Molecular & Cellular Toxicology

, Volume 7, Issue 4, pp 357–365 | Cite as

Comparative analysis of expressed sequence tags (ESTs) between normal group and softness syndrome group in Halocynthia roretzi

  • Ji Eun Jeong
  • Se Won Kang
  • Yun Kyung Shin
  • Je Cheon Jun
  • Young-Ok Kim
  • Young Baek Hur
  • Jae-Hyung Kim
  • Sung-Hwa Chae
  • Jun-Sang Lee
  • In ho Choi
  • Yeon Soo Han
  • Dae-Hyun Seog
  • Yong Seok Lee
Original Paper


To identify the cause of mass mortality in ascidians, we constructed cDNA libraries of both the normal and softness syndrome group in Halocynthia roretzi. To perform comparative analysis of transcripts between the two groups, we sequenced about 1,000 random clones. All the sequences obtained from the clones were processed to remove the vector region and low quality sequences through base calling and vector trimming. We collected 906 sequences with average length of 463 bp in the normal group and 1014 sequences in the softness syndrome group with an average length of 696 bp. Clustering and assembling of EST sequences using TGICL package resulted in 906 distinct sequences composed of 517 singletons and 77 contigs in 75 clusters in normal group and 1014 distinct sequences composed of 707 singletons and 120 contigs in 120 clusters in the softness syndrome group. All sequences derived from the two groups were compared against the NCBI Non-redundant database using BLASTX algorithms. As a result, 493 sequences in the normal group and 861 sequences in the softness syndrome group had significant hits within the database. In addition, we listed genes that showed differential expression in the softness syndrome group. Transcript levels of calponin increased by 11-fold and both E3 ubiquitin-protein ligase MARCH3 and selenium dependent salivary glutathione peroxidase by 5-fold in the softness syndrome group. Also, the expression of four genes including muscle actin increased by 4-fold. In contrast, we observed down-regulation of genes encoding trypsinogen 1, cathepsin D protein, serine protease, and halocidin precursor, decreasing by more than 6-fold. Herein, we identified differential expression of genes involved in the contraction and regulation of muscle cells and immune reaction in H. roretzi with softness syndrome. This study is the first report on gene expression changes occurring in H. roretzi with softness syndrome and would be useful in further studies.


Halocynthia roretzi Softness syndrome EST Mass mortality Smooth muscle 


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  1. 1.
    Rho, B. J. A Study on the Classification and the Distribution of the Korean Ascidians. Journal of Korean Research Institute for Better Living 6:103–166 (1971).Google Scholar
  2. 2.
    Baik, K. K., Chung, S. K. & Chung, Y. S. Studies on the spawning season of sea squirts, Halocynthia roretzi (von Drasche) and Halocynthia aurantium (Pallas) in the coast of Kangwon province. Bull Fish Res Dev Agency 37:179–183 (1986).Google Scholar
  3. 3.
    Na, G. H., Lee, C. S. & Choi, W. J. The effect of dissolved oxygen on the estival mass mortality of sea squirt, Halocynthia roretzi (Drasche). Bull Korean Fish Soc 24:52–58 (1991).Google Scholar
  4. 4.
    Chang, D. S., Chun, S. K., Cheong, S. C. & Seo, H. L. A study on the mortality of sea squirt, Halocyhthia roretzi (Drasche). Bull Nat Fish Res Dev Agency 29:7–40 (1982).Google Scholar
  5. 5.
    Hirose, E., Ohtake, S. I. & Azumi, K. Morphological characterization of the tunic in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on ’soft tunic syndrome’ in aquaculture. J Fish Dis 32:433–445 (2009).PubMedCrossRefGoogle Scholar
  6. 6.
    Choi, D. L., Jee, B. Y., Choi, H. J., Hwang, J. Y. & Kim, J. W. Infection of an intrahemocytic paramyxean parasite from tunicate Halocynthia roretzi in Korea. 2006 Joint Conference of Institute for Fisheries-Poster Presentation Substances 2006:457–458 (2006).Google Scholar
  7. 7.
    Choi, D. L. et al. First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea. Dis Aquat Organ 72:65–69 (2006).PubMedCrossRefGoogle Scholar
  8. 8.
    Jung, S. J., Oh, M. J., Date, K. & Suzuki, S. Isolation of marine birnavirus from sea squirts Halocynthia roretzi. The biology of ascidians (ed. by H. Sawada, H. Yokosawa & C. C. Lambert, editors):436–441 (2001).Google Scholar
  9. 9.
    Kitamura, S. I. et al. Tunic morphology and viral surveillance in diseased Korean ascidians: Soft tunic syndrome in the edible ascidian, Halocynthia roretzi (Drasche), in aquaculture. J Fish Dis 33:153–160 (2010).PubMedCrossRefGoogle Scholar
  10. 10.
    Azumi, K. et al. Accumulation of organotin compounds and marine birnavirus detection in Korean ascidians. Fish Sci 73:263–269 (2007).CrossRefGoogle Scholar
  11. 11.
    Rho, Y. G., Lee, Y. H. & Park, M. W. The environmental factors affecting mortality of cultured sea squirt, Halocynthia roretzi (Drasche). Bull Nat Fish Res Dev Agency (in Korea) 47:145–164 (1993).Google Scholar
  12. 12.
    Yoo, S. K., Chang, Y. J., Kang, K. H. & Kim, Y. K. Influence of water temperature on spawning induction, egg development and seed collection of sea squirt, Halocynthia roretzi. J Aquaculture 3:79–88 (1990).Google Scholar
  13. 13.
    Lee, Y. H. et al. The variation of water temperature and the mass mortalities of sea squirt, Halocynthia roretzi along Gyeongbuk Coasts. Journal of The Korean Society of Marine Environment & Safety 13:15–19 (2007).Google Scholar
  14. 14.
    Hong, J. P., Kim, Y. S. & Hur, S. B. Effect of temperature fluctuation and different stocking densities on mortality of sea squirt, Halocynthia roretzi (von Drasche). J Aquaculture 13:277–368 (2000).Google Scholar
  15. 15.
    Cho, H. K. et al. Identification of softness syndromeassociated candidate genes and DNA sequence variation in the sea squirt, Halocynthia roretzi. Mar Biotechnol (NY) 10:447–456 (2008).CrossRefGoogle Scholar
  16. 16.
    Pertea, G. et al. TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19:651–652 (2003).PubMedCrossRefGoogle Scholar
  17. 17.
    Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4:41 (2003).PubMedCrossRefGoogle Scholar
  18. 18.
    Shin, Y. P. et al. Antimicrobial activity of a halocidinderived peptide resistant to attacks by proteases. Antimicrob Agents Chemother 54:2855–2866 (2010).PubMedCrossRefGoogle Scholar
  19. 19.
    Jang, W. S. et al. Antimicrobial effect of halocidinderived peptide in a mouse model of Listeria infection. Antimicrob Agents Chemother 51:4148–4156 (2007).PubMedCrossRefGoogle Scholar
  20. 20.
    Jang, W. S. et al. Biological activities of synthetic analogs of halocidin, an antimicrobial peptide from the tunicate Halocynthia aurantium. Antimicrob Agents Chemother 47:2481–2486 (2003).PubMedCrossRefGoogle Scholar
  21. 21.
    Lee, K. H. et al. Functional porperties of sulfated polysaccharides in Asicidian (Halocynthia roretzi) tunic. J Korean Fish Soc 31:447–451 (1998).Google Scholar
  22. 22.
    Hong, B. I. et al. Utilization of pigments and tunic components of Ascidian as an improved feed aids for aquaculture 3. Functional porperties of sulfated polysaccharides from Asicidian (Halocynthia roretzi) tunic. J Korean Fish Soc 35:671–675 (2002).Google Scholar
  23. 23.
    Yook, H. S. et al. Changes of nutritional characteristics and serum cholesterol in Rats by the intake of dietary fiber isolated from Ascidian (Halocynthia roretzi) tunic. J Food Science and Nutrition 32:474–478 (2003).Google Scholar
  24. 24.
    Lee, S. J., Ha, W. H., Choi, H. J., Cho, S. Y. & Choi, J. W. Antihyperlipidemic and antidiabetic activities of the Ascidian tunic in Sprague-Dawley Rats. Kor J Fish Aquat Sci 43:567–572 (2010).Google Scholar
  25. 25.
    Mikami, N., Hosokawa, M. & Miyashita, K. Effects of sea squirt (Halocynthia roretzi) lipids on white adipose tissue weight and blood glucose in diabetic/obese KKAy mice. Mol Med Report 3:449–453 (2010).PubMedGoogle Scholar
  26. 26.
    Jin, J. P., Walsh, M. P., Sutherland, C. & Chen, W. A role for serine-175 in modulating the molecular conformation of calponin. Biochem J 350Pt 2:579–588 (2000).PubMedCrossRefGoogle Scholar
  27. 27.
    Szymanski, P. T., Dickie, R., Rogers, R. & Fredberg, J. J. Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers. Anal Biochem 321:8–21 (2003).PubMedCrossRefGoogle Scholar
  28. 28.
    Sobue, K., Hayashi, K. & Nishida, W. Molecular mechanism of phenotypic modulation of smooth muscle cells. Horm Res 50Suppl 2:15–24 (1998).PubMedCrossRefGoogle Scholar
  29. 29.
    Strasser, P., Gimona, M., Moessler, H., Herzog, M. & Small, J. V. Mammalian calponin. Identification and expression of genetic variants. FEBS Lett 330:13–18 (1993).PubMedCrossRefGoogle Scholar
  30. 30.
    Carmichael, J. D., Winder, S. J., Walsh, M. P. & Kargacin, G. J. Calponin and smooth muscle regulation. Can J Physiol Pharmacol 72:1415–1419 (1994).PubMedCrossRefGoogle Scholar
  31. 31.
    Gong, B. J., Mabuchi, K., Takahashi, K., Nadal-Ginard, B. & Tao, T. Characterization of wild type and mutant chicken gizzard alpha calponin expressed in E. coli. J Biochem 114:453–456 (1993).PubMedGoogle Scholar
  32. 32.
    Sobue, K., Hayashi, K. & Nishida, W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190:105–118 (1999).PubMedCrossRefGoogle Scholar
  33. 33.
    Applegate, D., Feng, W., Green, R. S. & Taubman, M. B. Cloning and expression of a novel acidic calponin isoform from rat aortic vascular smooth muscle. J Biol Chem 269:10683–10690 (1994).PubMedGoogle Scholar
  34. 34.
    Muller, F. L., Lustgarten, M. S., Jang, Y., Richardson, A. & Van Remmen, H. Trends in oxidative aging theories. Free Radic Biol Med 43:477–503 (2007).PubMedCrossRefGoogle Scholar
  35. 35.
    Ran, Q. et al. Reduction in glutathione peroxidase 4 increases life span through increased sensitivity to apoptosis. J Gerontol A Biol Sci Med Sci 62:932–942 (2007).PubMedCrossRefGoogle Scholar
  36. 36.
    Kumagai, A. et al. Mass mortality of cultured ascidians Halocynthia roretzi associated with softening of the tunic and flagellate-like cells. Dis Aquat Organ 90:223–234 (2010).PubMedCrossRefGoogle Scholar
  37. 37.
    Jang, W. S., Kim, K. N., Lee, Y. S., Nam, M. H. & Lee, I. H. Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium. FEBS Lett 521:81–86 (2002).PubMedCrossRefGoogle Scholar
  38. 38.
    Cha, I. S. et al. Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics. Dev Comp Immunol 35:809–816 (2011).PubMedCrossRefGoogle Scholar
  39. 39.
    Ewing, B., Hillier, L., Wendl, M. C. & Green, P. Basecalling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185 (1998).PubMedGoogle Scholar
  40. 40.
    Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194 (1998).PubMedGoogle Scholar
  41. 41.
    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J Mol Biol 215:403–410 (1990).PubMedGoogle Scholar

Copyright information

© The Korean Society of Toxicogenomics and Toxicoproteomics and Springer Netherlands 2011

Authors and Affiliations

  • Ji Eun Jeong
    • 1
  • Se Won Kang
    • 1
  • Yun Kyung Shin
    • 2
  • Je Cheon Jun
    • 2
  • Young-Ok Kim
    • 3
  • Young Baek Hur
    • 4
  • Jae-Hyung Kim
    • 5
  • Sung-Hwa Chae
    • 6
  • Jun-Sang Lee
    • 7
  • In ho Choi
    • 8
  • Yeon Soo Han
    • 9
  • Dae-Hyun Seog
    • 10
  • Yong Seok Lee
    • 1
  1. 1.Department of Parasitology, College of Medicine and UHRCInje UniversityBusanKorea
  2. 2.Aquaculture Management DivisionNFRDIBusanKorea
  3. 3.Biotechnology Research DivisionNFRDIBusanKorea
  4. 4.Southeast Sea Fisheries Research InstituteNFRDITongyeong, Gyeongsangnam-doKorea
  5. 5.Dong-il Shimadzu Biotech.DaejeonKorea
  6. 6.Research Institute, GnC BIO Co., LTD.DaejeonKorea
  7. 7.Institute of Environmental ResearchKangwon National UniversityGangwon-doKorea
  8. 8.School of BiotechnologyYeungnam UniversityGyeongsangbuk-doKorea
  9. 9.College of Agriculture and Life ScienceChonnam National UniversityGwangjuKorea
  10. 10.Department of Biochemistry, College of MedicineInje UniversityBusanKorea

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