Studies on the proposed role of Tubifex tubifex (Muller) as an intermediate host in the life cycle of Myxosoma cerebralis (Hofer, 1903)

Abstract. In a recently proposed hypothesis for the transmission of Myxosoma cerebralis , the causative agent of salmonid whirling disease, it was suggested that there was a developmental cycle in tubificid worms culminating in actinosporean spores, which were infective to the fish. Results are presented here which do not support the actinosporean involvement in the life cycle. On addition of M. cerebralis spores to Tubifex tubifex colonized in sterilized medium, no significant change in the prevalence of Triactinomyxon dubium (i.e. T. gyrosalmo ) was found. Although it is shown that these worms are capable of ingesting M. cerebralis spores, neither hatching of the spores nor further development within the worm has been observed. Field observations on the distribution of actinosporean species show no obvious correlation between the occurrence of T. dubium and M. cerebralis .     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

Salmonid whirling disease: myxosporean and actinosporean stages cross-react in direct fluorescent antibody test

Abstract. Serologic relatedness of the two life stages of the salmonid whirling disease parasite Myxosoma cerebralis Hofer, 1903 — myxosporean spores from fish cartilage and actinosporean triactinomyxon spores from aquatic tubificids — were investigated. When the direct fluorescent antibody technique was used, anti-triactinomyxon and anti- M. cerebralis rabbit sera conjugated with fluorescein isothiocyanate cross-reacted with the respective heterologous life stage. Both stages showed similar locations of specific fluorescence with conjugates of either homologous or heterologous serum. Thus, serology supports the relatedness of the myxosporean M. cerebralis and the actinosporean triactinomyxon stages.     

Lectin histochemical studies on Sphaerospora sp. (Myxosporea) from Italian brown trout, Salmo trutta L.

A Sphaerospora sp. (Myxosporea) infection (presumably S. truttae ) was identified on a trout farm in northeastern Italy. Parasites were detected in kidneys from infected brown trout, Salmo trutta L., over a 2-year period. Extrasporogonic, sporogonic stages and mature spores were simultaneously detected in the same fish. Traditional diagnostic methods for Sphaerospora spp. rely on the detection of the myxosporean developmental stages in Giemsa-stained kidney smears or haematoxylin-eosin stained tissue sections. A histochemical method was employed where 10 biotinylated lectins (Con-A, DBA, SBA, GS-I, PHA-P, LEA, PWM, RCA₁, WGA and UEA-I) and the avidin-biotin-peroxidase complex (ABC) were used on Sphaerospora -infected brown trout renal tissues and kidney imprints. Five monoclonal antibodies against PKX (Mab12, MabA3, MabC5, MabD4 and MabB4) were also tested. A lectin glycoconjugate binding pattern for Sphaerospora spp. is presented. This staining method shows that SBA lectin ( Glycine max agglutinin) is a useful tool for the detection of the Sphaerospora spp. Only MabB4 bound some of the most mature sporogonic stages. In contrast Mabs12, A3, C5 and D4, and GS-I lectin ( Griffonia simplicifolia agglutinin) did not stain any of the Sphaerospora spp. stages, but did bind very specifically to the sporogonic and extrasporogonic stages of PKX, the causative agent of proliferative kidney disease (PKD).     

Expanded geographical distribution of Myxobolus cerebralis: first detections from Alaska

The parasite responsible for salmonid whirling disease, Myxobolus cerebralis, was introduced to the USA in 1958. It has since spread across the country causing severe declines in wild trout populations, but has never been documented from Alaska. However, while assessing the risk of introduction of M. cerebralis into the state, we detected the parasite using a species-specific polymerase chain reaction (PCR) assay. Testing of 180 hatchery rainbow trout, Oncorhynchus mykiss (Walbaum), by pepsin trypsin digest (PTD) and quantitative PCR (QPCR) revealed 14 positive samples. Infection was confirmed by sequencing the parasite 18S rRNA gene and by a nested PCR assay based on the same gene. Sequence comparison of M. cerebralis from several locations demonstrated the Alaska isolates were genetically distinct and therefore not false-positives arising from contamination during processing. We were unable to visually identify myxospores, indicating that either infection was light or mature spores had not formed. A reference set of fish samples spiked with known numbers of myxospores verified the QPCR and PTD results. This paper presents DNA sequence data from the Alaska M. cerebralis isolates, provides a brief history of the fish and facility of origin, and discusses implications of different testing methods on asymptomatic fish populations.     

Development of proliferative kidney disease in rainbow trout, Oncorhynchus mykiss (Walbaum), following short-term exposure to Tetracapsula bryosalmonae infected bryozoans

The initial site of infection in the fish host for Tetracapsula bryosalmonae , causative agent of proliferative kidney disease (PKD) is poorly understood. Following the recent recognition that freshwater bryozoans harbour the infective stages to salmonid fish, experimental transmission studies were undertaken to investigate (1) the route of entry of the parasite into the fish host and (2) the minimum exposure time required to induce clinical signs of PKD. In-situ hybridization (ISH) studies were carried out on naïve rainbow trout exposed to the naturally infected bryozoan Fredericella sultana for up to 90 min. The sporoplasm of T. bryosalmonae was detected entering the fish via mucous cells in the skin epithelium within the first minute of exposure. In addition, T. bryosalmonae cells were infrequently detected in the skeletal musculature of exposed experimental fish up to 72 h post-exposure. The route of migration through the fish to the kidney and spleen was not determined. All fish exposed to infected, disrupted bryozoans for 10, 30 and 90 min and maintained for up to 8 weeks developed clinical PKD.     

Complete developmental cycle of Myxobolus pseudodispar (Gorbunova) (Myxosporea: Myxobolidae)

Myxobolus pseudodispar (Gorbunova) is a common parasite of the muscle of roach, Rutilus rutilus L., whereas its actinosporean development occurs in two oligochaete alternate hosts. This paper reports the complete developmental cycle of this parasite in the oligochaete alternate host Tubifex tubifex and the roach. In laboratory experiments, parasite-free T. tubifex specimens were infected by myxospores of M. pseudodispar collected from roach in Lake Balaton. Parasite-free roach fingerlings were infected with floating triactinospores (TAMs) released from oligochaetes on day 69 after challenge. Young plasmodia and spores in roach were first recorded on day 80 post-exposure (p.e.). Myxospores collected from experimentally infected roach initiated a new development in T. tubifex and the resulting TAMs infected roach. No infection of roach resulted from feeding oligochaetes containing mature triactinospores.     

Performance of different types of diets on experimental larval rearing of endangered Chitala chitala (Hamilton) in recirculatory system

This is the first report on the successful larval rearing of captive bred population of Chitala chitala (Hamilton). C. chitala is one of the endangered fresh water fish species in India for which the development of controlled larval rearing procedures are needed for stock enhancement. Fifteen days old post-hatchlings were stocked for 28 d in a 30 L recirculatory tanks using eight different diets i.e. live feed (tubifex worms, chironomous larvae, zooplanktons,), dry feed (dry tubifex, spirulina, daphnia) and other non-conventional feed (fish eggs and boiled egg-yolk). Fishes accepted all types of diets. The study revealed that specific growth rate (SGR) was higher in post-hatchlings fed on live tubifex worms (2.40 ± 0.72) followed by fish eggs (2.15 ± 0.71), dry tubifex (2.12 ± 0.40), chironomous larvae (1.91 ± 0.44), spirulina (1.79 ± 0.38), daphnia (1.42 ± 0.79) and planktons (1.37 ± 0.77) whereas minimum SGR was recorded with boiled egg-yolk (0.63 ± 0.5). A highly significant difference (p < 0.01) in SGR was observed in fish fed on live feed (tubifex worms, chironomous larvae, planktons, spirulina), dry tubifex and fish eggs whereas for daphnia and boiled egg-yolk it was only significant (p < 0.05). The final mean weight and weight gain showed highly significant difference (p < 0.01) in live tubifex, zooplanktons, spirulina, chironomous larvae, dry tubifex and fish eggs, whereas daphnia and boiled egg-yolk fed larvae showed significant difference (p < 0.05). Highest mean survival rate on day 28 was observed in live tubifex worms (94%) and chironomous larvae (92%). The post-hatchlings reared with spirulina and daphnia showed same survival rate of 88% whereas the lowest mean survival of 66% was recorded in boiled egg-yolk. The experiments showed that captive bred post-hatchlings of C.chitala could be reared in experimental recirculatory system for attaining higher growth and survival during early life stages. However, methods to improve the larval rearing have to be improved further for commercial farming of the species.     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

Experimental identification of the actinosporean stage of Sphaerospora renicola Dykova & Lom 1982 (Myxosporea: Sphaerosporidae) in oligochaete alternate hosts

The extrapiscine development of Sphaerospora renicola, a myxosporean parasite of the kidney of common carp, Cyprinus carpio L., was studied in the experimentally infected oligochaetes Tubifex tubifex (Müller) and Branchiura sowerbyi (Beddard). After the infection of these tubificids with homogenized common carp kidneys containing myxospores of S. renicola, the development of actinosporean stages was first observed under light microscopy 8 days after infection in pathogen-free T. tubifex. Infection of B. sowerbyi with mature actinosporean stages was first observed 91 days after infection. At that stage of development, pansporocysts containing neoactinospores filled the intestinal epithelium of the worm. Ninety-five days after infection, pansporocysts containing actinospores and free actinospores were found in the gut lumen of B. sowerbyi. Actinospores of S. renicola emerged from B. sowerbyi after 98 days of intraoligochaete development. These were floating in the water and showed the typical form of neoactinospores. The shape of the spores was triangular in apical view and elliptical in lateral view. The prevalence of infection reached 37%. Control specimens of B. sowerbyi proved to be free of neoactinospores. Except for a single specimen of B. sowerbyi, the only early developmental stages (pansporocysts) were found in T. tubifex.     

Morphological characterization and notes on the life cycle of a newly discovered variant of Loma salmonae (Putz, Hoffman & Dunbar) from a natural infection of chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Two variants of Loma salmonae occur in net-pen reared chinook salmon, Oncorhynchus tshawytscha. The typical variant (OA) has a host specificity for salmonids of the genus Oncorhynchus whereas the atypical variant (SV) has a host specificity for brook trout, Salvelinus fontinalis, and in this study, the ultrastructure of the two are compared. In fish at 8 weeks post-exposure xenomas of the SV variant have a very high proportion of mature spores compared with other developmental stages, while in xenomas of the OA variant there are fewer spores and many other developmental stages. Spores of the SV variant had up to 20 turns of their polar tube whereas those of the OA variant only had 17. Furthermore, the spores of the SV variant were significantly larger than those of the OA variant. The sporophorous vesicle for both variants appears to form around a sporogonial plasmodia, which results in many spores developing within the vesicle.     

The wheat germ agglutinin binding sites and development of the mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus)

By labelling with gold-colloid-conjugated wheat germ agglutinin (WGA), the WGA-binding sites were identified in the apical region of certain mitochondria-rich (MR) cells in gills of freshwater-adapted tilapia (Oreochromis mossambicus). Bromo-deoxyuridine (BrdU) injection was used to discern the differentiation of the WGA binding sites during development of MR cells. Double labeling of gill sections of fish 2, 4, 6, and 8 days after a single BrdU injection showed that the BrdU labeled MR cells 2 and 4 days after injection were mostly WGA negative, whereas 6 and 8 days after injection, WGA and BrdU-labeled MR cell increased gradually in number. Furthermore, the ratio of the WGA-positive to total MR-cells was higher in gills of tilapia adapted to low calcium ([Ca] = 0.015 mm) freshwater than in hard freshwater ([Ca] = 1.898 mm). The results indicate that WGA binding site may be a marker expressed late during differentiation of MR cells, and physiologically functional in assisting uptake of calcium when environmental calcium is low. According to their WGA binding and also characteristics their likely function in calcium absorption, the WGA positive MR cells shown in this study are considered to be similar to the cells described by Pisam et al. (1995).     

A comparative study of the myxosporeans Myxidium rhodei Léger, 1905 and Myxidium pfeifferi Auerbach, 1908 in roach, Rutilus rutilus L.

Abstract. The morphology and dimensions of Myxidium rhodei and M. pfeifferi were studied in roach originating from two different geographic locations (Greece and UK) using both scanning and light microscopy. The measurements and morphology of both mature spores and immature stages (trophozoites and spores) were studied and compared. No differences were observed in the morphology of mature or immature spores of both species when examined under light and SEM. Furthermore, no differences were found in the dimensions of the mature spores of both species in all fish. However, immature spores of both species were found to be smaller but wider than mature ones in all organs and in both habitats. Size differences between the two species were only seen in immature spores, M. rhodei immature spores being always larger than those of M. pfeifferi. When comparing the dimensions of the spores between the two habitats, these were larger and wider in the Greek fish than those in the British fish. The information in this study suggests that, in roach, M. pfeifferi is undistinguishable from M. rhodei and that similar studies should be carried out on the tench, Tinca tinca L., the type host of M. pfeifferi.     

Efficacy of different types of live and non-conventional diets in endangered clown knife fish Chitala chitala (Hamilton-Buchanan) during its early life stages

In the present study, the growth and survival of early life stages (ELS) of Chitala chitala were studied in nylon hapa for 28 days, followed by rearing in fibreglass reinforcement plastic (FRP) tanks for a period of 30 days. Ten‐day‐old ELS of C. chitala reared in hapa were fed with three different diets namely Indian Major Carp (IMC) spawn (<8 mm), live tubifex and fresh fish eggs. In the second phase, 28‐day‐old ELS were stocked in 200‐lit FRP tank and supplied four different live diets namely live tubifex worm, chironomous larvae, zooplanktons and mosquito larvae. Fish accepted all types of diets in the experimental rearing period in both the systems. The experiments conducted in hapa showed a higher specific growth rate (SGR), weight gain per cent and survival rate in larvae fed with live tubifex (SGR=1.76±0.02) than fish eggs (0.77±0.31) and IMC spawn (0.46±0.12). The study carried out in FRP tanks revealed that SGR was higher in ELS fed on chironomous larvae (4.44±0.61), followed by mosquito larvae (3.29±0.40) and live tubifex (3.28±0.36), whereas minimum SGR was recorded with zooplanktons (2.84±0.66). A significant difference (P<0.05) in SGR, final mean weight and weight gain (%) was also recorded. The highest mean survival rate (100%) of ELS in an FRP tank was observed in chironomous larvae and zooplanktons, whereas with live tubifex and mosquito larvae the same survival rate (80%) was recorded. The rate of survival of the ELS reared in hapa varied from 65% to 85%. The experiments showed that ELS of C. chitala could be reared successfully in hapas and fibreglass reinforcement tanks for attaining better survivability and growth.     

Development of Myxobolus bramae (Myxosporea: Myxobolidae) in an oligochaete alternate host, Tubifex tubifex

The development of Myxobolus bramae Reuss 1906, a myxosporean parasite of the gills of common bream Abramis brama L., was studied in experimentally infected oligochaetes. In five experiments, uninfected Tubifex tubifex (Müller) and Limnodrilus hoffmeisteri Claparéde were exposed to mature myxospores of M. bramae . In four experiments triactinomyxon type actinospores developed in Tubifex specimens but no infection was found in Limnodrilus . Actinospores were released from oligochaetes 70–81 days after initial exposure. At that time pansporocysts containing eight actinospores were located in the gut epithelium of experimental oligochaetes, but free actinosporean stages were also found in their gut lumen. Each actinospore had three pyriform polar capsules and a barrel-shaped sporoplasm with 32 secondary cells. The spore body joined the three caudal projections with a stout style. The total length of the actinospore was 139 μm on the average.     

The ecological impact of the Great Salinity Anomaly in the northern North-west Atlantic

In a number of recent papers Cushing has advocated that the Great Salinity Anomaly (GSA), which entered the northern North Atlantic in the early 1970s, adversely affected the recruitment of a number of deep-water fish stocks of this ocean. Cushing envisages that a temperature anomaly accompanying the GSA slowed and/or delayed growth in the spring phytoplankton bloom which sequentially affected zooplankton growth and fish recruitment. We test a number of hypotheses relating to Cushing's picture, focusing on the waters of the West Greenland, Labrador and Newfoundland Shelves. We find that, south of Greenland waters, there were no significant temperature anomalies corresponding to the GSA. In addition we show that stability of the shelf waters increased during the GSA, casting doubt on the contention that the phytoplankton bloom was delayed by retardation of the spring stabilization of the water column due to the influence of cold water. Our analysis indicates that the food chain coupling of environment to recruitment (climate to phytoplankton to zooplankton to fish) is not strong in the study region.     

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts,Anguilla anguilla and Cyprinus carpio

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named “rodlets.” These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i‐NOS), leu‐enkephalin, lysozyme, serotonin and tumour necrosis factor‐α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i‐NOS. The occurrence of the inflammatory peptides lysozyme and i‐NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.     

多子小瓜虫滋养体糖蛋白残基的种类和分布研究

应用伴刀豆凝集素A、麦胚凝集素、大豆凝集素和荆豆凝集素Ⅰ,检测寄生于金鱼皮肤、鳃和鳍上的多子小瓜虫滋养体糖蛋白残基的种类和分布。研究结果发现,伴刀豆凝集素A和麦胚凝集素免疫阳性染色在金鱼皮肤、鳃和鳍寄生的滋养体上均有分布,麦胚凝集素免疫阳性染色强于伴刀豆凝集素A,未见有大豆凝集素和荆豆凝集素Ⅰ免疫阳性染色。鳃上寄生的滋养体伴刀豆凝集素A免疫阳性染色最强,皮肤次之,鳍最弱。鳍上寄生的滋养体麦胚凝集素免疫阳性染色最强,皮肤次之,鳃最弱。研究结果表明,滋养体有单糖D-甘露糖和D-葡萄糖,以及氨基衍生物乙酰氨基葡萄糖。寄生于鳃上的滋养体D-甘露糖和D-葡萄糖可能较多,寄生在鳍上的滋养体乙酰氨基葡萄糖可能较多。     

Cell membrane glycoconjugates on virulent and avirulent strains of the haemoflagellate Cryptobia salmositica (Kinetoplastida)

The glycoconjugates on the cell membranes of Cryptobia salmositica (virulent and avirulent strains) were analysed using 13 highly purified lectins. The virulent strain of C. salmositica was agglutinated by two of these lectins (Concanavalin A (Con A) Canavalia ensiformis , specific for α-mannose and α- D -glucosyl residues; Pisum sativau agglutinin (PSA), specific for N-acetyl α- D -galactosaminyl) but the avirulent strain of C. salmositica was agglutinated by 11 lectins. No agglutination of C. salmositica (virulent and avirulent strains) was observed with lectin Tetragonolobus purpureas agglutinin (TPA, specific for α- L -fucose). Glycoprotein analysis with digoxigenin or biotin labelled lectins showed strain specific staining patterns for C. salmositica; with the avirulent strain showing stronger reactions than the virulent strain. These results indicate that the surface carbohydrate residues changed with attenuation of the pathogen and this change may be related to its loss of virulence.     

Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well-known genus

A new species of myxozoan, Kudoa trifolia sp. n., was found in various organs of the golden grey mullet, Liza aurata (Risso), and the thinlip mullet, L. ramada (Risso), from the western Mediterranean. Spores developed in subspherical plasmodia of 0.28-1 mm diameter within connective tissue, predominantly in the spleen, the outer wall of the gall bladder and the gut, the mesenteries and occasionally also in the gills. The spores of K. trifolia differ from the commonly known shape of Kudoa by considerable enlargement of one of the four valve cells, thus forming a 'spore body', which contains the major part of the binucleate sporoplasm. Scanning electron microscopy of the spores revealed the presence of grape-like appendages, which occur in bundles terminally on the valve cells. Phylogenetic analysis based on the 18S rDNA sequence of K. trifolia showed that this species is deeply embedded in the genus Kudoa despite its aberrant morphology and host tissue location. This suggests important amendments to the morphological diagnosis of the genus Kudoa.