Vol. 81: 173–176, 2008 DISEASES OF AQUATIC ORGANISMS Published August 27 doi: 10.3354/dao01949 Dis Aquat Org

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Emerging infectious disease in sea stars: castrating ciliate parasites in Patiria miniata

J. Sunday*, L. Raeburn, M. W. Hart

Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada

ABSTRACT: Orchitophrya stellarum is a holotrich ciliate that facultatively parasitizes and castrates male asteriid sea stars. We discovered a morphologically similar ciliate in testes of an asterinid sea star, the northeastern Pacific bat star Patiria miniata (Brandt, 1835). This parasite may represent a threat to Canadian populations of this iconic sea star. Confirmation that the parasite is O. stellarum would indicate a considerable host range expansion, and suggest that O. stellarum is a generalist sea star pathogen.

KEY WORDS: Scuticociliate · Asterias amurensis · Biocontrol · Sperm

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INTRODUCTION consequences for ecologically important sea stars. In North America, recently described pathogenic infec- The spread of infectious pathogens may be tions of asteriid hosts (Leighton et al. 1991, Stickle et enhanced by direct human dispersal or through indi- al. 2001, 2007a,b), including the keystone predator rect facilitation such as climate change (Dobson & Pisaster ochraceus Brandt, 1835, have been attributed Foufopoulos 2001). Such emergent diseases have to inadvertent anthropogenic introduction of O. stel- important implications for natural and managed larum; some P. ochraceus populations in Canada have ecosystems (Krkosek et al. 2007). When emergent dis- been devastated. Deliberate introduction of O. stel- eases interact with invasive host species, pathogens larum into Tasmania has been proposed as a biocontrol may limit the spread of these hosts or merely use them measure to eradicate invasive populations of A. as a reservoir for propagation into native host popula- amurensis Lütken, 1871 (Goggin & Bouland 1997), but tions (e.g. Beard & O’Neill 2005, Garner et al. 2006). the desirability of such an effort depends in large part The holotrich ciliate Orchitophrya stellarum Cépède, on the potential effect of O. stellarum on native Tas- 1907 (order Scuticociliata) is a facultative pathogen of manian sea stars (Byrne 1996, Byrne et al. 1997), espe- male sea stars (Bouland & Jangoux 1988, Clareboudt & cially endangered and endemic species such as Parvu- Bouland 1994). These ciliates seasonally invade devel- lastra vivipara (family Asterinidae) that represent much oping testes, consume the sperm, and ravage the ger- of the asteroid diversity of Australia (Byrne 2006). minal epithelium. Heavily infected males are function- Here we report the first occurrence of a ciliate ally castrated, and high castration rates can result in resembling Orchitophrya stellarum in an asterinid host effectively unisexual populations of females (Leighton species, the bat star Patiria miniata. Infection of P. et al. 1991, Byrne et al. 1997). Known hosts are mainly miniata represents a significant phylogenetic exten- Japanese and north Atlantic species of Asterias Lin- sion of the host range of such parasites from asteriids naeus, 1758 (family Asteriidae). Spread of O. stellarum (order Forcipulatida) to asterinids (order Valvatida). to other habitats and host species could have significant The generalist nature of host use by O. stellarum-like

*Email: [email protected] © Inter-Research 2008 · www.int-res.com 174 Dis Aquat Org 81: 173–176, 2008

parasites strongly argues against their deliberate intro- Pathological testes of infected males were small, duction into other faunas for biocontrol of invasive sea dark yellow-brown in color, and yielded few or no stars. sperm in comparison to healthy testes that were creamy white, rounded, and spontaneously released abundant swimming sperm (Fig. 1). This gross pathol- MATERIALS AND METHODS ogy was similar to previous descriptions of infected asteriid hosts (Bouland & Jangoux 1988, Byrne et al. As part of a continuing study of the reproduction and 1997). In most cases, single testes of infected males population structure of Patiria miniata, we routinely included both pathological and healthy tissue, as in collect and spawn adults from 2 intertidal locations at Fig. 1. In our 2007 samples, there was some gross Dixon Island and Grappler Inlet near the Bamfield pathology in 75% of males from Grappler Inlet and Marine Sciences Centre (48° 50’ N, 125° 8’ W) on Van- 50% of males from Dixon Island; in our 2008 sample couver Island, British Columbia, Canada. In June 2007, from Dixon Island, 1 of 8 males (12%) had gross testis we observed several males that responded to a hor- pathology. monal spawning inducer by producing seminal fluid Ciliates parasitizing male Patiria miniata varied in that had a slightly darker color than the usual milky form (e.g. Stickle et al. 2007a), but generally resem- white appearance of sea star sperm. On examination, bled Orchitophrya stellarum described from asteriid these spawns contained abundant small ciliates mixed sea star hosts, viz. prolate spheroidal cells approxi- with sperm. We confirmed these observations with 2 mately 45 µm long and 25 µm wide with cilia organized subsequent collections. In August 2007, we searched in longitudinal rows or kineties and with a prominent for ciliates on the external body wall of individuals by vacuole at one end (Fig. 2). A long caudal cilium was gently pipeting from the aboral surface around the visible in some specimens. Ciliates swam actively gonopores, and then examining this material for cili- among spawned sperm (Appendix 1, available as sup- ates that resembled those found in gonads. We then plementary material at: www.int-res.com/articles/ dissected the gonads to search for ciliates in thin suppl/d081p173_video/). In some specimens, we could smears of the gonadal tissue. We cultured ciliates col- clearly distinguish a buccal cavity with specialized cil- lected from gonads at 12°C in 50 ml plastic Falcon iation. Such forms had a more pronounced flattened tubes containing seawater with pieces of testis or aspect (nearer to disk-shaped than prolate), and aliquots of sperm from P. miniata. We photographed seemed to be more common among ciliates collected individual ciliates in thin wet mounts using bright field from the exterior body wall than from the testes. We or differential interference contrast (DIC) microscopy. For DIC, ciliates were fixed in 95% ethanol and imme- diately photographed. In February 2008, we collected 18 adults from Dixon Island and injected them with 1 to 3 ml of 100 µM 1-methyladenine (1-MA), which induces maturation of oocytes (in females) and spawn- ing (in both sexes, Strathmann 1987). We estimated the prevalence of infection by light microscopy of spawned material in wet mounts.

RESULTS

In August 2007, 50% of 12 males sampled from Grappler Inlet had ciliates resembling Orchitophrya stellarum on the external body around the gonopores. Ciliates were also identifiable in gonadal smears in 1 of these samples. In samples from Dixon Island (August 2007), 50% of 14 males and 66% of 9 females had O. stellarum-like ciliates from the aboral body wall. Four of the males sampled from Dixon Island (28%) had cil- iate infection in gonadal smears. In a later collection Fig. 1. Patiria miniata testis dissected from a male infected by ciliates (photographed in seawater). Healthy gonad tubules (t) from Dixon Island (February 2008), 8 males spawned are creamy white, and some have spawned a cloud of sperm in response to 1-MA injection. Of these, 6 (75%) had (s). Infected tubules (i) are darker, yellow-brown, firm, and abundant ciliates in the spawn. smaller in diameter. Scale bar: 500 µm Sunday et al.: Castrating sea star parasites 175

ton et al. 1991), and might do so in P. miniata as well. We found many examples of gross testis pathology. More extensive surveys are needed to determine whether these infections are chronic, cause significant long-term pathology, and result in skewed sex ratios in Patiria miniata populations. Injection of 1-MA is an effective and non-invasive method for rapidly survey- ing sea star host populations for ciliate infections (Stickle et al. 2007a). Byrne (1996) and Byrne et al. (1997) argued against the potential use of Orchitophrya stellarum as a bio- control agent against invasive Asterias amurensis pop- Fig. 2. Orchitophyra cf. stellarun. Differential interference contrast images of ciliate parasites from testes, with focal ulations due to the non-specific nature of O. stellarum plane (A) at the surface and (B) through the centre of cells. infections among asteriids. Our discovery of ciliates Brightness and contrast in both cells were increased (approxi- resembling O. stellarum in an asterinid host reinforces mately +40%) from the originals in Adobe Photoshop™. Mor- this argument. Use of such a generalist pathogen for phological features similar to Orchitophrya stellarum include biocontrol is more likely to threaten than to protect prolate shape, dimensions, tracks of cilia organized into kineties (k), ingested sea star sperm (s) in small phagosomes, endemic or endangered asterinid (and other) sea star and prominent contractile vacuole (cv). Scale bar = 10 µm species.

occasionally observed single ciliates capturing sperm Acknowledgements. Thanks to M. Byrne, E. Kozloff, and B. and sweeping them toward the buccal cavity. We often Leighton for discussion and to H. Hutter for help with observed sperm within small vacuoles in the ciliates or microscopy. R. Palmer and the staff of the Bamfield Marine Sciences Centre provided laboratory facilities and collection adhered to the outside of the ciliates (Fig. 2B). assistance. We are financially supported by Simon Fraser Uni- versity, the Natural Sciences and Engineering Research Council of Canada (M.W.H), and the Glen Geen Graduate DISCUSSION Scholarship in Marine Biology (J.S.).

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Editorial responsibility: Stephen Feist, Submitted: March 31, 2008; Accepted: June 11, 2008 Weymouth, UK Proofs received from author(s): August 5, 2008