Evaluation of Malacosporean Life Cycles Through Transmission Studies

DISEASES OF AQUATIC ORGANISMS Vol. 60: 109–121, 2004 Published August 9 Dis Aquat Org

Evaluation of malacosporean life cycles through transmission studies

S. Tops1, D. V. Baxa2, T. S. McDowell2, R. P. Hedrick2, B. Okamura1,*

1School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK 2School of Veterinary Medicine, Department of Medicine and Epidemiology, 2108 Tupper Hall, University of California, One Shields Avenue, Davis, California 95616, USA

ABSTRACT: Myxozoans, belonging to the recently described Class Malacosporea, parasitise freshwater bryozoans during at least part of their life cycle, but no complete malacosporean life cycle is known to date. One of the 2 described malacosporeans is Tetracapsuloides bryosalmonae, the causative agent of salmonid proliferative kidney disease. The other is Buddenbrockia plumatellae, so far only found in freshwater bryozoans. Our investigations evaluated malacosporean life cycles, focusing on transmission from fish to bryozoan and from bryozoan to bryozoan. We exposed bryozoans to possible infection from: stages of T. bryosalmonae in fish kidney and released in fish urine; spores of T. bryosalmonae that had developed in bryozoan hosts; and spores and sac stages of B. plumatellae that had developed in bryozoans. Infections were never observed by microscopic examination of post-exposure, cultured bryozoans and none were detected by PCR after culture. Our consistent negative results are compelling: trials incorporated a broad range of parasite stages and potential hosts, and failure of transmission across trials cannot be ascribed to low spore concentrations or immature infective stages. The absence of evidence for bryozoan to bryozoan transmissions for both malacosporeans strongly indicates that such transmission is precluded in malacosporean life cycles. Overall, our results imply that there may be another malacosporean host which remains unidentified, although transmission from fish to bryozoans requires further investigation. However, the highly clonal life history of freshwater bryozoans is likely to allow both long-term persistence and spread of infection within bryozoan populations, precluding the requirement for regular transmission from an alternate host.

KEY WORDS: Myxozoa · Malacosporea · Proliferative kidney disease · Freshwater bryozoans · Transmission · Potential hosts · Life cycles

Resale or republication not permitted without written consent of the publisher

INTRODUCTION known to date, and these involve annelid worms and fish as primary and secondary hosts (Okamura & Can- The Phylum Myxozoa is comprised of about 55 gen- ning 2003). Direct transmission has been demon- era and over 1300 species (Canning & Okamura 2004) strated between fish (Diamant 1997, Redondo et al. primarily parasitic in marine and freshwater fish. The 2002, Yasuda et al. 2002), but whether this process phylum contains 2 classes: the Myxosporea (Bütschli precludes the facultative incorporation of invertebrate 1881) and the Malacosporea (Canning et al. 2000, hosts and represents a reduction in life cycle complex- Kent et al. 2001). The former includes the majority of ity is unknown (Canning & Okamura 2004). Mala- species, some of which cause economically important cosporeans parasitise freshwater bryozoans (Bryozoa: diseases, such as Myxobolus cerebralis, the causative Phylactolaemata) during at least part of their life agent of salmonid whirling disease (Bartholomew & cycle, but no complete malacosporean life cycle is Reno 2002). Only some 25 myxosporean life cycles are known.

*Corresponding author. Email: [email protected] © Inter-Research 2004 · www.int-res.com 110 Dis Aquat Org 60: 109–121, 2004

Two species of malacosporean have been described 1979, D’Silva et al. 1984). In addition, the presence of T. so far. One is Buddenbrockia plumatellae (formerly bryosalmonae in bryozoan populations from sites de- Tetracapsula bryozoides; Canning et al. 2002), which void of salmonids (Okamura et al. 2001) suggests that occurs as both sac-like and vermiform stages in fresh- these fish are, at best, facultative hosts. D. C. Morris et water bryozoans (Monteiro et al. 2002, Okamura et al. al. (2002a) and D. J. Morris et al. (2002) reported possi- 2002). The other member of the Class Malacosporea is ble transmission of T. bryosalmonae and Budden- Tetracapsuloides bryosalmonae (formerly Tetracap- brockia plumatellae from fish to bryozoans. However, sula bryosalmonae; Canning et al. 2002). Only sac-like these results may have reflected previous development stages of T. bryosalmonae have been encountered in of infection in bryozoans in the field, or PCR amplifica- bryozoan hosts to date. T. bryosalmonae is the tion of residual DNA that had adhered to bryozoans or causative agent of salmonid proliferative kidney dis- of DNA from undetected, infected invertebrates. ease (PKD) (Anderson et al. 1999, Canning et al. 1999, Investigations into the pathogenesis of PKD have Feist et al. 2001) and was, until recently, referred to as shown that Tetracapsuloides bryosalmonae has 2 the PKX organism (Seagrave et al. 1980). The identifi- developmental stages in salmonids (Hedrick et al. cation of bryozoans as hosts of T. bryosalmonae 1993). The extrasporogonic stage, found in the blood (Anderson et al. 1999) represented a breakthrough, as and kidney interstitium of infected fish, produces the it finally determined the source of a disease which chronic inflammatory response which is characteristic causes significant financial losses to fish farms and of the disease (Kent & Hedrick 1986). The sporogonic hatcheries of Europe and North America (Hedrick et stage is found in the kidney tubule lumen 2 to 3 wk al. 1993, Bromage 1999). PKD has also been implicated after the extrasporogonic stages are first observed. in the decline of wild salmonid populations in Switzer- Sporogonic stages can persist in the kidney long after land (Wahli et al. 2002). the recovery of the fish from clinical disease (Kent & Freshwater bryozoans are sessile, colonial inverte- Hedrick 1986, Morris et al. 2000). Development of brates that are common in both lotic and lentic habi- sporogonic stages in at least some fish hosts proceeds tats. They undergo prolific colony growth in summer to the production of malacosporean-like spores con- and produce dormant, seed-like propagules (stato- taining 2 capsulogenic cells and 1 sporoplasm sur- blasts) that overwinter and hatch into small colonies rounded by 2 unstrengthened (soft) valve cells (Kent & when favourable conditions return (Okamura & Hat- Hedrick 1986, Hedrick et al. 2004). ton-Ellis 1995). The freshwater bryozoan Fredericella Certain observations have been cited as evidence sultana can also overwinter as colonies (Raddum & that salmonids may be aberrant hosts for Tetracapsu- Johnsen 1983, Gay et al. 2001). Malacosporeans loides bryosalmonae, including the severe inflamma- develop into freely circulating sacs and active vermi- tory response of fish and incomplete spore develop- form stages (Canning et al. 1996, 1999, 2002), which ment (Kent & Hedrick 1986). However, before can be observed within the body cavity of their bryo- description of the Class Malacosporea (Canning et al. zoan hosts by inspection using a dissection micro- 2000), researchers expected final spore development scope. Infective spores develop within sacs and worms. to terminate in stages with typical hardened valves There is relatively little host specificity. Molecular data consistent with those observed in other myxosporeans. have identified Tetracapsuloides bryosalmonae infec- The possibility that fish should be considered as poten- tions in 5 bryozoan species (Anderson et al. 1999, tial hosts is supported by the release of malaco- Longshaw et al. 1999, Okamura & Wood 2002), 2 of sporean-like spores in fish urine, as similar and func- which have been confirmed as hosts through transmis- tional spores of T. bryosalmonae develop in bryozoan sion studies (Feist et al. 2001, Gay et al. 2001). The ver- hosts (Hedrick et al. 2004). miform stage of Buddenbrockia plumatellae has been In view of our incomplete understanding of malaco- found in 3 species (Canning et al. 2002, Monteiro et sporean life cycles, we undertook a series of studies to al. 2002), but the sac-like stages have only been investigate potential routes of transmission of Tetra- found parasitising 1 bryozoan host—Cristatella mucedo capsuloides bryosalmonae and Buddenbrockia pluma- (Okamura 1996, Canning et al. 2002). tellae. In particular we focused on 2 unresolved issues: Knowledge of the ecology, development and life cy- whether transmission can occur from fish to bryozoan cles of malacosporeans is limited. Transmission studies and from bryozoan to bryozoan. from bryozoans to fish have confirmed bryozoans as true hosts of Tetracapsuloides bryosalmonae (Feist et al. 2001, Gay et al. 2001), but whether stages that de- MATERIALS AND METHODS velop in fish are capable of infecting new hosts has so far not been demonstrated. It is, however, known that We undertook the following types of transmis- PKD is not transmitted from fish to fish (Ferguson & Ball sion: (1) Fish to bryozoan: exposure of bryozoans to Tops et al.: Malacosporean transmission studies 111

Tetracapsuloides bryosalmonae stages in fish kidney; ambient laboratory conditions (exposure to normal (2) fish to bryozoan: cohabitation of bryozoans with fish daylight period, 20°C). The small colonies that infected with T. bryosalmonae; (3) bryozoan to bry- emerged from statoblasts were grown in the culture ozoan: exposure of bryozoans to T. bryosalmonae- system to provide material for trials. In keeping with infected Fredericella sultana; (4) bryozoan to bryozoan: the absence of Tetracapsuloides bryosalmonae during exposure of bryozoans to Buddenbrockia plumatellae- long-term monitoring of the source population (Tops & infected Plumatella fungosa and Cristatella mucedo. Okamura 2003), infections were never observed in any Below we describe the material used for transmission F. sultana material in the culture system. In addition, studies and the protocols for each type of transmission. PCR using T. bryosalmonae- and Buddenbrockia plu- Details of the individual trials are summarised in matellae-specific primers (see below) on portions of Table 1. F. sultana colonies used as source material in trans- Bryozoans used as targets for infection. Three spe- mission trials consistently provided negative results. cies of freshwater bryozoan (Fredericella sultana, Plu- This approach assumes that T. bryosalmonae is dis- matella fungosa and Plumatella emarginata) were cul- tributed throughout a colony and hence should be tured in aquarium tank systems composed of smaller detected by screening portions of colonies by PCR, but aquaria, housing the bryozoans, connected by recircu- there are no data to corroborate this assumption. lation to separate, larger aquarium tanks containing Source material was only used during Trials 1a and 1b. goldfish, according to the methods of Wood (1971) in For the remainder of the trials, statoblast-hatched conditions of constant light and temperature (20°C). F. sultana colonies were employed. The tank systems were topped up with deionised Plumatella fungosa: The source of all P. fungosa water every 2 to 3 d when required and periodically colonies was a single colony collected from Blenheim seeded with water from a small, concrete-lined pond Palace Lake (51° 51’ N, 01° 23’ W) in July 2000. The devoid of both bryozoans and fish with PKD. The incor- material used in the transmission trials was the result poration of goldfish and periodic seeding with pond- of several generations of statoblast-derived colonies water creates suitable environmental conditions and produced from this original colony, as described for food, allowing bryozoans to be maintained indefinitely Fredericella sultana above. No malacosporeans have in culture. Bryozoans in such culture systems have ever been observed in this P. fungosa material in cul- been grown for many years (T. Wood pers. comm., ture; however, as a further check that the material was S. Tops & B. Okamura pers. obs.) with no sign of uninfected, small portions of live bryozoans were cut malacosporean infections being transferred between from the stock material and subjected to PCR using bryozoans and goldfish. This cultured bryozoan mater- Tetracapsuloides bryosalmonae- and Buddenbrockia ial provided potential target hosts for infection in our plumatellae-specific primers (see below). Since none transmission studies and was regarded as infection- of these were positive for malacosporean infection, the free, as described below. The material was maintained material was deemed to be suitable for transmission in independent culture systems and was thus never in studies. Severed branches were induced to attach to contact with infected bryozoan material collected from surfaces for transmission studies as described above. the field for use in transmission studies (described Plumatella emarginata: P. emarginata colonies col- later). lected from Burghfield Lake, Berkshire (51° 26’ N, Fredericella sultana: Transmission trials were per- 01° 04’ W) in August 2002 released larvae in the labo- formed using F. sultana material from several different ratory. The larvae metamorphosed into small colonies derivations. The original material was collected from on plastic discs which were placed in culture for the Kennet and Avon Canal (51° 24’ N, 01° 08’ W) in onward growth, thus providing bryozoan colonies of October 2000. Colonies were induced to attach to sur- larval origin for transmission studies. As a check that faces of water-filled petri dishes or to submerged plas- the material was uninfected by malacosporeans, small tic discs (5.4 cm diameter) via their sticky, newly portions of colonies (n = 6 colonies) were subject to formed tips (Wood 1973, S. Tops pers. obs.). Subcul- PCR using Tetracapsuloides bryosalmonae- and Bud- tured material was derived by removing branches from denbrockia plumatellae-specific primers (see below). original material established in culture, and inducing Negative PCR results provided evidence that the the branches to attach to new surfaces. Statoblast- material was suitable for transmission studies. hatched material was obtained from statoblasts pro- Sources of potential infection. Fish used for expos- duced by subcultured F. sultana material. The stato- ing bryozoans to infected kidney included rainbow blasts were placed in deionised water at 4°C and kept trout Oncorhynchus mykiss obtained from a fish in the dark for a minimum period of 4 mo. They were farm on the Kennet and Avon Canal, Berkshire, UK then transferred to Petri dishes containing artificial (Table 1: Trial 1a), wild brown trout Salmo trutta col- pond water (Wood 1996) to induce hatching under lected during routine survey work of trout populations 112 Dis Aquat Org 60: 109–121, 2004

conducted by the Environment Agency on the River bryosalmonae and included rainbow trout from the Test, Hampshire, UK (Table 1: Trial 1b), and rainbow American River Hatchery, California (Table 1: Trial trout from a hatchery on the American River, Califor- 2b), and Chinook salmon Oncorhynchus tschawytscha nia, USA (Table 1: Trial 1c). Fish used in cohabitation from the Merced River Hatchery, California (Table 1: studies were naturally infected with Tetracapsuloides Trials 2a and 2c). Brown trout from Mount Shasta

Table 1. Summary of transmission trials, including identity of host and target species used in each trial, details of transmission including the exposure time of bryozoans to potential infection, the number (No.) of experimental (Exp) and control (Ctl) bryozoan colonies used in the trials (and subsequently subject to PCR), and the post-exposure culture period. Trials 1a to 3d involved transmission with Tetracapsuloides bryosalmonae; Trials 4a and 4b involved transmission with Buddenbrockia plumatellae

Trial Host species Target species Transmission No. experimental and control colonies in trial Culture (Date) details Fredericella Plumatella Plumatella period sultana fungosa emarginata

1a Oncorhynchus Fredericella sultana Exposure to Exp: 6 (0) 54 d (Oct 2000) mykiss macerated Ctl: 7 (0) kidney for 24 h 1b Salmo trutta Fredericella sultana Exposure to Exp: 4 (4) Exp: 10 (10) 37 d (Jan 2001) Plumatella fungosa macerated Ctl: 4 (0) Ctl: 10 (10) kidney for 24 h 1c Oncorhynchus Fredericella sultana Exposure to Exp: 6 (4) Exp: 1 (1) 14 d (Jul 2002) mykiss Plumatella fungosa macerated kidney for 8 h 2a Oncorhynchus Fredericella sultana Cohabitation with Exp: 20 (9) 22 d (Apr 2002) tshawytscha fish for 80 h Ctl: 20 (14) 2b Oncorhynchus Fredericella sultana Cohabitation with Exp: 20 (12) 22 d (Apr 2002) mykiss fish for 80 h 2c Oncorhynchus Fredericella sultana Cohabitation with Exp: 10 (8) Exp: 1 (0) 14 d (Jul 2002) tshawytscha Plumatella fungosa fish for 19.5 h Ctl: 6 (6) Ctl: 1 (1) 2d Salmo trutta Fredericella sultana Cohabitation with Exp: 10 (9) Exp: 1 (0) 14 d (Jul 2002) Plumatella fungosa fish for 19.5 h 3a Fredericella sultana Fredericella sultana Spores directed into Exp: 5 (5) Exp: 2 (2) 30 d (Jun 2002) Plumatella fungosa lophophores (4 h); retention in spore suspension (20 h) 3b Fredericella sultana Fredericella sultana Spores directed into Exp: 3 (3) Exp: 1 (1) Exp: 3 (3) (Oct 2002) Plumatella fungosa lophophores (4 h); 33 d Plumatella emarginata retention of spore suspension (16 h) 3c Fredericella sultana Fredericella sultana Exposure to Exp: 3 (2) 30 d (Jun 2002) shredded bryozoans Ctl: 2 (2) for 135 min 3d Fredericella sultana Plumatella fungosa Exposure to Exp: 10 (10) 21 d (Aug 2003) naturally released spores for 21 d 4a Plumatella fungosa Plumatella fungosa Spores directed into Exp: 2 (0) 52 d (Aug 2001) lophophores (4 h); Ctl: 2 (0) retention in spore suspension (12 h) 4b(i) Cristatella mucedo Fredericella sultana Spores directed into Exp: 5 (4) Exp: 5 (4) 60 d (Aug 2001) Plumatella fungosa lophophores (4 h); Ctl: 5 (5) retention in spore suspension (20 h) 4b(ii) Cristatella mucedo Fredericella sultana Exposure to Exp: 2 (2) Exp: 3 (2) 60 d (Aug 2001) Plumatella fungosa immature sacs for 24 h 4b(iii) Cristatella mucedo Fredericella sultana Spores directed into Exp: 2 (1) Exp: 1 (1) 60 d (Aug 2001) Plumatella fungosa lophophores (4 h); retention in spore suspension (20 h) Tops et al.: Malacosporean transmission studies 113

Hatchery, California, were hatched from eggs and (unless otherwise specified, see below) and were then maintained in well water at 15°C until intraperi- visually monitored every 2 to 3 d, using a dissection toneally injected as in previous studies (Feist et al. microscope, for signs of infection. For Trial 1c, the kid- 2001) with homogenised kidneys of Chinook salmon ney tissue was disrupted by passing it through a infected with extrasporogonic and sporogonic stages of 150 µm sieve with a minimal amount of PBS. Kidney T. bryosalmonae 6 wk prior to the transmission trials homogenates were then combined as above and added (Table 1: Trial 2d). to an aerated tank containing colonies of F. sultana and Colonies of Fredericella sultana infected with Tetra- Plumatella fungosa, and 9.5 l of well water. After 8 h capsuloides bryosalmonae collected from the River the bryozoans were transferred to aquaria with water Cerne, Dorset, UK, provided material for bryozoan to from a local pond at 15°C and visually monitored for bryozoan transmission (Table 1: Trials 3a to 3d). The signs of infection every 2 to 3 d. Controls for Trial 1a infection of a Plumatella fungosa colony by vermiform consisted of F. sultana colonies similarly treated but not stages of Buddenbrockia plumatellae during a field exposed to kidney homogenate, although PBS was experiment (Tops & Okamura 2003) provided material added. Trials 1b and 1c were constrained by availabil- for Trial 4a (Table 1), while infected colonies of ity of bryozoan material, and no separate colonies were Cristatella mucedo from Bonnerweiher, Bavaria, Ger- available to act as controls. Consequently, for Trial 1b, many, provided sac-like stages of B. plumatellae for portions of colonies collected prior to exposure to Trial 4b(i to iii) (Table 1). homogenate were subjected to PCR and served as con- Transmissions. Table 1 summarises details of the fol- trols. At the conclusion of the culture period, colonies lowing transmission trials and should be referred to for in Trials 1b and 1c were saved for PCR to assess poten- information on the number of experimental and control tial cryptic infection (Tops & Okamura 2003). Results of bryozoan colonies used in trials. Note that PCR of por- Trial 1a are based on visual inspection only. tions of experimental colonies prior to exposure in tri- (2) Fish to bryozoan. Cohabitation of bryozoans als served as an additional control unless otherwise with infected fish: Bryozoans were exposed to fish specified. As previously mentioned, this method recovering from PKD in cohabitation trials (see Trials assumes that Tetracapsuloides bryosalmonae is dis- 2a to d; Table 1) to determine whether sporogonic tributed throughout a colony and hence can be stages of Tetracapsuloides bryosalmonae that pass detected by screening portions of colonies by PCR. through the urinary tract of recovering fish are infec- Other controls are described below for individual tive to bryozoans. Four exposures were carried out trials. using naturally infected rainbow trout and Chinook (1) Fish to bryozoan. Exposure of bryozoans to salmon and artificially infected brown trout (see infected fish kidney: Fish were collected from sites Table 1). The fish were maintained in flow-through enzootic for PKD (Trials 1a to 1c; Table 1) and trans- tanks (133 l) supplied with well water at 15°C. ported to the laboratory. They were killed by a blow to Plastic discs (5.4 cm diameter) with attached bryo- the head, followed by severance of the spinal cord. For zoan colonies were randomly allocated positions Trials 1a and 1b, approximately 27 mm3 of tissue was within cylindrical plastic cages (Fig. 1). Twenty Fred- dissected from the posterior portion of the kidney and ericella sultana colonies were available for Trials 2a imprints were made for histological analysis to confirm and 2b, and 10 colonies for Trials 2c and 2d. Only 2 infection by Tetracapsuloides bryosalmonae. For Trial Plumatella fungosa colonies were available for Trials 1b, kidney tissue was fixed in absolute ethanol and 2c and 2d (see Table 1). Each cage could hold a maxi- stored prior to PCR at –20°C. For Trial 1c, wet mounts mum of 10 discs with colonies. Bryozoans were of the kidney material were prepared and examined exposed to fish for 16 h d–1 (Trial 2a and 2b) and 6.5 h according to Kent & Hedrick (1986) to confirm the d–1 (Trial 2c and 2d) over a period of 5 and 3 d respec- presence of sporogonic stages of T. bryosalmonae in tively. In between fish exposure periods, bryozoans kidney tubules. were returned to pond water to feed. This was deemed For Trials 1a and 1b, the remaining kidney tissue to be essential since no food was available in well was removed and placed in a minimal amount of water and it was important to ensure colonies did not 1Mphosphate buffered saline (PBS) (Oxoid™ tablets; degenerate before parasites might develop. For Trials pH 7.3) on ice. Kidney material from all fish collected 2a and 2b, bryozoans were cohabited with 50 fish per for each Trial was combined in a dish with a minimal treatment in flow-through tanks (133 l) supplied with amount of PBS, and disrupted using forceps. This mac- well water. For Trials 2c and 2d, bryozoans were erated kidney material was added to an aerated tank cohabited with fish in buckets filled with aerated well containing colonies of Fredericella sultana and 16 l of water (19 l) in order to increase the likelihood of expo- water from the bryozoan culture system. After 24 h the sure to potential infective stages, since these would bryozoans were transferred to the culture system have been continuously lost in the flow-through 114 Dis Aquat Org 60: 109–121, 2004

Fig. 1. Discs (5.4 cm diameter) and one half of the cage apparatus used in exposing bryozoans in Trials 2a to d and 3c. During trials bryozoans were enclosed within the fully assembled cage using cable ties, and were submersed in a downward facing position in experimental containers

arrangement used in Trials 2a and 2b. For Trials 2c and (3) Bryozoan to bryozoan. Exposure of bryozoans to 2d, 8 fish were caught at random each day from a large Tetracapsuloides bryosalmonae infected Fredericella group of infected individuals and were cohabited with sultana: For Trial 3a, colonies of F. sultana were the bryozoans in the bucket. At the end of each cohab- torn apart with forceps (‘shredded’) and observed with itation period the fish were returned to the flow- a dissection microscope. Sacs of T. bryosalmonae through system. Hence, different sets of fish were released by shredding were transferred to a small petri likely involved in the transmission study on different dish (with 10 µl disposable microcapillary tubes) and days. Bryozoans cohabited with a group of Chinook burst open using a sterile, mounted needle to release salmon hatched from eggs and reared in well water spores. Concentrated spores were pipetted up into acted as controls for all trials. clean microcapillary tubes and wafted into the open After completion of exposure, the bryozoans were lophophores of F. sultana and Plumatella fungosa returned to the laboratory culture system and moni- colonies (see Table 1) from our laboratory stock, in tored for the presence of Tetracapsuloides bryo- separate petri dishes containing 20 ml of culture water. salmonae stages every 2 to 3 d using a dissection The bryozoans were exposed to potential infection by microscope. Regular observations of colonies allowed spores in the petri dish for 24 h. The water was agitated us to fix moribund bryozoans in absolute ethanol prior every 30 min for 4 h to re-suspend any viable spores to their death to check for possible transmission of T. which may have settled to the bottom of the dish and bryosalmonae by PCR. Nonetheless, some mortality were thus unavailable for infection. After 24 h, the occurred. bryozoans were transferred to the culture system and The presence of spores and sporogonic stages in checked every 2 to 3 d for visual signs of infection. the kidney tubules of fish was assessed post-exposure Colonies which survived culture for 30 d were saved through histological examination of wet mounts as for PCR as a further check on infection status. Colonies described earlier. Examination of a subset of fish in that appeared to be moribund were fixed for PCR at Trials 2a and 2b indicated that extrasporogonic stages earlier dates prior to death. were present but sporogonic stages were rare. This A single colony of Fredericella sultana provided prompted us to undertake Trials 2c and 2d in hopes material for Trial 3b when it developed infection in of including more mature stages. Examination of a culture 10 wk after collection from the River Cerne, subset of fish indicated that sporogonic stages were Dorset. Sacs were collected as for Trial 3a. Target present in 3/10 fish in Trial 2c and 2/10 fish in Trial colonies of F. sultana, Plumatella fungosa and P. emar- 2d at low to moderate levels (based on subjective ginata (see Table 1) were exposed to spores for 20 h, scoring by R. P. Hedrick, assessing the relative abun- which were wafted into the lophophore every 30 min dance of parasite stages as a result of familiarity for 4 h. Colonies were subsequently checked for visual gained during previous studies: Kent & Hedrick 1986, signs of infection every d. After 33 d of culture the Foott & Hedrick 1987). colonies were saved for PCR. Tops et al.: Malacosporean transmission studies 115

For Trial 3c, Fredericella sultana colonies collected system never exposed to infected bryozoan colonies. from the River Cerne were shredded (see above) and After 5 d in the 2 l aquarium, colonies were transferred placed in a container with approximately 30 l of water to a culture system. containing naïve rainbow trout and cage-enclosed tar- For Trial 4b(i) (see Table 1), spore-filled sacs of Bud- get colonies of F. sultana for 135 min, as part of a larger denbrockia plumatellae were obtained by applying study (data not presented) on the transmission of gentle external pressure to infected Cristatella mucedo Tetracapsuloides bryosalmonae to fish. Target bryo- colonies. Colonies of Fredericella sultana and Pluma- zoans were then returned to culture and checked for tella fungosa were exposed to the spores in an aerated visual signs of infection every 2 d. After 30 d of labora- 2 l aquarium containing water from the culture system, tory culture the remaining live colonies were saved as described above for Tetracapsuloides bryosalmonae. for PCR. Spores were again resuspended every hour for 4 h, The development of persistent Tetracapsuloides and the total exposure time was 24 h. Colonies were bryosalmonae infections in colonies of Fredericella sul- then placed into culture. Controls for this experiment tana (originally collected from the River Cerne) in 1 were uninfected, laboratory-reared F. sultana colonies, culture system and the coincident development of new which were maintained in 2 l of aerated, culture- Plumatella fungosa colonies that hatched from stato- system water. Deionised water was wafted into the blasts in the same system allowed our fortuitous, lophophores and the water was agitated as described unplanned Trial 3d. The P. fungosa colonies became above. In addition, 20 C. mucedo colonies with imma- established adjacent to heavily infected F. sultana ture sacs (lacking spores) were disrupted (Trial 4b[ii]). colonies on the same petri dishes. The latter produced The disrupted material was added to 2 l of aerated cul- sacs over a prolonged period, and released mature ture water containing uninfected colonies of F. sultana spores and possibly sacs at a variety of developmental and P. fungosa. The water was stirred every hour for stages into the recirculating culture system. This 4h to maintain the material in suspension. After 24 h should have resulted in the relatively continuous expo- the colonies were transferred to culture. Finally, 1 sure to infection of the P. fungosa colonies during the colony of C. mucedo with mature, spore-filled sacs of period of spore viability. The P. fungosa colonies were B. plumatellae was placed in 500 ml culture water maintained for 21 d alongside the infected F. sultana along with uninfected colonies of F. sultana and P. colonies, were checked for visual signs of infection fungosa (Trial 4b[iii]). The water was gently agitated every 2 to 3 d, and were then fixed in absolute ethanol every hour for 4 h to suspend any spores which may for PCR. There were no controls for this trial. have been released by the infected C. mucedo colony. (4) Bryozoan to bryozoan. Exposure of bryozoans to The passive release of sacs has been observed for both Buddenbrockia plumatellae infected Plumatella fun- B. plumatellae (B. Okamura pers. obs.) and T. gosa and Cristatella mucedo: For Trial 4a, spore-filled bryosalmonae (M. Longshaw pers. comm.). After 24 h vermiform stages of B. plumatellae infecting a colony of exposure, the bryozoans were transferred to culture. of P. fungosa were collected by applying gentle exter- The bryozoans exposed to potential infection in Trials nal pressure to the infected portion of the bryozoan 4b (i to iii) were maintained in culture for 60 d, and colony. Spores were collected and wafted into the open were checked for visual signs of infection every 2 to 3 lophophores of uninfected P. fungosa colonies (see d. Appropriate culture conditions have not been iden- Table 1) in petri dishes as described above, and were tified for C. mucedo, so uninfected colonies of this spe- resuspended once per hour for 4 h. The P. fungosa cies were not included in the study. colonies were exposed to spores for 16 h and then DNA extractions and PCR. DNA extraction of both transferred to an aerated 2 l aquarium with water from bryozoan and fish material, that had been saved in the culture system (from a system never exposed to absolute ethanol and stored at –20°C, was conducted infected bryozoan colonies) for 5 d prior to transfer to using a standard CTAB (1% N-cetyl N,N,N-trimethyl- culture. Fifty percent of the volume in the 2 l tank was ammonium bromide)-Proteinase K method as de- replaced daily with fresh water from the culture sys- scribed by Winnepenninckx et al. (1993), with the tem. The 2 colonies were maintained in culture for 52 following modifications: (1) samples were stored at d, and were checked for visual signs of infection every –20°C, (2) the samples were not ground in liquid nitro- 2 to 5 d. Two uninfected, laboratory-reared P. fungosa gen prior to extraction, (3) samples were suspended in colonies were used as controls. These were initially 675 µl preheated (60°C) CTAB buffer, and (4) all steps maintained in petri dishes filled with culture-system were carried out using 1.5 ml plastic eppendorf tubes. water. Deionised water was wafted into the PCR amplification was conducted using either Tetra- lophophores and the water was agitated as described capsuloides bryosalmonae-specific primers (514F and above. The colonies were then transferred to an aer- 776R, from D. C. Morris et al. 2002b) or primers we de- ated 2 l aquarium containing water from the culture signed to specifically amplify Buddenbrockia plumatel- 116 Dis Aquat Org 60: 109–121, 2004

lae (BZ1 F: 5’-CGTAAGGCTTCAAGCGAAAG-3’ and myxosporeans (Diamant 1997, Redondo et al. 2002, BZ2R: 5’-CGAGCGTTTTAAATGCAACA-3’, product Yasuda et al. 2002). The consistent absence of evi- size 397 base pairs). The primers were not cross-reac- dence for bryozoan to bryozoan transmissions for tive between the 2 species of parasite. Conditions for both T. bryosalmonae and Buddenbrockia plumatellae amplification were according to D. C. Morris et al. strongly indicates that such transmission is precluded (2002b). Negative (adding ultrapure water as a tem- in malacosporean life cycles. plate) and positive controls were included in all amplifi- Our trials also provided no evidence for transmission cations. An internal, competitive-standard mimic mole- to bryozoans of Tetracapsuloides bryosalmonae stages cule developed by D. C. Morris et al. (2002b) was that develop in fish. This result supports the suggestion included as a means of checking the absence of PCR in- that fish are abnormal hosts, a view supported by the hibition in assessing T. bryosalmonae infection. No intensity of the immune response mounted by fish to internal competitive-standard was available for the the presence of T. bryosalmonae (Kent & Hedrick assessment of B. plumatellae infections. 1986) and by recent phylogeographic studies (Hender- son & Okamura 2004). The latter provide no indication that T. bryosalmonae was transferred to Europe during RESULTS the introduction of rainbow trout through fisheries activities nor subsequently within Europe, a result that We obtained no evidence that the stages of Tetra- strikingly contrasts with the introductions of pathogens capsuloides bryosalmonae that develop in fish and and parasites typically associated with aquaculture. bryozoans caused infections in target bryozoan For instance, such transfer is evident in the case of colonies, based on visual inspection of exposed Myxobolus cerebralis, where introduction to North colonies or by subsequent PCR. No infections were evi- America from Europe in the 1950s rapidly established dent in any controls. The conclusion that the T. this myxozoan parasite in native populations of the bryosalmonae stages found in bryozoans in our trans- obligate alternate host, the oligochaete worm Tubifex mission trials are incapable of infecting bryozoans is tubifex (Hoffman 1990). However, despite these bolstered by the finding that the naïve rainbow trout caveats, the possibility of fish to bryozoan transmission exposed to the parasite (Trial 3c) developed PKD; requires further attention, as discussed below. recipient bryozoans present during the same exposure While the availability of material and variation in period did not develop infections. Similarly, we protocols meant that each study lacked replication, our obtained no evidence that either the sac-like or consistent results are compelling. Indeed, a strength of vermiform stages of Buddenbrockia plumatellae that the work is the incorporation of both a broad range of develop in bryozoans are capable of producing new parasite stages and of potential new hosts. Thus, 3 infections in bryozoans. The identity of the parasites bryozoan species were assayed as potential malaco- used in transmission trials was confirmed by PCR using sporean hosts (Fredericella sultana, Plumatella fun- species-specific primers. gosa and P. emarginata) and we utilised parasite stages from rainbow trout, brown trout and Chinook salmon, and the bryozoans F. sultana, P. fungosa and DISCUSSION Cristatella mucedo. Also included were wild and farmed sources of fish, naturally and artificially Lack of transmissions infected fish, and fish from the UK and North America. In addition, we tested exposure to artificially and natu- Our studies provide evidence that the life cycles of rally released spores, and to parasitic sacs from the 2 malacosporean parasites known to date do not infected bryozoan colonies. In total we assessed infec- involve direct transmission from bryozoan to bryozoan, tion in 97 bryozoan colonies (a conservative estimate either by mature spores or by earlier sac stages. No based on number of colonies subjected to PCR follow- infections resulted from exposing bryozoans to con- ing post-exposure culture), and each bryozoan species centrated spores (Trials 3a to d, 4a, 4b[i and iii]), to tested as potential host material was sourced from dif- immature sacs (Trial 4b[ii]) or to long-term cohabita- ferent water bodies. Furthermore, transmission studies tion with infected colonies releasing spores and possi- indicate that our stock material of F. sultana was bly sacs at different stages of development (Trial 3d). susceptible to infection by Tetracapsuloides bryo- The lack of direct transmission from bryozoan to bry- salmonae in the field: colonies derived from our labo- ozoan by exposure to immature sac stages suggests ratory stock became infected when transplanted into that there is no parallel in the life cycle of Tetracapsu- the River Itchen, Hampshire, UK, while stock colonies loides bryosalmonae to the direct fish to fish trans- in culture did not develop infections (Tops & Okamura mission indicated for immature trophozoites of some 2003). Stock material of P. fungosa was similarly sus- Tops et al.: Malacosporean transmission studies 117

ceptible to infection by T. bryosalmonae in the River feed. Unfortunately, it was not feasible to predict Itchen and was also infected by Buddenbrockia pluma- before the transmission trials whether fish were releas- tellae in the Kennet and Avon Canal, Berkshire, UK ing spores. Post-transmission microscopic examination (Canning et al. 2002, Tops & Okamura 2003). These of kidney indicated that the fish used in Trials 2a and observations, together with the range of source locali- 2b had not yet reached the stage of recovery from PKD ties for infected material, suggest that lack of infection dominated by release of spores from sporogonic in our transmission trials is not a result of using resis- stages, although some sporogonic stages were de- tant host strains. It should also be pointed out that tected. Conducting these trials in flow-through sys- infection of bryozoans does not appear to be influ- tems may also have influenced transmission by greatly enced by bryozoan maturity since T. bryosalmonae reducing spore concentrations. Microscopic examina- infections have been encountered in, e.g., small tion indicated that a proportion of the fish used in Tri- (young) colonies of Pectinatella magnifica (Okamura et als 2c and 2d had sporogonic stages, and the conduc- al. 2001) and in established colonies of F. sultana which tion of these trials in buckets increased the possibility are producing statoblasts (S. Tops & B. Okamura of spore contact with bryozoans. However, random unpubl. data). Nonetheless, we offered a range of selection of fish for trials and lack of knowledge about stages of potential new hosts, including branches of actual spore release in urine during trials hamper the colonies derived from statoblasts (most trials), colonies interpretation of results. Similarly, infective stages may derived from statoblasts (Trial 3d), and colonies have been present in low concentration when bryo- derived from larvae (P. emarginata in Trial 3b). zoans were exposed to macerated fish kidney. Filtering of the water used in transmission studies to determine concentrations of infective stages was not conducted. Constraints of study This would not have been feasible in the trials conducted in the flow-through systems. In addition, the Negative results such as ours are justifiably subject soft, unprotected spores are likely to adhere and dis- to criticism on methodological grounds. Thus, failure to integrate on contact with filters, making their identifi- close the life cycle of either malacosporean could be cation and quantification exceedingly difficult. attributable to inappropriate conditions for transmis- Despite the above caveats, there is some evidence sion or for subsequent development of malacospore- that low concentrations of Tetracapsuloides bryo- ans. The latter is unlikely since the development and salmonae spores must be effective at achieving infec- persistence of infections in field-collected Fredericella tion. Concentrations of spores released from bryozoans sultana from the River Cerne in our laboratory culture should be exceedingly low for much of the year given system (Tops & Okamura 2003, current study) demon- the generally low prevalence of infections in bryozoan strate that the post-exposure culture conditions were populations (Okamura et al. 2001, Okamura & Wood appropriate for the development of Tetracapsuloides 2002, S. Tops & B. Okamura unpubl. data). In addition, bryosalmonae in bryozoan hosts. fish are routinely immunised by developing subclinical Constraints on transmission include the possibility infections through exposure to waters enzootic for PKD that spore concentrations may have been too low to be in autumn, when water temperatures are decreasing effective or that mature infective stages were not pres- (Longshaw et al. 2002) and when prevalence of infec- ent in the material used. We attempted to control for tion in bryozoan populations is low (S. Tops & B. Oka- such contingencies given the limitations of each trans- mura unpubl. data). Furthermore, Feist et al. (2001) mission trial. Thus, in our bryozoan to bryozoan trans- obtained transmission of T. bryosalmonae by cohabita- mission studies, we ensured physical contact between tion of fish with infected bryozoan material in a flow- bryozoans and spores by wafting spores directly into through system where the continuous loss of spores bryozoan lophophores, and we exposed bryozoans to should have resulted in low concentration. Hedrick et spores in small volumes of water to maximise spore al. (2004) estimated a maximum concentration of 120 concentrations. The inclusion of both bryozoans and T. bryosalmonae spores ml–1 fish urine, thus spores fish in Trial 3c demonstrated that functional spores should become highly dilute upon release from fish in were indeed present at least in this trial, since the fish natural populations. These observations suggest that subsequently developed PKD (M. Longshaw & S. Feist spores from both bryozoans and fish would have to be unpubl. data). effective in contacting and infecting hosts at very low There is a greater possibility that transmissions of concentrations. An ability to achieve transmission at Tetracapsuloides bryosalmonae from fish to bryozoans low concentration should be highly adaptive for dilute were hampered by lack of mature infective stages, low water-borne spores, and we would expect this to be concentrations of infective spores released in fish urine true for all infective stages in the life cycle of T. and short periods of exposure to allow bryozoans to bryosalmonae, be they stages achieving transmission 118 Dis Aquat Org 60: 109–121, 2004

from fish to bryozoans, from bryozoans to fish or from gated, including Gammarus sp., caddis flies, leeches any other potential host. and snails (Longshaw & Feist 2000, M. Longshaw & S. Despite the expected effectiveness of spores at low Feist unpubl. data cited in Feist et al. 2001). None of concentrations, given the uncertainties associated with these invertebrates tested positive for the parasite, as our fish to bryozoan transmissions, more work is determined by PCR investigation using specific required to assess this route of infection, particularly primers. In addition, our culture systems contain many given the possible infection of bryozoans by malaco- of the most cosmopolitan aquatic invertebrates, includ- sporeans through exposure to macerated fish kidney ing chironomids, cladocerans, rotifers, oligochaetes, (D. C. Morris et al. 2002a) and to stages released from flatworms and snails, which have been introduced fish with PKD (D. J. Morris et al. 2002). However, even along with bryozoans brought in from the field. If any if such transmission is eventually demonstrated, it may of these organisms were acting as hosts for T. bryo- only be facultative since infected bryozoans occur in salmonae, we would anticipate that infections would sites lacking salmonids in Ohio and Michigan (Oka- eventually have developed within the long-term cul- mura et al. 2001). Furthermore, even if spores released tured bryozoans that were cohabited with infected by fish are demonstrated to be infective for bryozoans, bryozoans introduced from the field. Such infection it is difficult to envision how such spores could regu- has never been observed. However, it is possible that larly infect bryozoan populations upstream from fish our culture conditions are unsuited to development in farms and hatcheries to cause the annual and massive another host or that such a host was lacking. PKD outbreaks that occur in many sites. For instance, So far there is no evidence that fish, other than very high prevalences of Tetracapsuloides bryo- salmonids and pike, act as hosts of Tetracapsuloides salmonae infections coincide with early seasonal bryosalmonae (Hedrick et al. 1993, Adams & Morris growth of the bryozoan population just upstream from 1999). A number of other potential fish hosts have been a fish farm which suffers PKD outbreaks on the River investigated, but none have been identified (Adams & Cerne, Dorset (Longshaw et al. 1999, S. Tops & B. Oka- Morris 1999). Furthermore, the broad diversity of habi- mura unpubl. data). The bryozoan population and fish tats associated with PKD outbreaks and T. bryo- farm are no more than 6 km from the headwaters of the salmonae infections in bryozoans provide evidence Cerne, and the only salmonids present in this stretch of that no single fish can be an obligate host. T. bryo- water are a few farm escapees and a small population salmonae has been recorded from sites ranging from of brown trout (M. Longshaw pers. comm.). It seems clear, cool streams to eutrophic lakes in North Amer- unlikely that the high prevalences of infections (over ica, and the number of resident non-salmonids range 40%; S. Tops & B. Okamura unpubl. data) in dense from single (e.g. the rare cyprinid at Hot Creek Hatch- stands of bryozoans on many willow roots could ery) to many species (Okamura et al. 2001). If fish hosts directly result from spores released by only a few fish. are obligate in the life cycle, this broad distribution of Another informative site is a salmonid hatchery in Cal- T. bryosalmonae suggests an unusual capacity to ifornia (Hot Creek Hatchery) which experiences PKD exploit a diversity of obligate fish hosts, although, as outbreaks. Fish from the hatchery have rarely been discussed below, the life cycle of bryozoans may com- collected upstream (probably due to difficulties in plicate the interpretation of distributional data. swimming through pipes) and rare cyprinids are the We anticipate that if another invertebrate or non- only resident fish in the short reach of stream between salmonid fish host is utilised in the life cycle of Tetra- the spring source and the diversion of flow-through capsuloides bryosalmonae, infection is likely to be pipes to the hatchery (Okamura & Wood 2002). patchy, as it is in bryozoan populations (Anderson et al. 1999, S. Tops & B. Okamura unpubl. data), and that this has led to the lack of detection. The best chance of Implications of study identifying what such a host might be rests in focusing on simple systems, as was done in the elucidation of Our results imply that there may be another host the life cycle of the oyster parasite Marteilia refringens incorporated in the life cycles of Tetracapsuloides (Audemard et al. 2002). The simplest system for T. bryosalmonae and Buddenbrockia plumatellae. Prior bryosalmonae would be the recirculating system in to the discovery of phylactolaemate bryozoans as hosts France (Gay et al. 2001), but PCR and transmission- of the ‘PKX’ parasite, researchers screened many based investigations have so far failed to determine aquatic invertebrates and other fish species from sites whether resident invertebrates in this system are enzootic for PKD in an attempt to locate the source of incorporated in the life cycle of T. bryosalmonae (de the infection. For instance, Morris et al. (1999) sur- Kinkelin et al. 1999, P. de Kinkelin & D. J. Morris veyed 35 000 oligochaete worms. In another study, 60 unpubl. data cited in Gay et al. 2001). If our transmis- to 80 species of aquatic invertebrates were investi- sion results are correct in demonstrating that transmis- Tops et al.: Malacosporean transmission studies 119

sion is not direct from bryozoan to bryozoan, and fur- sites within clonally reproducing bryozoan hosts. The ther work confirms that salmonids are dead-end hosts, growing body of evidence that waterfowl act as disper- the search for an unknown host might once again sal vectors of bryozoan statoblasts lends credence to become akin to looking for a needle in a haystack this possibility. Such evidence includes ongoing gene (Audemard et al. 2002). flow amongst sites traversed by migratory waterfowl (Freeland et al. 2000), the presence of intact statoblasts in waterfowl digestive tracts and faeces (Figuerola et Bryozoan life cycles and cryptic al. 2003), and the viability of some statoblasts following malacosporean stages ingestion and excretion by waterfowl (Charalambidou et al. 2003). Such waterfowl-mediated dispersal of It is now clear that malacosporeans can persist for at infected statoblasts, followed by the subsequent least 2 to 3 mo as cryptic stages in the body wall of spread of parasitism in the bryozoan population, bryozoans prior to proliferating as sacs in the body provides one explanation for the presence of infected cavity (Canning et al. 2002, Tops & Okamura 2003). bryozoan populations in sites lacking salmonids. Myxosporeans appear to behave in a similar manner. Unravelling the ecology of early cryptic stages of Myxobolus cerebralis maintains persistent infections malacosporeans, identification of the cues that pro- in oligochaetes characterised by periods of parasite mote proliferation of spore-producing sacs from these dormancy and intermittent shedding of actinospores cryptic stages and investigating the clonal structure of (Gilbert & Granath 2001), while at low temperatures bryozoan host populations are important areas for fur- cryptic immature forms undergo cycles of replication ther research. but do not produce actinospores (El-Matbouli et al. 1999). These recent studies suggest that persistent Acknowledgements. We thank A. Gómez for modified CTAB cryptic infection is a general myxozoan feature and protocols, W. Cox and A. Thomas for providing infected fish, that cryptic infections of malacosporeans may be main- D. Butterworth, A. Mercer and S. Leach for access to bryo- tained indefinitely within bryozoan populations. zoan populations and provision of fish, Prof. M. El-Matbouli for laboratory space in Germany, S. Feist and M. Longshaw We predict that the life history of bryozoans plays an for allowing cohabitation of bryozoans with fish during trans- important role in the maintenance and spread of cryp- mission studies, and W. Lockwood for assistance in the main- tic malacosporean infections without the necessity of tenance of bryozoan colonies. The work was supported by regular transmissions from any other hosts. If cryptic the Natural Environment Research Council (NER/A/S/1999/ stages proliferate as colonies grow, this would ensure 00075) and the Department for the Environment Food and Rural Affairs (FC1112). B.O. was supported by a Research that all regions of colonies become infected. Also, Fellowship from the Leverhulme Trust during a portion of the colonies of Fredericella sultana fragment as they work and preparation of the manuscript. become larger, and branches that drift downstream reattach (Wood 1973, S. Tops & B. Okamura pers. obs.). LITERATURE CITED Such fragmentation and reattachment could increase local prevalence of infection and introduce infected Adams A, Morris DJ (1999) Antibody development, PCR and colonies elsewhere. Furthermore, as F. sultana itself in-situ hybridisation. Annex 8 in: Proliferative kidney overwinters as live colonies (Wood 1973, Raddum & disease (PKD) in wild and farmed salmonids: draft report Johnsen 1983), cryptic infections can be maintained of the first workshop, 15–17 November 1999, CEFAS Laboratory, Weymouth, UK year-round in this species (Gay et al. 2001). Finally, if Anderson CL, Canning EU, Okamura B (1999) 18S rDNA cryptic stages are present in dormant bryozoan stato- sequences indicate that PKX organism parasitizes Bryo- blasts, infections could be passed to new colonies zoa. Bull Eur Ass of Fish Pathol 19:94–97 through hatching from statoblasts. All of these pro- Audemard C, Le Roux F, Barnaud A, Collins C and 6 others (2002) Needle in a haystack: involvement of the copepod cesses could promote long-term infections in bryozoan Paracartia grani in the life-cycle of the oyster pathogen populations without the necessity of regular transmis- Marteilia refringens. Parasitology 124:315–323 sions from any other hosts, thus explaining the high Bartholomew JL, Reno PW (2002) The history and dissemina- prevalences of mature infections in bryozoan population of whirling disease. In: Bartholomew JL, Wilson JC tions early in the growing season (Longshaw et al. (eds) Whirling disease: reviews and current topics. Am Fish Soc, Symp 29, Bethesda, MD, p 3–24 1999, S. T.ops & B. Okamura unpubl. data). Bromage N (1999) Impact of PKD on the UK trout industry. If cryptic stages of Tetracapsuloides bryosalmonae Annex 2 in: Proliferative kidney disease (PKD) in wild and are capable of undergoing dispersal in bryozoan stato- farmed salmonids: draft report of the first workshop, blasts, then the persistence of infections in some sites 15–17 November 1999, CEFAS Laboratory, Weymouth, UK may simply be explained by colonisation through Bütschli O (1881) Myxosporidien. Zool Jahrb 1:162–164 infected statoblasts and the subsequent spread of par- Canning EU, Okamura B (2004) Biodiversity and evolution of asitism in the population through proliferation of para- the Myxozoa. Adv Parasitol 56:43–131 120 Dis Aquat Org 60: 109–121, 2004

Canning EU, Okamura B, Curry A (1996) Development of a kidney disease of salmonid fish. Ann Rev Fish Dis 3: myxozoan parasite Tetracapsula bryozoides gen. n. et sp. 277–290 n. parasitic in the freshwater bryozoan Cristatella mucedo Hedrick RP, Baxa DV, de Kinkelin P, Okamura B (2004) Mala- (Bryozoa: Phylactolaemata). Folia Parasitol 43:249–261 cosporean-like spores in urine of rainbow trout react with Canning EU, Curry A, Feist SW, Longshaw M, Okamura B antibody and DNA probes to Tetracapsuloides bryo- (1999) Tetracapsula bryosalmonae n. sp. for PKX organ- salmonae. Parasitol Res 92:81–88 ism, the cause of PKD in salmonid fish. Bull Eur Ass Fish Henderson M, Okamura B (2004) The phylogeography of Pathol 19:203–206 salmonid proliferative kidney disease in Europe and North Canning EU, Curry A, Feist SW, Longshaw M, Okamura B America. Proc R Soc Lond B 271:1729–1736 (2000) A new class and order of myxozoans to accommo- Hoffman GL (1990) Myxobolus cerebralis, a worldwide cause date parasites of bryozoans with ultrastructural observa- of salmonid whirling disease. J Aquat Anim Health 2: tions on Tetracapsula bryosalmonae (PKX Organism). 20–37 J Eukaryot Microbiol 47:456–468 Kent ML, Hedrick RP (1986) Development of the PKX myx- Canning EU, Tops S, Curry A, Wood TS, Okamura B (2002) osporean in rainbow trout Salmo gairdneri. Dis Aquat Org Ecology, development and pathogenicity of Budden- 1:169–182 brockia plumatellae Schröder, 1910 (Myxozoa, Mala- Kent ML, Andree KB, Bartholomew JL, El-Matbouli M and 12 cosporea) (syn. Tetracapsula bryozoides) and establish- others (2001) Recent advances in our knowledge of the ment of Tetracapsuloides n. gen. for Tetracapsula Myxozoa. J Eukaryot Microbiol 48:395–413 bryosalmonae. J Eukaryot Microbiol 49:280–295 Longshaw M, Feist SW (2000) The agent of proliferative kid- Charalambidou I, Santamaría L, Figuerola J (2003) How far ney disease (PKD) requires a bryozoan in its lifecycle— can the freshwater bryozoan Cristatella mucedo disperse studies at CEFAS Weymouth on PKD and other myxo- in duck guts? Arch Hydrobiol 157:547–554 zoans. Trout News 29:24–27 de Kinkelin P, Chilmonczyk S, Bleux C, Dorson M (1999) Pro- Longshaw M, Feist SW, Canning EU, Okamura B (1999) First liferative kidney disease (PKD) in rainbow trout: 1. epi- identification of PKX in bryozoans from the United King- demiological investigations in an infected recirculating dom—molecular evidence. Bull Eur Ass Fish Pathol 19: system; 2. immune response of trout and protection 146–148 against clinical infection. Annex 9 in: Proliferative kidney Longshaw M, Le Deuff RM, Harris AF, Feist SW (2002) Devel- disease (PKD) in wild and farmed salmonids: draft report opment of proliferative kidney disease in rainbow trout, of the first workshop, 15–17 November 1999, CEFAS Lab- Oncorhynchus mykiss (Walbaum), following short-term oratory, Weymouth, UK exposure to Tetracapsula bryosalmonae infected bryo- Diamant A (1997) Fish-to-fish transmission of a marine myx- zoans. J Fish Dis 25:443–449 osporean. Dis Aquat Org 30:99–105 Monteiro AS, Okamura B, Holland PWH (2002) Orphan worm D’Silva J, Mulcahey MF, de Kinkelin P (1984) Experimental finds a home: Buddenbrockia is a myxozoan. Mol Biol Evol transmission of proliferative kidney disease in rainbow 19:968–971 trout, Salmo gairdneri Richardson. J Fish Dis 7:235–239 Morris DC, Morris DJ, Adams A (2002a) Molecular evidence El-Matbouli M, McDowell TS, Antonio DB, Andree KB, of release of Tetracapsula bryosalmonae, the causative Hedrick RP (1999) Effect of water temperature on the organism of proliferative kidney disease from infected development, release and survival of the triactinomyxon salmonids into the environment. J Fish Dis 25:501–504 stage of Myxobolus cerebralis in its oligochaete host. Int Morris DC, Morris DJ, Adams A (2002b) Development of J Parasitol 29:627–641 improved PCR to prevent false positives and false nega- Feist SW, Longshaw M, Canning EU, Okamura B (2001) tives in the detection of Tetracapsula bryosalmonae, the Induction of proliferative kidney disease (PKD) in rainbow causative agent of proliferative kidney disease (PKD). trout Oncorhynchus mykiss via the bryozoan Fredericella J Fish Dis 25:483–490 sultana infected with Tetracapsula bryosalmonae. Dis Morris DJ, Longshaw M, Adams A, Feist SW, Richards RH Aquat Org 45:61–68 (1999) Is an oligochaete involved in the life cycle of PKX? Ferguson HM, Ball HJ (1979) Epidemiological aspects of pro- In: Brit Soc Parasitol (BSP) Spring Meeting Abstracts, liferative kidney disease in rainbow trout, Salmo gairdneri University of Warwick, p 32 Richardson in Northern Ireland. J Fish Dis 2:219–225 Morris DJ, Adams A, Feist SW, McGeorge J, Richards RH Figuerola J, Green AJ, Santamaría L (2003) Passive internal (2000) Immunohistochemical and PCR studies of wild fish transport of aquatic organisms by waterfowl in Doñana, for Tetracapsula bryosalmonae (PKX), the causative south-west Spain. Global Ecol Biogeogr 12:427–436 organism of proliferative kidney disease. J Fish Dis 23: Foott JS, Hedrick RP (1987) Seasonal occurrence of the infec- 129–135 tious stage of proliferative kidney disease (PKD) and resis- Morris DJ, Morris DC, Adams A (2002) Development and tance of rainbow trout, Salmo gairdneri Richardson, to release of a malacosporean (Myxozoan) from Plumatella reinfection. J Fish Biol 30:477–483 repens (Bryozoa: Phylactolaemata). Folia Parasitol 49: Freeland JR, Noble LR, Okamura B (2000) Genetic conse- 25–34 quences of the metapopulation biology of a facultatively Okamura B (1996) Occurrence, prevalence and effects of the sexual freshwater invertebrate. J Evol Biol 13:383–395 myxozoan Tetracapsula bryozoides parasitic in the fresh- Gay M, Okamura B, de Kinkelin P (2001) Evidence that the water bryozoan Cristatella mucedo (Bryozoa: Phylactolae- infective stages of Tetracapsula bryosalmonae for rainbow mata). Folia Parasitol 43:262–266 trout, Oncorynchus mykiss, are present throughout the Okamura B, Canning EU (2003) Orphan worms and homeless year. Dis Aquat Org 46:31–40 parasites enhance bilaterian diversity. Trends Ecol Evol Gilbert MA, Granath WO Jr (2001) Persistent infection of 18:633–639 Myxobolus cerebralis, the causative agent of salmonid Okamura B, Hatton-Ellis T (1995) Population biology of whirling disease, in Tubifex tubifex. J Parasitol 87: bryozoans: correlates of sessile, colonial life histories in 101–107 freshwater habitats. Experientia 51:510–525 Hedrick RP, MacConnell E, de Kinkelin P (1993) Proliferative Okamura B, Wood TS (2002) Bryozoans as hosts for Tetracap- Tops et al.: Malacosporean transmission studies 121

sula bryosalmonae, the PKX organism. J Fish Dis 25: capsuloides bryosalmonae in sites endemic for salmonid 469–475 proliferative kidney disease. Dis Aquat Org 57:221–226 Okamura B, Anderson CL, Longshaw M, Feist SW, Canning Wahli T, Knuesel R, Bernet D, Segner H, Pugnovkin D, EU (2001) Patterns of occurrence and 18S rDNA sequence Burkhardt-Holm P, Escher M, Schmidt-Posthaus H (2002) variation of PKX (Tetracapsula bryosalmonae), the Proliferative kidney disease in Switzerland: current state causative agent of salmonid proliferative kidney disease. of knowledge. J Fish Dis 25:491–500 J Parasitol 87:379–385 Winnepenninckx B, Backeljau T, De Wachter R (1993) Extrac- Okamura B, Curry A, Wood TS, Canning EU (2002) Ultra- tion of high molecular weight DNA from molluscs. Trends structure of Buddenbrockia identifies it as a myxozoan Gen 9:407 and verifies the bilaterian origin of the Myxozoa. Para- Wood TS (1971) Laboratory culture of freshwater Ectoprocta. sitology 124:2115–223 Trans Am Microsc Soc. 90:229–231 Raddum GG, Johnsen TM (1983) Growth and feeding of Wood TS (1973) Colony development in species of Plu- Fredericella sultana (Bryozoa) in the outlet of a humic acid matella and Fredericella (Ectoprocta: Phylactolaemata). lake. Hydrobiologia 101:115–120 In: Boardman RS, Cheetham AH, Oliver WA Jr (eds) Redondo MJ, Palenzuela O, Álvarez-Pellitero P (2002) Exper- Development and function of animal colonies through imental transmission of Enteromyxum scopthalmi (Myxo- time. Dowden, Hutchinson and Ross, Stroudsberg, PA, p zoa), an enteric parasite of turbot, Scophthalmus maximus. 395–432 J Parasitol 88:482–488 Wood TS (1996) Aquarium culture of freshwater inverte- Seagrave CP, Bucke D, Alderman DJ (1980) Ultrastructure of brates. Am Biol Teach 58:46–50 a haplosporean-like organism: the possible causative Yasuda H, Ooyama T, Iwata K, Tun T, Yokoyama H, Ogawa K agent of proliferative kidney disease in rainbow trout. (2002) Fish-to-fish transmission of Myxidium spp. (Myxo- JFish Biol 16:453–459 zoa) in cultured tiger puffer suffering from emaciation dis- Tops S, Okamura B (2003) Infection of bryozoans by Tetra- ease. Fish Pathol 37:29–33

Editorial responsibility: Wolfgang Körting, Submitted: October 29, 2003; Accepted: March 12, 2004 Hannover, Germany Proofs received from author(s): July 22, 2004