PROCEEDINGS

OF THE

SECOND JOINT SYMPOSIUM

ON THE

NATURAL HISTORY AND GEOLOGY OF THE BAHAMAS

Edited by Tina M. Niemi and Kathleen Sullivan Sealey

ORGANIZER:

Troy A. Dexter

Executive Director Gerace Research Centre University of The Bahamas San Salvador, The Bahamas

2020

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Copyright 2020, Gerace Research Centre

All rights reserved. No part of this work may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopying, recording, or any data storage or retrieval system without the express written permission of the Gerace Research Centre.

ISBN: 978-0-935909-67-8

iv The 2nd Joint Symposium on the Natural History and Geology of The Bahamas

NATURAL HISTORY CHARACTERISTICS OF HYDRIFORMIS, AN APODID FROM OYSTER POND, SAN SALVADOR ISLAND, THE BAHAMAS

Eric S. Cole, Leigh Anne Hahn, Jessica Choquette, and Miranda Thacker Biology Department, St. Olaf College Northfield, MN 55057

ABSTRACT for colonizing the island’s inland, anchialine Synaptula hydriformis (Echinodermata, pond communities. Holothuroidea, , : Lesueur, 1824) is a small, hermaphroditic sea cucumber INTRODUCTION commonly found creeping over the benthic pearl oyster communities in Oyster Pond, San Salva- While investigating invertebrate communi- dor Island, The Bahamas. Unusual features in- ties in the anchialine ponds of San Salvador Island clude possession of an ovo-testis; internal self- in The Bahamas, we frequently encountered a fertilization; embryonic brooding accompanied small, "naked sea cucumber" identified as Synap- by matrophagy (juveniles feed on their mother’s tula hydriformis living in Oyster Pond. These soft- coleomic cells and tissues); and viviparous, live- bodied bear little resemblance to birth of juveniles with adult characteristics. other, hard-bodied members of the phylum, (sea Through live-culture and paraffin histology, life stars, brittle stars, and urchins) and have adapted a history stages were characterized. The ovo-testis life style that seems to have converged with that of was found to contain both sperm and eggs sup- small benthic worms. At first viewed as a nuisance porting its characterization as a simultaneous (they clung to wetsuits and booties with minute, hermaphrodite. Three unique color morphs were hook-like ossicles embedded in their gelatinous described: solid brown, brown striped, and trans- skins), we soon became fascinated by their natural lucent. In living transparent specimens, we are history and set out to investigate (and manipulate) able to observe juveniles ambulating and feeding their reproductive life cycle. within the mother’s body cavity. Autogamy Synaptula hydriformis was first described (self-fertilization) and vivipary (live-birth) mark by the accomplished French naturalist Charles-Al- the synaptid family as unusual among sea cucum- exandre Lesueur in 1824 during his association bers. Other families of sea cucumber tend to re- with William Maclure and Robert Owen, the re- lease gametes into the water column where they markable founders of an intellectual commune es- undergo external fertilization and develop into tablished in New Harmony, Indiana. Lesueur first free-swimming planktonic larvae. We developed encountered Synaptula hydriformis on an expedi- a method for inducing both synchronous spawn- tion he made with William Maclure in the winter ing and birthing. Subsequently, a successful of 1815-1816 to the West Indies, in a rich man- technique for culturing juvenile sea cucumbers grove community on the north side of the island of was established. Developmental stages were Guadeloupe. The species has been referred to by characterized for the larvae. We found evidence many different names, including Holuthuria hydri- for direct development, bypassing both auricu- formis, Holuthuria viridis, Synaptula vivipara, laria and doliolarian stages and skipping the met- Synapta viridis, Synapta pourtalesii, Leptosynapta amorphosis typical of other holothuroideans. We hydriformis, Leptosynapta pourtalesii, Heter- discuss Synaptula in the context of other her- osynapta viridis, Synapta vivipara, Synapta picta, maphroditic organisms that seem well-adapted and Chondrocloea vivipara (Heding, 1928).

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The second significant mention of Synap- and parturition developed, this organism offers tula hydriformis involves a specimen collected by promise as a novel model organism for the study of the U.S. Fish Commission on an expedition to The invertebrate () embryology. Their abil- Bahamas in 1886 from the very first ship ever de- ity to undergo live-birth also provides an intriguing signed specifically for marine research, the USS Al- novelty rarely seen outside of the chordate phylum: batross. While exploring San Salvador Island (for- a mechanism by which juveniles living within the merly Watling’s Island), the Albatross obtained a mesoderm-lined coelomic space of their mothers second specimen of this sea cucumber. This was are shunted to the ’s posterior, expressed stored in the Smithsonian Museum, and later iden- into the outside world within a mesoderm/ecto- tified as Synaptula hydriformis (Lesueur 1824) derm “birthing sac”, and released through a devel- when Hubert Clark uncovered it in the 1900s. opmentally programmed (reversible?) rupture of Clark performed extensive (beautifully illustrated) that sac during birth. It seems likely that juveniles research on this self-fertilizing, internally brooding from multiple, asynchronous reproductive cohorts species and wrote prolifically of its physiology, are delivered (exhibiting “superfetation”) as evi- , and embryology (Clark, 1897, 1907). denced by varying sizes and states of development The Smithsonian obtained a more recent sample of the progeny expressed at the time of birth from from The Bahamas in 1996, collected by Charlene a single individual. It would be interesting to learn D. Long (American Natural History Museum). whether or not larval forms can be sorted and re- The most-studied specimens were collected from tained when older juveniles are birthed, or if they Bermuda, Panama, Jamaica, and the Florida Keys. too are discharged without regard to their develop- Since Clark’s impressive monograph, there are mental maturity. only modest references to the species until Jennifer Frick (Smithsonian Marine Station, Fort Piece, FL) METHODS took up the study of their ovo-testis and matroph- agy (or matrotrophy: feeding on, or deriving nour- Collection and laboratory culture of ishment from ones' mother) in the late 1990s for Synaptula hydriformis her Ph.D. Frick collected specimens from Indian River Lagoon and Lake Surprise on Key Largo Specimens were collected by hand while (Frick et al., 1996; Frick, 1998). snorkeling over beds of scaly pearl oysters in Synaptids are interesting from a variety of Oyster Pond, San Salvador Island, The Bahamas. perspectives. Unlike most other sea cucumbers, It is perhaps worth noting, that synaptids insinu- the apodid species lack tube feet for motility. They ate themselves deeply into the internal spaces of adhere to the substrate through surprisingly clingy calcareous-algal aggregates that form biotic out- dermal hooks called ossicles and ambulate using crops which also house a variety of bivalves and their feeding tentacles to pull themselves along, living sponge. It is often, unfortunately neces- while feeding on the substrate. As self-fertilizing sary to open such outcrops in order to extract liv- hermaphrodites, they are well-adapted for colo- ing specimens. We would endorse an attitude of nizing novel habitats. A single specimen could the- respectful prudence when it is necessary or desir- oretically establish an entire population without a able to collect living specimens. Individual mating partner. This has implications for genetic specimens were isolated and maintained in finger diversity. It is possible that the entire population bowls with seawater, and fed small droppers-full within our field site is comprised of only a few ge- of sedimentary floc collected from Oyster Pond netic “clones”. The life-style of embryonic brood- (a loose bottom sediment rich in microbial mate- ing and matrophagy is also interesting. If labora- rial that the Synaptula appear to feed on). Floc tory cultures of the transparent adults can be estab- was maintained under full-spectrum lights to pro- lished, and experimental triggers for fertilization mote algal growth.

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Induction of birthing and spawning Olympus BH40 microscope using bright field op- tics and a DP-71 digital camera. Fixed histological Adult Synaptula were placed in 90 mm pe- specimens were examined using the Olympus tri dishes half-filled with seawater and floated on BH40 microscope. an ice-water bath for ten minutes. These gave birth within 6-18 hours (exhibiting a lot of individual variation). Juveniles of numerous developmental Paraffin Histology stages from a single adult were observed, confirm- ing “superfetation” (multiple broods maintained Sea cucumbers were collected in January within the same adult). We also used this cold- 2017. Specimens were fixed in Bouin’s fixative, shock treatment to induce ovulation and internal dehydrated, and embedded in paraffin as described spawning, which we confirmed by subsequently by Humason (1979). Eight to nine micron-thick decapitating and expressing the coelomic contents sections were made and slices were stained in onto a microscope slide. By doing this at various Gomori’s trichrome stain (Fisher Scientific Inc.). times after the cold shock, successive developmen- Slides were examined using an Olympus BH40 mi- tal of stages were recovered. It is not clear that this croscope using brightfield optics. Images were was a highly efficient method for obtaining larval taken with a DP-71 digital camera. stages, and we recently learned that Frick em- ployed a light/dark transition to induce spawning. She held them in continuous illumination and then RESULTS exposed them to darkness for approximately one hour. She then returned them to light, and egg re- Synaptula hydriformis color morphs lease began within 5 minutes (Fricke et al., 1996). Three distinct color morphs were identified Photography from Oyster Pond on San Salvador Island in The Bahamas: a solid brown, a “tiger-striped” form, Live images were taken with hand-held and a pale white or translucent (colorless) variety iPhones, focused through the microscope ocular (Figure 1). The relative abundances of the various lens, or deployed as makeshift macro-lens cameras. color morphs from a sample of 329 specimens were In one case the live image was taken with an 108 brown, 118 striped, and 103 translucent.

Figure 1: Three color morphs of Synpatula hydriformis: brown (left), striped (center), and translu- cent (right). Note, individuals are typically ~5 cm long.

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Figure 2: Paraffin histology, and Gomori's triple-stain sections of Synaptula ovo-testis. Red lines point to developing oocytes. Blue lines point to bundles of developing sperm cells. Scale bars = 50 um.

Histological observations of the gonad from their parental host (coelomocytes and possi- bly undeveloped eggs, Frick, 1998). Synaptula specimens were fixed, embed- ded in wax, sectioned, and stained using Gomori's tri-chrome. Examples appear in Figure 2. We could readily discern both developing sperm bundles and maturing oocytes within the same gonad, confirming this organ as an "ovo- testis".

Live observations of vivipary and matrotrophy

Translucent specimens allowed us to ob- serve live juveniles ambulating and feeding within the parent’s body cavity (Figure 3). Juveniles were fully formed and exhibited the classic holothurian Figure 3: Translucent bodies of Synaptula hy- “feed-walking” behavior in which they use their driformis under low magnification microscopy. sticky, para-oral tentacles to both pull themselves The left image is un-retouched. The right image along the substrate (in this case, their parent’s body highlights the outlines of juveniles in blue. cavity) and feed. After drawing themselves for- ward, each tentacle is inserted into the oral cavity, Parturition and adherent food/substrate is “wiped off” by ac- tion of an oral sphincter as the tentacle is retrieved Adult specimens were isolated into 90 mm for its next step. Given the variety of sizes of juve- petri dishes half-full of seawater. Individual dishes niles, from 200 microns up to nearly 1 cm, it was were floated on an ice-slurry for ten minutes and clear that the juveniles were indeed growing within restored to room temperature (76° F). Within 6-18 the parent’s body cavity, deriving nourishment hours, multiple juveniles were expressed from the

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posterior cloaca of the adults. The process of birth- cloacal opening. Eversion of this dual-membrane ing (at least by this artificial induction) appears to “birthing sac” is an extraordinary event. At some involve eversion of a membrane sack from the clo- point, an oval aperture appeared in the sac, through aca (Figure 4) that gradually fills with juveniles. which juveniles were expressed into the seawater medium. Juveniles expressed in this manner have been kept alive on Oyster Pond floc culture for up to 52 days (Figure 6). It should be noted that cold- shock induced birthing (especially appearance of the "birthing sac") may differ from birth that occurs in nature. A "natural birth" video by Alvaro Migotto (2017) from the University of Sao Paulo shows juveniles (and embryos) being expelled from the cloaca directly.

Figure 4: Five juveniles have been expressed into the birthing sac. Arrow indicates cloaca.

Figure 6: Two juveniles freshly born by cold- shock induced parturition. Image shows pos- terior of intact adult and two minute, live ju- veniles approximately 1-2 mm in length (ar- rows).

Figure 5: Complex double-membrane of birthing sac emerging from adult’s cloaca (paraffin histology). Superfetation

The membrane sack appears to be comprised of Cross-section histology revealed embryos two membranes (Figure 5). We suspect that these and juveniles of conspicuously different reproduc- twin membranes represent mesoderm and endo- tive cohorts within a common coelom. This sug- derm, respectively, as juveniles that are living in- gests that multiple rounds of ovulation and self-fer- side a mesodermally-lined coleomic space are sep- tilization can occur on a rolling schedule without arated from the outside world by both the coelomic intervening births—a process known as superfeta- lining and either ectoderm or endoderm near the tion (Figures 7 and 8).

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induction were obtained and photographed, re- sulting in a near complete gallery of developmen- tal stages (Figure 9). In one specimen, 75 juve- niles were found inside the adult body cavity. Although this technique of inducing gamete re- lease and fertilization appeared to work, the fre- quency of early (presumably “induced”) embryos produced seemed low. Since then, we have learned that Fricke deployed a light/dark entrain- ment to induce ovulation, triggered by a “lights- on” signal (Fricke et al., 1996).

Figure 7: An embryo (upper right) and a much larger juvenile (middle) within a single coelomic space.

Figure 8. Four (or more) embryos and juveniles of different sizes (ages?) being expressed during Figure 9: Embryos at various stages of devel- parturition (arrows). opment surgically expressed from shocked adults. Stages represent (beginning at center- left and traveling clockwise): (1) blastula, (2) Induction of ovulation, self-fertilization, and gastrula, (3) complete juvenile with 5 tentacle embryogenesis buds, (4) tentacle elongation, and (5) appear- ance of ossicles (tiny endoskeletal hooks in the To avoid multiple environmental shocks outer skin). Timing for stages: blastula: 3 hrs; accompanying collection and transport before lab gastrula: 6.5 hrs; tentacle buds: 12 hrs; ossicles: work, and the consequent confusion of timing, 24 hrs, post-fertilization. we treated isolated adult Synaptula to an ice- shock in the field at the time of capture. Back in DISCUSSION AND CONCLUSIONS the lab, at various times after this initial shock, Synaptula adults were decapitated and coelomic Synaptula hydriformis exhibit a broad fluids expressed by squeezing. In this way, juve- repertoire of fascinating reproductive habits in- niles (variable ages) and embryos from a variety cluding simultaneous hermaphroditism, internal of stages more tightly correlated with time of self-fertilization (they exhibit almost a clonal

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form of propagation), direct development from whether these isolated anchialine pond environ- gastrula to the adult body form without the inter- ments are unusually rich in self-fertilizing spe- vening metamorphosis seen in most echinoderm cies, reflecting their potentially rare episodes of larvae, matrotrophy (feeding on maternal cells or colonization. products), superfetation (brooding multiple co- horts of young from independent spawning ACKNOWLEDGEMENTS events), and live birth. If nothing else, this study promises to expand our vocabularies. Our find- This work was conducted in The Bahamas under a ings largely confirm earlier discoveries from the permit granted by The Bahamas Environment, Sci- elegant works of both Clark (1897, 1907, 1933), ence, and Technology (BEST) Commission. We Frick (1998) and Frick et al. (1992, 1996). Re- would like to thank Dr. Troy Dexter, Executive Di- garding matrophagy (or matrotrophy), a recent rector of the Gerace Research Centre, San Salva- review by Ostrovsky et al. (2016) suggests that dor, The Bahamas for his unfailing technical sup- matrophagy has actually arisen five times inde- port and encouragement. St. Olaf College pro- pendently within the holothuroidea, including the vided travel support, and we are grateful for field clade that encompasses both Synaptula and assistance from a number of St. Olaf undergradu- Chiridota (a second, less common apodid cu- ates including: Mark Li, Anthony Riesen, Tiffany cumber also found in Oyster Pond). Wong, Xiaoping Zhang, and Adrian Ripecky Since little other work exists on the spe- cies, it is nice to refresh these observations for a new generation of naturalists. The things that we LITERATURE CITED may have contributed uniquely to this body of work on Synaptula hydriformis include the de- velopment of an ice-shock technique for induc- Clark, H.L. 1897. Synapta vivipara: A contribution ing parturition, a photo-microscopic gallery of to the morphology of echinoderms. Memoirs of the embryonic stages, and images of the remarkable Boston Society of Natural History V(2): 53-93. “birthing sac” observed in our laboratory-in- duced cultures. Clark, H.L. 1907. The Apodus holothurians: A The discovery of a translucent morph of monograph of the Synaptidae and Molpadiidae. Synaptulid sea cucumbers, along with their direct Smithsonian Contributions to Knowledge 35: 42- pattern of development, raises the possibility of 131. recruiting this species as a new model organism for the study of invertebrate embryology. Its pat- Clark, H.L. 1933. Scientific survey of Porto Rico tern of self-fertilization also raises interesting and the Virgin Islands. New York Academy of questions about the genetic diversity of our pop- Sciences XVI. ulation and highlights its ability to colonize. This is interesting in that several organisms from the Frick, J.E. 1998. Evidence of matrophagy in the inland ponds appear to exhibit hermaphroditic Viviparous Holothuroid echinoderm Synaptula hy- (or even parthenogenetic) life history patterns driformis. Invertebrate Biology 117(2): 169-179. pre-adapting them for colonization. These in- clude the scaly pearl oyster (Pinctada longisqua- Frick, J.E., Ruppert, E.E., and Wourms, J.P. 1996. mosa: a sequential hermaphrodite), most of the Morphology of the ovotestis of Synaptula hydri- gastropods, and the mangrove killifish, Rivulus formis (Holothuroidea, Apoda): An evolutionary marmoratus (a self-fertilizing hermaphrodite we model of oogenesis and the origin of egg polarity found in Mermaid Pond and another of the inte- in echinoderms. Invertebrate Biology 115(1): 46- rior ponds). It would be interesting to explore 66.

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Frick, J., Ruppert, E.E., and Wourms, J.P. 1992. Migotti, A. 2017. A pregnant sea cucumber giving Nutrition of brooded young in a sea cucumber birth. YouTube video. https://www.youtube.com/ (Synaptula hydriformis). American Zoologist watch?v=Pz_O1rT1QVE. 32:113A. Ostrovsky, A.N., Lidgard, S., Gordon, D.P., Heding, S.G. 1928. Papers from Dr. Th. Morten- Schwaha, T., Genikhovich, G., and Ereskovsky, sen’s Pacific Expedition (1914-16). Smithsonian A.V. 2016. Matrotrophy and placentation in inver- Libraries. 154-203. tebrates: a new paradigm. Biological Reviews 91: 673–711. doi:10.1111/brv.12189 Humason, G.L. 1979. Animal Tissue Techniques. W.H. Freeman and Company, San Francisco.

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