Infestation of the Testes of the Japanese Sea Star Asterias

DISEASES OF AQUATIC ORGANISMS l Published March 27 Dis Aquat Org

NOTE

Infestation of the testes of the Japanese sea star Asterias amurensis by the ciliate Orchitophyra stellarum: a caution against the use of this ciliate for biological control

M. Byrnelv*, A. Cerra', T. ~ishigaki~,M. ~oshi~

'Department of Anatomy and Histology, F-13, University of Sydney, N.S.W. 2006, Australia 'Department ol Life Science, Tokyo Institute of Technology. 4259 Nagatsuta. Yokohama226, Japan

ABSTRACT: Male inferthty is a new phenomenon affecting noted to exhibit this condition has increased and now populations of the Japanese sea star Asterias amurensis. The male infertility is common in inany populations. agent causing partial or total castration of the testes was iden- The changes seen in the testes of Asterias amurensis tified to be Orchitophyra stellarum, a parasitic ciliate endemic are similar to those described for Asterias species from to the north Atlantic. A. amurensis is a new host for 0. stellarum, apparently due to the recent introduction of the ciliate the north Atlantic infested with the parasitic ciliate to the Pacific Ocean. This ciliate dlsrupts the germinal layer Orchitophyra stellarum (Cepede 1907, 1910, Burrows and phagocytoses sperm. Male infert~lityof A. amurensis is 1936, Vevers 1951, Jangoux 1987). 0. stellarum is largely effected by the phagocytic response of host's cells specific to the Asteriidae and, within this family, is with each cell engulfing numerous sperm. 0. stellarum 1s highly contagious in A. arnurensls with 100% of the males in known to parasitize 4 genera and 6 species (Jangoux some populations infested. The absence of male sea stars in 1987, Leighton et al. 1991, B. J. Leighton pers. comm.). some populations is also linked to ciliate disease. This report It usually infests male sea stars, where it causes regres- of 0. stellarum from the western Pacific Ocean completes the sion of the germinal epithelium, resulting in partial or circumboreal distribut~onof this parasite. The contagious spread of 0. stellarum in Japan and its abillty to parasitize complete castration of the testis (Bouland & Jangoux several asteroid genera cautions against its use for biological 1988). 0,stellarum was originally described at the turn control of A. amurensis in Japan and of recently established of the century from the gonads of A. rubens from the pest populations of this asteroid in Australia. northeast Atlantic Ocean (Cepede 1907) and was later found in asteroids from the northwest Atlantic and from KEYWORDS: Ciliate disease . Orchitophyra stellarum . Aste- rias amurensis . Biological control the Mediterranean Sea (Burrows 1936, Smith 1936, Febvre et al. 1981). This long-standing Atlantic distnb- ution of 0. stellarum changed with the recent discovery of this parasite in the gonads of Pisastel. ochraceus, a Asterias an~urensisis a locally abundant sea star in common asteroid in the northeast Pacific Ocean Japan and has long been an important research organ- (Leighton et al. 1991). In addition to causing regression ism for biologists investigating fertilization and de- of the testes, the ciliate caused morbidity and mortality velopment (Kume & Dan 1968, Longo et al. 1995). In of P. ochraceus, symptoms not normally associated with recent times, however, it has become difficult to obtain the disease. The greater virulence of the disease in P. sperm from A. amurensis even during the peak spawn- ochraceus was taken to suggest that this asteroid is a ing period of populations previously known to be a new host for 0. stellarum (Leighton et al. 1991). The reliable source of gametes. This change in the repro- range of 0. stellarum has increased in P. ochraceus duction of A. amurensis was first noted in 1990 when populations and a second northeast Pacific species, Ev- infertile males with discoloured and hardened testes asterias troscheli, has recently succumbed to the para- were encountered. These testes were observed at a site (B.J. Leighton pers. comm.). time in the reproductive cycle when copious amounts of The aim of this investigation was to identify the sperm should have been present (Ino et al. 1955, &m agent affecting the testes of Asterias amurensis. Ultra- 1968). Since 1990, the incidence of male A. arnurensis structural examination of the gonads revealed the presence of Orchitophyra stellarum. The effect of this ciliate on A. amurensis is described and the host's

O Inter-Research 1997 Resale of full article not permitted 236 Dis Aquat Org

response is compared to that documented for other 2a, b). Equally spaced longitudinal rows of cilia, called Asterias species (Vevers 1951, Bouland & Jangoux kinet~es,extend from the surface (Fig. le). 1988). This report from Japan, together with the pres- During the 1996 breeding season, ciliate infestation ence of 0. stellarurn in Canada (Leighton et al. 1991), was widespread in 2 of the Asterias arnurensis popula- shows that this species now has a circumboreal dis- tions examined. At Tokyo Bay 100% of the males (n = tribution. Our observations coincide with the dramatic 100) had parasitized gonads and at Otuchi Bay 38 % of increase of A, amurensis in Australia, where biological the males (n = 80) were infested. Male A. arnurensis control of this recently introduced pest is being con- were not present in the Ise Bay sample (n = 50). Field sidered (Byrne 1996, Byrne et al. 1996, Viney 1996). In surveys at Tokyo Bay revealed that ciliate disease is as- light of this new phenomenon, we present a cautionary sociated with a marked decrease in the proportion of assessment of 0. stellarum as an agent for biological male sea stars with sex-ratios of 1:l observed at the be- control. ginning of breeding and ratios 10:l or 20:l in favour of Materials and methods. During the 1996 breeding females observed at the end of breeding. A similar season, Asterias amurensis was collected by SCUBA decline in the male population is reported for other from populations in northern and central Japan. Speci- Asterias species infested by Orchitophyra stellarum mens were collected from Tokyo Bay (35" 15'N, (Claereboudt & Bouland 1994) and also for Pisaster 139" 45' E) and Ise Bay (34" 301S,136" 50' E) in January ochraceus (Leighton et al. 1991). The decrease in the and from Otuchi Bay (39" 20'S, 142" 00' E) in February. number of male A. amurensis in Tokyo Bay and the ab- At each site, samples of 50 to 200 A. amurensis were sence of males from Ise Bay indicate that ciliate disease collected at a depth of 2 to 3 m. The northern-most site, may cause host mortality, as suggested for A. vulgaris Otuchi Bay, is in northern Japan and the southern-most and P. ochraceus (Leighton et al. 1991, Claereboudt & site, Ise Bay, is in central Japan. These sites are ap- Bouland 1994). Although mortality of A. amurensis was proximately 740 km apart and are separated by over not observed in the field, diseased specimens were 1000 km of shoreline. The specimens were dissected more prone to autotomise their arms and die in aquaria, and examined for the presence of diseased gonads. Dis- compared with uninfected specimens. A. arnurensis eased gonads were examined by light (LM) and trans- migrates into shallow water for breeding and so the mission electron rnicroscopy (TEM).For histological ex- preponderance of females in the sampled populations amination, the gonads were fixed in 10 % formalin or in may be due to mortality of males in deep water. Bouin's fluid, dehydrated in a graded series of ethanols, Orchitophyra stellarum was only evident in mature embedded in paraffin and sectioned (7 pm thick). The testes. The disease developed progressively. In the sections were stained with haematoxylin and eosin. For early stages of the disease, the ciliates were inter- LM and TEM, pieces of gonad were fixed in 2.5% spersed with sperm (Fig. la, c) and, in later stages, glutaraldehyde in 0.45 pm filtered seawater for 1 h, completely occluded the gonadal lumen (Fig. lb). transferred to 70% alcohol and dehydrated in a graded Early in the breeding season infected testes had an series of ethanols. The tissues were then rinsed in active spermatogenic layer and contained viable sper- acetone and embedded in Spurr's resin. Thick sec- matozoa which could be used for fertilization. The tions (0.7 pm) were stained with 1 % toluidine blue in lumen of diseased testes contained spermatozoa, cili- 0.5 % ethanolamine. For TEM, ultrathin sections were ates and phagocytes (Figs. la-e, 2a-d). Disruption of stained with uranyl acetate and lead citrate and viewed the germinal layer was indicated by the presence of in a Phillips 400 transmission electron microscope. immature sperm in the lumen which had detached Results and discussion. D~seasedgonads of Asterias from a spermatocyte column (Fig. 2c). The ciliates amurensis were readily detected due to their atypical appeared to disrupt the germinal layer by their brown colour, shrivelled appearance and hard texture. locomotory activity. They occasionally inserted their In all cases the diseased specmens were male. Histo- anterior end between adjacent spermatocyte columns logical examination of the testes revealed the presence thereby causing the columns to separate (Fig. lc). As of small groups or extensive aggregations of ciliates in the parasite population increased, the amount of sperm the lumen (Fig. la-c). Ultrastructurally, these ciliates decreased until there were no germ cells evident. In were indistinguishable from the Orchitophyra stel- the advanced stage of the disease, A, amurensis have larum described in the testes of A, rubens and A, vul- hardened testes filled with ciliates and phagocytes. garis (Bouland et al. 1987, Bouland & Jangoux 1988, The symptoms exhibited by A. amurensis in response Claereboudt & Bouland 1994). 0, stellarum is fusiform to 0. stellarum infestation are characteristic of the dis- (40 pm long X 20 pm wide) and has a rounded posterior ease in other Astenas species (Cepede 1910, Vevers end and a pointed anterior end (Figs. lc-d, 2a). A 1951, Jangoux 1987, Bouland & Jangoux 1988). macronucleus, micronucleus, a Golgi conlplex and Orchitophyra stellarum phagocytoses the sperm of numerous vacuoles occupy the cytoplasm (Figs. lc-d, Asterias amurensis and may also utilise nutrients pre- Byrne et al.: Infestation of sea star testes by a ciliate

Fig. 1. Asterias amurensis. Light micrographs of parasitized testes. (a) Cross section of a mature testis containing Orchitophyra steUarum (C) and spermatozoa (Sz). The germinal epithelium contains columns of developing sperm (SC). (b) Mature testis with ciliates (C) occluding the lumen. Sz: spermatozoa. (c)0. stellarum (C) scattered in the testis lumen, one of which has its anterior end (arrow) inserted between adjacent spermatocyte columns. (d)0. stellarum and an adjacent host phagocyte (P)which contains sperm (S). (e) Grazing section of 0. steUarum showing 3 kineties (K).Scale bars: (a) = 50 pm; (b) = 30 pm; (c, d, e) = 8.0 pm sent in gonadal fluid though uptake of fluid into the process of engulfing sperm (Figs. lc, 2d). These cells numerous vacuoles seen in the cytoplasm (Figs. Id, contained numerous phagocytosed sperm (X = 4.3, 2a, b). The mean number of sperm engulfed by the SE = 0.84, range = 0 to 10, n = 10). This host's phago- ciliates was 0.1 (SE = 0.07, range = 0 to 1; n = 20). 0. cytic response is largely responsible for the marked stellarum also phagocytoses the sperm of A. rubens decrease and eventual absence of sperm in parasitized (Bouland et al. 1987, Bouland & Jangoux 1988). Asterias amurensis. Male infertility in A. amurensis is Phagocytic activity by the host's cells was prevalent thus induced by the disruptive activity of the ciliate in diseased testes with amoebocytes observed in the and is largely effected by the phagocytic response of 238 Dis Aquat Org 28 235-239, 1997

Fig 2. Astenas amurensls Electron micrographs of parasitlzed testes (a) Orchltophyra stellarum has a fusiform profile and contains a macronucleus (Mal and numerous vacuoles (V) Sz spermatozoa (b) Detall of macronucleus (Ma) and micronucleus (Mi). G Golgi complex Ic) Spermat~dsIn the lumen (d) Host's phagocyte contalnlng 3 engulfed sperm (arrows). Scale bars. (a, b) = 4 0 pm. [c. d) = 1.0 pm the host's cells. Disruption of the germinaI layer by 0. rubens, ~t1s surpnsing that these cells do not recognise stellarum followed by masslve lnvaslon of phagocytes the clllates as foreign inclusions to be engulfed lnto infested gonads IS also reported for A. rubens (Bouland & Jangoux 1988). (Bouland & Jangoux 1988). The phagocytes of A. The high infestation rate of ~~~~~~~~~~~~astellarum in dmurensrs dld not attack the clliates. As noted for A. Astenas dmurensjs, contrasts with the incidence of chate Byrne et al.: Infestation of sea star testes by a ciliate

disease in the Atlantic Ocean, where infestation rates are Acknorvledqements. We thank the Directors and staff of the generally low (0 to 1 %l, occasional exceptions Misakl ~arineBiological Station, Otuchi Marine Research in the literature (Jangoux 1987)' Center and Sugashima Marine Biological Station for assistance \Yith specimen collection. Mr R, Smith and the staff of infestation of male A anlurensis in Japan over a broad the Electron Microscope Unit of the Univers~tyof Sydney pro- geographical range indicates that the disease is highly vided technical assistance. contagious in this asteroid. Aquarium observations suggest that the spread of 0. stellarum is mediated by seawater or by direct contact. The more contagious and LITERATURE CITED debilitating aetiology of ciliate disease in A. amurensis and P. ochraceus compared with the disease in Atlantic Bouland C, De Puytorac P, Bricourt E (1987) Orch~tophyra sea stars is probably because these asteroids are new stellarum, c1116 pretendu astome, est un scuticocilie Ann hosts for stellarum (Leighton et al. 1991). Sci Nat Zool 8:249-257 0. Bouland C, Jangoux M (1988) Infestation of Asterias rubens Orchitophyra stellarum was first observed in the IEchinodermata) bv the ciliate Orchjto~hvrastellarum: ef- ,' ' a eastern Pacific Ocean in the mid 1980s (Leighton et al. fect on gonads and host reaction. Dis Aquat Org 5:239-242 1991) and was first noted in Japan in 1990. The method Burrows RB (1936) Further observations on parasitism in the of introduction of 0. stellarum to the Pacific Ocean is starfish. Science 84:329 Byrne M (1996) Starfish wanted, dead or alive. New Sci 2052: not known. Its introduction to Japan may have been 7 mediated through a seed source of 0. stellarum in Byrne M, Morrice MG, Wolf B (1996) lntroduction of the north- populations of Pisaster ochraceus in Canada. Asterias ern Pacific asteroid Asterias amurensis to Tasmania: repro- amurensis occurs across the north Pacific, and it would duction and current distribution. Mar Biol (in press) be interesting to determine the incidence of 0. stel- Cepede C (1907) La castration parasitaire des 6toiles de mer males par un nouvel infusoire astome: Orchitophyra larum in these asteroids where they CO-occur. stellarum n.a..d. n.sn. C R Seanc Acad Sci Paris 145: Extensive searches for mature male Asterias amu- 1305-1306 rensis over the last 5 yr at qeoqraphicallv- -- distant sites Cepede C (1910) Recherches sur les infusoires astomes: indicate that ciliate infestation has resulted in a marked anatomie, biologie, ethologie parasitaire et systematique. Arch Zool Exp Gen 3:341-609 by drop in sperm production Japanese populations of Claereboudt MR, Bouland C (1994) The effect of parasitic this asteroid. This reduction in male reproductive po- castration by a ciliate on a population of Asterias vulgaris. tential has most likely resulted in a decrease in the fer- J Invert Path01 63:172-177 tilization success of A. amurensis with potential long- Febvre M, Fredj-Reygrobellet D, Fredj G (1981) Reproduction term effects on its recruitment and population biology. sexuee d'une asterie fissipare, Sclerastenas richardi (Per- rier. 1882). Int J Invertebr Reprod 3:193-208 The epidemiology of ciliate disease in Japan highlights -,-,. Saoara d J, Hamada S, Tamakawa M r19551~, On the the need to monitor the spread of 0. stellarum in A. spawning season of the starfish, Asterias amurensis, in amurensis and in other potential asteroid hosts from Tokyo Bay. Bull Jap Soc Sci Fish 21:32-36 the family Asteriidae. Considering the role that asteriid Jangoux M (1987) Diseases of Echinodermata. 1. Agents microorganisms and protistans. Dis Aquat Org 2:147-162 sea stars as predators in many Jangoux M (1982) Food and feeding mechanisms: Asteroidea. nities (Jangoux 1982), this new situation in Japan may In: Jangoux M, Lawrence JM (eds) Echinoderm nutrition. also have serious ecological implications. Balkema, Rotterdam, p 117-159 The dietary preference of Asterias species for bivalve Kim YS (1968) Histological observations of the annual change molluscs often makes them a pest for fisheries and in the gonad of the starfish, Asterias amurensis Lutken. Bull Fac Fish Hokkaido Univ 19:97-108 aquaculture and several unsuccessful attempts have Kume M, Dan K (1968) Invertebrate embryology. NOLIT, been made to use Orchitophyra stellarum as an agent Publishing House, Belgrade for biological control of Aster~as(Cepede 1910, Piatt Leighton BJ, Boom JDC, Bouland C, Hartwick EB, Smith MJ 1935, Burrows 1936). Although the virulence of ciliate (1991) Castration and mortality in Pisaster ochraceus parasitized by Orchitophyra stellarum (Ciliophora). Dis Aquat disease in the Pacific might rekindle interest in the use Org 10:71-73 of 0. stellarum for biological control of A, amurensis in Longo FJ, Akira U, Kazuyoshi C, Hoshi M (1995) Ultra- Japan, and of recently established pest populations of structural localization of acrosome reaction-inducing sub- A. amurensis in Australia (Byrne 1996, Byrne et al. stances (ARIS) on sperm of the starfish Asterias amurensis. 1996, Viney 1996), the lack of species specificity of 0. Mol Reprod Dev 41:91-99 Piatt J (1935) An important parasite of starfish. Fish Serv Bull stellarum cautions against its use. The apparently rapid US Dept Commerce 247:3-4 spread of 0. stellarurn in Japan and its ability to para- Smith GF (1936) A gonad parasite of the starfish. Science sitize several asteroid qenera indicates that the use of 84:157 this parasite for biological control in Japan, or else- Vevers HG (1951) The biology of Asterjas rubens L. 11 Pm- sitization of the gonads by the ciliate Orchitophyra stel- where in the Pacific, might result in the infestation of a larum Cepede. J Mar Biol Assoc UK 29:169-624 range of endemic asteroids in the family Asteriidae Viney C (1996) Pest control in the deep. ECOS 89 (spring with serious consequences for their population biology. 1996):26-27

Responwble Subject Ed~tor:M. Jangoux, Brussels, Belgium Manuscript first received: June 5, 1996 Revised version accepted. October 30, 1996