İki çalı çekirgesi üzerinde doğal bağışıklık parametrelerinden fenoloksidaz aktivitesi ile litik aktivitenin ve hemolimfteki protein konsantrasyonunun yöntemsel olarak belirlenmesi

Yıl 2016, Cilt: 5 Sayı: 1, 51 - 62, 01.06.2016

Hasan Sevgili

Öz

Omurgasızlarda doğal bağışıklık sistemi patojenlere karşı aktive olan oldukça çeşitli ve özgün tepkiler içeren humoral aktivitelere sahiptir. Böceklerde, bu önemli anahtar immün parametrelerden ikisi fenoloksidaz yolağı ve litik aktivitedir. Lizozimler bakteri hücre duvarındaki peptidoglikanları çözen bir grup enzim olup pek çok böcek grubunda bakteri savunması ile bağlantılıdır. Litik aktivite bakteri hücrelerinin hemolimfteki tahrip yeteneğinin in vitro olarak ölçülmesiyle saptanabilir. Fenoloksidaz omurgasızların immün sisteminde anahtar bir enzim olup, hemolimfte yabancı bir cisim saptandığında salgılanır. Dolayısıyla fenoloksidaz aktivitesi (PO) organizmaların yabancı istilacılara karşı savunmada rol alan immün sistemin yeteneğiyle ilişkilidir. Bu çalışmada ilk kez barbitistin (Orthoptera: Phaneropterinae) çalıçekirgelerinden iki türde (Isophya speciosa, Poecilimon similis), bu iki anahtar immün parametrenin ölçülmesi yöntemi sınanmıştır. Her iki tür için aynı yaştaki 10 erkek 10 dişiden mikroşırınga ile çekilen farklı miktarlardaki hemolimf konsantrasyonundaki (3, 6, 8, 10 μl) PO ve litik aktiviteler optik yoğunluk bakımından mikroplaka okuyucu ile ölçülmüştür. Buna ilave olarak her iki tür için hemolimfteki protein konsantrasyonu da belirlenmiştir. Çalışma sonucunda daha önce grillid ve bazı diğer böcek gruplarında uygulanan yöntemlerin, küçük değişikliklerle çalıçekirgeleri için de kullanılabileceği önerilmiştir. Ancak, hemolifteki toplam litik aktivitenin ölçümünde daha iyi sonuçlar elde etmek için daha fazla hemolimf miktarına gerek duyulduğu anlaşılmıştır. Bu çalışma barbitistin çekirge türleri üzerinde ileride yapılacak immün çalışmalar için yol gösterici olacaktır.

Anahtar Kelimeler

Doğal bağışıklık, fenoloksidaz aktivitesi, litik aktivite, protein konsantrasyonu, hemolimf, çalı çekirgesi

Kaynakça

• Adamo, S. A., 2004. Estimating disease resistance in insects: phenoloxidase and lysozyme-like activity and disease resistance in the cricket Gryllus texensis. Journal of Insect Physiology, 50: 209-216.
• Adamo S. A, Jensen M., Younger M., 2001. Changes in lifetime immuno competence in male and female Gryllus texensis (formerly G. integer): trade-offs between immunity and reproduction. Animal Behavior. 62:417–425.
• Adamo, S. A., Lovett, M.M.E., 2011. Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket Gryllus texensis. Journal of Experimental Biology, 214: 1997-2004.
• Asada, N., Yokoyama, G., Kawamato, N., Norioka, S., Hatta, T., 2003. Prophenol oxidase A3 in Drosophila melanogaster: activation and the PCR based cDNA sequence. Biochemical Genetics, 41: 151-163.
• Asano, T., Ashida, M., 2001. Cuticular pro-phenoloxidase of the silk worm, Bombyx mori. The Journal of Biological Chemistry, 276: 111000-11112.
• Bailey, N. W., 2011. A test of the relationship between cuticular melanism and immune function in wild-caught Mormon crickets. Physiological Entomology, 36: 155-164.
• Beckage, N. E. 2008. Insect Immunology, Academic Press/Elsevier.
• Berggren, A. 2010. Testing the effect of individual color morphology on immune response in bush-crickets. Insect Science, 17: 400-405.
• Bradford, M. M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248–254.
• Cerenius L., Soderhall, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunological Reviews, 198: 116-126.
• Cerenius, L., Lee, B. L., Söderhall, K., 2008. The proPO-system: pros and cons for its role in invertebrate immunity. Trends in Immunology, 29: 263-271.
• Cooper, M. D., Alder, M. N., 2006. The evolution of adaptive immune system. Cell, 124: 815-822.
• Cotter, S.C., Kruk, L. E. B., Wilson, K., 2004. Cost of resistance: genetic correlations and potential trade-offs in an insect immune system. Journal of Evolutionary Biology, 17: 421–429.
• Eades, D.C., Otte, D., Cigliano, M. M., Braun, H., 2015. Orthoptera Species File. Version 5.0/5.0. [17.10.2015]. <http://Orthoptera.SpeciesFile.org>.
• Fedorka, M. K, Zuk, M., Mousseau, T. A., 2004. Immune suppression and the cost of reproduction in the ground cricket, Allonemobius socius. Evolution, 58: 2478-2485.
• Fedorka, K. M., Mousseau, T. A., 2007. Immune system activation affects the male sexual signal and reproductive potential in crickets. Behavioral Ecology, 18: 231-235.
• Fedorka, K. M., Copeland, E. K., Winterhalter, W. E., 2013. Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects. The Journal of Experimental Biology, 216: 4005-4010.
• Fedorka, K. M., Sevgili, H., 2014. The influence of nuptial feding and sperm transfer on the immunological cost of reproduction in the ground cricket Allonemobius socius. Physiological Entomology, 39: 89-93.
• Fredrick, W. S., Ravichandran, S., 2012. Hemolymph proteins in marine crustaceans. Asian Pacific Journal of Tropical Biomedicine, 2: 496-502.
• Fujimoto, K., Okino, N., Kawabata, S., Ohnishi, E., 1995. Nucleotide sequene of the cDNA encoding pro-phenoloxidase A1 of Drosophila melanogaster. Proceedings of the National Academy of Sciences, 92: 7769-7773.
• Ganz, T., 2003. The role of antimicrobial peptides in innate immunity. Integrative and Comparative Biology, 43: 300–304.
• Gershman, S. N., 2008. Sex-specific differences in immunological costs of multiple mating in Gryllus vocalis field crickets. Behavioral Ecology, 19 (4): 810-815.
• Gershman, S. N., Barnett, C. A. Pettinger, A: M., Weddl, C. B., Hunt, J., Sakaluk, S. K., 2010. Give ‘til it hurts: trade-offs between immunity and male reproductive effort in the decorated cricket, Gryllodes sigillatus. Journal of Evolutionary Biology, 23 (4): 829-839.
• Hoffmann, J. A., 2003. The immune response of Drosophila. Nature, 426: 33–38.
• Hoffmann, J. A., Reichart, J. M., 2002. Drosophila innate immunity: an evolutionary perspective. Nature Immunology, 3: 121-126.
• Hoffmann, J. A., Reichart, J. M., Hetru, C., 1996. Innate immunity in higher insects. Current Opinion in Microbiology, 8: 8-13.
• Imler, J. L., Hoffmann, J. A., 2000. Signaling mechanisms in the antimicrobial host defense of Drosophila. Current Opinion in Microbiology, 3: 16-22.
• Iwanaga, S., Lee, B. L., 2005. Recent advances in the innate immunity of invertebrate animals. Journal of Biochemistry and Molecular Biology, 38: 128-150.
• Jacot, A., Scheuber, H., Kurtz, J., Brinkhof, M. W. G., 2005. Juvenile immune system activation induces a costly up-regulation of adult immunity in field crickets Gryllus campestris. Proceedings of the Royal Society of London B: Biological Science, 272: 63–69.
• Janeway, C.A., Medzhitov, R., 2002. Innate immunity recognition. Annual Review of Immunology, 20: 197-216.
• Jiang, H., Wang, Y., Ma C., Kanost, M. R., 1997. Submit composition of pro-phenloxidase from Manduca sexta: molecular cloning of subunit proPO-P1. Insect Biochemistry and Molecular Biology, 27: 835-850.
• Jiang, H., Wang, Y., Yu, X., Kanost, M. R., 2003. Propehnoloxidase-activating proteinase-2 from hemolymph of Manduca sexta. The Journal of Biological Chemistry, 278: 3552-3561.
• Kawabata, T., Yashuara, Y., Ocha, M., Matsuura, S., Ashida, M., 1995. Molecular cloning of insect pro-phenoloxidase: a copper-containing protein homologous to arthropod hemocyanin. Proceedings of the National Academy of Sciences USA, 92: 7774-7778.
• Lee, K. P., Simpson, S. J., Wilson, K., 2008. Dietary protein-quality influences melanization and immune function in an insect. Functional Ecology, 6: 1052-1061.
• Lee, K. Y., Zhang, R., Kim, M. S., Park, J. W., Park, H. Y., Kawabata, S., Lee, B. L., 2002. A zymogen form of masquerade-like serin proteinase homologue is cleaved during pro-phenoloxidase activation by Ca++ in coleopteran and Tenebrio molitor larvae. European Journal of Biochemistry, 269: 4375-4383.
• Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., Hoffmann, J. A., 1996. The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 86: 973-983.
• Moret, Y., Schmid-Hempel, P., 2001. Immune defence in bumblebee offspring. Nature, 414: 506.
• Nappi, A. J., Christensen, B. M., 2005. Melanogenesis and associated cytotoxic reactions: Applications toinsect innate immunity. Insect Biochemistry and Molecular Biology, 35: 443-459.
• Park, Y., Kim, Y., Stanley, D., 2011. Cellular immunosenescense in adult male crickets, Gryllus assimilis. Archives of Insect Biochemistry and Physiology, 76: 185-194.
• Rantala, M.J., Kortet, R., 2003. Courtship song and immune function in the field cricket Gryllus bimaculatus? Biological Journal of the Linnean Society 79: 503–510.
• Rantala, M.J., Kortet, R., 2004. Male dominance and immunocompetence in a field cricket. Behavioral Ecology, 15 (2): 187-191.
• Rantala, M. J., Roff, D. A., 2005. An analysis of trade-offs in immune function, body size and development time in the Mediterranean Field Cricket, Gryllus bimaculatus. Functional Ecology, 19: 323-330.
• Rantala, M. J., Roff, D. A., 2006. Analysis of the importance of genotypic variation, metabolic rate, morphology, sex and developmenttime on immune function in the cricket, Gryllus firmus. Journal of Evolutionary Biology, 19: 834-843.
• Rolff, J., 2002. Bateman’s principle and immunity. Proceedings of the Royal Society of London B: Biological Science, 269: 867-872.
• Schneider, P. M., 1985. Purification and properties of three lysozymes from haemolymph of the cricket, Gryllus bimaculatus (De Geer). Insect Biochemistry, 15: 463-470.
• Schnitger, A. K., Kafatos, F. C., Osta, M. A., 2007. The melanizaton reaction is not required for survival of Anopheles gambiae mosqutoes after bacterial infections. The Journal of Biological Chemistry, 282: 21884-21888.
• Sevgili, H., 2004. Türkiye Isophya Brunner von Wattenwyl (Orthoptera: Tettigoniidae: Phaneropterinae) türlerinin revizyonu. Doktora Tezi, Hacettepe Üniversitesi, Ankara, 387 s.
• Silva, C. C. A., 2002. Activation of prophenoloxidase and removal of Bacillus subtilis from the hemolymph of Acheta domesticus (L.) (Orthoptera: Gryllidae). Neotropical Entomology, 31: 487-491.
• Silva, C. C. A., Dunphy, G. B., Rau, M. E., 2000. Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. Developmental & Comparative Immunology, 24: 367-379.
• Srygley, R. B., 2012. Age and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae). Environmental Entomology, 41: 166-171.
• Srygley, R. B., Lorch, P. D., Simpson, S. J., Sword, G. A., 2009. Immediate protein dietary effects on movement and the generalised immunocompetence of migrating Mormon crickets Anabrus simplex (Orthoptera: Tettigoniidae). Ecological Entomology, 34: 663-668.
• Srygley, R. B., Lorch P. D., 2011. Weakness in the band: nutrient mediated trade-offs between migration and immunity of Mormon crickets. Animal Behavior, 81: 395-400.
• Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C., 2006. Two proteases defining a melanization cascade in the immune system of Drosophila. The Journal of Biological Chemistry, 281: 28097-28104.
• Tregenza, T., Simmons, L. W., Wedell, N., Zuk, M., 2006. Female preference for male courtship song and its role as a signal of immune function and condition. Animal Behavior, 27: 809-818.
• Tsakas, S., Marmaras, V. J., 2010. Insect immunity and its signalling: an overview. Invertebrate Survival Journal, 7: 228-238.
• Tunaz, H., 2004. Böceklerde bağışıklık mekanizması. KSÜ Fen ve Mühendislik Dergisi, 7(2): 78-82.
• Zhao, P., Li, J., Wang, Y., Jiang, H., 2007. Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase. Insect Biochemistry and Molecular Biology, 37: 952-959.
• Zuk, M., Simmons, L. W., Rotenberry, J. T., Stoer, A. M., 2004. Sex differences in immunity in two species of field crickets. Canadian Journal of Zoology, 82: 627-634.