DISEASES OF AQUATIC ORGANISMS Vol. 40: 33-39.2000 Published February 24 Dis Aquat Org I The life cycle of Sphaerospora truttae (Myxozoa: Myxosporea) and some features of the biology of both the actinosporean and myxosporean stages 'University of Stirling, Institute of Aquaculture, Stirling FK9 4LA, Scotland. UK 20ndokuzmayis Universitesi. Su ~riinleriFakiiltesi. 57000 Sinop, Turkey ABSTRACT A previously undescnbed echinactinomyxon type actinosporean was shown experimen- tally to be the alternate stage of Sphaerospora truttae. The echinactinomyxon type spores were found to be released from Lumbriculus variegatus and occasionally Tubifex tubifex. Overall ~nfectionpreva- lence of the echinactinomyxon was 0.14%. Peak release was in March-May each year S. truttae spores developed in experimentally infected salmon 4.5 mo post-exposure to echinactinomyxon spores. Extrasporogonic stages of S. truttae first appeared in naturally infected salmon in early July and per- sisted for 8 to 10 wk. Correlation between peak release of echinactinomyxon spores and appearance of extrasporogonic stages of S. truttae is discussed. KEY WORDS: Sphaerospora truttae - Life cycle . Actinosporean . Myxosporean INTRODUCTION Sphaerospora truttae was first described by Fischer- Scherl et al. (1986) from brown trout Salmo trutta L. The life cycles of 17 different myxosporean species in Germany. Since then, Walter et al. (1991) and belonging to 7 different genera (Myxobolus, Thelo- McGeorge et al. (1996a,b) reported this parasite from hanellus, Myxidium, Zschokkella, Ceratomyxa, Hofer- grayling Thymallus thymallus L. in Germany and ellus and Sphaerospora) have been completed and Atlantic salmon Salmo salar L. in Scotland, respec- shown to involve members of 7 collective groups tively. The latter authors conducted extensive studies of actinosporeans (triactinomyxon, hexactinomyxon, on its morphology, ultrastructure and biology. raabeia, aurantiactinomyxon, neoactinomyxum, guy- McGeorge et al. (1996a) showed that Sphaerospora enotia and siedleckiella) (Markiw & Wolf 1983, El- truttae has 2 distinct stages, extrasporogonic and Matbouli & Hoffmann 1989, 1993, Rudish et al. 1991, sporogonic, in fish. The first detectable signs of infec- Styer et al. 1991, El-Matbouli et al. 1992, Grossheider tion occurred each year in late June/early July in fish & Korting 1992, Benajiba & Marques 1993, Kent et al. when extrasporogonic stages were found in the blood 1993, Uspenskaya 1995, Yokoyama et al. 1995, Troul- and kidney interstitium. These stages were present lier et al. 1996, Bartholomew et al. 1997, Yokoyama until early September and the sporogonic stages oc- 1997, El-Mansy et al. 1998a,b,c, Szekely et al. 1998, curred in the kidney tubules from early August, with Molnar et al. 1999).So far only 2 Sphaerospora species, mature spores first being present in September (Mc- S. ictaluri and S. renicola, have been shown to have an George et al. 1996a). They also showed that the tem- alternate actinosporean stage, with an aurantiactino- perature was an important factor influencing the infec- myxon and neoactinomyxum type, from the oligo- tion prevalence and intensity of S. truttae. chaetes Dero digitata and Branchiura sowerbyi, re- In the present study, the life cycle of Sphaerospora spectively (Styer et al. 1991, Molnar et al. 1999). truttae was completed using echinactinomyxon type actinospores and the biology of both the actinosporean 'Corresponding author. E-mail: [email protected] and myxosporean stages of S. truttae were studied, 8 Inter-Research 2000 Resale of full article not permitted 34 Dis Aquat Org 40: 33-39, 2000 + particularly in relation to the seasonal Min~rnurn -m- Maximum -A- Average pattern of release of echinactinomyxon /.1 spores and the Infection of salmon with S. truttae. MATERIALS AND METHODS Sampling programme. Fish and oligo- chaete samples were collected from a 4mL,-,s~r.s%m~~,,,-mm,,~mm, freshwater Atlantic salmon farm located SONDJFMAMJJASONDJFMAMJJA in Northern Scotland (British National Grid NC 387546). This was the same site Fig. 1. Monthly minimum, maximum and average water temperatures ("C) used by McGeorge et al. (1997). Sampling at the salmon farm was carried c\ct at in!ervals nf 4 wk in spring and summer and 6 wk in autumn and winter. using a micrometer calibrated eye-piece graticule. Whilst oligochaete sampling took place from 17 Octo- Dead, senescent or immature spores were not used for ber 1996 to 16 August 1998, fish samples were col- measurement. Spores were identified using the keys lected from 17 October 1996 to 6 September 1997 on and diagrams of Janiszewska (1955, 1957), Marques the same day that oligochaetes were collected. (1984),Lom et al. (1997) and by comparison with other Sampling procedure for oligochaetes. Oligochaetes published reports. The echinactinomyxon stage spore were collected from a settlement pond located at the of Sphaerospora truttae was designated as echinact- downstream end of the farm. Oligochaetes were sepa- inomyxon type5 in Ozer (1999). rated from substrate by passing the sediment through Experimental fish. Juvenile Atlantic salmon (length graded sieves of 1.5 mm, 1 mm and 500 pm mesh size. 10 to 13 cm) were collected from a farm where fish The contents of the sieves were then emptied into were reared entirely on bore-hole water. In the labora- shallow basins containing dechlorinated tap water and tory fish were kept in 100 1 rectangular tanks supplied observed under bright light to detect oligochaetes. At with charcoal filtered mains water at 12 to 16°C and least 1000 worms were obtained at each sampling fed ad libitum with a commercial pelleted feed. Thirty time. Oligochaetes were identified according to the fish were sacrificed and examined for the presence of key of Brinkhurst (1963). myxosporean infection by the methods outlined below. Sampling procedure for fish. Fish were randomly Exposure of fish to actinosporean spores. In trans- netted on each sampling date from the same tank mission experiments using actinosporean spores, the cohort of fish as far as possible. When the population water level of aquaria (13 X 12 X 21 cm) was reduced was graded, one of the resulting populations was sam- to 1-2 1 with no aeration for 30 min to 1 h. Echinactin- pled. At least 20 fish were collected at each sampling omyxon type spores were pipetted into the tanks at date. All fish samples were transferred alive to the the beginning of this period. Where sufficient spores Institute of Aquaculture, Stirling, in oxygenated river were available, the exposure of fish to echinactino- water. myxon type spores was repeated on up to 3 subse- Actinosporean samples. Actinosporean spores were quent days. After 1 h, fish and unexposed control fish collected from infected oligochaetes kept in 24-cell were transferred to aquaria containing charcoal fil- well plates according to the method of Yokoyama et al. tered mains water for 3.5 to 4.5 mo. After 3.5 mo, half (1991) and examined under an inverted microscope the number of infected and control fish were individu- after 24 h. ally examined as described below for the presence of Farm water temperature. Daily farm temperature myxosporean spores. Remaining f~shwere examined data was collected from the records of the fish farm after 4.5 mo. and plotted monthly with minimum, maximum and Examination of fish for myxosporeans. Fish were average levels (Fig. 1). Strict seasonal patterns were sacrificed by means of a sharp blow to the head. In fish observed each year, with a maximum of 18°C in July- from the salmon farm blood was obtained from the August and a minimum of 1°C in December-January. caudal artery by severing the tail and at least 3 blood Identification of actinosporean types released. Re- smears were made from each fish and stained with a leased actinosporeans were drawn and photographed Rapi-Diff I1 staining kit (Lamb, London, UK). The body under both phase contrast and bright field. Measure- cavity was dissected and the liver, heart, kidney, diges- ments were taken from at least 20 randomly selected tive tract, gall bladder, swimbladder, urinary bladder, spores from 1 infected oligochaete where possible, reproductive organs and brain removed for further Ozer & Wootten: Life cycle of Sphaerospora truttae 35 examination. All the organs used for detection of Table 1. Sphaerospora truttae infections in Atlantic salmon myxosporeans were examined using squash prepara- experimentally exposed to spores of echinactinomyxon tions. Kidneys were examined by squash preparations or Rapi-Diff I1 stained impression smears of anterior, Months post-infection Sample size No. of fish Infected mid and posterior kidney. Squash preparations were Expt l examined using standard light, phase contrast and Experimental group Nomarski optics. 3.5 mo 3 2 (sporogonic stages) Fish from experimental infections, including control 4.5 mo 3 1 (mature spores) fish were examined as described above except that Control group 3.5 m0 3 0 blood was not examined. 4.5 m0 3 0 Experimental exposure of fish to actinosporeans. Expt 1: Six Atlantic salmon were exposed to echin- Expt 2 actinomyxon type spores for 3 d starting 29 March Exper~mentalgroup 3.5 mo 2 2 (sporogonic stages) 1997 at a concentration of 10000 to 12 000 spores fish-' 4.5 m0 2 0 d-'. Six Atlantic salmon were used as a control. Control group Expt 2: Four Atlantic salmon were exposed to echin- 3.5 m0 2 0 actinomyxon type spores for 2 d starting on 9 August 4.5 m0 2 0 1997 at a concentration of 10 000 to 15 000 spores fish-' d-'. Four Atlantic salmon were used as a control. (Figs. 2a & 3). The spore body was 11.2 (10 to 12) pm in length and 8.1 (7 to 9) pm in width. Polar capsules were RESULTS 5 by 2 pm. The length of the caudal processes was 58 (38 to 75) um. Experimental infection of fish Thirty fish sampled from the stock of experimental Prevalence rate of the echinactinomyxon stage of fish prior to infection experiments were uninfected Sphaerospora truttae with myxosporeans.