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유세포 분석기와 Neutral Red Retention Assay를 이용한 자연산 참굴(Crassostrea gigas)의 면역력 측정

Assessment of Immune Parameters of the Wild Pacific Oyster (Crassostrea gigas) using a Flow Cytometry and Neutral Red Retention Assay

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초록

Hemocyte parameters of the wild Pacific oyster Crassostrea gigas inhabiting intertidal zones in small bays (Gwangyang and Jinhae Bay) on the southern coast of Korea were evaluated using flow cytometry and neutral red retention (NRR) assay. Morphological features, cell count, mortality, DNA damage, phagocytosis, and lysosomal membrane stability of hemocytes were analyzed. Three types of hemocytes were identified in the oyster hemolymph: granulocytes, hyalinocytes, and blast-like cells. Immune related functions of hemocyte including phagocytosis and lysosomal membrane stability were significantly different among the study areas (P<0.05), while cell count, mortality, and DNA damage of hemocytes were not significantly different. In Gwangyang Bay, phagocytosis of granulocytes and lysosomal membrane stability of oyster hemocytes inhabiting inside bay were significantly lower than those of oyster hemocytes in outside bay (P<0.05), indicating that oysters in inside bay of Gwangyang were relatively suppressed the immunological function in hemocytes. Contrary to Gwangyang Bay, immune parameters of oyster hemocytes in Jinhae Bay not showed the difference between sampling sites. In conclusion, flow cytometry and NRR assay using oyster hemocyte has a powerful tool to investigate the cell level in a short time due to no-preprocessing of material.

Abstract

Hemocyte parameters of the wild Pacific oyster Crassostrea gigas inhabiting intertidal zones in small bays (Gwangyang and Jinhae Bay) on the southern coast of Korea were evaluated using flow cytometry and neutral red retention (NRR) assay. Morphological features, cell count, mortality, DNA damage, phagocytosis, and lysosomal membrane stability of hemocytes were analyzed. Three types of hemocytes were identified in the oyster hemolymph: granulocytes, hyalinocytes, and blast-like cells. Immune related functions of hemocyte including phagocytosis and lysosomal membrane stability were significantly different among the study areas (P<0.05), while cell count, mortality, and DNA damage of hemocytes were not significantly different. In Gwangyang Bay, phagocytosis of granulocytes and lysosomal membrane stability of oyster hemocytes inhabiting inside bay were significantly lower than those of oyster hemocytes in outside bay (P<0.05), indicating that oysters in inside bay of Gwangyang were relatively suppressed the immunological function in hemocytes. Contrary to Gwangyang Bay, immune parameters of oyster hemocytes in Jinhae Bay not showed the difference between sampling sites. In conclusion, flow cytometry and NRR assay using oyster hemocyte has a powerful tool to investigate the cell level in a short time due to no-preprocessing of material.

참고문헌 (79)

  1. Aladaileh S, "Sydney rock oyster (Saccostrea glomerata) hemocytes: Morphology and function", J Invertebr Pathol, 96, pp.48~63 (2007)
  2. Ashton-Alcox KA, "Variability in molluscan hemocytes: A flow cytometric study", Tissue Cell, 30, pp.195~204 (1998)
  3. Auffret M, "Xenobiotic-induced immunomodulation in the European flat oyster, Ostrea edulis", Mar Environ Res, 54, pp.585~589 (2002)
  4. Bettencourt R, "Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus", Comp Biochem Phys B, 152(2), pp.278~289 (2009)
  5. Brousseau P, "Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals", Toxicology, 142, pp.145~156 (2000)
  6. Chávez-Villalba J, "Growth, condition, and survival of the Pacific oyster Crassostrea gigas cultivated within and outside a subtropical lagoon", Aquaculture, 300, pp.128~136 (2010)
  7. Cheng TC, "Bivalves, In Invertebrate blood cells", pp.233~300 (1981), Academic Press
  8. Cheng TC, "Cellular defense mechanisms in oysters, In Recent advances in marine biotechnology: Immunobiology and Pathology", pp.43~83 (2000), Science Publishers
  9. Cho SM, "Spawning impact on lysosomal stability of the Pacific Oyster, Crassostrea gigas", Aquaculture, 244, pp.383~387 (2005)
  10. Choquet G, "Reduction of adhesion properties of Ruditapesphilippinarum hemocytes exposed to Vibrio tapetis", DisAquat Organ, 57, pp.109~116 (2003)
  11. Chu FLE, "Defense mechanisms of marine bivalves, In Recent advances in marine biotechnology: Immunobiology and Pathology", pp.1~42 (2000), Science Publishers
  12. Cima F, "Biomarkers for TBT immunotoxicity studies onthe cultivated clam Tapes philippinarum (Adams & Reeve,1850): Morphofunctional characterization", Fish ShellfishImmun, 10, pp.677~693 (2000)
  13. Coles JA, "Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium", Dis Aquat Organ, 22, pp.59~65 (1995)
  14. da Silva PM, "Immunological responses of the Manila clam (Ruditapes philippinarum) with varying parasite (Perkinsus olseni) burden, during a long-term exposure to the harmful algae, Karenia selliformis, and possible interactions", Toxicon, 51, pp.563~573 (2008)
  15. da Silva PM, "Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule", Dis Aquat Organ, 67, pp.133~139 (2005)
  16. Darzynkiewicz Z, "Cytometry of the cell cycle: Cycling through history", Cytom A, 58, pp.21~32 (2004)
  17. Delaporte M, "Effet de la qualité lipidique du régime nutritif sur la composition des membranes des hémocytes de C. gigas. Impact sur les capacités de défense. Mémoire de fin d’ études. Diplome d’études approfondies Biologie et Productions Animales", 석사, pp.2001
  18. Donaghy L, "Hemocyte parameters of the Pacific oysterCrassostrea gigas a year after the Hebei Spirit oil spilloff the west coast of Korea", Helgoland Mar Res, 64(4), pp.349~355 (2010)
  19. 박흥식, "Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis", FISH & SHELLFISH IMMUNOLOGY, 27, pp.296~301, Elsevier
  20. Donaghy L, "Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum): Current knowledge and future prospects", Aquaculture, 297, pp.10~24 (2009)
  21. Dridi S, "Seasonal variation in weight and biochemical composition of the Pacific oyster, Crassostrea gigas in relation to the gametogenic cycle and environmental conditions of the Bizert lagoon, Tunisia", Aquaculture, 263, pp.238~248 (2007)
  22. Dyrynda EA, "Changes in immune parameters of natural mussel Mytilus edulis populations following a major oil spill (‘Sea Empress’, Wales, UK)", Mar Ecol- Prog Ser, 206, pp.155~170 (2000)
  23. Fisher WS, "A survey of oyster Crassostrea virginica from Tampa Bay, Florida: Association of internal defense measurements with contaminant burdens", Aquat Toxicol, 51, pp.115~138 (2000)
  24. Fisher WS, "Effects of tributyltin on defense-related activities of oyster hemocytes", Arch Environ Con Tox, 19, pp.354~360 (1990)
  25. Flye-Sainte-Marie J, "Variability of the hemocyte parameters of Ruditapes philippinarum in the field during an annual cycle", J Exp Mar Biol Ecol, 377, pp.1~11 (2009)
  26. Gagnaire B, "Diploid and triploid Pacific oyster, Crassostrea gigas (Thunber) at two heights above sediment in Marennes-Oleron Basin, France: Difference in mortality, sexual maturation and hemocyte parameters", Aquaculture, 254, pp.606~616 (2006)
  27. Givan AL, "Flow Cytometry: First Principles, 2nd ed", pp.273~ (2001), Wiley-Liss
  28. Goedken M, "Flow cytometry as a tool to quantify the oyster defence mechanisms", Fish Shellfish Immun, 16, pp.539~552 (2004)
  29. Gorinstein S, "Influence of mussels (Mytilus galloprovincialis) from polluted and non-polluted area on some atherosclerosis indices in rats fed cholesterol", Food Chem, 111, pp.381~386 (2008)
  30. Gosling E, "Reproduction, settlement and recruitment, In Bivalve Molluscs: Biology, Ecology and Culture", pp.131~168 (2003), Blackwell Science
  31. Grigoryan RS, "Flow cytometry analysis of single-strand DNA damage in nueroblastoma cell lines using the F7- 26 monoclonal antibody", Cytom Part A, 71, pp.951~960 (2007)
  32. Guidi P, "Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin", Aquat Toxicol, 100, pp.75~83 (2010)
  33. Hauton C, "The use of the neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European flat oyster Ostrea edulis (L)", Comp Biochem Phys B, 119, pp.619~623 (1998)
  34. Hauton C, "Response of haemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L)", Fish Shellfish Immun, 11, pp.143~153 (2001)
  35. Hégaret H, "Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum", J Exp Mar Biol Ecol, 371, pp.112~120 (2009)
  36. Hégaret H, "Flow-cytometric analysis of haemocytes from eastern oyster, Crassostrea virginica, subjected to a sudden temperature elevation I. Heamocyte type and morphology", J Exp Mar Biol Ecol, 293, pp.237~248 (2003)
  37. Hégaret H, "Immunological competence of eastern oysters, Crassostrea virginica, fed microalgal diets experimentally varied in both quantity and quality, before and after a sudden temperature elevation", Aquaculture, 234, pp.541~560 (2004)
  38. Hine PM, "The inter-relationships of bivalve haemocytes", Fish Shellfish Immun, 9, pp.367~385 (1999)
  39. Hong SH, "Polychlorinated biphenyls (PCBs) in a benthic ecosystem in Gwangyang Bay, South Korea", Mar Pollut Bull, 62, pp.2863~2868 (2011)
  40. Hong SH, "PCDD", F, PBDE, and nonylphenol contamination in a semi-enclosed bay (Masan Bay, South Korea) and a Mediterranean lagoon (Thau, France), 2009, pp.77, 854~862
  41. 강창근, "Condition, reproductive activity, and gross biochemical composition of the Manila clam, Tapes philippinarum in natural and newly created sandy habitats of the southern coast of Korea", JOURNAL OF SHELLFISH RESEARCH, 26(2), pp.401~412, NATL SHELLFISHERIES ASSOC
  42. 양현성, "GROWTH, REPRODUCTIVE CONDITION, AND DIGESTIVE TUBULE ATROPHY OF PACIFIC OYSTER CRASSOSTREA GIGAS IN GAMAKMAN BAY OFF THE SOUTHERN COAST OF KOREA", JOURNAL OF SHELLFISH RESEARCH, 29(4), pp.839~845, NATL SHELLFISHERIES ASSOC
  43. 강창근, "Physiological energetics and gross biochemical composition of the ascidian Styela clava cultured in suspension in a temperate bay of Korea", AQUACULTURE, 319(1-2), pp.168~177, ELSEVIER SCIENCE BV
  44. Labreuche Y, "Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01", 32, 2006, pp.8, 271~2724
  45. Lambert C, "Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites", Aquaculture, 270, pp.276~288 (2007)
  46. Lee RF, "Use of the single cell gel electrophoresis", comet assay for detecting DNA damage in aquatic (marine and freshwater) animals, 2003, pp.544, 43~64
  47. Li Y, "Assessment of metabolic and immune changes in postspawning Pacific oyster Crassostrea gigas: Identification of a critical period of vulnerability after spawning", Aquac Res, 1, pp.1~11 (2010)
  48. Liu J, "Immune responses, ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254", Fish Shellfish Immun, 26, pp.422~428 (2009)
  49. López C, "Morphological characterization of the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia)", J Invertebr Pathol, 69, pp.51~57 (1997)
  50. Lowe DM, "Contaminant induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from Venice Lagoon: An in vitro study", Mar Ecol-Prog Ser, 129, pp.189~196 (1995)
  51. Lowe DM, "Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from dab Limanda limanda", Mar Ecol-Prog Ser, 91, pp.135~140 (1992)
  52. Lowe DM, "Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene", Aquat Toxicol, 33, pp.105~112 (1995)
  53. Luzio JP, "Lysosomes: Fusion and function", Nat Rev Mol Cell Biol, 8(8), pp.622~632 (2007)
  54. MacKenzie NM, "Flow cytometry and its applications to veterinary medicine", Res Vet Sci, 42, pp.131~139 (1987)
  55. Matozzo V, "Effects of copper and cadmium exposure on functional responses of hemocytes in the clam, Tapes philippinarum", Arch Environ Con Tox, 41(2), pp.163~170 (2001)
  56. Mayrand E, "Haemocytes responses of blue mussels (Mytilus edulis L.) transferred from a contaminated site to a reference site: Can the immune system recuperate", Aquacult Res, 36, pp.962~971 (2005)
  57. Moore MN, "Cytochemical determination of cellular responses to environmental stressors in marine organisms", Rap Proces, 170, pp.7~15 (1980)
  58. Ngo TTT, "Prevalence and infection intensity of the ovarian parasite Marteilioides chungmuensis during an annual reproductive cycle of the oyster Crassostrea gigas", Dis Aquat Organ, 56, pp.259~267 (2003)
  59. Ngo TTT, "Seasonal changes of Perkinsus and Cercaria infections in the Manila clam Ruditapes philippinarum from Jeju, Korea", Aquaculture, 239, pp.57~68 (2004)
  60. Novas A, "After the Prestige oil spill modifications in NO production and other parameters related to the immune response were detected in hemocytes of Mytilus galloprovincialis", Aquat Toxicol, 85, pp.285~290 (2007)
  61. Oweson C, "A comparative study on the influence of manganese on the bactericidal response of marine invertebrates", Fish Shellfish Immun, 27, pp.500~507 (2009)
  62. Pampanin DM, "Physiological measurements from native and transplanted mussel (Mytilus galloprovincialis) in the canals of Venice. Survival in air and condition index", Comp Biochem Phys A, 140, pp.41~52 (2005)
  63. 박경일, "카드뮴에 노출된 바지락 (Ruditapes philippinarum)의 세포성 면역 반응", The Korean Journal of Malacology, 25(2), pp.135~143 (2009), 한국패류학회
  64. Park KI, "Flow cytometric assessment of immune parametersof the Manila clam (Ruditapes philippinarum)", J KoreanFish Soc, 39, pp.123~131 (2006)
  65. Park KI, "Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan", JOURNAL OF INVERTEBRATE PATHOLOGY, 99, pp.212~219 (2008), ACADEMIC PRESS INC ELSEVIER SCIENCE
  66. Pipe RK, "Environmental contaminants influencing immune function in marine bivalves molluscs", Fish Shellfish Immun, 5, pp.581~595 (1995)
  67. Pipe RK, "Copper induced immunomodulation in the marine mussel, Mytilus edulis", Aquat Toxicol, 46, pp.43~54 (1999)
  68. Raidal SL, "The flow cytometric evaluation of phagocytosis by equine peripheral blood neutrophils and pulmonary alveolar macrophages", Vet J, 156, pp.107~116 (1998)
  69. Raidal SL, "Flow cytometric determination of oxidative burst activity of equine peripheral blood bronchoalveolar lavage derived leucocytes", Vet J, 156, pp.117~126 (1998)
  70. Riley RS, "Clinical Applications of Flow Cytometry", pp.914~ (1993), Igaku-Shoin Press
  71. Sauvé S, "Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn)", Aquat Toxicol, 58, pp.189~200 (2002)
  72. Soudant P, "Impact of season and rearing site on the physiological and immunological parameters of the Manila clam Venerupis (=Tapes, =Ruditapes) philippinarum", Aquaculture, 229, pp.401~418 (2004)
  73. Tripp MR, "Phagocytosis by hemocytes of the hard clam, Mercenaria mercenaria", J Invertebr Pathol, 59, pp.222~227 (1992)
  74. 박경일, "Comparative reproductive biology of Yezo scallop, Patinopecten yessoensis, under two different culture systems on the east coast of Korea", AQUACULTURE, 265, pp.139~147, ELSEVIER SCIENCE BV
  75. Winstead JT, "Digestive tubule atrophy in eastern oysters, Crassostrea virginica (Gmelin, 1791), exposed to salinity and starvation stress", J Shellfish Res, 14, pp.105~111 (1995)
  76. Xue QG, "Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph", Fish Shellfish Immun, 11, pp.557~567 (2001)
  77. 양현성, "Pathologic Survey on the Manila Clam Ruditapes philippinarum (Adams and Reeve 1850) from Haeju off the Western Coastal Yellow Sea", Ocean Science Journal, 45(2), pp.93~100 (2010), 한국해양연구원
  78. Zhang Z, "Evaluation of the effects of grading and starvation on the lysosomal membrane stability in pacific oysters, Crassostrea gigas (Thunberg) by using neutral red retention assay", Aquaculture, 256, pp.537~541 (2006)
  79. Zhao C, "Assessments of lysosomal membrane responses to stresses with neutral red retention assay and its potential application in the improvement of bivalve aquaculture", Afr J Biotechnol, 10, pp.13968~13973 (2011)

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