شناسایی باکتری‌های انتروکوکوس ازدستگاه گوارش زنبور عسل کوچک Apis florea Fabricius, 1973 (Hymenoptera: Apidae)

نوع مقاله: مقاله کامل، انگلیسی

نویسندگان

1 Ph. D Student, Department of Agricultural Entomology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Animal Science Research Institute of Iran, Agricultural Research, Edcucation and Extention Organization (AREEO), Karaj, Iran.

3 Department of Insect Taxonomy Research, Institute of Plant Protection, Tehran , Iran

4 Department of Agricultural Entomology, Science and Research Branch, Islamic Azad University, Tehran, Iran

5 Department of honeybee, Animal Science Research Institute of Iran, Agricultural Research, Edcucation and Extention Organization (AREEO), Karaj, Iran

چکیده

باکتری‌هایلاکتیک اسید با گونه‌های مختلف جنسApis  روابط همزیستی دارند که می‌توانند اثرات زیادی بر روی میزبانشان داشته باشند. در این پژوهش 1400 زنبور عسل کارگر مربوط به 14 کلنی از استانهای سیستان و بلوچستان، کرمان، هرمزگان، فارس، کهکیلویه و بویر احمد، بوشهر، خوزستان و ایلام جمع آوری گردید. نمونه‌ها در لوله‌های استریل حاوی نرمال سالین قرار داده شد، سپس جداسازی باکتری‌ها از دستگاه گوارش زنبور عسل کوچک با استفاده از محیط کشت‌های اختصاصی انجام گرفت. برای شناسایی، تست‌های بیوشیمیایی و سپس استخراج DNA کلنی‌های جدا شده انجام شد. تشخیص مولکولی کلنی‌ها با روش تعیین توالی ژن 16S rDNA و با استفاده از پرایمرهای اختصاصی انجام گرفت. پس از تعیین توالی و شناسایی نمونه‌ها، با به کار بردن نرم افزارهای مختلف ژنتیکی باکتری‌هایی که با هم از نظر ژنتیکی فاصله داشتند در بانک جهانی ژن (NCBI) ثبت گردید. در این آزمایش با تعیین توالی 30 کلنی از باکتری‌های جداسازی شده از دستگاه گوارش زنبور عسل کوچک، در مجموع 5 سویه مختلف متعلق به 3 گونه Enterococcus faecalis، Enterococcus faeciumوEnterococcus hiraeشناسایی شدند. با توجه به نتایج بدست آمده زنبورهای عسل کوچک در ایران از لحاظ باکتری‌های انتروکوکوس موجود در دستگاه گوارش به 4 گروه طبقه‌بندی شدند. به طور کلی جداسازی و شناسایی این باکتری‌ها می تواند قدم مثبتی در جهت افزایش درجه سلامت مواد غذایی مورد استفاده انسان‌ها و حیوانات باشد. 

کلیدواژه‌ها


عنوان مقاله [English]

Identification of Enterococcus bacteria in gastrointestinal tract of dwarf honey bee, Apis florea Fabricius, 1973 (Hymenoptera: Apidae)

نویسندگان [English]

  • Shabnam Parichehreh 1
  • Gholamhosein Tahmasbi 2
  • Alimorad Sarafrazi 3
  • Sohrab Imani 4
  • Naser Tajabadi 5
1 دانشگاه آزاد اسلامی واحد علوم و تحقیقات تهران، ، ایران تهران
2 بخش زنبور عسل، موسسه تحقیقات علوم دامی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران
3 موسسه تحقیقات گیاهپزشکی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران
4 دانشگاه آزاد اسلامی واحد علوم و تحقیقات تهران، تهران، ایران
5 بخش زنبور عسل، موسسه تحقیقات علوم دامی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران
چکیده [English]

Apis species that engage in symbiotic association with Lactic Acid Bacteria (LAB), have diverse functions on their hosts. This study was intended to isolate and identify aeoccus bacteria living in the gastrointestinal tract of Asian dwarf honey bee, Apis florea,in Iran. One hundred isolates were Gram-stained and tested for catalase reaction. By using bacterial universal primers, the 16S rDNA gene of bacterial colonies was amplified. 16S rDNA genes from thirty bacteria were sequenced. Phylogenetic analysis showed that Enterococcus flora in the gastrointestinal tract of A. florea, contained five phenotypes which classified in the species E. faecium, E. faecalis and E. hirae.  Based on the specific association between bacteria and A. florea, we divided the Asian dwarf honey bee populations into four categories. 

کلیدواژه‌ها [English]

  • Apis florea
  • Enterococcus bacteria
  • LAB
  • symbiotic association

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Bauer, S., Tholen, A., Overmann, J. & Brune, A. (2000) Characterization of abundance and diversity of LAB in the hindgut of wood-and soil-feeding termites by molecular and culture-dependent techniques. Archives of Microbiology 173(2), 126-137.

Casaus, M. P., Herranz, C., Nes, I. F., Hernández, P. E. & Cintas,L. M. (2001) Bacteriocins of Lactic Acid Bacteria. Food Science and Technology 7(4), 281-305.

Castro, M. S., Molina, M. A., Azpiroz, M. B., Díaz, A. M., Ponzio, R., Sparo, M. D., Manghi, M. A. & Canellada, A. M. (2016) Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells. Journal of Applied Microbiology 121(4), 1117-1129.

Dillon, R. J. & Dillon, V. M. (2004) The gut bacteria of insects: nonpathogenic interactions. Annual Reviews in Entomology 49(1), 71-92.

Engel, P. & Moran, N. A. (2013) The gut microbiota of insects–diversity in structure and function. FEMS Microbiology Reviews 37(5), 699-735.

FAO/WHO. (2002) Guidelines for the Evaluation of Probiotics in Food: Joint FAO/WHO Working Group meeting. London Ontario, Canada. Available: http://www.who.int/foodsafety/publications/fs_management/probiotics2/en

Franz, C. M., Huch, M., Abriouel, H., Holzapfel, W. & Gálvez, A. (2011) Enterococci as probiotics and their implications in food safety. International Journal of Food Microbiology 151(2), 125-140.

Gilliam, M. (1979) Microbiology of pollen and bee bread: the genus Bacillus. Apidologie 10(3), 269-274.

Hirose, E., Panizzi, A. R., De Souza, J. T., Cattelan, A. J. & Aldrich, J. R. 2006. Bacteria in the gut of southern green stink bug (Heteroptera: Pentatomidae). Annals of the Entomological Society of America 99, 91-95.

Hooper, L. V., Littman, D. R. & Macpherson, A. J. (2012) Interactions between the microbiota and the immune system. Science 336(6086), 1268-1273.

Hroncova, Z., Havlik, J., Killer, J., Doskocil, I., Tyl, J., Kamler, M., Titera, D., Hakl, J., Mrazek, J., Bunesova, V. & Rada, V. (2015) Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location. PloS One 10(3), 1-17.

Ibarguren, C., Raya, R. R., Apella, M. C. & Audisio, M. C. (2010) Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains. The Journal of Microbiology 48(1), 44-52.

Kačániová, M., Chlebo, R., Kopernický, M. & Trakovicka, A. (2004) Microflora of the honeybee gastrointestinal tract. Folia microbiologica 49(2), 169-171.

Kerry, M., Oliver, L., Patrick, H., Degnan, T., Gaelen, R. & Nancy, A. (2010) Facultative symbionts in aphid and horizontal transfer of ecologically important, Annual Review of Entomology 55, 247-266.

Kwong, W. K. and Moran, N. A. (2016) Gut microbial communities of social bees. Nature Reviews Microbiology 14(6), 374-384.

Lane, D. (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt E and Goodfellow M, eds). J Wiley and Sons, Chichester 115-175.

Olofsson, T. C., Vásquez, A., Sammataro, D. & Macharia, J. (2011) A scientific note on the lactic acid bacterial flora within the honeybee subspecies Apis mellifera (Buckfast), A. m. scutellata, A. m. mellifera, and A. m. monticola. Apidologie 42(6), 696-699.

Olofsson, T. C. & Vásquez, A. (2008) Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Current Microbiology 57(4), 356-363.

Parichehreh, S., Farshineh, A. M. & Fallahzadeh, M. (2014) Study and comparison of morphological characteristics of dwarf honey bees, Apis florea F. (Hymenoptera, Apidae) in Iran.Journal of Entomological Research 5( 4), 315-330.

Pidiyar, V. J., Jangid, K., Patole, M. S. & Shouche, Y. S. (2004) Studies on cultured and uncultured microbiota of wild Culex quinquefasciatus mosquito midgut based on 16S ribosomal RNA gene analysis. The American Journal of Tropical Medicine and Hygiene 70(6), 597-603.

Sepúlveda, D. A., ZepedaPaulo, F., Ramírez, C. C., Lavandero, B. & Figueroa, C. C. (2016) Diversity, frequency, and geographic distribution of facultative bacterial endosymbionts in introduced aphid pests. Insect Science 0,1-11.

Singh, S. T., Priya, N. G., Kumar, J., Rana, V. S., Ellango, R., Joshi, A., Priyadarshini, G., Asokan, R. & Rajagopal, R. (2012) Diversity and phylogenetic analysis of endosymbiotic bacteria from field caught Bemisia tabaci from different locations of North India based on 16S rDNA library screening. Infection, Genetics and Evolution 12(2), 411-419.

Tahmasebi, G., Ebadi, R., Tajabadi, N., Akhondi, M. & Faraj, S. (2002) The Effects of Geographical and Climatological Conditions on the morphological Variation and Separation of Iranian Small Honeybee (Apis florea F.) Populations. JWSS-Isfahan University of Technology 6(2), 169-176.

Tajabadi, N., Mardan, M., Saari, N., Mustafa, S., Bahreini, R. & Manap, M. Y. A. (2013) Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee. Brazilian Journal of Microbiology 44(3), 717-722.

Tajabadi, N., Mardan, M., Manap, M. Y. A., Shuhaimi, M., Meimandipour, A. & Nateghi, L. (2011) Detection and identification of Lactobacillus bacteria found in the honey stomach of the giant honeybee Apis dorsataApidologie 42(5), 642-649.

Tajabadi, M., Heidari Nasrabadi, M. & Jafari, P. (2010) Health and Iran traditional dairy products. Proceedings of 1st National Conference on Probiotics and Functional foods 30-39.

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24(8), 1596-1599.

Bauer, S., Tholen, A., Overmann, J. & Brune, A. (2000) Characterization of abundance and diversity of LAB in the hindgut of wood-and soil-feeding termites by molecular and culture-dependent techniques. Archives of Microbiology 173(2), 126-137.

Casaus, M. P., Herranz, C., Nes, I. F., Hernández, P. E. & Cintas,L. M. (2001) Bacteriocins of Lactic Acid Bacteria. Food Science and Technology 7(4), 281-305.

Castro, M. S., Molina, M. A., Azpiroz, M. B., Díaz, A. M., Ponzio, R., Sparo, M. D., Manghi, M. A. & Canellada, A. M. (2016) Probiotic activity of Enterococcus faecalis CECT7121: effects on mucosal immunity and intestinal epithelial cells. Journal of Applied Microbiology 121(4), 1117-1129.

Dillon, R. J. & Dillon, V. M. (2004) The gut bacteria of insects: nonpathogenic interactions. Annual Reviews in Entomology 49(1), 71-92.

Engel, P. & Moran, N. A. (2013) The gut microbiota of insects–diversity in structure and function. FEMS Microbiology Reviews 37(5), 699-735.

FAO/WHO. (2002) Guidelines for the Evaluation of Probiotics in Food: Joint FAO/WHO Working Group meeting. London Ontario, Canada. Available: http://www.who.int/foodsafety/publications/fs_management/probiotics2/en

Franz, C. M., Huch, M., Abriouel, H., Holzapfel, W. & Gálvez, A. (2011) Enterococci as probiotics and their implications in food safety. International Journal of Food Microbiology 151(2), 125-140.

Gilliam, M. (1979) Microbiology of pollen and bee bread: the genus Bacillus. Apidologie 10(3), 269-274.

Hirose, E., Panizzi, A. R., De Souza, J. T., Cattelan, A. J. & Aldrich, J. R. 2006. Bacteria in the gut of southern green stink bug (Heteroptera: Pentatomidae). Annals of the Entomological Society of America 99, 91-95.

Hooper, L. V., Littman, D. R. & Macpherson, A. J. (2012) Interactions between the microbiota and the immune system. Science 336(6086), 1268-1273.

Hroncova, Z., Havlik, J., Killer, J., Doskocil, I., Tyl, J., Kamler, M., Titera, D., Hakl, J., Mrazek, J., Bunesova, V. & Rada, V. (2015) Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location. PloS One 10(3), 1-17.

Ibarguren, C., Raya, R. R., Apella, M. C. & Audisio, M. C. (2010) Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains. The Journal of Microbiology 48(1), 44-52.

Kačániová, M., Chlebo, R., Kopernický, M. & Trakovicka, A. (2004) Microflora of the honeybee gastrointestinal tract. Folia microbiologica 49(2), 169-171.

Kerry, M., Oliver, L., Patrick, H., Degnan, T., Gaelen, R. & Nancy, A. (2010) Facultative symbionts in aphid and horizontal transfer of ecologically important, Annual Review of Entomology 55, 247-266.

Kwong, W. K. and Moran, N. A. (2016) Gut microbial communities of social bees. Nature Reviews Microbiology 14(6), 374-384.

Lane, D. (1991) 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt E and Goodfellow M, eds). J Wiley and Sons, Chichester 115-175.

Olofsson, T. C., Vásquez, A., Sammataro, D. & Macharia, J. (2011) A scientific note on the lactic acid bacterial flora within the honeybee subspecies Apis mellifera (Buckfast), A. m. scutellata, A. m. mellifera, and A. m. monticola. Apidologie 42(6), 696-699.

Olofsson, T. C. & Vásquez, A. (2008) Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Current Microbiology 57(4), 356-363.

Parichehreh, S., Farshineh, A. M. & Fallahzadeh, M. (2014) Study and comparison of morphological characteristics of dwarf honey bees, Apis florea F. (Hymenoptera, Apidae) in Iran.Journal of Entomological Research 5( 4), 315-330.

Pidiyar, V. J., Jangid, K., Patole, M. S. & Shouche, Y. S. (2004) Studies on cultured and uncultured microbiota of wild Culex quinquefasciatus mosquito midgut based on 16S ribosomal RNA gene analysis. The American Journal of Tropical Medicine and Hygiene 70(6), 597-603.

Sepúlveda, D. A., ZepedaPaulo, F., Ramírez, C. C., Lavandero, B. & Figueroa, C. C. (2016) Diversity, frequency, and geographic distribution of facultative bacterial endosymbionts in introduced aphid pests. Insect Science 0,1-11.

Singh, S. T., Priya, N. G., Kumar, J., Rana, V. S., Ellango, R., Joshi, A., Priyadarshini, G., Asokan, R. & Rajagopal, R. (2012) Diversity and phylogenetic analysis of endosymbiotic bacteria from field caught Bemisia tabaci from different locations of North India based on 16S rDNA library screening. Infection, Genetics and Evolution 12(2), 411-419.

Tahmasebi, G., Ebadi, R., Tajabadi, N., Akhondi, M. & Faraj, S. (2002) The Effects of Geographical and Climatological Conditions on the morphological Variation and Separation of Iranian Small Honeybee (Apis florea F.) Populations. JWSS-Isfahan University of Technology 6(2), 169-176.

Tajabadi, N., Mardan, M., Saari, N., Mustafa, S., Bahreini, R. & Manap, M. Y. A. (2013) Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee. Brazilian Journal of Microbiology 44(3), 717-722.

Tajabadi, N., Mardan, M., Manap, M. Y. A., Shuhaimi, M., Meimandipour, A. & Nateghi, L. (2011) Detection and identification of Lactobacillus bacteria found in the honey stomach of the giant honeybee Apis dorsataApidologie 42(5), 642-649.

Tajabadi, M., Heidari Nasrabadi, M. & Jafari, P. (2010) Health and Iran traditional dairy products. Proceedings of 1st National Conference on Probiotics and Functional foods 30-39.

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24(8), 1596-1599.