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Molecular identification and transmission studies of X-cell parasites from Atlantic cod Gadus morhua (Gadiformes: Gadidae) and the northern black flounder Pseudopleuronectes obscurus (Pleuronectiformes: Pleuronectidae).

Freeman MA, Eydal M, Yoshimizu M, Watanabe K, Shinn AP, Miura K, Ogawa K - Parasit Vectors (2011)

Bottom Line: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites.Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum.Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Ocean and Earth Sciences & Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia. mark@um.edu.my

ABSTRACT

Background: Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata.Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan.

Results: Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body.

Conclusions: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

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Epidermal X-cell pseudotumours on formalin-fixed Pseudopleuronectes obscurus from Hokkaido, Japan. a) A large central dorsal pseudotumour, seen in cross section (b). c) Pseudotumours can extend from the dorsal surface to the ventral surface and remain pigmented. d) Juvenile fish are also infected and ventral pseudotumours can also be unpigmented. Scale bars a, c & d = 3 cm, b = 10 mm.
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Figure 2: Epidermal X-cell pseudotumours on formalin-fixed Pseudopleuronectes obscurus from Hokkaido, Japan. a) A large central dorsal pseudotumour, seen in cross section (b). c) Pseudotumours can extend from the dorsal surface to the ventral surface and remain pigmented. d) Juvenile fish are also infected and ventral pseudotumours can also be unpigmented. Scale bars a, c & d = 3 cm, b = 10 mm.

Mentions: Pseudopleuronectes obscurus with epithelial pseudotumours caused by X-cell infection were caught in Notsuke Bay, Hokkaido (Figure 2a-d). Epithelial pseudotumours were typical of those described from numerous flatfish species from Hokkaido and were often large and significantly raised from the body surface, but did not penetrate the underlying musculature (Figure 2a &2b), making them easy to remove from the fish to prepare the homogenate for the transmission experiment. Pseudotumours were always pigmented on the ocular (eyed) side, but could be either pigmented or non-pigmented on the abocular (non-eyed) side (Figure 2c &2d) and when sampled, juvenile fish were also sometimes infected (Figure 2d). Pseudotumours often extended to the edge of the fins (Figure 2c & Figure 3a). Here, and at other margins of the pseudotumour, sac-like structures were observed that could be detached, intact, with mild pressure (Figure 3b). The sacs were relatively fragile, easy to rupture and contained X-cells and other cellular debris (Figure 3c). X-cells from ruptured sacs that had been maintained in seawater for 24 hrs showed no signs of adhering to the plastic tissue culture plates, had no pseudopodia and were not amoeboid in form. After 24 hrs in seawater they appeared similar to ones that were freshly removed from sacs and had not suffered any noticeable shrinkage (Figure 3d).


Molecular identification and transmission studies of X-cell parasites from Atlantic cod Gadus morhua (Gadiformes: Gadidae) and the northern black flounder Pseudopleuronectes obscurus (Pleuronectiformes: Pleuronectidae).

Freeman MA, Eydal M, Yoshimizu M, Watanabe K, Shinn AP, Miura K, Ogawa K - Parasit Vectors (2011)

Epidermal X-cell pseudotumours on formalin-fixed Pseudopleuronectes obscurus from Hokkaido, Japan. a) A large central dorsal pseudotumour, seen in cross section (b). c) Pseudotumours can extend from the dorsal surface to the ventral surface and remain pigmented. d) Juvenile fish are also infected and ventral pseudotumours can also be unpigmented. Scale bars a, c & d = 3 cm, b = 10 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045979&req=5

Figure 2: Epidermal X-cell pseudotumours on formalin-fixed Pseudopleuronectes obscurus from Hokkaido, Japan. a) A large central dorsal pseudotumour, seen in cross section (b). c) Pseudotumours can extend from the dorsal surface to the ventral surface and remain pigmented. d) Juvenile fish are also infected and ventral pseudotumours can also be unpigmented. Scale bars a, c & d = 3 cm, b = 10 mm.
Mentions: Pseudopleuronectes obscurus with epithelial pseudotumours caused by X-cell infection were caught in Notsuke Bay, Hokkaido (Figure 2a-d). Epithelial pseudotumours were typical of those described from numerous flatfish species from Hokkaido and were often large and significantly raised from the body surface, but did not penetrate the underlying musculature (Figure 2a &2b), making them easy to remove from the fish to prepare the homogenate for the transmission experiment. Pseudotumours were always pigmented on the ocular (eyed) side, but could be either pigmented or non-pigmented on the abocular (non-eyed) side (Figure 2c &2d) and when sampled, juvenile fish were also sometimes infected (Figure 2d). Pseudotumours often extended to the edge of the fins (Figure 2c & Figure 3a). Here, and at other margins of the pseudotumour, sac-like structures were observed that could be detached, intact, with mild pressure (Figure 3b). The sacs were relatively fragile, easy to rupture and contained X-cells and other cellular debris (Figure 3c). X-cells from ruptured sacs that had been maintained in seawater for 24 hrs showed no signs of adhering to the plastic tissue culture plates, had no pseudopodia and were not amoeboid in form. After 24 hrs in seawater they appeared similar to ones that were freshly removed from sacs and had not suffered any noticeable shrinkage (Figure 3d).

Bottom Line: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites.Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum.Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Ocean and Earth Sciences & Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia. mark@um.edu.my

ABSTRACT

Background: Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata.Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan.

Results: Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body.

Conclusions: The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

Show MeSH
Related in: MedlinePlus