Evidence for Sequential Hermaphroditism in Sabellastarte spectabilis (Polychaeta: ) in Hawai‘i1

David R. Bybee,2,4 Julie H. Bailey-Brock,2 and Clyde S. Tamaru3

Abstract: Understanding the reproductive characteristics of Sabellastarte specta- bilis (Grube, 1878), an economically important worm collected for the aquarium trade, is essential to the development of artificial propagation and conservation of coral reefs. The purpose of this study was to determine whether S. spectabilis is hermaphroditic. Using histological techniques, 180 indi- viduals were examined for gametes. Gametes were present only in abdominal segments. Primary oocytes were 7–8 mm in diameter in histologically prepared sections. Sperm appeared as round black dots about 2 mm in diameter on histo- logically prepared slides. Most individuals sampled had only one type of gamete in the coelom, but both eggs and sperm were seen in the coelom of 15% of individuals, demonstrating the occurrence of hermaphroditism in Hawaiian populations of S. spectabilis. The sex ratio of males to females was skewed signif- icantly toward males in both the small (6–8 mm diameter) and medium (9–10 mm diameter) sized worms. Among the largest worms (11–13 mm diameter), the sex ratio did not diverge significantly from 1:1. There was a significantly higher proportion of hermaphrodites (30%) in the large size class. Worms of unknown gender, although present in all size classes examined, were most fre- quent (33%) in the medium size class. These patterns are consistent with se- quential (protandrous) hermaphroditism.

The sabellid polychaete Sabellastarte pal collection site for this commercially im- spectabilis (Grube, 1878) is common in bays portant ornamental (Walsh et al. and harbors in the main Hawaiian Islands 2003). These worms settle in cracks and and particularly in Ka¯ne‘ohe Bay, on the crevices of living and dead reef corals, mak- windward coast of O‘ahu. Ka¯ne‘ohe Bay is ing collection difficult and damaging to the partially cut off from the ocean by a barrier reef community. Reproduction is currently reef, which protects it from large waves being studied in this species (Bybee et al. in and provides an ideal habitat for these soft- press) with the goal of facilitating artificial tubed filter feeders. Sabellastarte spectabilis is propagation to reduce pressure on coral reefs very abundant in the bay, which is the princi- due to destructive collecting practices.

1 The PADI Foundation, the Edmondson Fund (Uni- from the NOAA Office of Sea Grant, U.S. Department versity of Hawai‘i), and The Center for Tropical and of Commerce, and the State of Hawai‘i Department of Subtropical Aquaculture provided financial support for Agriculture, Aquaculture Development Program, Con- this project. This paper is funded in part by grants/ tracts No. 49855 and 52663. The views expressed herein cooperative agreements from the U.S. Department of are those of the authors and do not necessarily reflect the Agriculture, CSREES Agreement No. 2002-38500- views of NOAA, USDA, the State of Hawai‘i, or any of 12039; National Oceanic and Atmospheric Administra- their subagencies. UNIHI-SEAGRANT-JC-04-09. Fa- tion, Project No. A/AS-1, which is sponsored by the cilities were generously made available for this research University of Hawai‘i Sea Grant College Program, at the Hawai‘i Institute of Marine Biology (contribution SOEST, under Institutional Grant No. NA16RG2254 no. 1216). Manuscript accepted 9 December 2005. 2 Department of Zoology, University of Hawai‘i at Ma¯noa, Honolulu, Hawai‘i. 3 College Sea Grant Program, University of Hawai‘i Pacific Science (2006), vol. 60, no. 4:541–547 at Ma¯noa, Honolulu, Hawai‘i. : 2006 by University of Hawai‘i Press 4 Corresponding author (fax: 808-956-9812; e-mail: All rights reserved [email protected]).

541 542 PACIFIC SCIENCE . October 2006

Polychaetes are predominately gonochoric ditic and the nature of the hermaphroditism (separate sexes) (Schroeder and Hermans formed the basis for this investigation. 1975) with hermaphroditism occurring in a minority of families. Of the 9,000 known materials and methods species belonging to 81 families of (Rouse and Pleijel 2001), hermaphroditism Worms were collected at monthly intervals has been reported in a total of 67 species (2002–2003) from the reefs surrounding the among 25 families (Giangrande 1997). About Hawai‘i Institute of Marine Biology on Moku half of the reported hermaphroditic species O Lo‘e (also known as Coconut Island) in are sequential hermaphrodites (36); the ma- Ka¯ne‘ohe Bay for histological analyses of ga- jority of those (32) are protandrous forms metogenesis and examination of their inter- (Giangrande 1997). The remainder (31) are nal anatomy. Whole specimens were fixed in simultaneous hermaphrodites (Schroeder and 10% seawater formalin for a minimum of 72 Hermans 1975, Giangrande 1997). hr, after which they were transferred to 80% Gonochorism is also the predominant con- ethanol for long-term storage. Transverse dition within the Sabellidae, though her- sections were taken from both the thorax maphroditism is not uncommon. Most of the and abdomen of whole worms. Tissue sam- reported sabellid hermaphrodites are listed ples were placed in tissue cassettes and were as simultaneous, and sequential (protandrous) then processed using a tissue processor (Shan- forms seem to be rare (Rouse and Fitzhugh don Citadel 2000) and embedded in paraplast 1994). However, it has been hypothesized with an embedding center (Tissue Tek III). that sequential hermaphroditism is probably The paraplast blocks were sectioned at a more widespread than we know and that thickness of 4 mm using a microtome (Ameri- more detailed studies are needed to sup- can Optical) and placed on microscope slides. port this supposition (Rouse and Fitzhugh The slides with adhered sections were then 1994). cleared with xylene and dehydrated through There are eight accepted species in the an alcohol series and finally stained with genus Sabellastarte according to the most hematoxylin/eosin stain. Individuals were recent revision (Knight-Jones and Mackie classified as male if only sperm were present 2003). Of those eight species, only two have and female if only oocytes were found. Those any published information on reproductive with both male and female gametes were clas- mode. Runganathan (1943) observed S. mag- sified as hermaphrodites. Worms with only nifica (Shaw, 1800) [later identified as S. indica coelomocytes were classified as unknown. (Savigny, 1822)] (Rouse and Fitzhugh 1994) Fresh samples of coelomic contents were also to be a simultaneous hermaphrodite. Sabellas- extracted from live worms, and gametes were tarte indica has since been synonymized with digitally photographed using a compound S. spectabilis (Knight-Jones and Mackie 2003). light microscope (Nikon Eclipse E200). Rouse and Fitzhugh (1994) observed three The body diameter of worms (removed preserved specimens of the same species, two from tubes) collected during months in which ‘‘small’’ individuals from Kiribati and one gametogenesis is known to occur (May– from Palau (Belau). All three were reportedly November) was measured with calipers to gonochoric. They also examined two pre- the nearest millimeter, and worms were di- served specimens of S. magnifica. One speci- vided into three different size classes (small, men (from the West Indies) was found to be 6–8 mm; medium, 9–10 mm; and large, 11– a simultaneous hermaphrodite and the other 13 mm). Worms were further placed into the (from Puerto Rico) was gonochoric. Under- following reproductive groups: male, female, standing the reproductive characteristics of S. hermaphrodite, and unknown, based on ob- spectabilis, an economically important worm, servations made from the histologically pre- is a prelude to the development of means to pared slides. A chi square (X 2) test was used artificially propagate the species. Determining to assess the statistical significance of various whether or not this polychaete is hermaphro- levels of abundance between groups. Sequential Hermaphroditism in Sabellastarte spectabilis . Bybee et al. 543

Scanning Electron Microscopy 1D). A more detailed description of S. specta- bilis larval morphology and discussion of life Scanning electron microscopy (SEM) was history implications is found in Bybee et al. used to confirm spermatocytes. This was (in press). For a review of polychaete sperma- done by first extracting coelomic contents tozoa morphology see Jamieson and Rouse from several individuals until a male was (1989). found. The contents were pipetted from the Most worms (85%) had only one type of coelom, then fixed with 4% gluteraldehyde gamete in the coelom and were designated in 0.1 molar sodium cacodylate buffer (7.6) as being male or female. Several individuals with 0.35 molar sucrose. They were washed (15%) clearly possessed both oocytes and in 0.1 molar sodium cacodylate buffer with spermatocytes (Figure 1A,B) and were classi- 0.44 molar sucrose. Coelomic contents were fied as hermaphrodites (Table 1). Individuals then postfixed with 1% osmium tetroxide in in which no gametes could be identified were buffer and dehydrated in a graded series of classified as unknown. Observations of coelo- ethanol, critical point dried, and mounted mic contents of live worms were consistent on aluminum stubs. They were then sputter with histological observations. A histogram coated with gold palladium and viewed in a (Figure 2) revealed that the sex ratio of males field emission scanning electron microscope to females was skewed significantly toward (Hitatchi S-800) at 15 kv accelerating voltage. males (P < 0:01, X 2 test) in both the small The abdomen and thorax were also sectioned and medium size classes. In the large size and prepared for histological processing as class, however, the sex ratio did not diverge just described. significantly from 1:1. The proportion of hermaphrodites found in the large size class 2 results was significantly higher (P < 0:05, X test) than what was observed in the small and me- To date, gender in S. spectabilis cannot be de- dium size classes. Worms that were charac- termined visually on whole specimens from terized as unknown were most abundant in external appearance. This makes extraction the medium size class. and wet-mount microscopic examination of coelomic contents or the use of histological discussion preparation and examination of thin sections necessary. Gametes were present only in ab- From the data presented in the current inves- dominal sections. Oocytes were first visible tigation it appears that many of the smaller on histologically prepared slides at about 7–8 worms in the population sampled were males. mm in diameter using a compound micro- All four groups (male, female, hermaphrodite, scope at 40 magnification. These cells were and unknown) were present in all of the size composed mostly of a large nucleus contain- classes examined. It is not clear whether all ing a dark nucleolus and surrounded by a individuals first become sexually mature as grainy-textured ooplasm (Figure 1A). Wet males as is the case with protandrous her- mounts of oocytes showed little more than a maphrodites. However, within the size range spherical, gray structure (Figure 1B) though of individuals examined, there is a clear shift the nucleus and nucleolus were sometimes in the percentage composition of the sexes, visible (Figure 1C). Sperm cells appeared on particularly as the worms attain the large size histologically prepared slides as round black class (11–13 mm diameter). At this size many dots about 2 mm in diameter (Figure 1A). of the males apparently change gender, be- Sperm on wet mounts appeared as small, coming females. This is indicated by the translucent, circular cells with a faint but long significant increase in the proportion of her- flagellum (Figure 1B,E), which were often maphrodites that is found in the large size very active. A scanning electron micrograph class. One clear result of the observed transi- of an individual sperm cell clearly shows the tion in gender is that the sex ratio is more round sperm head and flagellum (Figure evenly distributed between both sexes. It is 544 PACIFIC SCIENCE . October 2006

Figure 1. Gametes of Sabellastarte spectabilis. A. Both oocytes (O) and sperm (S) in the coelom. Scale bar ¼ 10 mm. B. Wet mount of coelomic contents showing one egg (O) and several sperm cells (S). Scale bar ¼ 25 mm. C. An unfertil- ized egg taken from the coelom showing ooplasm (oo), nucleus (n), and nucleolus (nc). Scale bar ¼ 50 mm. D. Mature sperm extracted from the coelom showing head and a portion of its flagellum. Scale bar ¼ 10 mm. E. Sperm cells show- ing long flagella (arrows). Scale bar ¼ 1.7 mm.

TABLE 1 determined morphologically whether they Number of Individuals of Sabellastarte spectabilis are indeed capable of undergoing final matu- Collected in Each Size Category ration, spawning, and production of viable larvae. Small Medium Large Our observation of hermaphroditic indi- Reproductive (6–8 (9–10 (11–13 viduals in this Hawaiian population of S. spec- Group mm) mm) mm) Totals tabilis agrees with published observations of Male 25 30 16 71 the same species in the Indian Ocean (Runga- Female 6 13 19 38 nathan 1943 [see introduction]) but differs Unknown 11 24 9 44 from observations in the Pacific (appendix 1 Hermaphrodites 3 4 20 27 in Rouse and Fitzhugh 1994). There are five Totals 45 71 64 180 Sabellastarte species in the Indo-Pacific; some are widespread (Knight-Jones and Mackie 2003). Records and museum identifications not clear whether all of the males will eventu- before 2003 may well be S. spectabilis but ally change to females as they continue to need to be confirmed. grow/age. Likewise, although gametes found There are also differing observations in males and females of the smaller size classes within Caribbean species. Sabellastare magnif- appear to be sexually mature, it remains to be ica from the West Indies was reported as a Sequential Hermaphroditism in Sabellastarte spectabilis . Bybee et al. 545

Figure 2. Size classes (from body diameter in mm) of Sabellastarte spectabilis collected in this study. simultaneous hermaphrodite, but the same broadcast spawners. This does not seem to species from Puerto Rico was determined to be the case with S. spectabilis, which is clearly be a gonochoric female (Rouse and Fitzhugh a broadcast spawner with planktonic develop- 1994). Sabellastarte magnifica is probably the ment of lecithotrophic larvae (Bybee et al. in only species of the genus in the Caribbean, press). yet there seems to be a difference in repro- According to Ghiselin (1974, 1987), her- ductive mode between the two museum maphroditism should evolve under three con- holdings (Rouse and Fitzhugh 1994). It is ditions: (1) when finding a mate is difficult, possible that S. magnifica and S. spectabilis dis- (2) in small, genetically isolated populations, play different reproductive modes in different and (3) when one sex benefits from being locations (or under different environmental larger or smaller than the other. With regard conditions). It is more likely, however, that to how these conditions may apply to Ha- this tubeworm is a sequential (protandrous) wai‘i’s population, finding a mate may be dif- hermaphrodite, which is difficult to detect ficult for sessile species like these tubeworms. with so few preserved (museum) specimens. Because S. spectabilis is believed to be an in- No quantitative size information was available troduced species to Hawai‘i (Coles and El- for the reported specimens. dredge 2002), it is difficult to know what Heath (1977, 1979) hypothesized that ecological or genetic selective forces may hermaphroditism is associated with internal have influenced the evolution of reproductive fertilization and tube brooding and that gon- mode at its location of origin. Females ochorism is found in externally fertilizing may benefit from being large because eggs 546 PACIFIC SCIENCE . October 2006 are larger and require more food resources to reefs: A need for information. Pac. Sci. produce than sperm. Females with large 56:191–209. bodies (more coelomic space) can hold more Cotter, E., R. M. O’Riordan, and A. A. eggs, thus enhancing their chances of provid- Meyers. 2003. A histological study of re- ing larvae with an adequate yolk supply to production in the serpulids Pomatoceros ensure successful reproduction. The younger, triqueter and Pomatoceros lamarckii (Annel- smaller-bodied S. spectabilis may be able to re- ida: Polychaeta). Mar. Biol. (Berl.) produce most effectively by producing sperm. 142:905–914. As these worms grow bigger, some may Fleminger, A. 1985. Dimorphism and possi- change sex, evening out the sex ratio and en- ble sex change in copepods of the family abling the production of a large number of Calanidae. Mar. Biol. (Berl.) 88:273–294. eggs, which is true of many marine organisms Ghiselin, M. T. 1974. The economy of na- (Fleminger 1985, Brook et al. 1994, Rouse ture and the evolution of sex. University and Fitzhugh 1994, Sewell 1994, Giangrande of California Press, Berkeley. 1997, Svensen and Tande 1999, Cotter et al. ———. 1987. Evolutionary aspects of marine 2003, Munoz and Warner 2003). invertebrate reproduction. Pages 609–655 Results from the current investigation in A. C. Giese, J. S. Pearse, and V. B. clearly demonstrate that the Hawaiian popu- Pearse, eds. Reproduction of marine inver- lation of S. spectabilis sampled in this study is tebrates. Vol. IX. General aspects: Seeking hermaphroditic and can be tentatively charac- unity in diversity. Blackwell Scientific terized as being a protandrous hermaphro- Publications and The Boxwood Press, dite. This is useful information for future California. phylogenetic and reproductive studies as well Giangrande, A. M. 1997. Polychaete repro- as helping to facilitate the culture of this in- duction patterns, life cycles, and life his- tensely collected marine ornamental species, tories: An overview. Oceanogr. Mar. Biol. which we hope will decrease the damage cur- Annu. Rev. 35:323–386. rently done to coral reefs during collection. Heath, D. J. 1977. Simultaneous hermaphro- ditism: Costs and benefits. J. Theor. Biol. acknowledgments 64:363–373. ———. 1979. Brooding and the evolution of We are most grateful to Ron Shimojo and Lei hermaphroditism. J. Theor. Biol. 81:151– Yamasaki at ‘A¯ nuenue Fisheries, Honolulu, 155. for assistance with the histology. We are also Jamieson, B. G. M., and G. W. Rouse. 1989. grateful for the help of I. Carvalho and the The spermatozoa of the Polychaeta (Annel- Biological Electron Microscope Facility of ida): An ultrastructural review. Biol. Rev. the University of Hawaii. 64:93–157. Knight-Jones, P., and A. S. Y. Mackie. 2003. Literature Cited A revision of Sabellastarte (Polychaeta: Sa- bellidae). J. Nat. Hist. 19:2269–2301. Brook, H. J., T. A. Rawlins, and R. W. Munoz, R. C., and R. R. Warner. 2003. A Davies. 1994. Protogynous sex change in new version of the size-advantage hypoth- the intertidal isopod Gnorimosphaeroma or- esis for sex change: Incorporating sperm egonense (Crustacea: Isopoda). Biol. Bull. competition and size-fecundity skew. Am. (Woods Hole) 187:99–111. Nat. 161:749–761. Bybee, D. R., J. H. Bailey-Brock, and C. S. Rouse, G. W., and K. Fitzhugh. 1994. Broad- Tamaru. In Press. Larval development of cast fables: Is external fertilization really Sabellastarte spectabilis (Grube 1878) (Poly- primitive: Sex, size and larvae in sabellid chaeta: Sabellidae) in Hawaiian waters. Sci. polychaetes. Zool. Scr. 23:273–301. Mar. Rouse, G. W., and F. Pleijel. 2001. Poly- Coles, S. L., and L. G. Eldredge. 2002. Non- chaetes. Oxford University Press, New indigenous species introductions on coral York. Sequential Hermaphroditism in Sabellastarte spectabilis . Bybee et al. 547

Runganathan, V. 1943. On the occurrence of Svensen, C., and K. Tande. 1999. Sex change hermaphroditism in Sabellastarte magnifica and female dimorphism in Calanus fin- Shaw together with an account of the ex- marchicus. Mar. Ecol. Prog. Ser. 176:93– cretory organs in the species. Proc. Indian 102. Sci. Congr. 29:150–151. Walsh, W. J., S. P. Cotton, J. Dierking, and I. Schroeder, P. C., and C. O. Hermans. 1975. D. Williams. 2003. The commercial ma- Annelida: Polychaeta. Pages 1–169 in A. rine aquarium fishery in Hawai‘i 1976– C. Geise and J. S. Pearse, eds. Reproduc- 2003. Pages 132–159 in A. M. Friedlander, tion of marine invertebrates. Academic ed. Status of Hawai‘i’s coastal fisheries Press, New York. in the new millennium. Proceedings of a Sewell, M. A. 1994. Small size, brooding and symposium sponsored by the American protandry in the apodid sea cucumber Lep- Fisheries Society, Hawai‘i chapter. Hono- tosynapta clarki. Biol. Bull. (Woods Hole) lulu. 187:112–123.