Pebrine disease is the most important and dangerous disease of silkworm caused by Nosema bombycis as an obligate intracellular parasitic fungus. It has caused tremendous economic losses in the silk industry in recent years. Given the fact that light microscopy method (with low accuracy) is the only method for diagnosing pebrine disease in the country, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) methods were adopted in this study for accurate morphological identification of the spores causing pebrine disease. Infected larvae and mother moth samples were collected from several farms (Parand, Parnian, Shaft, and Iran Silk Research Center in Gilan province, Iran). The spores were then purified using the sucrose gradient method. From each region, 20 and 10 samples were prepared for SEM and TEM analysis, respectively. In addition, an experiment was performed to evaluate the symptoms of pebrine disease by treating fourth instars with the spores purified for the present study, along with a control group. The results of SEM analysis showed that the mean±SD length and width of spores were 1.99±0.25 to 2.81±0.32 μm, respectively. Based on the obtained results, the size of spores was smaller than the Nosema bombycis (N. bombycis) as the classic species that cause pebrine disease. In addition, transmission electron microscopy (TEM) pictures showed that the grooves of the adult spores were deeper than those of other Nosema species, Vairomorpha, and Pleistophora, and resembled N. bombycis in other studies. Examination of pathogenicity of the studied spores indicated that the disease symptoms in controlled conditions were similar to those in the sampled farms. The most important symptom in fourth and fifth instrars were the small size and no growth in the treatment group compared with the control group. Findings of SEM and TEM analysis showed better morphological and structural details of parasite compared with light microscopy, and demonstrated that the studied species were a native strain of N. bombycis specific to Iran, whose size and other characteristics were unique and introduced for the first time in this study. |
- Bhat IA, Buhroo ZI, Bhat MDA. Microsporidiosis in silkworms with particular reference to mulberry silkworm (Bombyx Mori L.). Int J Entomol Res. 2017;2:01-9.
- Abedi Parijani A, Motamed M, Kavusi Kalashmi M, editors. The role of silkworm breeding in job creation. Proceedings of Rural Development. 2015: Mashhad, Iran.
- Biabani M, Nematollahian S. A review of the prevalence of Pebrine disease in different provinces in 2017 and its prevention and control methods in Iran. In: Mirhoseini S, editor. Proceedings of the first national silk conference of Iran. Rasht, Iran: University of Guilan; 2017. p. 52-60.
- Keeling PJ, Luker MA, Palmer JD. Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol. 2000;17(1):23-31.
- Franzen C. How do microsporidia invade cells? Folia Parasitol (Praha). 2005;52(1-2):36-40.
- Wang JY, Chambon C, Lu CD, Huang KW, Vivares CP, Texier C. A proteomic-based approach for the characterization of some major structural proteins involved in host-parasite relationships from the silkworm parasite Nosema bombycis (Microsporidia). Proteomics. 2007;7(9):1461-72.
- Hanumappa H. Sericulture for Rural Development. Bombay, India: Himalaya Publishing House; 1968.
- Nataraju B, Stahyaprasad K, Manjunath D, Aswani Kumar C. Silkworm crop protection. Member Secretary ed. Bangalore, India: Central Silk Board; 2005.
- Sato R, Kobayashi M, Watanabe H. Internal ultrastructure of spores of microsporidans isolated from the Silkworm, Bombyx mori. J Invertebr Pathol. 1982;40(2):260-5.
- Sato R, Kobayashi M, Watanabe H, Fujiwara T. Serological discrimination of several kinds of microsporidian spores isolated from the silkworm, Bombyx mori, by an indirect fluorescent antibody technique. J Sericult Sci Jpn. 1981;50(3):180-4.
- Undeen AH. Microsporidia (Protozoa): A Handbook of Biology and Research Technniques: Oklahoma State University; 1997.
- Chakrabarty S, Saha A, Manna B, Bindroo B. Light and electron microscopy of nosema ricini (microsporidia: Nosematidae), the causal pathogen of pebrine disease in eri silkworm: Life cycle and cross-infectivity. Appl Biol Res. 2012;14(1):1-14.
- Ptaszyńska AA, Borsuk G, Mułenko W, Demetraki-Paleolog J. Differentiation of Nosema apis and Nosema ceranae spores under Scanning Electron Microscopy (SEM). J Apic Res. 2014;53(5):537-44.
- Fries I, Feng F, da Silva A, Slemenda SB, Pieniazek NJ. Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eur J Protisto. 1996;32(3):356-65.
- Fries I, Martín R, Meana A, García-Palencia P, Higes M. Natural infections of Nosema ceranae in European honey bees. J Apic Res. 2006;45(4):230-3.
- Chen YP, Evans JD, Murphy C, Gutell R, Zuker M, Gundensen-Rindal D, et al. Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. J Eukaryot Microbiol. 2009;56(2):142-7.
- Fries I, Chauzat M-P, Chen Y-P, Doublet V, Genersch E, Gisder S, et al. Standard methods for Nosema research. J Apic Res. 2013;52(1):1-28.
- Vavra J, Barker R. The observation of microsporidian spores using the scanning electron microscope: an evaluation of techniques. Folia Parasitol. 1980;27(2):97-102.
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