reproduction: widespread cloning in echinoderm larvae

1
Reproduction Widespread cloning in echinoderm larvae A sexual reproduction by free-living invertebrate larvae is a rare and enig- matic phenomenon and, although it is known to occur in sea stars 1–4 and brittle stars 5,6 , it has not been detected in other echinoderms despite more than a century of intensive study 6,7 . Here we describe spontaneous larval cloning in three species from two more echinoderm classes: a sea cucumber (Holothuroidea), a sand dollar and a sea urchin (Echinoidea). Larval cloning may therefore be an ancient ability of echinoderms and possibly of deutero- stomes — the group that includes echino- derms, acorn worms, sea squirts and vertebrates. To confirm that genuine cloning was occurring, we reared cloning larvae and sep- arated clones individually (Fig. 1, legend). Although most sea cucumber larvae meta- morphosed normally after a month, 5 of 41 (12.2%) free-swimming doliolaria larvae were half the length of their siblings and had a constriction around the penultimate ciliary band. These buds retained a ciliary band and were still attached by a thin tether into the benthic pentacula stage 12 h later (Fig. 1a). Eventually, after separation, buds developed into normal auricularia larvae with a juvenile rudiment (Fig. 1b). Most sand dollar larvae metamorphosed within 7 weeks, but 6 of 170 (3.5%) formed a hollow bud near the future juvenile mouth (results not shown). Once separated, the evenly ciliated buds developed into an almost solid gastrula with bilateral spicules. Within two days, a tripartite gut and further skeletal spicules formed, and the clones began to feed. After 4 weeks, nearly 5% of sea urchin larvae (n>500) showed constrictions at their posterior end (Fig. 1c). These eventually yielded uniformly ciliated buds that began feeding within four days, developed paired skeletal rods within a week, and acquired a normal larval form within two weeks. Juve- nile rudiments began to form within three weeks (Fig. 1d), accompanied by further larval arms. Larval cloning is therefore known to occur in all classes except crinoids (feather stars and sea lilies), supporting an earlier conjecture that it might be an ancestral ability of echinoderms 8 . The mechanisms by which this cloning occurs, however, are unexpect- edly diverse. First, clones may arise from var- ious larval body regions, including arms (sea stars 1–4 , brittle stars 5 ), the oral hood (sea stars 2–4 ), the posterior end (sea stars 3 , sea cucumbers (Fig. 1a), sea urchins (Fig. 1c)) and the lateral body wall (sea stars 2 , sand dol- lars (our results, not shown)). Second, the developmental stage of clones at separation ranges from blastulae 2 to fully formed lar- vae 3 . Third, some clones may not separate until after the primary larva has begun to metamorphose 5 (Fig. 1a). This indicates either that larval cloning evolved indepen- dently on several occasions or that its mecha- nisms have diverged widely from an ances- tral mode. Cloning can be surprisingly frequent: up to 12% in laboratory-reared sea cucumber larvae and 10–90% in samples of field-col- lected sea star larvae 1,4 . The fact that such a common phenomenon should have been overlooked in heavily studied organisms seems remarkable. Were preconceptions about ‘normal’ development so strong that cloning was simply dismissed as aberrant? From a theoretical perspective, larval cloning — particularly in sea urchins — challenges a central tenet of the ‘set-aside’ cell theory 9 because, contrary to prediction, larval body cells are not “essentially eutelic” 9 , but can differentiate into juvenile structures (Fig. 1b, d). Nevertheless, such cloning offers an opportunity to study the well-characterized developmental regula- tory networks of sea urchins 10 in a new ontogenetic context. Larval cloning represents an intriguing new dimension to invertebrate life histo- ries. The process confers three potential ecological advantages: increased fecundity under optimal growth conditions 2,3 , increased chances of settlement after a protracted larval life 1,2,5 , and recycling of otherwise discarded or reabsorbed larval tissue 5 . Larval cloning may also be evolu- tionarily significant, for two reasons. First, clones may subsequently clone 5 , potentially leading to a new, entirely pelagic bauplan. Second, although it may be merely another idiosyncrasy of echinoderm development, larval cloning might also be more taxo- nomically widespread. As nearest relatives to the echinoderms 11 , acorn worms offer a critical test. If their tornaria larvae clone, then ancient deuterosomes may have had this ability. Alexandra A. Eaves*, A. Richard Palmer† *Physiology and Cell Biology Group, and Systematics and Evolution Group, Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada, and Bamfield Marine Sciences Centre, Bamfield, British Columbia V0R 1B0, Canada e-mail: [email protected] 1. Bosh, I., Rivkin, R. B. & Alexander, S. P. Nature 337, 169–170 (1989). 2. Jaeckle, W. B. Biol. Bull. 186, 62–71 (1994). 3. Vickery, M. S. & McClintock, J. B. Biol. Bull. 199, 298–304 (2000). 4. Knott, K. E., Balser, E. J., Jaeckle, W. B. & Wray, G. A. Biol. Bull. 204, 246–255 (2003). 5. Balser, E. J. Biol. Bull. 194, 187–193 (1998). 6. Mortensen, T. Studies of the Development and Larval Forms of Echinoderms (Gad, Copenhagen, 1921). 7. MacBride, E. W. Nature 108, 529–530 (1921). 8. Lacalli, T. C. Invertebr. Biol. 119, 234–241 (2000). 9. Davidson, E. H., Cameron, R. A. & Ransick, A. Development 125, 3269–3290 (1998). 10. Davidson, E. H. et al. Science 295, 1669–1678 (2002). 11. Cameron, C. B., Garey, J. R. & Swalla, B. J. Proc. Natl Acad. Sci. USA 97, 4469–4474 (2000). Competing financial interests: declared none. brief communications 146 NATURE | VOL 425 | 11 SEPTEMBER 2003 | www.nature.com/nature Figure 1 Asexual budding by echinoderm larvae. a, Sea cucumber (Parastichopus californicus; Holothuroidea; Aspidochirotida) early pen- tacula with clone attached at posterior end (arrowhead). b, Sea cucumber 18 days after separation as a complete auricularia larva, with juvenile rudiment (arrow). c, Sea urchin (Strongylocentrotus purpuratus; Echinoidea, Echinacea) larva with a clone (arrow) constricting from the posterior end around the robust ciliary band. d, Sea urchin clone three weeks after separation, with early juvenile rudiment (arrow). Scale bars, 50 Ȗm. Adult P. californicus were collected sub-tidally from San Juan Island, Washington, and gametes were col- lected by dissection. Larvae were maintained at 11 C under natural lighting in filtered sea water and were fed a red alga Rhodomonas salina, ad libitum. Adult S. purpuratus from Prasiola Point (Vancouver Island, Canada) were spawned by intracoelomic injection of 0.55 M potassium chloride. Larvae were maintained in a 19-h light cycle at 14 C in pasteurized sea water and were fed >10 6 cells per ml of a golden brown alga Isochrysis galbana and R. salina; food and water were replaced every 72 h. To monitor their development, cloning larvae and separated clones were isolated in 7-ml glass vials and cultured; most developed to metamorphosis. Cultures were deemed to be healthy as no larval tissue necrosis was seen. Further details are available from the authors. brief communications is intended to provide a forum for brief, topical reports of general scientific interest and for technical discussion of recently published material of particular interest to non-specialist readers (communi- cations arising). Priority will be given to contributions that have fewer than 500 words, 10 references and only one figure. Detailed guidelines are available on Nature’s website (www.nature.com/nature). © 2003 Nature Publishing Group

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Page 1: Reproduction: Widespread cloning in echinoderm larvae

Reproduction

Widespread cloning inechinoderm larvae

Asexual reproduction by free-livinginvertebrate larvae is a rare and enig-matic phenomenon and, although it

is known to occur in sea stars1–4 and brittlestars5,6, it has not been detected in otherechinoderms despite more than a centuryof intensive study6,7. Here we describespontaneous larval cloning in three speciesfrom two more echinoderm classes: a seacucumber (Holothuroidea), a sand dollarand a sea urchin (Echinoidea). Larvalcloning may therefore be an ancient abilityof echinoderms and possibly of deutero-stomes — the group that includes echino-derms, acorn worms, sea squirts andvertebrates.

To confirm that genuine cloning wasoccurring, we reared cloning larvae and sep-arated clones individually (Fig. 1, legend).Although most sea cucumber larvae meta-morphosed normally after a month, 5 of 41(12.2%) free-swimming doliolaria larvaewere half the length of their siblings andhad a constriction around the penultimateciliary band. These buds retained a ciliaryband and were still attached by a thin tetherinto the benthic pentacula stage 12 h later(Fig. 1a). Eventually, after separation, budsdeveloped into normal auricularia larvaewith a juvenile rudiment (Fig.1b).

Most sand dollar larvae metamorphosedwithin 7 weeks, but 6 of 170 (3.5%) formed ahollow bud near the future juvenile mouth(results not shown). Once separated, theevenly ciliated buds developed into analmost solid gastrula with bilateral spicules.Within two days, a tripartite gut and furtherskeletal spicules formed, and the clonesbegan to feed.

After 4 weeks, nearly 5% of sea urchinlarvae (n�500) showed constrictions attheir posterior end (Fig.1c).These eventuallyyielded uniformly ciliated buds that beganfeeding within four days, developed pairedskeletal rods within a week, and acquired anormal larval form within two weeks. Juve-nile rudiments began to form within threeweeks (Fig. 1d), accompanied by furtherlarval arms.

Larval cloning is therefore known tooccur in all classes except crinoids (featherstars and sea lilies), supporting an earlierconjecture that it might be an ancestral abilityof echinoderms8. The mechanisms by whichthis cloning occurs, however, are unexpect-edly diverse.First, clones may arise from var-ious larval body regions, including arms (seastars1–4, brittle stars5), the oral hood (seastars2–4), the posterior end (sea stars3, seacucumbers (Fig. 1a), sea urchins (Fig. 1c))and the lateral body wall (sea stars2, sand dol-lars (our results, not shown)). Second, the

developmental stage of clones at separationranges from blastulae2 to fully formed lar-vae3. Third, some clones may not separateuntil after the primary larva has begun tometamorphose5 (Fig. 1a). This indicateseither that larval cloning evolved indepen-dently on several occasions or that its mecha-nisms have diverged widely from an ances-tral mode.

Cloning can be surprisingly frequent: upto 12% in laboratory-reared sea cucumberlarvae and 10–90% in samples of field-col-lected sea star larvae1,4. The fact that such acommon phenomenon should have beenoverlooked in heavily studied organismsseems remarkable. Were preconceptionsabout ‘normal’ development so strong thatcloning was simply dismissed as aberrant?From a theoretical perspective, larvalcloning — particularly in sea urchins —challenges a central tenet of the ‘set-aside’cell theory9 because, contrary to prediction,larval body cells are not “essentiallyeutelic”9, but can differentiate into juvenilestructures (Fig. 1b, d). Nevertheless, suchcloning offers an opportunity to study thewell-characterized developmental regula-tory networks of sea urchins10 in a newontogenetic context.

Larval cloning represents an intriguingnew dimension to invertebrate life histo-ries. The process confers three potentialecological advantages: increased fecundityunder optimal growth conditions2,3,increased chances of settlement after aprotracted larval life1,2,5, and recycling ofotherwise discarded or reabsorbed larvaltissue5. Larval cloning may also be evolu-tionarily significant, for two reasons. First,clones may subsequently clone5, potentiallyleading to a new, entirely pelagic bauplan.Second, although it may be merely another

idiosyncrasy of echinoderm development,larval cloning might also be more taxo-nomically widespread. As nearest relativesto the echinoderms11, acorn worms offer acritical test. If their tornaria larvae clone,then ancient deuterosomes may have hadthis ability.Alexandra A. Eaves*, A. Richard Palmer†*Physiology and Cell Biology Group, and†Systematics and Evolution Group, Department ofBiological Sciences, University of Alberta,Edmonton, Alberta T6G 2E9, Canada, andBamfield Marine Sciences Centre, Bamfield, BritishColumbia V0R 1B0, Canadae-mail: [email protected]. Bosh, I., Rivkin, R. B. & Alexander, S. P. Nature 337, 169–170

(1989).

2. Jaeckle, W. B. Biol. Bull. 186, 62–71 (1994).

3. Vickery, M. S. & McClintock, J. B. Biol. Bull. 199, 298–304

(2000).

4. Knott, K. E., Balser, E. J., Jaeckle, W. B. & Wray, G. A. Biol. Bull.

204, 246–255 (2003).

5. Balser, E. J. Biol. Bull. 194, 187–193 (1998).

6. Mortensen, T. Studies of the Development and Larval Forms of

Echinoderms (Gad, Copenhagen, 1921).

7. MacBride, E. W. Nature 108, 529–530 (1921).

8. Lacalli, T. C. Invertebr. Biol. 119, 234–241 (2000).

9. Davidson, E. H., Cameron, R. A. & Ransick, A. Development

125, 3269–3290 (1998).

10.Davidson, E. H. et al. Science 295, 1669–1678 (2002).

11.Cameron, C. B., Garey, J. R. & Swalla, B. J. Proc. Natl Acad. Sci.

USA 97, 4469–4474 (2000).

Competing financial interests: declared none.

brief communications

146 NATURE | VOL 425 | 11 SEPTEMBER 2003 | www.nature.com/nature

Figure 1 Asexual budding by echinoderm larvae. a, Sea cucumber (Parastichopus californicus; Holothuroidea; Aspidochirotida) early pen-

tacula with clone attached at posterior end (arrowhead). b, Sea cucumber 18 days after separation as a complete auricularia larva, with

juvenile rudiment (arrow). c, Sea urchin (Strongylocentrotus purpuratus; Echinoidea, Echinacea) larva with a clone (arrow) constricting

from the posterior end around the robust ciliary band. d, Sea urchin clone three weeks after separation, with early juvenile rudiment

(arrow). Scale bars, 50 �m. Adult P. californicus were collected sub-tidally from San Juan Island, Washington, and gametes were col-

lected by dissection. Larvae were maintained at 11 �C under natural lighting in filtered sea water and were fed a red alga Rhodomonas

salina, ad libitum. Adult S. purpuratus from Prasiola Point (Vancouver Island, Canada) were spawned by intracoelomic injection of 0.55 M

potassium chloride. Larvae were maintained in a 19-h light cycle at 14 �C in pasteurized sea water and were fed �106 cells per ml of a

golden brown alga Isochrysis galbana and R. salina; food and water were replaced every 72 h. To monitor their development, cloning larvae

and separated clones were isolated in 7-ml glass vials and cultured; most developed to metamorphosis. Cultures were deemed to be

healthy as no larval tissue necrosis was seen. Further details are available from the authors.

brief communications is intended to provide a forumfor brief, topical reports of general scientific interest andfor technical discussion of recently published material ofparticular interest to non-specialist readers (communi-cations arising). Priority will be given to contributionsthat have fewer than 500 words, 10 references and onlyone figure. Detailed guidelines are available on Nature’swebsite (www.nature.com/nature).

© 2003 Nature Publishing Group