Molecular and Microscopic Detection of Microsporidia in Some

Yıl 2014, Cilt: 1 Sayı: 4, 450 - 453, 28.10.2014

Olgay Tekın Duran Sezer Kubra Sogancı Rahsan Ivgın Tunca

Öz

 

The silkworm, an easy to grow and quite precious insect, is affected by many diseases like other organisms. The microsporidia spores caused to Pebrine which is quite dangerous and causes serious economic damage. In this study, 450 silkworm eggs (Bombyx mori L.) collected from Bursa province in Turkey were fed into ten different groups until the 4th and 5th larval stages. Nine groups were fed with unwashed mulberry leaves whereas only one group was fed with washed ones. The samples at the 4th and 5th larval stages from each group were screened for N. bombycis and Vairimorpha sp. using both microscopic and molecular methods. There were no nosema spores detected in dyed samples under microscope (100Xobjective). The primers, specific for N. bombycis NBEF35F, NBEF957R and Vairimorpha sp. M1196F, M11822R classified into microsporidia, were used for detection of disease from isolated DNAs. There was no pathogens detected using molecular technique. In conclusion, considering that infection is due to horizontal transmission, the study results may demonstrate that food materials and silkworm from breeding region are safe for disease pathogens. This study is the first molecular application for detection of N. bombycis and Vairimorpha sp. in Turkey.

Anahtar Kelimeler

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Türkiye’de Bazı İpek Böceği (Bombyx mori L.) Populasyonlarında Microsporadianın Moleküler ve Mikroskobik Tespiti

Öz

 

Yetiştirilmesi kolay ve oldukça kıymetli olan ipek böceği, diğer canlılarda da olduğu gibi birçok hastalıktan etkilenmektedir. Bunlardan birisi microsporadia sporlarının neden olduğu oldukça tehlikeli ve ciddi anlamda ekonomik zarara neden olan Pebrine hastalığıdır. Bu çalışmada, Bursa ilinden toplanan 450 adet ipek böceği (Bombyx mori L.) yumurtaları 4. ve 5. larval döneme kadar 10 farklı gruba ayrılarak beslenmiştir. İlk 9 grup yıkanmamış dut yaprakları ile beslenirken 1 grup yıkanmış dut yaprakları ile beslenmiştir. 4. ve 5. larval dönemdeki örnekler N. bombycis ve Vairimorpha sp. tespiti için mikroskobik ve moleküler metotlar kullanılarak taranmıştır. Boyanmış örnekler mikroskop (100Xbüyütme) altında incelendiğinde nosema sporuna rastlanmamıştır. İzole edilmiş DNA örneklerinde Nosema bombycis’e özgü NBEF35F, NBEF957R ve aynı zamanda microsporidia içinde yer alan Vairimorpha sp. M1196F, M11822R primerleri kullanılarak hastalık taraması yapılmıştır ve hastalık patojenine rastlanmamıştır. Sonuç olarak, horizontal bulaşma göz önüne alındığında, çalışma yetiştirmenin yapıldığı bölgenin ve yiyecek materyallerinin patojen bakımından güvenli olduğunu göstermiştir.

Anahtar Kelimeler

Microsporidia, Vairimorpha, Nosema bombycis, PZR, İpekböceği (Bombyx mori L.)

Kaynakça

• Aydın, L., Gulegen, E., Girisgin, O., Kurtaraner, L., 2007. Occurrence of Nosema bombycis (Naegeli, 1857) in silkworms in Turkey, Turkish Society for Parasitology, 31 (1): 72-74
• Han, M. S. and Watanabe, H., 1987. Immunoperoxidasestaining methods of discrimination of microsporidian spores in the pebrine inspection of silkworm mother moths. Journal of Sericulture Science of Japan, 56: 431–435.
• Hayasaka, S. and Ayuzawa, C., 1987. Diagnosis of microsporidians, Nosema bombycis and closely related species by antibody-sensitized latex. Journal of Sericulture Science of Japan, 56: 169–170.
• Hatakeyama, Y. and Hayasaka S., 2002. Specific amplification of microsporidian DNA fragments using multiprimer PCR, Japan Agricultural Research Quarterly, 36 (2): 97-102.
• Hatakeyama, Y and Hayasaka, S., 2003. A new method of pebrine inspection of silkworm egg using multiprimer PCR, Journal of Invertebrate Pathology, 82:148-151.
• Iwano, H. and Ishihara, R., 1981. Inhibitions effect of several chemicals against hatching of Nosema bombycis spores, Journal of Sericulture Science of Japan, 50: 276- 281.
• Jyothi, N. B., Patil, C.S., Dass, C.M.S., 2005. Action of C arbedazmin on the development of Nosema bombycis Naegeli in silkworm Bombyx mori L, Journal of Applied Entmology, 129: 205-210.
• Kawarabata, T. and Ishihara, R., 1984. Infection and development of Nosema bombycis in cell lines of Antheraea eucalypti. Journal of Invertebrate Pathology, 44: 52-62.
• Keeling, P.J and Fast, N. M., 2002. Microsporidian: Biology and evolution of highly reduced intracellular parasites. Annual Review of Microbiology, 56: 93-116.
• Patil, C.S., Jyothi, N.B., Dass, C.M.S., 2001. Faecal pellet examination of silkworm, Bombyx mori as a diagnostic method to detect pebrine disease. Journal of Experimental Zoology, India, 4(2): 311- 315.
• Patil, C.S., Jyothi, N.B., Dass, C.M.S., 2002. Role of Nosema bombycis infected male silk moths in the venereal transmission of pebrine disease in Bombyx mori (Lep., Bombycidae). Journal of Applied Entomology, 126: 563-566.
• Ravikumar, G., Raje, U.S., Vijaya, Prakash, N.B., Rao, C.G.P., Vardhana, K. V., 2011. Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms. Journal of Invertebrate Pathology, 10(3): 193-197.
• Saharan, S. K., Bansal, A. K., Shukla, R.M., Thangavelu, K., 1992. A new method of detection of Pebrine Disease in Tasar silk moth, Antheraea mylitta Drury (Saturniidae). Journal of Research on the Lepidoptera, 31(1-2): 12-15
• Sato, R., Kobayashi, M., Watabane, H., 1981. Internal ultrastructure of spores of microsporidians isolated from the silkworm, Bombyx mori L. Journal of Invertebrate Pathology, 40: 260-265.
• Sato, R., Masaiko, K., Watanabe, H, Fujiwara, T., 1982. Serological discrimination of several kinds of microsporidian spores isolated from the silkworm, Bombyx mori. Journal of Sericulture Science of Japan, 50: 180–184.
• Singh, T., Bhat, M. M., Khan, M.A., 2012. Microsporidiosis in the Silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Pertanika Journal of Tropical Agricultural Science, 35 (3): 387 – 406.
• Şahan, U., 2011. İpek Böcekçiliği: İpekböceği yetiştirme ve ıslahı, koza üretimi, ham ipek, yumurta üretimi, hastalıklar ve dut yetiştirme. 1.Baskı, Dora yayınları, Bursa, 150 p. (Lecturer notebook. In Turkish).
• Undeen, A. H., Solter, L. F., 1996. The Sugar Content and Density of Living and Dead Microsporidian (Protozoa: Microspora) Spores. Journal of Invertebrate Pathology, 67(1): 80–91.