A new cell line derived from the kidney of European eel, Anguilla anguilla, has been established and characterized. This cell line, designated as EK (eel kidney), has been maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum for over 24 months, and subcultured more than 60 times. This cell line consists predominantly of fibroblast-like cells, and can grow at 15–37°C under an optimum temperature of 26°C. The origin of this cell line was confirmed by polymerase chain reaction (PCR) amplification and 18s recombinant (r)RNA sequencing. The chromosome analysis of EK cells at passage 58 revealed an ananeuploid karyotype. The EK cells were successfully transfected with the Pegfp-N1 plasmid, suggesting its potential in genetic studies. The susceptibility test showed a significant cytopathic effect (CPE) in EK cells for Rana grylio virus, and the viral replication was evidenced with quantitative real-time PCR (qRT-PCR) assay. After poly (I:C) stimulation, the expression of the immune-related molecules including interferon regulatory factor-3 (irf3), interferon regulatory factor-7 (irf7) and cytochrome P450 (CYP450) were significantly upregulated in EK cells, while the expression of transforming growth factor (TGF-β) was downregulated. These results suggested the potential of EK cell line as a model in gene engineering, virus identification and environmental toxicology.
The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.
The ciliated protozoan
Cryptocaryon irritans
infects a wide range of marine fish and causes the highly lethal white spot disease. This parasite possesses three morphologically and physiologically distinct life stages: an infectious theront, a parasitic trophont, and an asexually reproductive tomont. In the past few years, several attempts have been made to help elucidate how C. irritans transforms from one stage to another using transcriptomic or proteomic approaches. However, there has been no research studying changes in transcription profiles between different time points of a single
C
.
irritans
life stage—the development of this parasite. Here we use RNA-seq and compare gene expression profiles of theront cells collected by 1 and 10 hrs after they emerged from tomonts. It has been shown that infectivity of theront cells declines 6–8 hours post-emergence, and we used this characteristic as a physiological marker to confirm the aging of theront cells. We identified a total of 41 upregulated and 90 downregulated genes that were differentially expressed between young and aging theront cells. Using Blast2Go to further analyze functions of these genes, we show that genes related to energy production are downregulated, but quite surprisingly many genes involved in transcription/translation processes are upregulated. We also show that expression of all nine detectable agglutination/immobilization antigen genes, with great sequence divergence, is invariably downregulated. Functions of other differentially expressed genes and indications are also discussed in our study.
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