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Avrupa Yaban Arısı Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae)'in Türkiye'de Microsporidian Patojeni Kaydı

Year 2023, Volume: 6 Issue: 2, 30 - 40, 30.12.2023
https://doi.org/10.35206/jan.1362309

Abstract

Microsporidia, hymenopteranların yaygın gizemli patojenleridir. Bu türler daha çok Apidae (özellikle bal arıları) ile ilgili olsa da Vespidae üyelerini de enfekte ettikleri bilinmektedir. Bu türlerin dışında literatürde Vespidae bireylerini enfekte eden tanımlanmış ve tanımlanmamış birçok microsporidia enfeksiyonu tespit edilmiştir. Özellikle Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae), Nosema bombi, Vavraia culicis, Nosema vespula, vb. gibi farklı mikrosporidian türleri ile enfekte olmuştur. V. vulgaris gibi yırtıcı türlerdeki moleküler tanımlamaya dayalı mikrosporidian kayıtları oldukça şüphelidir. Bu tür yırtıcı böceklerde mikrosporidia enfeksiyonları, patojenin yaşam döngüsünün karakteristik görselleriyle desteklenmesi gerekir. Bu bakış açısıyla bu çalışma, V. vulgaris'ten izole edilen bir microsporidium'un yaşam döngüsü aşamalarını ve spor morfometrik verilerini sunan ilk ve tek çalışmadır. V. vulgaris örnekleri Temmuz-Eylül 2021 tarihleri arasında Trabzon ilinde toplandı. Gözlemler sırasında 415 örnek incelendi ve bunlardan 5 örnekte enfeksiyon pozitif çıktı (mikrosporidiosis oranı %1.20). Enfeksiyon çoğunlukla konağın orta bağırsağında bulundu ve çoğunlukla kronikti. Taze olgun sporlar oval şekilliydi ve 4.57±0.54 (3.26–5.95; n=200) µm uzunluğunda ve 2.43±0.33 (1.43–3.35; n=200) µm genişliğinde ölçüldü. Mevcut microsporidium, Nosema benzeri bir dissporoblastik merogoni ve sporogoniye sahiptir.

References

  • Baki, H., & Bekircan, Ç. (2018). A new microsporidium, Vairimorpha subcoccinellae n. sp. (Microsporidia: Burenellidae), isolated from Subcoccinella vigintiquatuorpunctata L. (Coleoptera: Coccinellidae). Journal of Invertebrate Pathology, 151, 182-190.
  • Bekircan, Ç. (2020). Assignment of Vairimorpha leptinotarsae comb. nov. on the basis of molecular characterization of Nosema leptinotarsae Lipa, 1968 (Microsporidia: Nosematidae). Parasitology, 147(9), 1019-1025.
  • Bekircan, Ç., Bülbül, U., Güler, H. İ., & Becnel, J. J. (2016). Description and phylogeny of a new microsporidium from the elm leaf beetle, Xanthogaleruca luteola Muller, 1766 (Coleoptera: Chrysomelidae). Parasitology Research, 116(2), 773-780.
  • Bekircan, Ç., & Tosun, O. (2021). First record and distribution of Adelina sp. (Coccidia: Adeleidae) in populations of the Anisoplia segetum Herbst (Coleoptera: Scarabaeidae) in Turkey. Microbial Pathogenesis, 154, 104848.
  • Biganski, S., Wennmann, J. T., Vossbrinck, C. R., Kaur, R., Jehle, J. A., & Kleespies, R. G. (2020). Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae). Journal of Invertebrate Pathology, 174, 107440.
  • Boeve, J. L., Honraet, K., & Rossel, B. (2014). Screening of Repellents against Vespid Wasps. Insects, 5, 272-286.
  • Canning, E. U., & Vávra, J. (2000). Phylum microsporida, In The Illustrated Guide to the Protozoa, J. J. Lee, G. F. Leedale & P. Bradburry (Eds.), Allen Press Inc., (Vol. 1, pp. 39-126). Lawrence.
  • Chen, Y. P., Evans, J. D., Murphy, C., Gutell, R., Zuker, M., Gundensen-Rindal, D., & Pettis, J.S. (2009). Morphological, Molecular, and Phylogenetic Characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. Journal of Eukaryotic Microbiology, 56(2), 142-147.
  • Da Silva, A. J., Maleszka, R., Slemenda, S. B., & Pieniazek, N. J. (1994). Nosema vespula small subunit ribosomal RNA gene. https://www.ncbi.nlm.nih.gov/nuccore/U11047 (Date accessed: 20.09.2022).
  • Erdoğan, Y., & Dodoloğlu, A. (2013). The determination of the efficiency of some traps and feeds on wasps (Vespa sp.) in the bee yards. Uludag Bee Journal, 13(2), 63-69.
  • Felden, A., Baty, J. W., Bulgarella, M., Brown, R. L., Dobelmann, J., Gruber, M. A. M., Quinn, O., & Lester, P. J. (2020). Viral and fungal pathogens associated with Pneumolaelaps niutirani (Acari: Laelapidae): a mite found in diseased nests of Vespula wasps. Insectes Sociaux, 67(1), 83-93.
  • Fries, I. (1993). Nosema apis—A parasite in the honey bee colony. Bee World, 74(1), 5-19.
  • Fries, I., Feng, F., Silva, A. D., Slemenda, S. B., & Pieniazek, N. J. (1996). Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). European Journal of Protistology, 32, 356-365.
  • Gabín-García, L. B., Bartolomé, C., Guerra-Tort, C., Rojas-Nossa, S. V., Llovo, J., & Maside, X. (2021). Identification of pathogens in the invasive hornet Vespa velutina and in native Hymenoptera (Apidae, Vespidae) from SW‑Europe. Scientific Reports, 11, 11233.
  • Goulet, H., & Huber, J. T. (1993). Hymenoptera of the world: An identification guide to families (pp. 668). Agriculture Canada, Canada.
  • Graystock, P., Yates, K., Darvill, B., Goulson, D., & Hughes, W. O. H. (2013). Emerging dangers: deadly effects of an emergent parasite in a new pollinator host. Journal of Invertebrate Pathology, 114(2), 114-119.
  • Gruber, M. A. M., Quinn, O., Baty, J. W., Dobelmann, J., Haywood, J., Wenseleers, T., & Lester, P. J. (2019). Fitness and microbial networks of the common wasp, Vespula vulgaris {Hymenoptera: Vespidae), in its nativ and introduced ranges. Ecological Entomology, 44(4), 512-523.
  • Lester, P. J., Bosch, P. J., Gruber, M. A., Kapp, E. A., Peng, L., Brenton-Rule, E. C., Buchanan, J., Stanislawek, W. L., Archer, M., Corley, J. C., Masciocchi, M., Van Oystaeyen, A., & Wenseleers, T. (2015). No evidence of enemy release in pathogen and microbial communities of common wasps (Vespula vulgaris) in their native and introduced range. PLoS One, 10(3), e0121358.
  • Lester, P. J., Gruber, M. A. M., Brenton-Rule, E. C., Archer, M., Corley, J. C., Dvořák, L., Masciocchi, M., Van Oystaeyen, A., & Roura-Pascual, N. (2014). Determining the origin of invasions and demonstrating a lack of enemy release from microsporidian pathogens in common wasps (Vespula vulgaris). Diversity and Distributions, 20(8), 964-974.
  • McIvor, C. A., & Malone, L. A. (1995). Nosema bombi, a microsporidian pathogen of the bumble bee Bombus terrestris (L.). New Zealand Journal of Zoology, 25(1), 25-31.
  • Nägeli, K. W. (1857). Überdie neue Krankheit der Seidenraupe und verwandte Organismen. Botanische Zeitung, 15, 760-761.
  • Plischuk, S., Martín-Hernández, R., Prieto, L., Lucía, M., Botías, C., Meana, A., Abrahamovich, A. H., Lange, C., & Higes, M. (2009). South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environmental Microbiology Reports, 1(2), 131-135.
  • Quinn, O., Gruber, M. A. M., Brown, R. L., Baty, J. W., Bulgarella, M., & Lester, P. J. (2018). A metatranscriptomic analysis of diseased social wasps (Vespula vulgaris) for pathogens, with an experimental infection of larvae and nests. PLoS One, 13(12), e0209589.
  • Rose, E. A. F., Harris, R. J., & Glare, T. R. (1999). Possible pathogens of social wasps (Hymenoptera: Vespidae) and their potential as biological control agents. New Zealand Journal of Zoology, 26(3), 179-190.
  • Sprague, V., Becnel, J. J., & Hazard, E. I. (1992). Taxonomy of Phylum Microspora. Critical Reviews in Microbiology, 18(5/6), 285-395.
  • Tosun, O., & Bekircan, Ç. (2021). Molecular characterization of the Vairimorpha (Nosema) ceranae infection from Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae) in Turkey. Journal of the Entomological Research Society, 23(3), 305-319.
  • Vega, F. E., & Kaya, H. K. (2012). Insect Pathology (Second ed.). UK: Elsevier.
  • Vossbrinck, C. R., & Debrunner-Vossbrinck, B. A. (2005). Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitologica, 52, 131-142.
  • Weiss, L. M., & Becnel, J. J. (2014). Microsporidia Pathogens of Opportunity. USA: (pp. 747) John Wiley & Sons, Inc.
  • Weiser, J. (1947). A key for the determination of microsporidia. Práce Mor. Přír. Spol., 18, 1–64. (In Czech.)
  • Yıldırım, E., & Gusenleitner, J. (2012). Contribution to the knowledge of the Vespidae (Hymenoptera, Aculeata) of Turkey, with a checklist of the Turkish species. Turkish Journal of Zoology, 36(3), 361-374.
  • Yıldırım, H., & Bekircan, Ç. (2020). Ultrastructural and molecular characterization of Nosema alticae sp. nov. (Microsporidia: Nosematidae), pathogen of the flea beetle, Altica hampei Allard, 1867 (Coleoptera: Chrysomelidae). Journal of Invertebrate Pathology, 170, 107302.
  • Yıldırım, H., Tosun, O., & Bekircan, Ç. (2022). Ophryocystis sitonae sp. nov., (Neogregarinida: Ophryocystidae) parasitizing Sitona humeralis Stephens, 1831 (Coleoptera: Curculionidae). Microbial Pathogenesis, 162, 105305. Zander, E. (1909). Tierische parasiten als krankenheitserreger bei der biene. Münchener Bienenzeitung, 31, 196-204.

A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey

Year 2023, Volume: 6 Issue: 2, 30 - 40, 30.12.2023
https://doi.org/10.35206/jan.1362309

Abstract

Microsporidia are common enigmatic pathogens of hymenopterans. Although these species are more concerned with Apidae (especially honeybees), they are also known to infect members of Vespidae. Apart from these species, many defined and undefined microsporidia infections were detected infecting Vespidae individuals in the literature. Especially Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) infected different microsporidian species like a Nosema bombi, Vavraia culicis, Nosema vespula, etc. Molecular identification-based microsporidian records in predator species such as V. vulgaris are highly suspicious. In such predator insects, microsporidian infections should be supported by characteristic visuals of the pathogen’s life cycle. With this perspective this study is the first and only study that presents the life-cycle stages and spore morphometrics data of a microsporidium isolated from V. vulgaris. V. vulgaris samples were collected from July to September 2021 in Trabzon, Turkey. During the observations, 415 samples were examined, and five of them were infection positive (microsporidiosis prevalence 1.20%). Infection was found mostly in the midgut of the host, and infection was mostly chronic. Fresh mature spores were oval in shape and measured 4.57±0.54 (3.26–5.95; n=200) µm in length and 2.43±0.33 (1.43–3.35; n=200) µm in width. The current microsporidium has a Nosema-like disporoblastic merogony and sporogony.

References

  • Baki, H., & Bekircan, Ç. (2018). A new microsporidium, Vairimorpha subcoccinellae n. sp. (Microsporidia: Burenellidae), isolated from Subcoccinella vigintiquatuorpunctata L. (Coleoptera: Coccinellidae). Journal of Invertebrate Pathology, 151, 182-190.
  • Bekircan, Ç. (2020). Assignment of Vairimorpha leptinotarsae comb. nov. on the basis of molecular characterization of Nosema leptinotarsae Lipa, 1968 (Microsporidia: Nosematidae). Parasitology, 147(9), 1019-1025.
  • Bekircan, Ç., Bülbül, U., Güler, H. İ., & Becnel, J. J. (2016). Description and phylogeny of a new microsporidium from the elm leaf beetle, Xanthogaleruca luteola Muller, 1766 (Coleoptera: Chrysomelidae). Parasitology Research, 116(2), 773-780.
  • Bekircan, Ç., & Tosun, O. (2021). First record and distribution of Adelina sp. (Coccidia: Adeleidae) in populations of the Anisoplia segetum Herbst (Coleoptera: Scarabaeidae) in Turkey. Microbial Pathogenesis, 154, 104848.
  • Biganski, S., Wennmann, J. T., Vossbrinck, C. R., Kaur, R., Jehle, J. A., & Kleespies, R. G. (2020). Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae). Journal of Invertebrate Pathology, 174, 107440.
  • Boeve, J. L., Honraet, K., & Rossel, B. (2014). Screening of Repellents against Vespid Wasps. Insects, 5, 272-286.
  • Canning, E. U., & Vávra, J. (2000). Phylum microsporida, In The Illustrated Guide to the Protozoa, J. J. Lee, G. F. Leedale & P. Bradburry (Eds.), Allen Press Inc., (Vol. 1, pp. 39-126). Lawrence.
  • Chen, Y. P., Evans, J. D., Murphy, C., Gutell, R., Zuker, M., Gundensen-Rindal, D., & Pettis, J.S. (2009). Morphological, Molecular, and Phylogenetic Characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. Journal of Eukaryotic Microbiology, 56(2), 142-147.
  • Da Silva, A. J., Maleszka, R., Slemenda, S. B., & Pieniazek, N. J. (1994). Nosema vespula small subunit ribosomal RNA gene. https://www.ncbi.nlm.nih.gov/nuccore/U11047 (Date accessed: 20.09.2022).
  • Erdoğan, Y., & Dodoloğlu, A. (2013). The determination of the efficiency of some traps and feeds on wasps (Vespa sp.) in the bee yards. Uludag Bee Journal, 13(2), 63-69.
  • Felden, A., Baty, J. W., Bulgarella, M., Brown, R. L., Dobelmann, J., Gruber, M. A. M., Quinn, O., & Lester, P. J. (2020). Viral and fungal pathogens associated with Pneumolaelaps niutirani (Acari: Laelapidae): a mite found in diseased nests of Vespula wasps. Insectes Sociaux, 67(1), 83-93.
  • Fries, I. (1993). Nosema apis—A parasite in the honey bee colony. Bee World, 74(1), 5-19.
  • Fries, I., Feng, F., Silva, A. D., Slemenda, S. B., & Pieniazek, N. J. (1996). Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). European Journal of Protistology, 32, 356-365.
  • Gabín-García, L. B., Bartolomé, C., Guerra-Tort, C., Rojas-Nossa, S. V., Llovo, J., & Maside, X. (2021). Identification of pathogens in the invasive hornet Vespa velutina and in native Hymenoptera (Apidae, Vespidae) from SW‑Europe. Scientific Reports, 11, 11233.
  • Goulet, H., & Huber, J. T. (1993). Hymenoptera of the world: An identification guide to families (pp. 668). Agriculture Canada, Canada.
  • Graystock, P., Yates, K., Darvill, B., Goulson, D., & Hughes, W. O. H. (2013). Emerging dangers: deadly effects of an emergent parasite in a new pollinator host. Journal of Invertebrate Pathology, 114(2), 114-119.
  • Gruber, M. A. M., Quinn, O., Baty, J. W., Dobelmann, J., Haywood, J., Wenseleers, T., & Lester, P. J. (2019). Fitness and microbial networks of the common wasp, Vespula vulgaris {Hymenoptera: Vespidae), in its nativ and introduced ranges. Ecological Entomology, 44(4), 512-523.
  • Lester, P. J., Bosch, P. J., Gruber, M. A., Kapp, E. A., Peng, L., Brenton-Rule, E. C., Buchanan, J., Stanislawek, W. L., Archer, M., Corley, J. C., Masciocchi, M., Van Oystaeyen, A., & Wenseleers, T. (2015). No evidence of enemy release in pathogen and microbial communities of common wasps (Vespula vulgaris) in their native and introduced range. PLoS One, 10(3), e0121358.
  • Lester, P. J., Gruber, M. A. M., Brenton-Rule, E. C., Archer, M., Corley, J. C., Dvořák, L., Masciocchi, M., Van Oystaeyen, A., & Roura-Pascual, N. (2014). Determining the origin of invasions and demonstrating a lack of enemy release from microsporidian pathogens in common wasps (Vespula vulgaris). Diversity and Distributions, 20(8), 964-974.
  • McIvor, C. A., & Malone, L. A. (1995). Nosema bombi, a microsporidian pathogen of the bumble bee Bombus terrestris (L.). New Zealand Journal of Zoology, 25(1), 25-31.
  • Nägeli, K. W. (1857). Überdie neue Krankheit der Seidenraupe und verwandte Organismen. Botanische Zeitung, 15, 760-761.
  • Plischuk, S., Martín-Hernández, R., Prieto, L., Lucía, M., Botías, C., Meana, A., Abrahamovich, A. H., Lange, C., & Higes, M. (2009). South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environmental Microbiology Reports, 1(2), 131-135.
  • Quinn, O., Gruber, M. A. M., Brown, R. L., Baty, J. W., Bulgarella, M., & Lester, P. J. (2018). A metatranscriptomic analysis of diseased social wasps (Vespula vulgaris) for pathogens, with an experimental infection of larvae and nests. PLoS One, 13(12), e0209589.
  • Rose, E. A. F., Harris, R. J., & Glare, T. R. (1999). Possible pathogens of social wasps (Hymenoptera: Vespidae) and their potential as biological control agents. New Zealand Journal of Zoology, 26(3), 179-190.
  • Sprague, V., Becnel, J. J., & Hazard, E. I. (1992). Taxonomy of Phylum Microspora. Critical Reviews in Microbiology, 18(5/6), 285-395.
  • Tosun, O., & Bekircan, Ç. (2021). Molecular characterization of the Vairimorpha (Nosema) ceranae infection from Bombus terrestris (Linnaeus, 1758) (Hymenoptera: Apidae) in Turkey. Journal of the Entomological Research Society, 23(3), 305-319.
  • Vega, F. E., & Kaya, H. K. (2012). Insect Pathology (Second ed.). UK: Elsevier.
  • Vossbrinck, C. R., & Debrunner-Vossbrinck, B. A. (2005). Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitologica, 52, 131-142.
  • Weiss, L. M., & Becnel, J. J. (2014). Microsporidia Pathogens of Opportunity. USA: (pp. 747) John Wiley & Sons, Inc.
  • Weiser, J. (1947). A key for the determination of microsporidia. Práce Mor. Přír. Spol., 18, 1–64. (In Czech.)
  • Yıldırım, E., & Gusenleitner, J. (2012). Contribution to the knowledge of the Vespidae (Hymenoptera, Aculeata) of Turkey, with a checklist of the Turkish species. Turkish Journal of Zoology, 36(3), 361-374.
  • Yıldırım, H., & Bekircan, Ç. (2020). Ultrastructural and molecular characterization of Nosema alticae sp. nov. (Microsporidia: Nosematidae), pathogen of the flea beetle, Altica hampei Allard, 1867 (Coleoptera: Chrysomelidae). Journal of Invertebrate Pathology, 170, 107302.
  • Yıldırım, H., Tosun, O., & Bekircan, Ç. (2022). Ophryocystis sitonae sp. nov., (Neogregarinida: Ophryocystidae) parasitizing Sitona humeralis Stephens, 1831 (Coleoptera: Curculionidae). Microbial Pathogenesis, 162, 105305. Zander, E. (1909). Tierische parasiten als krankenheitserreger bei der biene. Münchener Bienenzeitung, 31, 196-204.
There are 33 citations in total.

Details

Primary Language English
Subjects Food Engineering
Journal Section Research Articles
Authors

Çağrı Bekircan 0000-0002-5968-7359

Onur Tosun 0000-0002-6763-5671

Hilal Yıldırım 0000-0002-6072-5543

Publication Date December 30, 2023
Published in Issue Year 2023 Volume: 6 Issue: 2

Cite

APA Bekircan, Ç., Tosun, O., & Yıldırım, H. (2023). A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey. Journal of Apitherapy and Nature, 6(2), 30-40. https://doi.org/10.35206/jan.1362309
AMA Bekircan Ç, Tosun O, Yıldırım H. A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey. J.Apit.Nat. December 2023;6(2):30-40. doi:10.35206/jan.1362309
Chicago Bekircan, Çağrı, Onur Tosun, and Hilal Yıldırım. “A Record of Microsporidian Pathogen of the European Wasp, Vespula Vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey”. Journal of Apitherapy and Nature 6, no. 2 (December 2023): 30-40. https://doi.org/10.35206/jan.1362309.
EndNote Bekircan Ç, Tosun O, Yıldırım H (December 1, 2023) A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey. Journal of Apitherapy and Nature 6 2 30–40.
IEEE Ç. Bekircan, O. Tosun, and H. Yıldırım, “A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey”, J.Apit.Nat., vol. 6, no. 2, pp. 30–40, 2023, doi: 10.35206/jan.1362309.
ISNAD Bekircan, Çağrı et al. “A Record of Microsporidian Pathogen of the European Wasp, Vespula Vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey”. Journal of Apitherapy and Nature 6/2 (December 2023), 30-40. https://doi.org/10.35206/jan.1362309.
JAMA Bekircan Ç, Tosun O, Yıldırım H. A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey. J.Apit.Nat. 2023;6:30–40.
MLA Bekircan, Çağrı et al. “A Record of Microsporidian Pathogen of the European Wasp, Vespula Vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey”. Journal of Apitherapy and Nature, vol. 6, no. 2, 2023, pp. 30-40, doi:10.35206/jan.1362309.
Vancouver Bekircan Ç, Tosun O, Yıldırım H. A Record of Microsporidian Pathogen of the European wasp, Vespula vulgaris Linnaeus, 1758 (Hymenoptera: Vespidae) in Turkey. J.Apit.Nat. 2023;6(2):30-4.

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