A new approach for classification of major larval hemocytes (prohemocytes, plasmatocytes and granulocytes) in the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) by acridine orange staining

Year 2012, Volume: 36 Issue: 2, 163 - 168, 01.04.2012

Savaş İzzetoğlu 163-168

Abstract

Larval hemocytes of the greater wax moth, Galleria mellonella L. 1758 (Lepidoptera: Pyralidae), were classified by fluorescence microscopy staining with acridine orange. Based on their fluorescence characteristics, plasmatocytes and prohemocytes could be easily distinguished from granulocytes because they appear faint green and do not contain any fluorescent granules in their cytoplasm. Nuclei of granulocytes emit bright green fluorescence when observed under fluorescence microscope. Prohemocytes are round and their nuclei are bright. Plasmatocytes are irregularly shaped. Therefore, all three types of the wax moth’s major hemocytes that are difficult to identify by bright field or phase contrast microscopy can be easily classified by fluorescence microscopy with staining acridine orange. Furthermore, this technique will assist further studies to understand how hemocyte differentiation and regeneration proceeds in larval hematopoietic organs and during hemocyte transformation. The fluorescent method for hemocyte classification is more precise than the common method of hemocyte identification using bright field or phase contrast microscopy.

Keywords

Galleria mellonella, hemocyte, acridine orange, fluorescence microscopy

Büyük bal mumu güvesi Galleria mellonella L. (Lepidoptera: Pyralidae)'nın başlıca larval hemositlerinin (prohemositler, plazmatositler ve granülositler) akridin oranj boyama ile sınıflandırılması için yeni bir yaklaşım

Abstract

Büyük mum güvesi, Galleria mellonella L. 1758 (Lepidoptera: Pyralidae)’nın larval hemositleri acridine orange ile boyanarak floresans mikroskopta sınıflandırılmıştır. Hemositlerin floresan özelliklerine göre, plasmatositler ve prohemositler, granulositlerden kolaylıkla ayırt edilebilir. Çünkü plasmatositler ve prohemositler soluk yeşil görünürler ve sitoplazmalarında herhangi bir floresan granülleri içermezler. Granulositler floresans mikroskop ile incelendiğinde nukleusları parlak yeşil ışıma yaparlar. Prohemositler yuvarlaktır ve nukleusları parlaktır. Plasmatositlerin düzensiz şekilleri vardır. Büyük mum güvesinin en önemli üç tip hemositlerin parlak alan ve faz kontrast mikroskop ile tanımlanmaları zor olduğundan dolayı, hemositler acridine orange boyası ile floresans mikroskop altında sınıflandırılabilir. Ayrıca, bu teknik hemositlerin dönüşümü sırasında ve hematopoietik organda hemositlerin nasıl farklılaştığının anlaşılması için yapılan ileri çalışmalara destek sağlayacaktır. Hemositlerin sınıflandırılmasında kullanılan floresan tekniği, genel bir yöntem olan parlak alan ve faz kontrast mikroskop ile yapılan hemosit belirlenmesine göre daha hassas bir yöntemdir

Keywords

Galleria mellonella, hemosit, akridin oranj, floresan mikroskobu

References

• Akai, H. & S. Sato, 1973. Ultrastructure of the larval Hemocytes of the Silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). International Journal of Insect Morphology and Embryology, 2 (3): 207-231.
• Andrejko, M. & M. Mizerska-Dudka, 2011. Elastase B of Pseudomonas aeruginosa Stimulates the humoral Immune Response in the greater Wax Moth, Galleria mellonella. Journal of Invertebrate Pathology, doi:10.1016/j.jip.2010.12.015.
• Canete, M., A. Juarranz, P. Lopez-Nieva, C. Alonso-Torcal, A. Villanueva & J. C. Stockert, 2001. Fixation and permanent mounting of Fluorescent Probes After Vital Labelling of Cultured Cells. Acta Histochemistry, 103: 117-126.
• Chain, B. M., K. Leyshon-Sorland & M.T. Siva-Jothy, 1992. Haemocyte heterogeneity in the Cockroach Periplaneta americana analysed using monoclonal antibodies. Journal of Cell Science, 103 (4): 1261-1267.
• Foglieni, C., C. Meoni & A. M. Davalli, 2001. Fluorescent dyes for cell viability: An Application on prefixed conditions. Histochemistry of Cell Biology, 115: 223-229.
• Gardiner, E. M. M. & M. R. Strand, 1999. Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includes. Journal of Insect Physiology, 45: 113-126.
• Gillespie, J. P., M. R. Kanost & T. Trenczek, 1997. Biological mediators of insect immunity. Annual Review of Entomology, 42: 611-43.
• Huang, F., Y. Yang, M. Shi, J. Li, Z. Chen, F. Chen & X. Chen, 2010. Ultrastructural and functional characterization of circulating hemocytes from Plutella xylostella: Cell types and their role in phagocytosis. Tissue and Cell, 42: 360-364.
• İzzetoğlu, S. & S. Karaçalı, 2010. A novel site for hematopoietic organ in Bombyx mori L. (Lepidoptera: Bombycidae). The Journal of Faculty of Veterinary Medicine, Kafkas University, 16 (Suppl-B): 243-247.
• Jarosz, C., 1998. Active resistance of Entomophagous Rhabditid Heterorhabditis bacteriophora to insect immunity. Parasitology, 117: 201-208.
• Karaçalı, S., R. Deveci, S. Pehlivan & A. Özcan, 2000. Adhesion of hemocytes to desialylated prothoracic glands of Galleria mellonella (Lepidoptera) in larval stage. Invertebrate Reproduction and Development, 37 (2): 167-170.
• Lackie, A. M., 1988. Haemocyte behavior. Advances in Insect Physiolgy, 21: 85-178.
• Levin, D. M., L. N. Breuer, S. Zhuang, S. A. Anderson, J. B. Nardi & M. R. Kanost, 2005. A Hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 35 (5): 369-380.
• Ling, E, K. Shirai, R. Kanekatsu & K. Kiguchi, 2003. Classification of larval circulating hemocytes of the Silkworm, Bombyx mori, by acridine orange and propidium iodide staining. Histochemistry and Cell Biology,120 (6): 505-511.
• Ling, E., K. Shirai, R. Kanekatsu & K. Kiguchi, 2005. Hemocyte Differentiation in the hematopoietic organs of the silkworm, Bombyx mori: Prohemocytes have the function of phagocytosis. Cell and Tissue Research, 320 (3): 535-43.
• Ling, E. & X. Q., Yu, 2006. Hemocytes from the tobocco hornworm Manduca sexta have distinct functions in phagocytosis of foreign particules and self-dead cells. Developmental and Comparative Immunology, 30: 301- 309.
• Mullett, H., N. A. Ratcliffe & A. F. Rowley, 1993. The generation and characterization of anti-insect blood cell monoclonal antibodies. Journal of Cell Science, 105: 93-100.
• Nakahara, Y., H. Matsumoto, Y. Kanamori, H. Kataoka, A. Mizoguchi, M. Kiuchi & M. Kamimura, 2006. Insulin signaling is involved in hematopoietic regulation in an insect hematopoietic organ. Journal of Insect Physiology, 52 (1): 105-11.
• Nardi, J. B., B. Pilas, E. Ujhelyi, K. Garsha & M. R. Kanost, 2003. Hematopoietic organs of Manduca sexta and hemocyte lineages. Development Genes and Evolution 213 (10): 477-91.
• Nardi, J. B., 2004. Embryonic origins of the two main classes of hemocytes granular cells and plasmatocytes in Manduca sexta. Development Genes and Evolution, 214 (1): 19-28.
• Oberlander, H., C. E. Leach & E. Shaaya, 2000. Juvenile hormone and juvenile hormone mimics inhibit proloferation in a lepidopteran imaginal disc cell line. Journal of Insect Physiology, 46: 259-265.
• Pech, L. L., & M. R. Strand, 1996. Granular cells are required for encapsulation of foreign targets by insect haemocytes. Journal of Cell Science, 109: 2053-60.
• Ratcliffe, N. A., A. F. Rowley, S. W. Fitzgerald & C. P. Rhodes, 1985. Invertebrate immunity: Basic Concepts and Recent Advances. International Review of Cytology, 97: 183-350.
• Shrivastava, S. C. & A. G. Richards, 1965. An autoradiographic study of the relation between hemocytes and connective tissue in the wax moth, Galleria mellonella. The Biological Bulletin 128: 337–345.
• Strand, M. R., 2008. The insect cellular immune response. Insect Science 15: 1-14.
• Tojo, S., F. Naganuma, K. Arakawa & S. Yokoo, 2000. Involvement of both granular cells and plasmatocytes in phagocytic reactions in the greater wax moth, Galleria mellonella. Journal of Insect Physiology, 46: 1129- 1135.
• Vilcinskas, A & P. Götz, 1999. Parasitic fungi and their interactions with the insect immune system. Advances in Parasitology, 43: 267-313.
• Willott, E., T. Trenczek, L. W. Thrwer & M. R. Kanost, 1994. Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in Manduca sexta. European Journal of Cell Biology, 65: 417-423.
• Yokoo, S., P. Götz & S. Tojo, 1995. Phagocytic activities of haemocytes separated by two simple methods from larvae of two lepidopteran species, Agrotis segetum and Galleria mellonella. Applied Entomology and Zoology, 30 (2): 343-350.