Yanhua Zhai

Morphological and molecular characterization of Thelohanellus macrovacuolaris n. sp. (Myxosporea: Bivalvulida) infecting the palate in the mouth of common carp Cyprinus carpio L. in China

Thelohanellus macrovacuolaris n. sp. is described during a survey on myxozoan diversity of common carp Cyprinus carpio L. in China. It is characterized by the presence of round or ellipsoidal plasmodium in the palate in the mouth of host. Mature spores were pyriform in frontal view, lemon shaped in lateral view, measuring 21.6±0.9 (19.3-23.8) long, 12.5±0.7 (10.3-13.6) wide, and 10.2±0.4 (9.8-11.8) thick. Most spores were surrounded by the membrane sheath. Single polar capsule was round with an apophysis at its top end presented close to apex of spore, measuring 9.1±0.6 (8.0-10.0) in length, 8.6±0.5 (7.8-9.6) in width. Polar filaments coiled, with 7 to 8 turns. A large, round iodinophilous vacuole was present, with 5.8-7.5 in diameter. The present species is morphologically distinct from all other Thelohanellus species. The BLAST search indicated that the newly obtained small subunit ribosomal RNA (ssrRNA) gene sequence of T. macrovacuolaris n. sp. did not match any available sequences in GenBank. Phylogenetically, T. macrovacuolaris n. sp. was placed sister to Thelohanellus wangi in the Thelohanellus clade. Both morphology and ssrRNA gene sequence data revealed that the present parasite is a new species of genus Thelohanellus.

New type of pathogenicity of Thelohanellus kitauei Egusa & Nakajima, 1981 infecting the skin of common carp Cyprinus carpio L.

Thelohanellus kitauei Egusa & Nakajima, 1981 is a common parasite infecting the intestine of common carp Cyprinus carpio L., resulting in mass mortality or loss of economic value of cultured carp. In the present study, T. kitauei infecting host skin was detected. The morphological, molecular and histological data of this parasite in the new organ record are presented. Morphological analysis showed the current specimen morphologically similar to T. kitauei from the intestine. Despite the spore length and polar capsule length of the current specimen larger than those of T. kitauei from the intestine, ranges of dimensions overlap, which is more suggestive of intraspecific variation than distinct species. BLAST search revealed that the present small subunit ribosomal DNA gene sequence is identical to those of T. kitauei. Histologically, most of spores distributed in the stratum spongiosum of dermis, and some spores in the strata compactum of host skin were also observed. Above all, both morphology and molecular analysis indicated that the current species from the skin of common carp is conspecific with T. kitauei from the intestine of carp and organ habitats transfer of T. kitauei from host intestine to skin may have occurred.

First record of Chilodonella piscicola (Ciliophora: Chilodonellidae) from two endangered fishes, Schizothorax o ’ connori and Oxygymnocypris stewartii in Tibet

Schizothorax o’connori and Oxygymnocypris stewartii are two endangered endemic Tibetan fishes that thrive in the Lhasa River at an average altitude over 4000xa0m. During artificial reproduction of endemic Tibetan fishes, the juvenile fish of S. o’connori and O. stewartii experienced mass mortality event. The causative agent is diagnosed to be a ciliate parasite, Chilodonella piscicola (syn. C. cyprini), which is common in various fishes. Here, we supplemented its description based on the morphological and molecular data. The body of C. piscicola is oval, 30–60u2009×u200925–40xa0μm in vivo. Cyrtos is hook-like, composed of 9–10 toothed nematodesmal rods. Somatic kineties usually contain seven right kineties and nine left kineties. Two parallel circumoral kineties revolve round the cyrtos, and one preoral kinety extends to the anterior end of the fourth left-most kinety. Terminal fragment kinety is linear and on the top left of dorsal side. Sequence alignments revealed that the present SSU rDNA and ITS1-5.8S-ITS2 sequences are both most similar to the sequences of C. uncinata with the similarities of 98.2 and 99.5xa0%. The phylogenetic analyses showed that C. piscicola is sister to other Chilodonella species, whereas C. cyprini (FJ873805) cluster with Tetrahymena species. Molecular analysis shows that the ITS1-5.8S-ITS2 sequence of C. cyprini in GenBank is unreliable. Our study extended the host range of C. piscicola and supplemented and revised the molecular data. Besides, as far as we know, this is the first record of C. piscicola in Tibetan plateau.

Myxobolus pseudowulii sp. n. (Myxozoa: Myxosporea), a new skin parasite of yellow catfish Tachysurus fulvidraco (Richardson) and redescription of Myxobolus voremkhai (Akhmerov, 1960)

Two species of Myxobolus Bütschli, 1882 were found in yellow catfish Tachysurus fulvidraco (Richardson). A species of Myxobolus infecting the gills was morphologically identified as Myxobolus voremkhai (Akhmerov, 1960) and it was characterised here with additional morphological and molecular data. The other species of Myxobolus infecting the hosts skin did not conform to any known myxosporean species. It is characterised by the presence of round, black or milky white plasmodia with black spots. Myxospores are pyriform in frontal view and lemon-shaped in lateral view, measuring 12.9-16.2 μm (14.6 ± 0.7 μm) in length, 8.1-10.8 μm (9.4 ± 0.5 μm) in width, and 6.1-8.1 μm (7.0 ± 0.4 μm) in thickness. Two ampullaceous polar capsules are slightly unequal in size, larger polar capsule 7.2-9.5 μm (7.9 ± 0.4 μm) long by 3.0-3.9 μm (3.5 ± 0.2 μm) wide, smaller capsule 6.9-8.0 μm (7.4 ± 0.3 μm) long by 2.9-3.9 μm (3.4 ± 0.2 μm) wide. Polar filaments are coiled with seven to nine turns. Histologically, the plasmodia develop in the stratum spongiosum of skin dermis, resulting in epithelial cell shedding and immunological cell infiltration. Given the morphological and molecular differences between this species and other species of Myxobolus, we proposed the name of Myxobolus pseudowulii sp. n. for this parasite from the skin of yellow catfish. Interestingly, some spores of the new species possess Henneguya-like caudal appendages. Phylogenetically, M. pseudowulii sp. n. and M. voremkhai infecting yellow catfish group together in one clade with other parasites of Siluriformes, indicating that parasites clustering according to the fish host order may be an important factor affecting the evolution of species within the Myxobolus clade.

Intraspecific morphometric variation in myxosporeans.

Morphometric data from spores of ten myxosporean species were statistically analysed to explore myxosporean intraspecific variation in measurements when obtained from a sample from: (1) the same plasmodium, (2) different plasmodia from the same host and (3) different host individuals and localities. In some cases, significant differences in spore dimensions were found between samples from the same plasmodium, highlighting the difficulty of obtaining representative measurements of myxosporean spore. In addition, significant differences in spore dimensions were found when plasmodia from the same site of infection were compared, suggesting that measurements of spores should come from several different plasmodia of the sampling to increase the reliability of the morphology data. Moreover, significant differences in spore dimensions were observed for most spore dimensions when data were compared between localities. In all cases, there was clear overlap in ranges of dimensions even when means differed significantly. The present statistical analysis shows that intraspecific morphometric variation of myxosporean species commonly occurs, highlighting the importance of reporting ranges of measurements for a species, not just the mean dimensions, and taking into account all evidence when assigning or describing myxosporean species.

Supplementary studies on Myxobolus tsangwuensis Chen, 1954 (Myxozoa: Myxosporea) infecting the gills of common carp Cyprinus carpio (L.): molecular and histological data

Myxobolus tsangwuensis Chen, 1954 is a common parasite infecting the gills of common carp Cyprinus carpio (L.). It was described simply in the original description and its molecular data was absent, which makes the accurate diagnosis challenging. Here we supplemented its description based on the morphological, histological and molecular data. It was characterized by the presence of small round or ellipsoidal plasmodia in the gills of host and histology showed the plasmodia developed in the capillary network of the gill lamella. Mature spores of M. tsangwuensis were ellipsodal in frontal view and lemon shaped in lateral view, averaging 11.2 ± 0.7 (10.2–12.5) μm × 9.3 ± 0.3 (8.5–10.0) μm × 6.2 ± 0.4 (5.5–7.0) μm. Spores valves were symmetrical and smooth. Occasionally, a small intercapsular appendix was observed. Two polar capsules were pyriform with different sizes, measuring 5.0 ± 0.2 (4.4–5.3) μm × 3.1 ± 0.1 (2.9–3.5) μm and 3.9 ± 0.2 (3.4–4.3) μm × 2.5 ± 0.2 (2.0–2.8) μm, respectively. Polar filaments coiled five to seven turns in large polar capsule and three to four turns in the small polar capsule. Some spores were surrounded by the mucous envelope that was not recorded in the original description. Molecular analysis revealed that the present SSU rDNA sequences did not match any available sequences in GenBank and phylogenetic analysis showed M. tsangwuensis was sister to M. basilamellaris and M. musseliusae.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048xa0bp) and Aurantiactinomyxon type 2 (2031xa0bp) share 99.2–99.4xa0%, 99.8–100xa0% similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4xa0% similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

Histopathological and ultrastructural studies of Myxobolus turpisrotundus from allogynogenetic gibel carp Carassius auratus gibelio in China

During an ongoing systematic survey on species diversity of myxozoans parasitising allogynogenetic gibel carp Carassius auratus gibelio (Bloch) in China, plasmodia were detected in the fins, lip, jaw, gill chamber, gill arches, operculum and oral cavity of infected fish. Combining the morphological and molecular data, the present species was identified as Myxobolus turpisrotundus Zhang, Wang, Li et Gong, 2010. Histopathological examination revealed that despite infecting different organs, M. turpisrotundus always occurred in dermis, demonstrating its affinity to this tissue. Histopathological effect of M. turpisrotundus on the host is relatively mild except parasites in the gill arches producing compression of the adipose tissue and heavy adductor muscles deformation with lymphohistiocytic infiltrates. In addition, the plasmodia in different sites were with the same complex structure arrangement: cup-like cells with unknown derivation, a thin collagenous fibril layer, areolar connective tissue, basement membrane and host epithelial cell. Ultrastructural analysis showed that the parasite has monosporic pansporoblast and sporogenesis followed the usual pattern of most of the myxosporeans.

Development of monoclonal antibodies against polar filaments and spore valves of Myxobolus honghuensis (Myxosporea: Bivalvulida).

Myxobolus honghuensis infects the pharynx of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) and can cause high mortality. Only morphology-based diagnostic methods are currently available for clinical samples, but these methods are laborious and have low efficiency of detection. To overcome this problem, we designed a more sensitive diagnostic method. Two monoclonal antibodies (MAbs 1C7 and 3B7) were prepared by immunizing mice with soluble protein from sonicated M. honghuensis spores. Immunofluorescence analysis revealed that MAb 1C7 specifically reacts with polar filaments from spores, whereas MAb 3B7 identified protein localized on the spore valves. The isotypes of MAb 1C7 and MAb 3B7 were IgM and IgG1, respectively. Results of Western blot analysis revealed that MAb 1C7 recognized 2 prominent protein bands with molecular weights of 130 and 180 kDa, while MAb 3B7 recognized a protein band of 28 kDa. Thus, in this study we have developed 2 MAbs that have the potential for efficient detection of M. honghuensis. Moreover, identification of MAb 1C7 and MAb 3B7 allows for further studies of the functions and biochemical composition of polar filament and spore surface antigens.

Morphological, histological and molecular characterization of Myxobolus kingchowensis and Thelohanellus cf. sinensis infecting gibel carp Carassius auratus gibelio (Bloch, 1782).

A Myxobolus species and a Thelohanellus species infecting Carassius auratus gibelio (Bloch, 1782) were redescribed by their morphological, histological and molecular characterization. In the present study, the Myxobolus species infecting the muscle was identified as Myxobolus kingchowensis Chen et Ma, 1998 by the morphological and molecular data. Histologically, mature spores of M. kingchowensis were observed in the intercellular and connective tissue of muscle, though the plasmodia were not found. In addition, scattered spores also occurred in the intercellular of haematopoietic cells, intraepithelial of the renal tubules and interior of the melano-macrophage centres. Phylogenetic analysis showed that M. kingchowensis clustered in the clade of muscle-infecting Myxobolus species, further supporting muscle as the infection site of M. kingchowensis. The present Thelohanellus species infecting the gills was identified conspecific as Thelohanellus sinensis reported in Sun (2006) (mark it as T. sinensis-Sun) based on spore morphology, biological traits (host specificity and organ specificity), and molecular data. However, compared with the original description of T. sinensis Chen et Hsieh, 1960, the present Thelohanellus species and T. sinensis-Sun both infecting the gills of gibel carp are distinguishable from the original description in the host and infection site, which made the validity of T. sinensis-Sun dubious. Due to the absence of molecular data in the original description of T. sinensis, we suggest marking the present species and T. sinensis-Sun as T. cf. sinensis to avoid the confusion until T. sinensis is obtained from the type host and type infection site.