Table Of ContentReference: Biol. Bull. 194: 1S7-193. (Apnl. 1998)
Cloning by Ophiuroid Echinoderm Larvae
ELIZABETH BALSER
J.
Illinois Wesleyan University. Department ofBiolo(;y. Bloomington. Illinois 61702-2900
Abstract. Larvae of the brittle star Ophiopholis acu- parentage in future generations without additional repro-
leata. common to the North Pacific coast of the United ductive input from the adult.
States, and an unidentified species ofophiuroid, collected
from waters off the eastern coast of Florida, undergo Introduction
asexual reproduction of the primary larva to produce a
In 1921, Mortensen reported the appearance ofunusual
secondary larval clone. Generation of a secondary larva
brittle star larvae in plankton tows taken from the waters
begins with the release of the larval posterolateral arms,
surrounding the islands ofthe West Indies. He speculated
which are initially retained by the settled juvenile. In O.
that these larvae were asexual clones originating from the
aculeata, the released arms regenerate all the structures released posterolateral arms of settled juveniles. Mac-
typical of the primary ophiopluteus. Tissue and energy
Bride (1921), renowned for his contributions to echino-
reserves required for formation of the secondary feeding
derm embryology, strongly rejected this interpretation,
larva appear to be supplied by the absorption and reorga-
and Mortensen's observation remained unconfirmed for
nization ofpart ofthe posterolateral arms. Various devel-
the next 75 years (Mladenov and Burke, 1994).
opmental stages of the unidentified ophiopluteus were
Larval cloning is a rare event in the life history of
collected from plankton samples taken off the coast of
nonparasiticplanktonic invertebratelarvae,butisreported
Florida. These includedjust-released posterolateral arms,
for a number of species of asteroid echinoderms (Bosch
plutei, and metamorphosedjuveniles with the posterolat-
et ai. 1989; Rao et ai. 1993; Jaeckle, 1994). In asteroid
eral arms still attached. The identification ofregenerating larvae, the formation ofthe asexually produced secondary
arms from the plankton demonstrates that asexual repro-
propagule results from the differentiation and release of
duction by ophiuroid larvae is not restricted to a single, some part of the primary larval body. The propagule de-
laboratory-cultured species. In both O. aculeata and the
velops into a secondary larva that is morphologically
unidentified Atlantic ophiopluteus, cloning involves the
identical to the primary larva. It is not known if the sec-
dedifferentiation of primary larval tissue and a develop-
ondary larvae of asteroids are capable of settlement and
mental progression similarto that followed by the zygote,
metamorphosis or if continued cycles of asexual repro-
although it is not known whether the formation of the duction are possible.
secondary larva follows the same pathway utilized by the
The work presented here confirms Mortensen's (1921)
primary larva or a novel developmental program. Asexu-
earlierobservationsthatophiuroidlarvae undergoasexual
ally produced secondary larvae of O. aculeata undergo
reproduction to produce secondary clones. Like asteroids,
metamorphosis, settletothebenthos, andinitiate a tertiary
secondary larvae of Ophiopholis aculeata and of an un-
larval generation, indicating that cloned larvae could be
identified ophiopluteus collected fromthe Western Atlan-
added to the population as long as environmental condi-
tic Ocean off the coast of Florida originate from the
tionscould supportaplanktonicexistence. Thisphenome-
release and development ofprimary larval structures, spe-
non represents an unusual potential to increase the geo-
cifically, the posterolateral arms. The released arms un-
graphic range and the number of juveniles of a given
dergo gastrulation and development similar to that ofthe
primary embryo, but development of secondary larvae
Received 15 August 1997; accepted 22 December 1997. follows from the reorganization of larval tissues instead
E-mail: [email protected] offrom embryonic cells. This raises significant questions
187
188 E. J. BALSER
concerning the flexibility ofdevelopmental pathways and by the appearance of the five hydrocoelic lobes of the
the initiation and control of morphogenesis. presumptive primary podia. Metamorphosis continues
In O. Liciileata. asexually produced secondary larvae while the larva remains in the water column, eventually
undergo metamorphosis, settle to the benthos, and initiate resulting in a juvenile suspended between the two outer
atertiary larval generation. Asexual reproduction oflarval posterolateral arms (Fig. lA). The juvenile retains the
propagules has potential evolutionary and ecological ram- posterolateral arms for some undetermined period after
ifications. For each juvenile that survives to settlement, settlement. During this time the transverse rods appear to
an additional larva is produced that not only increases the shorten and contact is made between the proximal tip
number of individuals ofa given genetic lineage, but also of each posterolateral arm. The posterolateral arms are
enhances the dispersal potential of the species. released from the settled juvenile (Fig. IB) and, with
Methods and Materials
Adult specimens of Ophiopholis aculcata were col-
lected during the summer of 1996 from the intertidal and
subtidal zones of various sites along the shores of San
Juan Island, Washington. The animals were transported
to the Friday Harbor Laboratories, Friday Harbor, Wash-
ington, where spawning was induced by alternating light
and dark exposure. Fertilization and culturing of the em-
bryos and larvae followed methods outlined by Strath-
mann (1987). Feeding ophioplutei were kept in 2 1 of 5-
pm-hltered seawater in glass jars at 10C°, stirred gently
by paddles, andfedculturedcellsofthe algaRhodomonas
sp. Juveniles suspended between the posterolateral arms
and free-swimming released arms were removed from
the jars and maintained in culture dishes. Clean filtered
seawater and algae were provided every other day, and
the cultures were stirred gently with a pipette several
times a day to suspend food and larvae in the water col-
umn. A Wild M-5 dissecting scope and a Nikon Optiphot-
2 compound microscope were used to take photographs
on T-Max 100 (Kodak) film.
During May and June of 1997, plankton was collected
approximately 1 mile off the coast of Ft. Pierce, Florida,
by towing a 202-/jm-mesh plankton net at a depth ofless
than 10 m. The samples were transported tothe Smithson-
ian Marine Station in Ft. Pierce and examined for ophi-
oplutei. In some samples, an unidentified ophiopluteus
was the most abundant organism present. Plutei,juveniles
with attached posterolateral arms, and free-swimming
posterolateral armsin various stages ofdevelopmentwere
Figure 1. (A) Light photomicrograph of a juvenile Ophiopholis
collected and maintained in culture dishes in filtered sea- aculeatasuspendedbetween theouterposterolateralarms. (B)Recently
water. Larvae were fed cells of Rhodomonas sp. Larvae releasedposterolateralarmsfromasettledjuvenile.(C)Adevelopmental
and regenerating arms were photographed with a Zeiss series ofthree different asexually produced secondary larvae arranged
Photomicroscope II and T-Max 100 film. chronologically from top to bottom. (D) Gastrulation of released pos-
terolateralarms. Aftersettlement,thejuvenilereleasestheposterolateral
arms (B) which swim using the ciliated epidermis covering the arms
Results (originally part of the primary larval ciliated hand). The posterolateral
arms (pa) are supported internally by skeletal rods (sr). seen in D. An
Larvae o/Ophiopholis aculeata event similarto gastrulation begins with the invaginationofthe epider-
mis (arrowhead) in the vee of the arms. Formation of the secondary
The development ofprimary larvae of O. aculeata fol- pluteus (shown in series in C), is characterized by a shortening ofthe
lows that described for other planktotrophic ophioplutei outer arms and a concentration oftissue between the arms. Scale bars
(Hendler, 1991). The onset ofmetamorphosis is indicated = 0.1 mm.
CLONING BY OPHIUROID LARVAE 189
ciliary band intact, swim off the bottom of the culture anterior coelom that divides and grows to produce the
dish and into the water column. right and left axohydrocoels (Fig. 2B). The formation of
The freearmsconsistoftheskeletal rods, mesenchymal the somatocoels in both primary and secondary larvae is,
cells, a ciliated epidermis, and the blastocoel of the pri- at present, unclear. Over the course of 3 to 4 days, the
mary larva's posterolateral arms. Within 24 h of release, posterolateral arms regenerate the structures typical ofan
the tissue forming the inner vee ofthe arms undergoes a ophiopluteus, including the gut, the coeloms, and addi-
process similar to gastrulation. This results in the produc- tional feeding arms (Figs. IC, 2C).
tion ofan archenteron that continues to develop, forming Prior to feeding in the secondary larva, tissue and en-
a new larval gut (Figs. IC, D and 2A). As in development ergy resources appear to be supplied by the posterolateral
of the primary larva, the archenteron gives rise to an arms, as indicated by a decrease in the length of the arms
B
Figure 2. (A, B) Light photomicrographs ofthe formation ofthe larval gut in 2-arm clones ofOphio-
plwlis aculeata. (C) Photomicrograph of a 4-arm pluteus produced from released posterolateral arms of
primary juvenile. (D) Photomicrograph of a primary pluteus resulting from fertilization of an ovum and
developmentofthe zygote. In cloned larvae, the archenteron (ar) gives rise to theesophagus (es), stomach
(SI), and intestine (not shown). The mouth (asterisk) opens from the exterior into the esophagus, but it is
not known if it forms at the opening of the archenteron (blastopore analog) or elsewhere. As in primary
larvae, the antenor coelom arises from the tip of the archenteron and later divides to form the right and
left axohydrocoels (arrowheads). The 4-arm asexually produced pluteus (C) is morphologically similar to
aprimary pluteus (D)exceptforincrea.sedpigment in theepidermis (seen asdark spots in this micrograph)
and disproportionately longer posterolateral arms (pa), aa, anterolateral arm; bl, blastocoel; ep, epidermis;
sr, skeletal rod. Scale bars = 0.1 mm.
190 E. J BALSER
(Fig. IC). The average total length (sum of left and right peared by the 4-arm stage. In plankton-collected clones,
arm lengths) for armsjust released from thejuvenile was no asymmetry in arm length was observed in 4-arm sec-
620 ± 71 /jm (mean ± 1 SD. n = 6); in contrast, the mean ondary larvae.
total arm lengthof2-arm prefeeding secondary larvae was Gastrulation by posterolateral arms of this east coast
374 ± 82 /jm (« = 6). This 40% difference represents a species may not occur as it does in O. aculeata. At the
significant decrease (f = 5.53, P < 0.001) in the average junction of the two posterolateral arms, the transverse
total arm length. During reformation of the secondary rods form a spherical cage (Fig. 3C). Although not clearly
pluteus. the epidermis covering the arms appears to be seen in Figure 3C. the lumen of this cage is filled with
pulled towards the inner vee of the arms, presumably cells. It is not known from where these cells originate or
providing the cells for invagination and formation of the if they reform the larval gut, and subsequently the larval
archenteron. The epidermis appears buckled along the coeloms. Asexually produced 4-arm plutei appeared to
length of the arms and is pulled away from the ends of have disproportionately longer posterolateral arms than
the skeletal rods. In some larvae, the reduction in arm those expected for primary plutei at a similar develop-
length was not symmetrical, with one arm being shorter mental stage, but without direct comparison, this remains
than the other (Fig. IC). The reason for this asymmetry a subjective observation. Beyond the 4-ann stage, second-
is not known, but it apparently has no adverse effect on ary plutei were indistinguishable from primary larvae.
development. These larvae formed normal plutei and the
asymmetry disappeared as development progressed. Discussion
Within 5 days, a feeding 4-arm pluteus is formed (Fig.
2C). This larval clone is morphologically indistinct from In O. aculeata. tissues in the asexually produced larvae
a primary larva except for having more ofa characteristic appear to originate from dedifferentiation and redifferen-
orange pigment in its epidermis (seen in fully developed tiation of the epidermis covering the posterolateral arms.
primary larvae) and disproportionately long posterolateral Mesenchymal cells are present in the blastocoel (of the
arms (compare Fig. 2C with 2D). Continued growth and arms), but are associated with the skeletal rods and do
morphogenesis results in an 8-armpluteus that is indistin- not seem to contribute to the formation ofthe archenteron
guishable from the primary larva at the same develop- or the axohydrocoels. This developmental sequence is
mental stage. Metamorphosis of the secondary larva be- similar to that seen in the blastula of O. aculeata and
gins with the formation of the hydrocoelic lobes and fol- raises questions concerning the ontogeny of larval mor-
lows a morphogenic pattern similar to that ofthe primary phogenesis. Principally, do the clones follow the same
larva. At the time of settlement, the secondary juvenile genetic and morphogenetic pathways as the primary larva
retains the posterolateral arms but, as in the primary larva, or do they use a novel developmental pattern?
eventually releasesthem. The free armsreturn tothewater Specific questions about the development of a.sexually
column and begin a tertiary cycle of development that produced larvae are how the larval body axes are estab-
results in another planktonic ophiopluteus. lished in the clone and what mechanisms are involved in
differentiation of endoderm and subsequently mesoderm
from the ectodermal epithelium covering the free-swim-
Ophiopliitei collected from plankton tows
ming arms. In both primary and secondary larvae, the
Plankton tows taken offthe coast ofFt. Pierce, Florida, archenteron forms along the long axis ofthe larval skele-
contained a series ofdevelopmental stages ofan unknown ton (anterior-posterior axis), but it is not yet known if
ophiuroid larva. These stages included 8-arm plutei (Fig. the mouth in the secondary larva forms at the site of
3A), metamorphosed juveniles suspended between the invagination or at a point 90 degrees from the advancing
posterolateral arms, free-swimming posterolateral arms tip of the archenteron, as in the primary larva. If, after
(Fig. 3B), and various stages of regenerating posterolat- invagination of the arm epithelium, the cells forming the
eral arms (Fig. 3C, D). Juveniles maintained in culture archenteron in the cloned pluteus are embryologically
dishes eventually settled and released the posterolateral equivalent to those of the primary gastrula. formation
arms which, as in O. aculeata, began morphogenetic of endodermal and mesodermal structures could simply
changes consistent with regeneration of the larva. repeatthe developmental programofthe primary embryo.
Stages collected from the plankton showed a progres- O. aculeata and the ophiuroid species collected from
sive series from recently released arms (Fig. 3B) to an the plankton may not follow the same—developmental se-
early 4-arm pluteus (Fig. 3D). In some, but not all, ofthe quence in forming a secondary larva specifically with
recently released stages the two arms were of different respect to gastrulation. Although multiple modes ofasex-
lengths (Fig. 3B). This asymmetry had no noticeable ad- ual reproduction are reported for asteroid larvae (Jaeckle,
verse effect on swimming or development and disap- 1994), gastrulation appears to proceed from an invagi-
CLONING BY OPHIUROID LARVAE 191
B
Figure 3. Light micrographs ofan ophiopluteus (A), released posterolateral arms (B). and apparently
regenerating larvae (C. D) collected from plankton samples taken offthe eastcoast ofFlorida. Similarities
inthelarvalskeletonandinpigmentationsuggestthatthesespecimensrepresentonespecies,aa.anterolateral
arm; pa, posterolateral arm; st, stomach; sr. skeletal rod; tr, transverse rod; arrowhead, concentration of
tissue at the point ofcontact ofthe two posterolateral arms. Scale bars = 0.1 mm.
nation of the outer (epidermal) epithelium of the propa- arms by the settled juveniles of Ophiothrix oersted!. In
gule (Bosch et ai, 1989; Jaeckle, 1994). The origin of this species, however, the arms did not develop and even-
thecells inthe veeofthereleasedarmsofthis unidentified tually died. The unidentified pluteus collected off the
secondary ophiopluteus is at present unknown. They may coast ofFlorida may be the same larva described by Mor-
have originated from invagination ofthe epidermis ofthe tensen (1921) from the West Indies and designated as O.
arms or, alternatively, from mesenchymal cells of the opulentus species c. Similarities in the larval skeleton of
primary larva. Ifthe latter hypothesis is true, the develop- both larvae and the confluence between the West Indies
ment of the secondary larva deviates from that described and the Florida coastline provided by the flow of the
for asteroids and for O. aculeata. FloridaCurrentofthe GulfStream system raisethe possi-
In ophiuroids, asexually produced larvae can complete bilitythatthe unidentifiedlarvais O. opulentus. Neverthe-
metamorphosis and initiate another generation of plank- less, the potential for larval cloning is evident in possibly
tonic larvae. Theformationoftertiary larvae suggests that three species of planktotrophic ophioplutei (although O.
the production of asexual larval clones could continue opulentus is known only from larval stages), and may
indefinitely as long as environmental conditions could represent an important life history strategy for planktonic
support a planktonic existence. Development of second- ophiuroid larvae.
ary larvae is known for Ophiopluteus opiilentus (Mor- The impact of asexual reproduction by larvae on the
tensen, 1921) and Ophiopholisaculeata (this manuscript). life history of a species is dependent on the cloned indi-
Mladenov (1979)reportedtherelease oftheposterolateral viduals' ability to produce viable gametes, which, in turn.
—
192 E. J. BALSER
requires the formation of germ cells. The origin of the hydromedusapredator, agreaterpercentage oflater larval
primary gemi cells in ophiuroids, and in general in echi- stages was lost to predation (Rumrill, 1987). Neverthe-
noderms. is equivocal. Germ cells in echinoderms are less, decreased predation resulting from morphological or
thought to arise either from secondary mesenchymal cells behavioral characteristics of more advanced larval stages
or from a proliferation or outgrowth of cells from the (Pennington et al.. 1986) may in some cases improve the
epithelial lining of the somatocoel (Gemmill. 1914; survivorship of asexually produced plutei.
Nieuwkoop and Sutasurya, 1981; Delavault. 1966; Houk For each juvenile that survives to settlement, an addi-
and Hinegardner, 1980, 1981; Hendler. 1991; Holland, tional larva is produced; this not only increases the num-
1991). In ophiuroids, as in otherechinoderms. the primary ber of individuals of a given genetic lineage, but also
germ cells are not evident in the larval body until late in enhances the dispersal potential of the species. Subse-
development, usuallyjust priorto or after metamorphosis quent generations of asexually produced larvae have the
(Gemmill. 1914; Houk and Hinegardner, 1980, 1981; potential to increase the distance of dispersal by at least
Smiley, 1986; Hendler, 1991; Holland, 1991). The "ab- as much as the primary larva. Dispersal promotes genetic
sence"" ofgerni cells in earlierdevelopmental stages sug- connectivity between populations geographically isolated
gests, concunently or alternatively, a delay in the induc- by distance and allows recruitment to new habitats (Chia,
tion of the expression of germ cell characteristics and an 1974; Crisp, 1974; Strathmann, 1974; Scheltema, 1986).
origin of the primary germ cells from structures not Asexual reproduction by planktonic larvae increases the
formed until later in development. life span of a genetically identical cohort and distributes
The formation ofprimary germcells in asexually repro- the timing of the attainment of competence to settle. If
duced larvae has not been demonstrated, but the following appropriate settlement sites are randomly d—istributed
three hypotheses are possible. The cloned larvae lack the along a dispersal axis, then asexual propagation by pro-
ability to produce germ cells and, therefore, form sterile ducing more larvae that can spread over a wider area
adults. Primary germ cells are sequestered from a popula- increases the likelihood that acompetent larva will locate
tion ofstem cells in the primary larva and are transferred a suitable settlement site.
to the secondary (or subsequent) propagule prior to its
release from the primary larva. Finally, the primary germ Acknowledgments
cells could arise de novo from the somatocoelic epithe-
thank Bruno Pernet, Chris Lowe, and Dr. William
lium, which apparently arises anew in the cloned larva. 1
Jaeckle for theirassistance in collecting aduUs and cultur-
The latter two hypotheses are consistent with the forma- ing primary larvae of O. aculeata. My appreciation is
tion ofthegerm cells in primary larvaeeitherfrom mesen-
also extended to Dr. Dennis Willows and Friday Harbor
chyme or from the epithelium of the somatocoel.
Laboratories, Friday Harbor, Washington, for laboratory
canPrporvoidduecdethvaitabtlheejguavmeentielse,s aresseuxlutailngrefprroomduccltoinoendolfarlvaare- space and technical support. I am grateful to Dr. Mary
Rice and the Smithsonian Marine Station, Ft. Pierce, Flor-
val propagules increases the effective population size
ida, for use of the Marine Station facilities. I offer a
without additional reproductive cost to the primary adult.
special thanksto Sheny Reed forherassistance incollect-
Further, asexual reproduction by larvae may produce a
ing plankton. This work was supported, in part, by an
larger increase in the number ofjuveniles than could be
Artistic and Scholarly Development Grant provided to
realized from an equivalent increase in the number of
the author by Illinois Wesleyan University. Smithsonian
eggs released by the female. The loss of individuals dur-
ing larval development (mortality rate > 0) requires that Contribution Number 437.
the number of eggs released by the female exceed the
Literature Cited
number ofjuveniles produced.
Cloned larvae, which enter the water column at a size Bosch, I., R.B. Rivkin, and S.P. Alexander. 1989. Asexual repro-
equivalent tothearm-span ofan 8-aiTn pluteus, mayexpe- duction by oceanic planktotrophic echinoderm larvae. Nuliiif 337:
169-170.
rience a decrease in predation (Rumrill et al.. 1985; Pen-
Chia, F.S. 1974. Classification and adaptive significance ofdevelop-
nington et al., 1986; Rumrill, 1987), and thus a lower mental patterns ofmarine invertebrates. ThahissioJugost. 10: 121-
mortality rate, compared to less developed stages. The 130.
reduction may, however, depend on the type of predator Crisp,D.J. 1974. Theroleofpelagiclarvae. Pp. 145-155inPcnyifc-
encountered. In the presence of arthropod and chaeto- tivcsonExpciimcmalBiology. Vol. 1.P. Spencer-Davies,ed. Perga-
gnath predators, blastulae and prism stages ofO. aculeata DelamvoanultP,reRss.,1O9x6f6o.rd.Determinism ofsex. Pp. 615-63S m Physiology
were more susceptible than 4-arm plutei, which were ofEchinodermma. R. A. Boolootian, ed. Wiley-Interscience, New
more susceptible than 8-arm plutei (Rumrill, 1987). In York.
contrast, when larvae were presented to a copepod or Gemmill,J.F. 1914. Thedevelopment andcertain points in the adult
CLONING BY OPHIUROID LARVAE 193
structure ofAslerias ruhens. Philos. Trans. Zool. Soc. London 20: Cells in the Inveriehrates: From Epigenesis to Prefornuition. Cam-
1-17. bridge University Press, Cambridge. 258 pp.
Hendler,G. 1991. Echinodermata: Ophiuroidea. Pp. 355-511 in Re- Pennington, J.T., S.S. Rumrill, and F.S. Chia. 1986. Stage-spe-
parnodduV.ctBi.onPeoafrsMea,riednse.ITnhveerBteobxrawtoeos.dVPorle.ss6.,PAa.ciCfi.cGGireosve,e,J.CaS.liPfeoarrnsiea., cDiefnicdrparsetdeartieo.nueuiiptornicuesm.bbryyo1s1 sapnedcileasrvoafecoofmtmhoenPazcoiofpiclasnakntdondoplrleadr-,
HollpaarnnoddduV,.ctNBi..oDnP.eoaf1r9sM9ea1,r.iednse.EcIThnhvieenroBtdeoebxrrmwataoetosa.d:VPCorrlei.snso6.,iPdAae.cai.Cf.iPcpG.Gire2os4ve,e7,-J.2Ca9Sl.9iPfieonarrnRsieae-.. Raoa,ttiooPrn.s.So.fB,ulKbl.u.dHdM.ianRrgaoSi,cni.abni3pd9i:nKn2.a3rS4iha-y2al4na0ri.vaaseun(dAasrteir.oi1d9e9a3).. CuArrr.areScci.ond6i5:
792-793.
Houdki,ffMer.enSt.i,atainodn oRf.sTe.aHuirncehgianrgdonncard.s.19B8i0ol.. BTulhle. f1o5r9m:at2i2o0n-2an9d4.early Rumroifltle,mpSe.rSa.te19P8a7c.ificDiefcfheirneontdiearlmsp.rePdha.tiDo.nDiuspsoenrteatmiborn.yoUsniavnedrsliatryvaoef
Houskp.ecMif.icS.t,oatnhedsRe.aTu.rcHhiinneggearrdmnelirn.e.1D9e8v1..BioCly.to8p6l:a9s4mi-c99i.nclusions RumrAilblelr,taS..4S.3,2J.ppT.. Pennington, and F.S. Chia. 1985. Differential
Jaeckle,W.B. 1994. Multiplemodesofasexualreproductionbytrop- susceptibility ofmarine invertebrate larvae: Laboratory predationof
ical and subtropical sea star larvae; an unusual adaptation forgenet sand dollar. Dendraster e.xcentricus. embryos by zoeae of the red
dispersal and survival. Biol. Bull. 186: 62-71. crab. Cancerproductus. J. E.xp. Mar Biol. Ecol. 90: 193-208.
MacBride,E.W. 1921. Echinodermlarvae and theirheanngonclas- Scheltema, R.S. 1986. Ondispersal andplanktonic larvae ofbenthic
sification. Nature 108: 529-530. invertebrates: an eclectic overview and summary ofproblems. Bull.
Mladenov, P.V. 1979. Unusual lecithotrophic development of the Mar Sci. 39: 290-322.
Caribbean brittle star Ophiothrix oerstedi. Mar. Biol. 55: 55-62. Smiley, S. 1986. Metamorphosis of Stichopus californicus (Echino-
Mladenov, P.V., and R.D. Burke. 1994. Echinodermata: asexual dermata: Holothuroidea) and its phylogenetic implications. Biol.
propagation. Pp. 339-383 inReproductiveBiologyofInvertebrates. Bull. 171: 611-631.
K.G. and R.G. Adiyodi. eds., Oxford and IBH Publishing. New Strathmann, M. F. 1987. Reproduction andDevelopmentofMarine
Delhi InvertebratesoftheNorthernPacificCoast. University ofWashing-
Mortensen, T. 1921. Studies ofthe development and larval forms of ton Press, Seattle. 670 pp.
echinodemis. G.E.C. Gad.. Copenhagen. 1-261 pp. Strathmann. R.R. 1974. The spread of sibling larvae of sedentary
Nieuwkoop. P.D., and L.A. Sutasurya. 1981. Primordial Germ marine invertebrates. Am. Nat. 108: 29-44.
