Phylogenetic analyses of the myxosporeans parasitic in freshwater fish of China based on 18S ribosomal DNA

Partial 18S rDNA sequences of five myxosporean species, Myxobolus rotundus, M. rotundatus, Thelohanellus wuhanensis, Henneguya weishanensis and M. cubanicum were amplified utilizing two common primers myxoF (5'-CGCGGTAATTCCAGCTCCAGTAG-3') and myxoR (5'-ACCAGGTAAGTTTTCCCGTGTTGA-3') and stored in the GenBank with the access number AY165179-AY165183. By combining the 18S rDNA sequences of other 13 species available in the GenBank a parsimonious tree of 18 species was built with Polypodium hydriforme used as the outgroup. Results show Tetracapsula bryozoides and "PKX" organism lie on the root within the Myxozoa before the divergence of other major groups which form two clades as T. wuhanensis-T. hovorkai-M. rotundatus-M. rotundus-M. bibullatus-M. pellicides-M. pendula-H. salminicola and H. exilis-H. ictaluri-M. spinacurvatura-M. osburni-H. lesteri-H. weishanensis-M. kubanicum. Among the genus myxobolus, 3 species found in China did not form a Glade but grouped with the European species, indicating that the geographic divergence of Myxobolus may not be distinct. Neither Myxobolus nor Henneguya was monophyletic and these two genera can not be distinguished using the molecular data. It is further proposed that the caudal protrusion of Henneguya may not be a valid taxonomic character but an appending structure homologous with the "V" projections in Myxobolus

Population distribution and seasonal alternation of two species of monogeneans on the gills of Hypophthalmichthys molitrix and Ctenopharyngodon idellus

The population distribution and seasonal alternation of Dactylogyrus hypophthalmichthys on gills of silver carp Hypophthalmichthys molitrix, and D. lamellatus on grass carp Ctemopharyngodon idellus were studied. Two species of fish were collected in a fish pond in the Institute of Hydrobiology, Chinese Academy of Sciences in Wuhan, China from December 1995 to November 1996. The population dynamics of abundance and prevalence were similar for the two species of monogeneans. The prevalence was higher than 70% for D. hypophthalmichthys and higher than 60% for D. lamellatus, and no significant difference ( G = ?, P 0.05) was found between months for the two species of monogeneans. The abundance of D. hypophthalmichthys increased gradually from February to March, and reached the highest level in April, then declined gradually and remained at a low level in autumn and winter; while D. lamellatus increased from November to March, and reached the highest value in April, then decreased rapidly and maintained a low level in summer and autumn. Statistical analysis indicated that changes in abundance of D. hypophthalmichthys and D. lamellatus were both significantly seasonal (F = ?, P 0.05). The pattern of distribution of D. hypophthalmichthys was over-dispersed, as the variance to mean ratio was more than 1 and the degree of aggregation was high in spring and autumn. The pattern of D. lamellatus was also over-dispersed, with its the degree of aggregation being high in spring and winter

Intestinal pathology of the mandarin fish Siniperca chuatsi infected naturally with the acanthocephalan Hebesomea violentum

Siniperca chuatsi carrying heavy infections of Hebesomea Violentum in the gut were obtained from Liangzi Lake, Hubei Province. For light microscopical examination, the gut were fixed overnight in MFAA (Mathanol : Formalin: Acetic acid = 85: 10: 5). After embedding in Histowax, the tissues were serially sectioned at Stint and stained with May-Grunwald Giemsa using standard techniques. For transmission electronic microscopical examination, small pieces of gut with attached parasites were immersed in ice-cooled fixative and minced in approximately 2mm(3) pieces and then they were fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS; 0.1 M; pH 7.2) for 4 hours at 4 degrees C and postfixed for 1h in 1% osmium tetroxide in the same buffer at 4 degrees C, and embedded in Epson-812. Semithin sections were stained with 1% aqueous solution of toluidine blue to select the most suitable areas. Ultrathin sections were double-stained with uranil acetate and lead citrate, and observed in a JEOL JEM-1230 electron microscope at 80kV. The parasite-induced histopathology was related to the penetration of H. violentum proboscis in the intestine wall. The host responded to the parasites with connective tissue hyperplasia and with an infiltration of leukocytes, predominantly eosinophilic granulocytes. Eosinophils were accumulated in the epithelium and lamina propria of the infected gut and some adhere to the parasite tegument and proboscis attachment. These eosinophils were subdivided into immature eosinophils, mature eosinophils and degranulating eosinophils. Fibre cells, fibroblast cells, and eosinophils were orderly distributed at the interface of the parasite-host. In addition, H. violentum elicits the formation of nodule in tunica propria. These nodules comprised a tri-layered structure to wrap the H. violentum. Tissue cava infiltrated by host cells were observed in the lamina propria and they are presumed to be the degenerated nodules

A new parasitic copepod, Ergasilus danjiangensis sp. nov. (Poecelostomatoida, Ergasilidae) on gills of two cyprinid fish Opsariichthys bidens and Zacco platypus

A new parasitic copepod, Ergasilus danjiangensis sp. nov. was found on gills of two cyprinid fish Opsariichthys bidens and Zacco platypus in Danjiangkou Reservoir of Hubei Province, China. Morphological examinations revealed that E. danjiangensis resembled the Ergasilus species in many characters, especially Ergasilus briam and Ergasilus tumidus, while E - danjiangensis can be distinguished from other Ergasilus species by the antenna morphology: a slippery inner edge and a cuniform-like second segment with a prominent arc outer edge. Based on the internal transcribed spacer (ITS) rDNA sequences, phylogenetic study revealed that E. danjiangensis was much more closely related to E. briani than to E. tumidus. The morphological and phylogenetic analyses therefore suggested that E. danjiangensis was an independent and new species in the genus Ergasilus

Ontogenetic development of the pronephros and the distribution of B-lymphocytes in adult pronephros of the mandarin fish Siniperca chuatsi

Ontogenetic development of the pronephros was examined in the mandarin fish Siniperca chuatsi, collected from the first to the 35th day post hatching (dph). Renal tubules and undifferentiated haemopoietic cells were observed on the 1st dph. The development of the pronephros was subjectively divided into three stages according to changes in its composition. At the first stage ( from the 1st to 7 dph), the pronephros mainly consisted of renal tubules and some haemopoietic cells; at the second stage (from the 7th to 35th dph) with the composition of lymphocytes and renal tubules. Afterwards, the number of renal tubules decreased and then disappeared, and the number of lymphocytes increased and adrenal gland was found surrounding the blood vessels. In situ PCR technique was successfully used to detect the distribution of B-lymphocytes expressing IgM mRNA in the pronephros of adult mandarin fish. It is shown that lymphocytes are distributed in the whole pronephros, with some of them located around the blood vessel

Ultrastructural examination on the tegument of procercoid of Bothriocephalus acheilognathi

The tegument of the procercoid of Bothriocephalus acheilognathi was studied by transmission electron microscopy (TEM) The tegument of the procercoid consists of an outer syncytial layer with underlaying perikarya. The outer portion of the tegument is composed of a dense layer of microtriches and some tumuli. The microtriches are longer and thicker than those on the adult worm, which are monomorphic and might be stripped off during their establishment in definite hosts and then re-occur on the tegument surface of adult worms. The tumuli are spaced on the anterior part of the procercoid, containing electron-dense inclusions. Three types of secretion processes are observed in the tegumental cytoplasm, i. e., apocrine, eccrine and microapocrine processes. The ducts of the penetration gland open at the hook region at the posterior end of the procercoid. Muscle bundles can be seen within the perinuclear region and just below the fibrous layer. The function of tumuli of procercoid also is discussed in this paper

Endohelminths and their community characteristics in fish of the Lhasa River in Tibet of China

Endohelminths were investigated in seven species of fish, including six in the Schizothoracinae and one in the Coitidae in Lhasa River in Tibet of China in July 2004. A total of 9 species of endohelminths were recorded in addition to digeneans in the Allocreadium, including 3 species of acanthocephalans, i. e. Neoechinorhynchus qinghaiensis, Echinorhynchus gymnocyprii and the unidentified species Neoechinorhynchus sp. 3 species of nematodes, i. e. Rhabdochona hellichi, Contracaccum sp., Eustrongylides sp., 3 species of cestodes, i. e. Breviscolex orientalis, Ligula sp., Diphyllobothrium sp. Morphological characters of N. qinghaiensis, R. hellichi, B. orientalis, digeneans in the Allocreadium spp. and the unidentified acanthocephalans Neoechinorhyachus sp. were described. Specimens of Allocreadium could not be identified to species due to their morphological variations in different fish hosts of Schizothoracinae. Other helminths which use fish as intermediate hosts were also unable to be identified to species level. Since N. qinghaiensis and Allocreadium. spp. were reported in other species of fish in the Schizothoracinae, findings of the acanthocephalan and digencans in Lhasa River may Suggest that they be specialists in fish of the Schizothoracinae. 4 to 6! helminth species were found in the fish species examined in the Lhasa River, indicating a high species richness, with the exception of no single endohelminth parasite detected in Schizothorax oconnori. The Shannon-Wiener index for 5 species in the Schizothoracinae ( 0. 613 1 - 1.543 4) were much higher than in Triplophysa stenura (0.291 1. Similarities among the 5 species were relatively high (0. 173 2 +/- 0.230 3) - (0.375 1 +/- 0.275 8) whereas similarities between the Schizothoracinae and T. stenura were rather low (0.028 8 +/- 0.087 7) (0. 091 6 +/- 0. 161 8). In the present study, 4 allogenie parasite species which mature in final bird hosts were recorded,which may at least to some extent reflects the fauna composition in this high land area of Tibet

EFFECT OF FORMALIN INACTIVATED FLAVOBACTERIUM COLUMNARE ON EXPRESSION OF IMMUNE RELATED GENES IN GRASS CARP, CTENOPHARYNGODON IDELLUS

Immune responses of fish are normally regulated by various cytokines and their encoding genes, which are generally called immune related genes. Expressions of different immune related genes of fish were previously proved to be effected by vaccination with various pathogens,including bacteria viruses, parasite, and so on. In the present study,the expressions of six immune related genes in liver,spleen and head kidney of grass carp, i. e. C-reactive protein (CRP) major histocompability complex I (MHC I), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta) interleukin-8 (IL-8) and interferon I (IFN I) as well, were quantified using quantitative real-time polymerase chain reaction at 1, 7, 15 and 28 days post. intraperitoneal injection (dpi) with formalin killed Flavobacterium columnare G(4) (FKG(4)), respectively. Control fish were injected intraperitoneally with sterile phosphate buffer solution (PBS). The gene expression of CRP in liver of grass carp was detected at a higher level than that in either spleen or head kidney of fish, and was elevated significantly by vaccination with FKG(4) at 1 and 7 dpi, with the highest expression being observed at 1 dpi. By contrast, the gene expression of CRP in either spleen or head kidney of fish was detected at a relatively low level, with no significant difference being observed between vaccinated and control fish at all four detected time points. Significant higher gene expression of MHC I was observed at 1, 7 and 15 dpi in all three detected tissues of vaccinated fish when compared with control fish while its gene expression at 28 dpi showed no significant difference between vaccinated and control fish. Gene expression of TNF alpha in all three detected tissues of fish was detected at a quite low level, with a significant higher gene expression being observed at 1 and 7 dpi in detected tissues of vaccinated fish compared with control fish and its gene expression in three tissues of vaccinated fish at 15 and 28 dpi are at the same level as that of control fish. Gene expressions of IL-1 beta in liver and spleen of vaccinated fish were significantly enhanced at 1 dpi and then down-regulated to control level at 7, 15 and 28 dpi while its gene expression in head kidney was significantly elevated at 1 and 7 dpi and then decreased to control level at 15 and 28 dpi. Gene expressions of IL-8 in three tissues of vaccinated fish were significantly up regulated at 1 dpi, and then down regulated to control expression level in all detected tissues at 7, 15 and 28 dpi. However, the gene expression of IFN I, on the other hand, did not change significantly in all three detected tissues of vaccinated fish at four time points,although a slight increase of gene expression of IFN I in these tissues was also noticed at 1, 7 and 15 dpi. It is suggested that vaccination of FKG(4) be rather effective on increasing expressions of immune related genes in grass carp, which are mainly related to the fish immunity against infection of bacterial pathogen