Molecular Characterization of $Micrococcus$ sp. Associated with Insects and Their Virulence Against $Galleria$ $mellonella$ (Lepidoptera: Pyralidae)

Yıl 2024, , 14 - 25, 29.06.2024

Ali Sevim

https://doi.org/10.33484/sinopfbd.1344047

Öz

So far, more than 100 species of bacteria are known to cause disease in arthropods. Entomopathogenic bacteria have been used for many years in the microbial control of insect pests due to reasons such as cheapness, ease of mass production, host specificity, safety, and persistence in the environment. In this study, six (6) bacterial strains isolated from various insect samples ($Malacosoma$ sp. (Lepidoptera: Lasiocampidae), $Ogcodocera$ sp. (Diptera: Bombyliidae) and $Orgyia$ sp. (Lepidoptera: Erebidae)) were initially identified as $Micrococcus$ sp. at morphological level. These bacterial strains were then identified by 16S rRNA sequence analysis at the molecular level. In addition, the mortality effects of these bacterial strains against $Galleria$ $mellonella$ (Lepidoptera: Pyralidae) larvae were determined. Six bacterial strains (MK-5, AS-2, AS-3, AS-4, BB-1, and BB-5) were identified at the genus level as $Micrococcus$ sp. As a result of pathogenicity tests against $G.$ $mellonella$ larvae, only the MK-5 strain caused a 70% mortality rate, while the other strains did not cause a significant mortality rate. It is thought that the obtained results could be useful in identifying symbiotic bacteria associated with insects and determining their pathogenic properties

Anahtar Kelimeler

$Malacosoma$ sp., $Ogcodocera$ sp., $Orgyia$ sp., $Micrococcus$, 16S rRNA, virulence

Böceklerle İlişkili $Micrococcus$ sp. Türlerinin Moleküler Karakterizasyonu ve $Galleria$ $mellonella$ (Lepidoptera: Pyralidae)’ya Karşı Virülansları

Öz

Şimdiye kadar 100’den fazla bakteri türünün eklem bacaklılarda hastalık oluşturduğu bilinmektedir. Entomopatojenik bakteriler ucuz olmaları, kitle üretimindeki kolaylık, konak spesifikliği, güvenlik ve çevrede kalıcılık gibi nedenlerden ötürü zararlı böceklerle mikrobiyal mücadelede uzun yıllardan beri kullanılmaktadır. Bu çalışmada çeşitli böcek örneklerinden ($Malacosoma$ sp. (Lepidoptera: Lasiocampidae), $Ogcodocera$ sp. (Diptera: Bombyliidae) ve $Orgyia$ sp. (Lepidoptera: Erebidae)) izole edilen altı (6) adet bakteri suşu ilk etapta morfolojik olarak $Micrococcus$ sp. olarak tanımlanmıştır. Daha sonra bu bakteri suşlarının 16S rRNA sekans analizi ile moleküler seviyede tanımlanmaları gerçekleştirilmiştir. Ayrıca bu bakteri suşlarının $Galleria$ $mellonella$ (Lepidoptera: Pyralidae) larvalarına karşı öldürücülük etkileri belirlenmiştir. Altı adet bakteri suşu da (MK-5, AS-2, AS-3, AS-4, BB-1 ve BB-5) $Micrococcus$ sp. olarak cins düzeyinde tanımlanmıştır. $G.$ $mellonella$ larvalarına karşı patojenite testleri sonucunda ise sadece MK-5 suşu %70 ölüm oranına neden olmuş diğer suşlar önemli derecede ölüm oranına neden olmamıştır. Elde edilen sonuçların böceklerle ilişkili simbiyotik bakterilerin tanımlanmasında ve patojenik özelliklerinin belirlenmesinde faydalı olacağı düşünülmektedir.

Anahtar Kelimeler

$Malacosoma$ sp., $Ogcodocera$ sp., $Orgyia$ sp., $Micrococcus$, 16S rRNA, virülans

Kaynakça

• Gurung, K., Wertheim, B., & Salles, J. (2019). The microbiome of pest insects: It is not just bacteria. Entomologia Experimentalis et Applicata, 167, 156-170. https://doi.org/10.1111/eea.12768
• Boucias, D. G., & Pendland, J. C. (1998). Insect-Pathogen Relationships. In D. G. Boucias, J. C. Pendland, (Eds), Principles of Insect Pathology, (pp. 1-30). Springer.
• Zhao, M., Lin, X., & Guo, X. (2022). The role of insect symbiotic bacteria in metabolizing phytochemicals and agrochemicals. Insects, 13(7), 583. https://doi.org/10.3390/insects13070583
• Salcedo-Porras, N., Umaña-Diaz, C., de Oliveira Barbosa Bitencourt, R., & Lowenberger, C. (2020) The role of bacterial symbionts in triatomines: An evolutionary perspective. Microorganisms, 8(9), 1438. https://doi.org/10.3390/microorganisms8091438
• Paniagua Voirol, L. R., Frago, E., Kaltenpoth, M., Hilker, M., & Fatouros, N. E. (2018) Bacterial symbionts in Lepidoptera: Their diversity, transmission, and impact on the host. Frontiers in Microbiology, 9, 556. https://doi.org/10.3389/fmicb.2018.00556
• Ruiu, L. (2015) Insect pathogenic bacteria in integrated pest management. Insects, 6(2), 352-367. https://doi.org/10.3390/insects6020352
• Glare, T. R., Jurat-Fuentes, J. L., & O’Callaghan, M. (2017) Basic and Applied Research: Entomopathogenic Bacteria. In L. A. Lacey, (Ed), Microbial Control of Insect and Mite Pests. (pp. 47-67), Academic Press.
• Kalha, C. S., Singh, P. P., Kang, S. S., Hunjan, M. S., Gupta V., & Sharma R. (2014). Entomopathogenic Viruses and Bacteria for Insect-Pest Control. In Abrol D. P., (Ed), Integrated Pest Management, (pp. 225-244). Academic Press.
• Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41(7), 423-431. https://doi.org/10.1016/j.ibmb.2011.02.006
• Kocur, M., Kloos, W. E., & Schleifer, K. H. (2006). The Genus Micrococcus. In Dworkin, M., Falkow, S., Rosenberg, E., & Schleifer, K. H., Stackebrandt, E., (Eds), The Prokaryotes, (pp. 961-971). Springer.
• [Nunez, M. (2014). Micrococcus. In Batt C. A., Tortorello M. L., (Eds), Encyclopedia of Food Microbiology, (pp. 627-633). Academic Press.
• Zhu, M., Zhu, Q., Yang, Z., & Liang, Z. (2021). Clinical characteristics of patients with Micrococcus luteus bloodstream infection in a Chinese tertiary-care hospital. Polish Journal of Microbiology, 70(3), 321-326. https://doi.org/10.33073/pjm-2021-030
• Demirci, M., Sevim, E., Demir, İ., & Sevim, A. (2013). Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains. Folia Microbiologica, 58(3), 201-210. https://doi.org/10.1007/s12223-012-0199-1
• Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning. Cold Spring Harbor Laboratory Press, p 19.
• Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
• Benson, D. A., Karsch-Mizrachi, I., Clark, K., Lipman, D. J., Ostell, J., & Sayers, E. W. (2012). GenBank. Nucleic Acids Research, 40 (Database issue), D48-D53. https://doi.org/10.1093/nar/gks1195
• Moar, W. J., Pusztzai-Carey, M., & Mack, T. P. (1995). Toxicity of purified proteins and the HD-1 strain from Bacillus thuringiensis against lesser cornstalk borer (Lepidoptera: Pyralidae). Journal of Economic Entomology, 88, 606-609. https://doi.org/10.1093/jee/88.3.606
• Meyling, N. N. (2007). Methods for isolation of entomopathogenic fungi from the soil environment. Laboratory manual, DARCOF III: Research in Organic Food and Farming (FØJO III).
• Sevim, E., Çocar, M., Sezgin, F. M., & Sevim, A. (2018). Aerobic gut bacterial flora of Cydia pomonella (L.) (Lepidoptera: Tortricidae) and their virulence to the host. Egyptian Journal of Biological Pest Control, 28, 30. https://doi.org/10.1186/s41938-018-0036-1
• Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium, 41, 95-98.
• Thomson, J. D., Higgins, D. G., & Gibson T. J. (1994). Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-4680.
• Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution, 38(7), 3022-3027. https://doi.org/10.1093/molbev/msab120
• Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18, 265-267.
• Ayilara, M. S., Adeleke, B. S., Akinola, S. A., Fayose, C. A., Adeyemi, U. T., Gbadegesin, L. A., Omole, R. K., Johnson, R. M., Uthman, Q. O., & Babalola, O. O. (2023). Biopesticides as a promising alternative to synthetic pesticides: A case for microbial pesticides, phytopesticides, and nanobiopesticides. Frontiers in Microbiology, 14, 1040901. https://doi.org/10.3389/fmicb.2023.1040901
• Jouzani, G. S., Valijanian, E., & Sharafi, R. (2017). Bacillus thuringiensis: A successful insecticide with new environmental features and tidings. Applied Microbiology and Biotechnology, 101(7), 2691-2711. https://doi.org/10.1007/s00253-017-8175-y
• Chattopadhyay, P., Banerjee, G., & Mukherjee, S. (2017). Recent trends of modern bacterial insecticides for pest control practice in integrated crop management system. 3 Biotech, 7(1), 60. https://doi.org/ 10.1007/s13205-017-0717-6
• Çelebi, Ö., Sevim, E., & Sevim, A. (2014) Investigation of the internal bacterial flora of Eurygaster integriceps (Hemiptera: Scutelleridae) and pathogenicity of the flora members. Biologia, 69, 1365-1375. https://doi.org/10.2478/s11756-014-0445-x
• Sezen, K., & Demirbag, Z. (2006). Insecticidal effects of some biological agents on Agelastica alni (Coleoptera: Chrysomelidae). Biologia, 61, 687-692. https://doi.org/10.2478/s11756-006-0141-6
• Indiragandhi, P., Yoon, C., Yang, J. O., Cho, S., Sa, T. M., & Kim, G. H. (2010) Microbial communities in the developmental stages of B and Q biotypes of sweetpotato whitefly, Bemisia tabaci (hemiptera: Aleyrodidae). Journal of The Korean Society for Applied. Biological Chemistry, 53, 605–617. https://doi.org/10.3839/jksabc.2010.093
• Lipa, J. J., & Wiland, E. (1972). Bacteria isolated from cutworms and their infectivity to Agrotis sp. Acta Microbiologicas Polonica, 4, 127.140.
• Sezen, K., & Demirbag, Z. (1999). Isolation and insecticidal activity of some bacteria from the hazelnut beetle (Balaninus nucum L.). Applied Entomology and Zoology, 34, 85-89.
• Yaman, M., Nalcacioglu, R., & Demirbag, Z. (2002). Studies on bacterial flora in the population of the fall webworm, Hyphantria cunea Drury. (Lep., Arctiidae). Journal of Applied Entomology, 126, 470–474. https://doi.org/10.1046/j.1439-0418.2002.00681.x
• Vaneechoutte, M., & Heyndrickx, M. (2001). Application and Analysis of ARDRA Patterns in Bacterial Identification, Taxonomy and Phylogeny. In Dijkshoorn, L., Towner, K. J., Struelens, M., (Eds), New Approaches for the Generation and Analysis of Microbial Typing Data, (pp. 211-247). Elsevier Science B.V.
• Raina, V., Nayak, T., Ray, L., Kumari, K., & Suar, M. (2019). A Polyphasic Taxonomic Approach for Designation and Description of Novel Microbial Species. In. Das, S., Dash, H. R., (Eds), Microbial Diversity in the Genomic Era, (pp. 137-152). Academic Press
• Hugenholtz, P., Chuvochina, M., Oren, A., Parks, D. H., & Soo, R. M. (2021) Prokaryotic taxonomy and nomenclature in the age of big sequence data. The ISME Journal, 15, 1879-1892. https://doi.org/10.1038/s41396-021-00941-x