Tetracapsula renicola n. sp. (Myxozoa:Saccosporidae); The PKX Myxozoan: The Cause of Proliferative Kidney Disease of Salmonid Fishes

191

124

The importance of tissue-specificity was studied in Myxobolus spp. infecting the gills of 7 cyprinid species. The 18S rDNA of 10 Myxobolus species was amplified by optimised nested-PCR, resulting in ~1600 bp PCR products. Phylogenetic trees generated by distance matrix and parsimony analyses revealed 4 main groups. Muscle-infecting species all belonged to the same group, while members of 2 gill-infecting groups were clearly distinguishable on the basis of tissue-specificity, and were also recognisable by differences in spore morphology. On the basis of tissue tropism, phylogenetic relationships among the species examined indicate that genetic separation is a more ancient evolutionary feature than host-specificity.KEY WORDS: Myxobolus · Myxosporeans · Phylogeny · 18S rDNA · Tissue tropism · Cyprinids · Gills Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: 35-40, 2004 (2002). Myxobolus species parasitising the gills were also differentiated using a combined polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method by Xiao & Desser (2000) and Eszterbauer et al. (2001). Using this method, Eszterbauer (2002) confirmed that the morphologically indistinguishable M. elegans from orfe and M. hungaricus from common bream are indeed valid species. Furthermore, Eszterbauer et al. (2002) proved that M. pavlovskii is also present in bighead and silver carp.The present paper reports the results of phylogenetic analyses of 10 Myxobolus species infecting the gills of cyprinids in order to determine their genetic relationship and examine the importance of tissuespecificity in the fish hosts. MATERIALS AND METHODS Collection of spores. Myxospores of 10Myxobolus species were collected from 7 fish species in Hungarian natural lakes or rivers and fish farms between 1999 and 2002 (Table 1). After collection and transportation to the laboratory, the 2 to 4 yr old fish were sacrificed using an overdose of the anaesthetic MS 222, followed by severance of the spinal cord.For studying the occurrence of plasmodia in the gills, samples were examined with a stereomicroscope at 10 × magnification. Spores isolated from the plasmodia were carefully examined under coverslip at 100 × magnification with a compound microscope to identify the parasites. Digital pictures of the spore samples were also taken.In most cases, we freed the plasmodia from the host cells or from the capsule formed by the connective tissue of the host under a stereomicroscope, and froze them at -20°C in 1.5 ml centrifuge tubes until further use. The samples contained different numbers of spores (10 2 to 10 6 ). At least 2 different samples of myxospores collected from different fish specimens at different times were used for molecular biological examinations. In the case of Myxobolus bramae, 4 samples of spores were collected from different fish specimens at different times.DNA extraction. After defrosting the spores, samples were centrifuged at low speed (1000 × g). The spores were suspen...

Section: Introductionsupporting

confidence: 49%

Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity

Eszterbauer¹

2004

191

124

“…Concerning the molecular based division of my xosporeans into the major freshwater and marine clades (Kent et al 2000, Fiala 2006, Bartošová et al 2009, Holzer et al 2010, we note that the clade comprising Zschokkella auratis sp. nov. constitutes yet another exception to this main division.…”

Section: Discussionmentioning

confidence: 99%

“…A 17th SSU rDNA sequence ter med Z. mugilis Sitjà-Bobadilla & Al va rez-Pellitero, 1993 remains in GenBank; however, this species was recently revised to Ellipsomyxa mugilis (Køie & Karlsbakk 2009), having also been linked to its correspondent actinosporean stage developing in an estuarine polychaete (Rangel et al 2009(Rangel et al , 2012. Overall, the molecular information regarding Myxo sporea has revealed the existence of 2 main clades dividing freshwater and marine species (Kent et al 2000, Fiala 2006, Bartošová et al 2009, Holzer et al 2010, for which myxosporean parasites infecting anadromous hosts, as well as some species of Henneguya, Chloromyxum, Ceratomyxa, Myxo bolus, Myxidium, Parvicapsula, and Sphaero myxa, constitute exceptions (Diamant et al 2004, Fiala 2006, Rocha et al 2013). Phylogenetic analyses have also shown that most myxosporeans cluster according to their host tissue tropism rather than by spore morphology or host species (Eszterbauer 2004, Holzer et al 2004, Fiala 2006, Bartošová et al 2009, Fiala & Bartošová 2010, and the genus Zschokkella is no ex ception (Holzer et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Ultrastructural and phylogenetic description of Zschokkella auratis sp. nov. (Myxozoa), a parasite of the gilthead seabream Sparus aurata

Rocha¹,

Casal²,

Rangel³

et al. 2013

A new myxosporean, Zschokkella auratis sp. nov., infecting the gall bladder of the gilthead seabream Sparus aurata in a southern Portuguese fish farm, is described using microscopic and molecular procedures. Plasmodia and mature spores were observed floating free in the bile. Plasmodia, containing immature and mature spores, were characterized by the formation of branched glycostyles, apparently due to the release of segregated material contained within numerous cytoplasmic vesicles. Mature spores were ellipsoidal in sutural view and slightly semicircular in valvular view, with rounded ends, measuring 9.5 ± 0.3 SD (8.7−10.3) µm in length and 7.1 ± 0.4 (6.5−8.0) µm in width/thickness. The spore wall was composed of 2 symmetrical valves united along a slightly curved suture line, each displaying 10 to 11 elevated surface ridges. Two equal subspherical polar capsules, 3.7 ± 0.3 (3.0−4.1) µm long and 3.0 ± 0.2 (2.6−3.2) µm wide, were located separately at the spore's extremities. Each polar capsule contained a polar filament forming 4 to 5 coils. The sporoplasm was binucleate and contained numerous sporoplasmosomes. Morphological data, tissue tropism, and molecular analysis of the small subunit rDNA gene identified this parasite as a new species of Zschokkella. Maximum parsimony, neighbor-joining, and maximum likelihood inferences clustered the parasite in a subclade containing other Zschokkella species parasitizing the gall bladder of brackish and marine fish hosts, located within the coelozoic clade of the major freshwater clade; this supports the existence of a marine subclade within the 'freshwater' clade, as well as the existence of a correlation between tissue tropism and myxosporean phylogeny.

“…AF201375, Kent et al 2000), and in combination with a general ssu PCR primer, Ecf, they were used to pick out the first part of the ssu from P. pseudobranchicola (Table 2). In addition, PCR primers and diagnostic primers for Parvicapsula spp.…”

Section: Methodsmentioning

confidence: 99%

Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon Salmo salar: tissue distribution, diagnosis and phylogeny

Nylund¹,

Karlsbakk²,

Sæther³

et al. 2005

Parvicapsula pseudobranchicola infections in farmed Atlantic salmon in Norway are associated with low-grade to significant mortalities. The parasite is found as mature spores in pseudobranchs, but has also been detected in the gills, liver and kidney. Diagnosis has relied on the detection of Parvicapsula spores, with the pseudobranch being the preferred organ. A better understanding of the epizootiology of this myxosporean is a prerequisite for appropriate management and control. Hence, early detection of infections and life cycle studies are needed. We sequenced the small subunit (ssu) rDNA (18S) from P. pseudobranchicola and developed a sensitive diagnostic PCR protocol. This allowed us to (1) identify appropriate tissues for diagnostic assays, (2) examine the intraspecific variation in ssu rDNA in the parasite's Norwegian range, (3) examine annelid potential primary hosts and (4) obtain additional ssu rDNA sequences of marine Parvicapsula species to perform a phylogenetic study. Primers were constructed targeting the ssu rDNA from P. minibicornis. With these we obtained a partial ssu sequence of the P. pseudobranchicola type isolate. A new set of primers (PCF3/PCR3) was constructed for diagnostic purposes. These were tested against DNA from the host and several myxozoan species infecting Norwegian salmon. The primers give a positive product of 203 bp and pick out P. pseudobranchicola in salmonids. They also amplify the congeners P. unicornis and P. asymmetrica infecting unrelated fish. The PCR protocol developed showed a greater sensitivity than light microscopy. The pseudobranchs were always positive and are the recommended organ for PCR diagnostics. There was no sequence variation between geographic isolates from farmed salmon. Preliminary examinations of marine polychaetes and oligochaetes collected from farm sites with parvicapsulose problems were negative. A comparison of the sequence of the ssu rDNA from P. pseudobranchicola with that of other myxozoans shows that it groups closely together with P. unicornis and P. asymmetrica. The closest relative to this group is P. minibicornis.