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Assessment of Immune Parameters of the Wild Pacific Oyster (Crassostrea gigas) using a Flow Cytometry and Neutral Red Retention Assay
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  • Journal title : Ocean and Polar Research
  • Volume 34, Issue 2,  2012, pp.137-149
  • Publisher : Korea Institute of Ocean Science & Technology
  • DOI : 10.4217/OPR.2012.34.2.137
 Title & Authors
Assessment of Immune Parameters of the Wild Pacific Oyster (Crassostrea gigas) using a Flow Cytometry and Neutral Red Retention Assay
Hong, Hyun-Ki; Kang, Hyun-Sil; Kim, Young-Ok; Choi, Kwang-Sik;
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 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.
 Keywords
Crassostrea gigas;flow cytometry;neutral red retention assay;hemocyte;DNA damage;phagocytosis;lysosomal membrane stability;
 Language
Korean
 Cited by
 References
1.
Aladaileh S, Nair SV, Birch D, Raftos DA (2007) Sydney rock oyster (Saccostrea glomerata) hemocytes: Morphology and function. J Invertebr Pathol 96:48-63 crossref(new window)

2.
Ashton-Alcox KA, Ford SE (1998) Variability in molluscan hemocytes: A flow cytometric study. Tissue Cell 30:195-204 crossref(new window)

3.
Auffret M, Mujdzic N, Corporeau C, Moraga D (2002) Xenobiotic-induced immunomodulation in the European flat oyster, Ostrea edulis. Mar Environ Res 54:585-589 crossref(new window)

4.
Bettencourt R, Dando P, Collins P, Costa V, Allam B, Serrão Santos R (2009) Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Comp Biochem Phys B 152(2):278-289 crossref(new window)

5.
Brousseau P, Pellerin J, Morin Y, Cyr D, Blakley B, Boermans H, Fournier M (2000) 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 142:145-156

6.
Chavez-Villalba J, Arreloa-Lizarraga A, Burrola-Sanchez S, Hoyos-Chairez F (2010) Growth, condition, and survival of the Pacific oyster Crassostrea gigas cultivated within and outside a subtropical lagoon. Aquaculture 300:128-136 crossref(new window)

7.
Cheng TC (1981) Bivalves. In: Ratcliffe NA, Rowly AF (eds) Invertebrate blood cells. Academic Press, London, pp 233-300

8.
Cheng TC (2000) Cellular defense mechanisms in oysters. In: Fingerman N, Nagabhushanam (eds) Recent advances in marine biotechnology: Immunobiology and Pathology. Science Publishers, Enfield, NH, pp 43-83

9.
Cho SM, Jeong WG (2005) Spawning impact on lysosomal stability of the Pacific Oyster, Crassostrea gigas. Aquaculture 244:383-387 crossref(new window)

10.
Choquet G, Soudant P, Lambert C, Nicolas JL, Paillard C (2003) Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis. Dis Aquat Organ 57:109-116 crossref(new window)

11.
Chu FLE (2000) Defense mechanisms of marine bivalves. In: Fingerman N, Nagabhushanam R (eds) Recent advances in marine biotechnology: Immunobiology and Pathology. Science Publishers, Enfield, NH, pp 1-42

12.
Cima F, Marin MG, Matozzo V, Da Ros L, Ballarin, K (2000) Biomarkers for TBT immunotoxicity studies on the cultivated clam Tapes philippinarum (Adams & Reeve, 1850): Morphofunctional characterization. Fish Shellfish Immun 10:677-693 crossref(new window)

13.
Coles JA, Farley SR, Pipe RK (1995) Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium. Dis Aquat Organ 22:59-65 crossref(new window)

14.
da Silva PM, Hégaret H, Lambert C, Wikfors GH, Le Goïc N, Shumway SE, Soudant P (2008) 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 51:563-573 crossref(new window)

15.
da Silva PM, Soudant P, Carballal MJ, Lambert C, Villalba A (2005) Flow cytometric DNA content analysis of neoplastic cells in haemolymph of the cockle Cerastoderma edule. Dis Aquat Organ 67:133-139 crossref(new window)

16.
Darzynkiewicz Z, Crissman H, Jacobberger JW (2004) Cytometry of the cell cycle: Cycling through history. Cytom A 58:21-32

17.
Delaporte M (2001) Effet de la qualite lipidique du regime nutritif sur la composition des membranes des hemocytes de C. gigas. Impact sur les capacites de defense. Memoire de fin d'etudes. Diplome d'etudes approfondies Biologie et Productions Animales, Option: Biologie Aquacole (Ms dissertation) Universite Rennes I-Ecole Nationale Superieure Agronomique de Rennes, 36 p

18.
Donaghy L, Hong HK, Lee HJ, Jun JC, Park YJ, Choi KS (2010) Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea. Helgoland Mar Res 64(4): 349-355 crossref(new window)

19.
Donaghy L, Kim BK, Hong HK, Park HS, Choi KS (2009a) Flow cytometry studies on the populations and immune parameters of the hemocytes of the Sumino oyster, Crassostrea ariakensis. Fish Shellfish Immun 27:296-301 crossref(new window)

20.
Donaghy L, Lambert C, Choi KS, Soudant P (2009b) Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum): Current knowledge and future prospects. Aquaculture 297:10-24 crossref(new window)

21.
Dridi S, Romdhane MS, Elcafsi M (2007) 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 263:238-248 crossref(new window)

22.
Dyrynda EA, Law RJ, Dyrynda PEJ, Kelly CA, Pipe RK, Ratcliffe NA (2000) Changes in immune parameters of natural mussel Mytilus edulis populations following a major oil spill ('Sea Empress', Wales, UK). Mar Ecol-Prog Ser 206:155-170 crossref(new window)

23.
Fisher WS, Oliver LM, Winstead JT, Long ER (2000) A survey of oyster Crassostrea virginica from Tampa Bay, Florida: Association of internal defense measurements with contaminant burdens. Aquat Toxicol 51:115-138 crossref(new window)

24.
Fisher WS, Wishkovsky A, Chu FLE (1990) Effects of tributyltin on defense-related activities of oyster hemocytes. Arch Environ Con Tox 19:354-360 crossref(new window)

25.
Flye-Sainte-Marie J, Soudant P, Lambert C, Le Goïc N, Goncalvez M, Travers MA, Paillard C, Jean F (2009) Variability of the hemocyte parameters of Ruditapes philippinarum in the field during an annual cycle. J Exp Mar Biol Ecol 377:1-11 crossref(new window)

26.
Gagnaire B, Soletchnik P, Madec P, Geairon P, Le Moine O, Renault T (2006) 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 254:606-616 crossref(new window)

27.
Givan AL (2001) Flow Cytometry: First Principles, 2nd ed. Wiley-Liss, Somerset, 273 p

28.
Goedken M, De Guise S (2004) Flow cytometry as a tool to quantify the oyster defence mechanisms. Fish Shellfish Immun 16:539-552 crossref(new window)

29.
Gorinstein S, Leontowicz M, Leontowicz H, Namiesnik J, Jastrzebski Z, Drzewiecki J, Park YS, Ham KS, Heo BG, Trakhtenberg S (2008) Influence of mussels (Mytilus galloprovincialis) from polluted and non-polluted area on some atherosclerosis indices in rats fed cholesterol. Food Chem 111:381-386 crossref(new window)

30.
Gosling E (2003) Reproduction, settlement and recruitment In: Elizabeth G (ed) Bivalve Molluscs: Biology, Ecology and Culture. Blackwell Science, UK, pp 131-168

31.
Grigoryan RS, Yang B, Keshelava N, Barnhart JR, Reynolds CP (2007) Flow cytometry analysis of single-strand DNA damage in nueroblastoma cell lines using the F7-26 monoclonal antibody. Cytom Part A 71:951-960

32.
Guidi P, Frenzilli G, Benedetti M, Bernardeschi M, Falleni A, Fattorini D, Regoli F, Scarcelli V, Nigro M (2010) Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin. Aquat Toxicol 100:75-83 crossref(new window)

33.
Hauton C, Hawkins LE, Hutchinson S (1998) 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 119:619-623 crossref(new window)

34.
Hauton C, Hawkins LE, Hutchinson S (2001) 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 11:143-153 crossref(new window)

35.
Hegaret H, da Silva P, Sunila I, Shumway SE, Dixon MS, Alix J, Wikfors GH, Soudant P (2009) Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum. J Exp Mar Biol Ecol 371:112-120 crossref(new window)

36.
Hegaret H, Wikfors GH, Soudant P (2003) 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 293:237-248 crossref(new window)

37.
Hegaret H, Wikfors GH, Soudant P, Delaporte M, Alix JH, Smith BC, Dixon MS, Quere C, Le Coz JR, Paillard C, Moal J, Samain JF (2004) 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 234:541-560 crossref(new window)

38.
Hine PM (1999) The inter-relationships of bivalve haemocytes. Fish Shellfish Immun 9:367-385 crossref(new window)

39.
Hong SH, Kannan N, Yim UH, Choi JW, Shim WJ (2011) Polychlorinated biphenyls (PCBs) in a benthic ecosystem in Gwangyang Bay, South Korea. Mar Pollut Bull 62: 2863-2868 crossref(new window)

40.
Hong SH, Munschy C, Kannan N, Tixier C, Tronczynski J, Heas-Moisan K, Shim WJ (2009) PCDD/F, PBDE, and nonylphenol contamination in a semi-enclosed bay (Masan Bay, South Korea) and a Mediterranean lagoon (Thau, France). Chemosphere 77:854-862 crossref(new window)

41.
Kang CK, Kang YS, Choy EJ, Kim DS, Shim BT, Lee PY (2007) 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. J Shellfish Res 26:401-412 crossref(new window)

42.
Kang DH, Chu FL, Yang HS, Lee CH, Koh HB, Choi KS (2010) Growth, reproductive condition, and digestive tubule atrophy of Pacific oyster Crassostrea gigas in Gamakman Bay off the southern coast of Korea. J Shellfish Res 29(4):839-845 crossref(new window)

43.
Kang CK, Choy EJ, Lee WC, Kim NJ, Park HJ, Choi KS (2011) Physiological energetics and gross biochemical composition of the ascidian Styela clava cultured in suspension in a temperate bay of Korea. Aquaculture 319:168-177 crossref(new window)

44.
Labreuche Y, Lambert C, Soudant P, Boulo V, Huvet A, Nicolas JL (2006) Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01/32. Microb Infect 8:271-2724

45.
Lambert C, Soudant P, Dégremont L, Delaporte M, Moal J, Boudry P, Jean F, Huvet A, Samain JF (2007) Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites. Aquaculture 270:276-288 crossref(new window)

46.
Lee RF, Steinert S (2003) Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals. Mutat Res-Rev Mutat 544:43-64 crossref(new window)

47.
Li Y, Qin JG, Li X, Benkendorff K (2010) Assessment of metabolic and immune changes in postspawning Pacific oyster Crassostrea gigas: Identification of a critical period of vulnerability after spawning. Aquac Res 1:1-11

48.
Liu J, Pan LQ, Zhang L, Miao J, Wang J (2009) Immune responses, ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254. Fish Shellfish Immun 26:422-428 crossref(new window)

49.
Lopez C, Carballal MJ, Azevedo C, Villalba A (1997) Morphological characterization of the hemocytes of the clam, Ruditapes decussatus (Mollusca: Bivalvia). J Invertebr Pathol 69:51-57 crossref(new window)

50.
Lowe DM, Fossato VU, Depledge MH (1995a) Contaminant induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from Venice Lagoon:An in vitro study. Mar Ecol-Prog Ser 129:189-196 crossref(new window)

51.
Lowe DM, Moore MN, Evans BM (1992) Contaminant impact on interactions of molecular probes with lysosomes in living hepatocytes from dab Limanda limanda. Mar Ecol-Prog Ser 91:135-140 crossref(new window)

52.
Lowe DM, Soverchia C, Moore MN (1995b) Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to fluoranthene. Aquat Toxicol 33:105-112 crossref(new window)

53.
Luzio JP, Pryor PR, Bright NA (2007) Lysosomes: Fusion and function. Nat Rev Mol Cell Biol 8(8):622-632 crossref(new window)

54.
MacKenzie NM, Pinder AC (1987) Flow cytometry and its applications to veterinary medicine. Res Vet Sci 42:131-139

55.
Matozzo V, Ballarin L, Pampanin DM, Marin MG (2001) Effects of copper and cadmium exposure on functional responses of hemocytes in the clam, Tapes philippinarum. Arch Environ Con Tox 41(2):163-170 crossref(new window)

56.
Mayrand E, St-Jean SD, Courtenay SC (2005) Haemocytes responses of blue mussels (Mytilus edulis L.) transferred from a contaminated site to a reference site: Can the immune system recuperate? Aquacult Res 36:962-971 crossref(new window)

57.
Moore MN (1980) Cytochemical determination of cellular responses to environmental stressors in marine organisms. Rap Proces 170:7-15

58.
Ngo TTT, Berthe FCJ, Choi KS (2003) Prevalence and infection intensity of the ovarian parasite Marteilioides chungmuensis during an annual reproductive cycle of the oyster Crassostrea gigas. Dis Aquat Organ 56:259-267 crossref(new window)

59.
Ngo TTT, Choi KS (2004) Seasonal changes of Perkinsus and Cercaria infections in the Manila clam Ruditapes philippinarum from Jeju, Korea. Aquaculture 239:57-68 crossref(new window)

60.
Novas A, Barcia R, Ramos-Martínez JI (2007) 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 85:285-290 crossref(new window)

61.
Oweson C, Hernroth B (2009) A comparative study on the influence of manganese on the bactericidal response of marine invertebrates. Fish Shellfish Immun 27:500-507 crossref(new window)

62.
Pampanin DM, Volpato E, Marangon I, Nasci C (2005) Physiological measurements from native and transplanted mussel (Mytilus galloprovincialis) in the canals of Venice. Survival in air and condition index. Comp Biochem Phys A 140:41-52 crossref(new window)

63.
Park KI, Choi MS, Park SW, Park KH, Choi SH, Yoon JM (2009) Cellular immune responses of the Manila clam, Ruditapes philippinarum, exposed to cadimium chloride. Korean J Malacol 25:135-143

64.
Park KI, Park HS, Kim JM, Park YJ, Hong JS, Choi KS (2006) Flow cytometric assessment of immune parameters of the Manila clam (Ruditapes philippinarum). J Korean Fish Soc 39:123-131

65.
Park KI, Tsutsumi H, Hong JS, Choi KS (2008) Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan. J Invertebr Pathol 99:212-219 crossref(new window)

66.
Pipe RK, Coles JA (1995) Environmental contaminants influencing immune function in marine bivalves molluscs. Fish Shellfish Immun 5:581-595 crossref(new window)

67.
Pipe RK, Coles JA, Carissan FMM, Ramanathan K (1999) Copper induced immunomodulation in the marine mussel, Mytilus edulis. Aquat Toxicol 46:43-54 crossref(new window)

68.
Raidal SL, Bailey GD, Love DN (1998a) The flow cytometric evaluation of phagocytosis by equine peripheral blood neutrophils and pulmonary alveolar macrophages. Vet J 156:107-116 crossref(new window)

69.
Raidal SL, Bailey GD, Love DN (1998b) Flow cytometric determination of oxidative burst activity of equine peripheral blood bronchoalveolar lavage derived leucocytes. Vet J 156:117-126 crossref(new window)

70.
Riley RS, Mahin EJ, Ross W (1993) Clinical Applications of Flow Cytometry. Igaku-Shoin Press, New York, 914 p

71.
Sauve S, Brousseau P, Pellerin J, Pmorin Y, Senecal L, Goudreau P, Fournier M (2002) Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn). Aquat Toxicol 58:189-200 crossref(new window)

72.
Soudant P, Paillard C, Choquet G, Lambert C, Reid HI, Marhic A, Donaghy L, Birkbeck TH (2004) Impact of season and rearing site on the physiological and immunological parameters of the Manila clam Venerupis (=Tapes, =Ruditapes) philippinarum. Aquaculture 229:401-418 crossref(new window)

73.
Tripp MR (1992) Phagocytosis by hemocytes of the hard clam, Mercenaria mercenaria. J Invertebr Pathol 59:222-227 crossref(new window)

74.
Uddin MJ, Park KI, Kang DH, Park YJ, Choi KS (2007) Comparative reproductive biology of Yezo scallop, Patinopecten yessoensis, under two different culture systems on the east coast of Korea. Aquaculture 265:139-147 crossref(new window)

75.
Winstead JT (1995) Digestive tubule atrophy in eastern oysters, Crassostrea virginica (Gmelin, 1791), exposed to salinity and starvation stress. J Shellfish Res 14:105-111

76.
Xue QG, Renault T, Chilmonczyk S (2001) Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph. Fish Shellfish Immun 11:557-567 crossref(new window)

77.
Yang HS, Park KJ, Choi KS (2010) Pathologic survey on the Manila clam Ruditapes philippinarum (Adams and Reeve 1850) from Haeju off the western coastal Yellow Sea. Ocean Sci J 45(2):93-100 crossref(new window)

78.
Zhang Z, Li X (2006) 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 256:537-541 crossref(new window)

79.
Zhao C, Li X, Luo S, Chang Y (2011) 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 10:13968-13973