution of the larval body and sim­ plification of the sinuous looping of the ciliary band. By the time a spherical body shape is assumed, the ciliary band has rearranged into five transverse rings of cilia. Further reduction in body size results in a compact barrel­ shaped doliolarfa. Five primary tentacles, containing evaginations of the hydrocoel, lengthen and push out through the oral indentation. Their protru­ sion marks the onset of the pentactula stage. The knobbed, "sticky" tips of the tentacles attach to the substratum at settlement. During or just after settle­ ment, aspidochirotid pentactulae grow a single ventroposterior podium and a covering coat of ossicles. Planktotrophic development and metamorphosis in holothuroids has been described and reviewed by Metschnikoff (1869), Selenka (1876), Semon (1888), Bury (1889, 1895), Mortensen (1921, 1937, 1938), Chia & Burke (1978), Maruyama (1980), Holland (1981), Cameron (1985), and Smiley (1986a,b).

Fig. 28.1 - 28.6. Holothuroid larvae: (1) Eupentacta quinquesemita larva at 4 days (530-650 urn length j.ventral view showing even ciliation, oral indentation (0) and one podial pit (P) (the other podial pit is still positioned on the larva's right side (arrow) ; (2) Pentamera populifera doliolaria at 5.75 days (just prior to emergence of tentacles) (465-510 um length ), ventral view showing evenly ciliated preoral lobe (L); (3) Psolus chitonoides doliolaria at 6.3 days (925 x 400 urn), dorsal view (posterior end has not yet lost all the cilia); (4 ) Molpadia intermedia doliolaria at 77 hours (390 urn length). dorsal view showing coelomic pouches (C); (5 ) Parastichopus californicus late auricularia at 45-50 days (1120 urn length ). ventral view showing lobe of hydrocoel (H) and ciliary band (dark stippling); (6) P. californicus late doliolaria at 55-60 days (450­ 500 urn length), showing tentacles (D and a midventral water vascular canal (w) that will elongate and protrude posteriorly to form the first podium. -

HOLOTIillROIDEA 577

"':"""'"~--L

1 2

3 4

T

H "'---r-- W

5 6

• • 1 I

578 F. S. McEuEN

Pelagic lecithotrophic development. Large yolky eggs (267-627 urn in diameter), often brightly pigmented, are released in mass spawnings, usually in spring. Approximate periods of peak spawning are listed in Table 28.1. Eggs are released freely as separate eggs, or they are bound in pellets or ropes with a mucoid substance that disperses 10-20 minutes after release. Larger eggs, 500-625 urn in diameter, float at the surface, whereas smaller eggs are neutrally or slightly negatively buoyant. Cleavage is normally equal and holoblastic; slightly unequal blastomeres are not uncommon in embryos originating from large eggs. Wrinkled blastulae, sometimes with large egres­ sion tracts (furrows), occur in some species. Gastrulation is by invagination. Coelomic pouches are formed as outpocketings of the archenteron that even­ tually separate from the archenteron (Runnstrom 1927; Inaba 1930, 1968). Gastrulae of most lecithotrophic species elongate into evenly ciliated early larvae. The' archenteron bends and makes contact with a ventral indentation. Larvae do not feed. Formation of the mouth is often delayed, and the blasto­ pore retains its posterior terminal position as the future anus. Two podial pits (through which the first podia will emerge) appear (in dendrochirotids) in the posterior two-thirds of the larva; one is posterior to the oral indentation and the other is on the right side. The latter eventually assumes a ventral position opposite the left pit. Yolky holothuroid larvae later either remain evenly ciliated or acquire 2-3 rings of cilia. For those larvae with transverse rings, epidermal ridges first form on the posterior two-thirds of the evenly ciliated larval body. Zones of cilia on either side of the ridges are later lost, but the preoral lobe (the pigmented yolk reservoir anterior to the oral indentation) retains its uniform ciliation. Ciliary ring formation and appearance of the five primary tentacles just precede settlement in most species. Major metamorphic changes immediately after settlement include loss of external ciliation, appearance of the first two podia (in dendrochirotids) , loss of the preoral lobe through resorption or differential growth, and growth of protective ossicles. Size at metamorphosis is correlated with egg size (McEuen 1986). Primary works describing lecithotrophic development of holothuroid larvae are by Ohshima (1921), Runnstrom & Runnstrom (1919), Runnstrom (1927), Inaba (1930), Nyholm (1951), Chia & Buchanan (1969), McEuen & Chia (1985 and in press) and McEuen (1986).

Brooded development. Internal and external brooding are known among local holothuroids. Peak spawning and brooding periods occur in late fall and winter (see Table 28.1). Internal, or ovarian, brooding occurs locally in Leptosynapta clarki and possibly L. transgressor (Everingham 1961; Brooks 1973). Egg diameters range from 240 to 404 urn. Young develop within the ovarian tubules; tenta­ cled juveniles consume nurse eggs in the ovary. External brooding occurs in two local cucumariids, Cucumaria lubrica and C. pseudocurata. Large eggs (970-1050 urn in diameter) are placed beneath the trivium (the ventral body region encompassed by the three ventral ambulacra) and kept in position by maternal podia. Larval stages are lacking; -

HOLOTIIUROIDEA 579

TABLE 28.1 DEVELOPMENTAL MODES AND SPAWNING SEASONS OF LOCAL SPECIES

Species Developmental Mode Peak Spawning Season

Parastichopus californicus Pelagic Planktotrophic late May - mid Jul Cucumaria minuua Pelagic Lecithotrophic mid Mar - late Apr Cucumaria piperata Pelagic Lecithotrophic mid Mar - late Apr Cucumaria fallax Pelagic Lecithotrophic mid or late Mar - early May Eupeniactaquinquesemita Pelagic Lecithotrophic late Mar - early or mid May Peniamera populi/era Pelagic Lecithotrophic mid Feb - late Mar Psolidiwn bullatum Pelagic Lecithotrophic late Mar - early May Psolus chitonoides Pelagic Lecithotrophic mid Mar - mid or late May Molpadiaintermedia Pelagic Lecithotrophic mid Nov - mid or late Dec Cucumaria lubrica Externally Brooded mid Nov - mid or late Dec Cucumaria pseudocurata Externally Brooded mid or late Dec - early or mid Jan Leptosynaptaclarki Internally Brooded mid Nov- early Dec (Brooding begin s)

young hatch as pentactulae. Accounts and reviews of holothuroid brooding have been provided by Clark (1907, 1910), Vaney (1925), John (1939 ), Wootton (1949), Hyman (1955), Everingham (1961), Boolootian (1966), Rutherford (1973), Engstrom (1982), and McEuen (1986).

METHODS

Identification. General classification used in this chapter follows Pawson (1982). Keys to shallow- water sea cucumbers from Alaska to California are given by Rutherford (1975), Lambert (1986), and Kozloff (1987). Austin & Deutsch (1978), Lambert (1984) , and Austin (1985) give systematic lists of species, species distributions, and lists of early taxonomic references. Photo­ graphs of some common Pacific coast species , with notes on biology and natural history are provided by Ricketts et ai. (1968), Morris et al. (1980), Kozloff (1983), and Lambert (1986). Habitats of some species are described by Shelford & Towler (1925) and Shelford et al. (1935). No thorough, com­ prehensive monograph exists for northeast Pacific holothuroids. Nomencla­ ture used in this chapter follows that used by authors cited above, but some groups (e.g. dendrochirotids) are in need of revision (D.L. Pawson & M. Bergen pers. comm.). Cucumaria curata and C. pseudocurata are both included under the latter species, according to Lambert (1985).

Collection and Maintenance. Many of the cucumariid species are found on upper and lower surfaces of rocks in high-current areas. These species keep well in flow-through sea water systems, particularly when provided with natural substrata . The relatively plankton-free sea water in aquarium systems 580 F. S. McEuEN cannot sustain specimens in a normal healthy state for long periods. Mud­ dwelling forms do well when mud or sand from the habitat is included in the aquaria. Without mud, Molpadia intermedia begins sloughing body wall after 4-5 days. The delicate Leptosynapta clarki is very likely to autotomize pos­ terior body portions if not handled carefully and if not given sand in which to burrow. Parastichopus californicus must also be handled with care to prevent evisceration; this species is sensitive to warming and hypoxic conditions in crowded collecting buckets. Providing adequate food for the deposit-feeding P. californicus in aquaria presents logistical problems and unfed individuals gradually reduce in size after several weeks. Ripe animals normally will not spawn during collection and transport, ex­ cept for Pentamera populi/era. Spawning can be delayed at least two months in Psolus chitonoides by keeping ripe specimens in dark tanks . Chances of mass spawning can be reduced by removing spawning individuals, particularly males.

Inducing spawning. Reliable methods for inducing spawning have not been established for holothuroids. Many of the local species, if collected early in their spawning season , will release gametes within 1-2 weeks in the labora­ tory (McEuen 1986). Some simulated environmental conditions that may induce spawning by ripe animals in the laboratory are described below. During the height of the spawning season, Psolus chitonoides frequently spawns in early morning hours, apparently in response to increases in light intensity. Allowing early morning sunlight to fall on aquaria normally brings on gamete release within 30-45 minutes. Spawning can be enhanced and can also be induced during afternoon or evening hours by shutting off water flow and allowing warming. Mass spawnings in this and other species are enhanced by addition of "sperm water." Cucumaria miniata discharges gametes in late morning and afternoon. Jordan (1972) found that decreasing sea water temperature by 2°C then warming in a dense concentration of Dunaliella sp. evoked gamete release in C. frondosa , an Atlantic coast form similar in size and ecology to C. miniata. Warming in large bowls sometimes induces spawning by ripe adults of C. piperata. Late afternoon, evening and early night hours are times of reproductive activity in Eupentacta quinquesemita. Good numbers of gametes have been obtained by isolating mature specimens of E. quinquesemita and allowing them to warm in bowls of seawater in the evening. Specimens of Pentamera populi/era should be placed in a large dish with sediment and allowed to burrow; seawater flow is turned off for 10-30 minutes during daylight hours and then resumed with a slow, steady current passing over the sea cucumbers. Often, a few males will discharge sperm during the period of no current, and other individuals follow with spawning when water flow is resumed. r

582 F. S. McEuEN

Oocytes of holothuroids from Japan were artificially induced to mature with disulfide reducing agents (Maruyama 1980). Oocytes that were excised from ripe ovaries and placed in solutions of dithiothreitol (5 x 10-4 to 1 x 10-2 M) for 10 minutes; 2,3-dimercapto-l-paropanol (5 x 10-3 M) and L-cysteine (5 x 10-2 to 1 x 10-1 M) also gave high percentages of oocyte maturation. In unfertilized oocytes matured by dithiothreitol, rupture (by pipetting) of the geminal vesicle before its normal breakdown was followed by polar body formation and parthenogenetic development to the auricularia larva (Maru­ yama 1981). Of these agents, low concentrations of dithiothreitol (10 mM for 10 minutes or less) are recommended (S. Smiley pers. comm.), but only a low percentage of normal development is obtained even with this low dosage. Active sperm may be obtained by opening a testis tubule with forceps, then pipetting sperm into recently filtered sea water. Sperm can be activated by adding aqueous NH4Cl to a final concentration of7-10 mM (Smiley 1986b).

Insemination. The optimal time for insemination is thought to be immedi­ ately following GVBD. For Parastichopus californicus, higher levels of poly­ spermy result when sperm are added to ripe oocytes after formation of the first polar body (Smiley 1986b). Polyspermy also occurred in Stichopus tremulu s eggs inseminated at high temperatures (15°C ; Holland 1981). Wash eggs in filtered sea water; large, yolky eggs are less likely to rupture during washing if they are kept submerged and not allowed to dra in com­ pletely. Procedures for sperm dilution and insemination are the same as those for Asteroidea (see p. 542). Spermatozoa of Cucumaria pseudocurata and C. lubrica are spawned in mucous strands and bundles and are resistant to immediate dilution; diluted C. pseudocurata sperm are particularly slow to be activated.

Cultures. See Chapter 1. Rate of development is variable and influenced by maturation rate , time of fertilization, water temperature, density of embyros or larvae, quantity and quality of food, and individual variation in develop­ mental rate. Gentle stirring of cultures is recommended although larvae can be reared successfully through metamorphosis in standing cultures if sea water is changed every 1-5 days (McEuen unpubl.), The planktotrophic larvae of Parastichopus californicus have been reared on mixtures of the algae Dunaliella tertiolecta , D. salina, lsochrysis galbana, Pavlova lutheri , and Phaeodactylum tricornutum (Strathmann 1971; Cameron 1985; Smiley 1986b; McEuen unpubl.): Mortensen (1921) used unfiltered sea water. The volume of water cleared of algae per time declined at concentra­ tions of D. tertiolecta above 1000 cells per ml, although ingestion rates increased with increasing concentrations up to 5000 cells per ml (Strathmann 1971). Cultures of positively buoyant eggs or embryos must be stirred occa­ sionally; otherwise, coalescence of early embryos in the dense aggregations at the edges of the bowl is not uncommon. T HOL01HUROIDEA 583

For externally brooding species, fertilization levels are high when groups of 15-20 or more adults are kept in a large bowl and allowed to spawn natural­ ly. Both Cucumaria lubrica and C. pseudocurata can be reared from fertiliza­ tion to the juvenile stage in clean conditions without the broodcare of a female.

Settlement and metamorphosis. Feeding larvae remain planktonic for about 1-2 months and lecithotrophic larvae are pelagic for 3.5 -14 days. Most species metamorphose and settle readily in glass bowls without natural sub­ strata. Molpadia intermedia, however, delayed settlement without fine mud from its usual habitat (McEuen & Chia 1985). Psolidium bullatum settled readily in laboratory cultures, but did not complete metamorphosis with formation of the sole (McEuen & Chia, in press). Crowded cultures result in prolonged settlement times and incomplete or abnormal metamorphosis. Chia & Spaulding (1972) observed settlement of Psolus chitonoides on tubes of Phyllochaetopterus sp. in the laboratory. Aspects of holothuroid larval settle­ ment have been reviewed by Strathmann (1978b).

SEM methods. McEuen & Chia (1985) describe procedures that work well with yolky developmental stages. Sati sfactory methods have yet to be developed for critical-point drying of fragile auricularia larvae.

SELECTED LOCAL SPECIES

Distributions and habitats are described by Shelford & Towler (1925) , Shel­ ford et al. (1935), and McEuen (1986). Most data on spawning behavior, egg size, and fecundity given below are from McEuen (1986 and in press). For a more comprehensive review of literature on breeding seasons , see McEuen (1986).

Order Dendrochirotida Cucumaria fallax Ludwig. Occurs with C. miniata in subtidal cobble fields to 35 m and appears to be more abundant in areas of slower current under rocks and in cracks and crevices at greater depths than C. miniata. The adult is tan, light brown, or light yellow, with a white or pinkish introvert, and light pinkish flesh-colored or sometimes light yellow tentacles. Maximum length is 23 em, not including tentacles. The body wall is slimy. In spawning, eggs are released in a long strand or short segments. Tan, or occasionally brown or olive green, the floating eggs are 456-547 urn in diameter (mean = 504 urn) ; fecundity can reach 8,800. Laboratory and field spawnings have been observed mid March - late May (McEuen 1986 and in prep.). The pre­ oral lobe of the early doliolaria larva is orange , and the posterior body is a light yellow. Development is similar to that of C. piperata, with a three­ ringed doliolaria. Recently settled C.fallax, C. miniata, and C. piperata form aggregations in cultures away from light sources and are probably negatively phototactic. A developmental schedule at 10.5-11.5°C is given in Table 28.2.

- 584 F. S. McEUEN

TABLE 28.2 DEVELOPMENTAL SCHEDULES OF HOLOTHUROIDS FROM SAN JUAN ARCHIPELAGO IN CULTURE AT 11 ± O.5°C. (from McEuen & Chia 1985, in press; McEuen 1986, unpubl.) Cl =Cucumaria lubrica, Cm=C. miniata, Cp =C.piperata, Cps =C. pseudocurata, Cf=Cifallax, Eq = Eupeniacta quinquesemita, Pp =Pentamerapopulifera, Pb =Pso/idiumbullatum, Pch =Psoluschitonaides, Pc =Parastichopus califomicus, Mi =Molpadia intermedia. Dashes indicate no data available.

2-cell 4-cell 8-cell 16-cell 32-cell 64-cell blastula a 9-12 h 11-23 h 12-26 h 14-33 h 20-38 h 25-41 h

On 2.75 h 4.5 h 5.5 h 6.75 h 9h llh 16.75 h

Cp 2h 3h 4h 5h 6.75 h 8.75 h 13.75 h

Cps 1O-16h 15-21 h 16-28 h 23-30 h 29-37 h 30-42 h

q 3h 3.75 h 4.5 h 5.25 h 6.5 h 8h

F'.LJ 5h 6h 7h 8h 9.5 h llh 18 h

Pp 3h 4h 5.25 h 6.25 h 7.25 h 8.5 h 14.5 h

Pb 3.5 h 5h 6.5 h 8h 9h 10.25 h 19.75h

Pch 2h 3.75 h 5h 6.25 h 8h 10.25 h 18 h

Pc 2.75 h 10.75 h 12 h 19 h

Mi 2.25 h 3.5 h 4.75 h 6h 7.25 h 8.75 h 13h HOLOTIIUROIDEA 585

TABLE 28.2 DEVELOPMENTAL SCHEDULES OF HOLOTHUROIDS FROM SAN JUAN ARCHIPELAGO IN CULTURE AT 11 ± O.5°C . (Continued) Early larva is the evenly ciliated post-gastrula larva; the early doliolaria has evident epidermal ridges. "Armored" refers to the stage when settled young are covered with ossicle plates or tables. Dashe s indicate no data are available. Numbers in parenthese s are sizes of embryo , larva, or young (in urn), "na" is for not applicable.

early early settle- hatching gastrula larva doliolaria ment "armored"

a 32-35 d 3.5-6 .5 d na na na na (975-1190)

On 33 h 41 h 57 h 3.5 d 8-13 d 29-30 d (600-885) (675-765) (700-800) (875-1230)

Cp 32 h 41 h 62h 3.5 d 8-9.5 d 22-25 d (620-815) (680-740) (740-770) (925-1325)

Cps 36-37 d 7.5-9 .5 d na na na na (945-1250)

q 62h 3.75 d 9.5-10.75 d 18-21 d (625-670) (675-725) (600-710) (700-1125)

Eq 28 h 33 h 51 h 2.75 d 6.5-7.5 d 11-16.5 d (555-660) (510-570) (495-675) (540-700)

Pp 23 h 30 h 44h 2.25 d 6-7.5 d 9-15 d (540-630) (470-550) (490-600) (545-630)

Ph 28.5 h 40h 55 h 70-75 h 7.75 -8.5 d 22 d (410) (430) (600) (445) (670)

Pch 40h 60-70 h 80-90 h 11-12 d 26-32 d (1125) (925) (730-855)

Pc 27 h *3.25-4.25 d 52 d 60-61 d 61-65 d (290) **(1120) (410-550) (245-365) (299)

Mi 24-25 h 32.5 h 46h 70 h 95-96 h (350-390) (345)

* = early auricularia ** = size of full auricularia just prior to onset of metamorphosis -

586 F. S. McEuEN

Cueumaria lubriea Clark. Occasionally intertidal; abundant subtidally in dense populations on subtidal boulders and rock walls in areas of high current. This species is a suspension-feeder. Adults are frequently covered with shell, wood , detritus, or algae (CallophylIis sp.). Adults are brownish black, but there are white, dark speckled, and light brown morphs. Spawning occurs mainly mid November- mid December; young are brooded to January-March (Engstrom 1974, 1982; McEuen 1986). Large eggs (877-1078 urn in diame­ ter, mean =973 urn) give the small, slender ovarian tubules a knobby appear­ ance. Egg color is normally dull yellow, but is variable (tan, light or bright yellow, light or dull green) . Spawning males lift the anterior end and release strands of sperm; the elongate spermatozoa are packaged in bundles. Spawn­ ing females tuck the anterior end under to lay up to 373 eggs beneath the tri­ vium. Cleavage is superficial and irregular. Young are brooded to juveniles; brooding lasts 2-3 months, with markedly asynchronous development within a single brood. Brooding females of C. lubrica and C. pseudocurata do not feed. At hatching, young have five primary tentacles and are still under broodcare; two posterior first podia appear within a few days of hatching. White juveniles with numerous tube feet emerge from under the female and disperse rapidly. A developmental schedule at 10.5-11SC is given in Table 28.2.

Cueumaria miniata Brandt. Primarily subtidal, to 225 m, under rocks and in cobble fields adjacent to current-swept channels; often low intertidal and in algal holdfasts on the Monterey Peninsula, California (Rutherford 1975). This species suspension feeds on small organisms and detritus using bright orange or brown tentacles (Brumbaugh 1980). Ripe testis is brownish orange ; ripe ovary is dark brown. Gamete release occurs during intervals of slack water between tide cycles from early March to mid May, with most populations spawning mid March to late April. In spawning, the anterior half of the body is stretched into the water, and tentacles are held half-contracted, while white or pink sperm are broadcast (McEuen 1986). This species has also been observed spawning in tide pools after prolonged exposure during low tide (M. Strathmann pers . comm.). Pellets of positively buoyant green eggs (488-573 urn in diameter; mean =520 urn) float to the surface and fall apart. The jelly coat surrounding each egg is 30-35 urn thick. Fecundity can reach 1.318 x 105• Pelagic, lecithotrophic development includes a rounded conical gastrula and a doliolaria with three ciliary rings. Many larvae lack an oral indentation , and tentacles arise from the ventral surface (McEuen 1986); the oral indentation can also be large and cavernous. Larvae are sluggish swimmers with an olive green preoral lobe and light green posterior. Ohshima (1921) describes details of the sequence of appearance and growth of tentacles and tube feet in dendrochirotids. Pentactulae settle on undersides of rocks in adult habitats (McEuen 1986). A developmental schedule at 10.5-11.5°C is given in Table 28.2. z

HOLOTIlUROIDEA 587

Cue umaria piperata (Stimpson). Intertidal and subtidal to 137 m; quite variable in habitat. Large subtidal populations lie in V-shaped burrows in firm mud or on the mud surface; also sympatric with C. miniata under or on rocks and in crevices. Height of the spawning season is mid March - late April. Floating, olive-green eggs (480-601 urn in diameter; mean =532 urn) are packed in pellets when first released . Fecundity may reach 1.08 x 104. Spawning behavior is similar to that of C. miniata. Pelagic lecithotrophic development includes a doliolaria with three ciliary rings that is difficult to distinguish from that of C. miniata. The doliolaria has an olive green preoral lobe and a light green larval body (McEuen 1986). The preoral lobe of the settling pentactula is yellowish green and later fades to a light yellow . The "armored" juvenile is covered with five rows of imbricat­ ing ossicles. Recently settled juveniles have been found in July under rocks with conspecific adults and C. miniata. A developmental schedule at 10.5­ 11.5°C is given in Table 28.2.

Cueumaria pseudoeurata Deichmann. In mussel zone (Mytilus califor­ nianus) to zero tide level; also in coralline algae and mats ofsea weeds (Rhodo­ mela and Odonthalia ) (Brumbaugh 1980) and on rock in surge channels. Adults are dark brownish black, but can also be gray or mottled gray on black. This species is a suspension- and deposit-feeder on phytoplankton and attached diatoms (Brumbaugh 1964, 1980). The spawning season is mainly mid December to early or mid January. Spawning males raise the anterior end and release elongate sperm bound in strings of mucus that sink to the bottom. Bright orange eggs (916-1237 urn in diameter; mean = 1051 urn), each enclosed in a tough cellophane-like membrane, are placed under the trivium and held tightly against rock or mussel-shell substratum by the female's tube feet. Maximum fecundity is 340 (Rutherford 1973). Eggs adhere to one another and to maternal tube feet. Dry weight is 169­ 190 ug per egg, and egg organic composition is 54% protein, 33% lipid, 5% carbohydrate, and 8% ash; reproductive effort is 75-243 calories per brood (Turner & Rutherford 1976a, b). Fertilized eggs pinch off large globules of cytoplasm. Cleavage is sometimes equal up to the 4-cell stage, but is most often unequal , superficial, and asynchronous within a brood. Pigmentation in developing embryos changes to dark brown by the time of hatching. Hatch­ lings have two first podia and five primary tentacles; two pairs of podia emerge anterior to the first pair within four days of hatching. Juveniles crawl away from the females at approximately 2-3 months. Smith (1962) reports a much faster development rate in central California where 10-tentacled juve­ niles disperse in the second week after fertilization. A developmental schedule at 1O.5-11.5°Cis given in Table 28.2.

Eupentaeta quinquesemita (Selenka). Subtidal to 55 m, in high densities in areas exposed to swift currents (rocky points, walls, and on Balanus nubil­ is); also abundant on docks and pilings; sometimes intertidal and under rocks and in crevices . Gonads appear ripe (testis is yellow with up to 204 tubules, a

588 F. S. McEuEN and ovary is green) and spawnings have been observed in the laboratory from mid March to mid May (Byrne 1983; McEuen 1986). Late afternoon and evening are the best times for inducing spawning. Slightly negatively or neutrally buoyant, light green eggs measure 370-416 urn in diameter (mean = 397 urn) and are released in a loose mucus. Fecundity is as high as 1.05 x 105. Pelagic lecithotrophic development includes an evenly ciliated larva with­ out ciliary rings (Fig. 28.1) that does not appear to exhibit phototactic behavior (McEuen 1986). The blastula is wrinkled and is quartered by four main egression tracts or furrows. The larval preoral lobe is light green and the posterior body is a semi-translucent white. Resorption of the preoral lobe is rapid, occurring within 1-1.5 days after settlement, and the benthic pentactula is a semi-opaque white color. A developmental schedule at 10.5­ 11.5°C is given in Table 28.2. Juveniles 23-days old have been observed to feed on detritus and defecate pellets. In the field, 0.25-1.0 em long juveniles are found on hydroids and small algae in high-current areas; larger young (> 1.0 ern) are found in nearby cracks on rocks (McEuen 1986 and unpubl.). Filamentous hydroids on docks also harbor newly settled juveniles.

Pentamera populifera (Stimpson). Subtidal to 257 m. Population densi­ ties to 1400 per m2 occur in the upper 5-7 em of muddy areas. This species is a suspension- and deposit-feeder. Spawning occurs mid February-late March, earlier than in other local dendrochirotids. Ripe gonads (creamy orange testes or off-green ovaries) are visible through the thin body walls. During spawn­ ing, females entangle the spawned, negatively buoyant eggs and lift them into the current. Eggs are light green, sometimes off-white, and 339-439 urn in diameter (mean =372 urn). A female may spawn up to 3.53 x 1()4 eggs . Pelagic lecithotrophic development includes a doliolaria with three ciliary rings (Fig. 28.2). The preoral lobe is light green and the posterior two-thirds of the larval body is a semi-opaque white. Doliolarias in culture swim on the bottom and gather in small clumps toward light sources. The preoral lobe is resorbed 1-2 days after settlement and the pentactulae lie on their sides or dorsal surfaces (McEuen 1986). A developmental schedule at 10.5-II.5°C is given in Table 28.2. Settled young fed on Dunaliella tertiolecta and Isochrysis ga/bana defecate pellets when 17 days old. Two first podia appear 2-3 days after settlement. These may extend 2.5 times the body length. Density of newly settled juveniles can reach 1.5 x 105 per m2 in the adult habitat.

Psolidium bullatum Ohshima. Occurs in small depressions on upper sur­ faces of rock covered with a thin layer of detritus and silt, commonly sympa­ tric with Pso/us chitonoides. Ripe gonads (tan ovary and white testis) are visi­ ble through the thin sole from March to early May (Hadfield 1961; McEuen & Chia, in press). In spawning, the anterior end is lifted. A female may spawn as many as 3,074 golden yellow to light brownish orange eggs. Egg size varies from 293 to 354 urn in diameter (mean =330 urn). F

HOWTIIUROIDEA 589

Pelagic lecithotrophic development includes a doliolaria with three ciliary rings (McEuen & Chia, in press). The doliolaria is orange at the apical end and occasionally has small brown splotches or streaks in the whitish opaque posterior. The preoral lobe turns light yellowish orange after settlement. A developmental schedule at 10.5-11.5°C is given in Table 28.2. Complete metamorphosis is difficult to obtain in the laboratory; settled, incompletely metamorphosed "juveniles" survive in culture as long as 50 weeks.

Psolus chitonoides Clark. Sometimes intertidal, but primarily subtidal to 137 m on upper and lower surfaces of rocks and on rock walls in areas with good water flow. This species uses the tentacles to feed on suspended particu­ late matter (Fankboner 1978). Spawning, in the field and in the laboratory, and planktonic developing stages are observed principally mid March-mid or late May (Johnson & Johnson 1950; Jones 1960; Young & Chia 1982; McEuen & Chia, in press). Spawning commonly occurs during early morning. Spawning males swab the genital papilla with their tentacles, then wave the tentacles to disperse sperm; long ropes of brick-red eggs are released by females. Up to 3.47 x 1()4 eggs may be spawned. Eggs are 570-708 urn in diameter (mean =627Ilm). Pelagic lecithotrophic development includes a doliolaria with three ciliary rings (Fig. 28.3). The preoral lobe is reddish orange and the posterior larval body is yellow. This species is easily cultured through metamorphosis. Development proceeds according to the following approximate schedule at 10°C initially then at 12-14°C (Jones 1960): at 10°C 0.25 hrs First polar body 0.5-0.75 hrs Second polar body 2 hrs First cleavage 4 hrs Second cleavage 6 hrs Third cleavage 7 hrs Fourth cleavage 8 hrs Fifth cleavage 10 hrs Sixth cleavage at 12-14°C 17-22 hrs Blastula 25 hrs Gastral plate formation 27 hrs Invagination 55 hrs Hatching A developmental schedule at 1O.5-11.5°C is given in Table 28.2. Young & Chia (1982) found late larvae and early juveniles to be strongly negatively phototactic and to settle gregariously.

Thyone benti Deichmann. Subtidal to 135 m in firm mud or sand. This 10-15 ern long species makes a rounded V-shaped burrow and is occasionally found on top of the substratum. The whitish body wall is covered with small , pinkish orange tube feet. Tentacles are light orange or light brown. This dendrochirote is a nocturnal suspension feeder (McEuen unpubl.). Spawning 590 F. S. MCEuEN season may be winter (January-February). Positively buoyant, opaque , light brown relict oocytes in a spent ovary from a specimen collected the first week of March were 512-560 urn in diameter (mean = 535 urn, n = 9) (McEuen unpubl.). Egg size suggests that development includes a pelagic 1ecithotrophic larva resembling those of cucumariids with similar size eggs. Sperm type is unknown.

Order Aspidochirotida Parastichopus californicus (Stimpson). Subtidal to 249 m, sometimes in low intertidal. On rock, sand-shell, or kelp bed and sand substrata (Feder et al. 1974; Fankboner & Cameron 1985). Adults are usually brown, but sometimes are pinto or albino. Ripe ovary is plump and translucent glassy orange; testis is white. Adults may migrate to shallower water for spawning (Courtney 1927). The spawning season extends from late April into August (Courtney 1927; R. Miller pers. comm.); the height of the season at Friday Harbor is late May-mid July (Bovard & Osterud 1918; Johnson & Johnson 1950; Smiley 1984a, 1986a,b; McEuen 1986). MacGinitie & MacGinitie (1949) report spawning during August in Newport Bay, California; field spawning in southern British Columbia populations occurs June through August (Cameron & Fankboner 1986). Large females have fecundities up to 8.92 x 106. In spawning, the anterior one-third to one-half of the body is raised. The light orange eggs are small (185-210 urn in diameter; mean = 204 urn) and negatively buoyant. Pelagic planktotrophic development includes a feeding auricularia larva (Fig. 28.5) that normally swims 35-52 days (Mortensen 1921; Strathmann 1971, 1978a; Cameron 1981, 1985; McEuen 1986). The larva has a light yellow tint on the ciliary band and an irregular star-shaped ossicle in the left posterolateral lobe. At 20-23°C, the pelagic period (to settlement) is as brief as 14 days (McEuen unpubl.). An approximate schedule of early devel­ opment at 14.3°C is as follows (A.H. Whiteley unpubl.): omin Spawning (germinal vesicle present) 12-18 min Germinal vesicle breakdown 53 min First polar body 3 hrs First cleavage 24 hrs Swimming blastulae 42 hrs Gastrulae (archenteron 1/3 body length) A developmental schedule at 10.5-11.5°C is given in Table 28.2. Partial metamorphosis produces a doliolaria with five ciliary rings (Fig. 28.6) (see Smiley 1984b, 1986a,b; Cameron 1985); posterior rings are frequently tinted reddish orange. Some doliolarias swim up in the water column , but most remain on the culture dish bottom. Pentactulae settle and rapidly lose cilia. They become covered with ossicles in 1-2 days, taking on a spiny appearance. The period to settlement followed by completion of metamorphosis may be as long as 131 days (McEuen 1986). Settling larvae probably attach to under- HOWlHUROIDEA 591

sides of rock in calm coves, bays, and fjords, since that is where juveniles 1.0­ 11.0 cm long are found (McEuen unpubl.). In southern British Columbia, juveniles are regularly encountered in dense mats of filamentous red algae (Sarcodiotheca gaudichaudii and S. furcata), on tubes of the polychaete Phyllochaetopterus prolifica, and in fissures and crevices on vertical rock walls (Cameron 1985).

Order Apodida Leptosynapta clarki Heding. Intertidal to 73 m in sandy silt to gravel, and among rhizomes in Zostera beds. The male:female ratio in late fall is 2.6:1.0. Everingham (1961) thought this synaptid might be protandrous. Ripe ovary is pink or yellowish white; testis is white. Males free-spawn mid November­ early or mid December. Fertilization is internal, but how sperm enter females is unknown. Everingham (1961) and Brooks (1973) give egg sizes of 240-404 urn. At the onset of the spawning season in False Bay, San Juan Island, eggs are 323-382 urn in diameter (mean = 345 urn) and maximum fecundity is 2,495 (McEuen 1986). Eggs are pink or white. Internal brooding occurs November-April. Development proceeds within the ovary , where juveniles engulf whole nurse eggs (termed "inert cells" by Everingham); nurse eggs often break up into smaller globules and granules which are also consumed by the young. Juveniles with 12 tentacles attain lengths up to 12 rom within the ovary and are probably expelled through the circumanal ducts (Anderson 1966; Shinn 1985). The approximate durations of developmental stages are as follows; most are estimated from a census of developmental stages present in females collected September-June (Everingham 1961): Duration of stage Stage unknown Fertilization 1 day Early cleavage (holoblastic & equal) 1 day Coeloblastula 3 days Gastrula 1-2 weeks Pre-pentactula 1 month Early pentactula 2 weeks Mid pentactula 1 month Late pentactula 1 month 7-tentacled juvenile (2-3.6 mm long) 1 month 8-tentacled juvenile (3.2-5.4 rom long)

Leptosynapta transgressor Heding. Subtidal, 6-40 m in sandy silt to medium sand (finer sediments than L. clarki) in bays and channels of Puget Sound and inside waters of southern British Columbia. Large adults occur a few centimeters below the sediment surface; small specimens are frequently entwined around tube masses of the polychaetes Phyllochaetopterus prolifica and Prionospio sp. Spermatogenesis is apparent in July and August , with 592 F. S. McEuEN sperm beginning to mature during fall and winter. Brooks (1973) found males with ripe testes in March, while in April most males had partially empty testes. A maximum egg size of 272 urn was reached in March, and fertiliza­ tion occurred between March 19 and April 21. Gastrula and post-gastrula stages were first encountered in females in April. Brooks' observations indicate fertilization is internal and that young may be brooded, but mode of development is not clear.

Order Molpadiida Molpadia intermedia (Ludwig). Subtidal to 2925 m (Clark 1907). Bur­ rows as deep as 35 cm in mud. Ingests mud. Ripe gonads and spawning in the laboratory have been seen mid November- mid or late December. Ripe ovary is pinkish orange . Telolecithal eggs have orangish pink yolk caps and sink. The egg diameter is 240-285 urn (mean =267 urn). Females forcefully eject up to 3.592 x 105 eggs at one time; males emit puffs of sperm for 1.5-3 hours. Pelagic lecithotrophic development occurs (McEuen & Chia 1985). The larva has two transverse ciliary rings and a uniformly ciliated , orangish pink preoral lobe (Fig. 28.6). The semi-transparency of the larval body allows some features of internal development to be observed. Early juveniles feed in the top few millimeters of mud and are completely transparent except for semi-opaque triradiate ossicles and a calcareous ring. A developmental schedule at 1O.5-11.5°C is given in Table 28.2.

REFERENCES

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