First Report on Secondary Endosymbiont Bacteria of Pomegranate Aphid Aphis punicae Passerini, 1863 (Hemiptera: Aphididae)

Öz

The pomegranate aphid, Aphis punicae (Hemiptera: Aphididae), is an important pest affecting pomegranate trees in various regions. Although its ecological impact is well documented, no previous studies have been conducted on its association with secondary bacterial endosymbionts. The study is the first report on the distribution and presence of four important secondary endosymbionts, Wolbachia, Arsenophonus, Rickettsia and Hamiltonella defensa, in field populations of A. punicae collected from Antalya, Turkey. Using species-specific primers and a descriptive Touchdown PCR protocol, endosymbiont infection was detected in all sampled individuals (n = 50) individually. Arsenophonus was the most common (74%), while Wolbachia was the least common (52%) endosymbiont. No aphid individual was infected with Wolbachia or Rickettsia alone; most individuals hosted co-infections and multiple infections. Our results suggest complex infection patterns with implications for aphid fitness, resistance and potential biological control strategies. These results will contribute to the figuring out of the microbial ecology of A. punicae and provide a foundation for future research into symbiont-mediated pest control.

Anahtar Kelimeler

• Aphis punicae
• Arsenophonus
• Hamiltonella
• Rickettsia
• Wolbachia

Nar Afidi Aphis punicae Passerini, 1863 (Hemiptera: Aphididae)’nın Sekonder Endosimbiyont Bakterileri Üzerine İlk Rapor

Öz

Nar yaprakbiti, Aphis punicae (Hemiptera: Aphididae), çeşitli bölgelerdeki nar ağaçlarını etkileyen önemli bir zararlıdır. Ekolojik etkisi iyi belgelenmiş olmasına rağmen, daha önce ikincil bakteriyel endosimbiyontlarla ilişkisi hakkında hiçbir çalışma yapılmamıştır. Bu çalışma, Türkiye, Antalya'dan toplanan A. punicae popülasyonlarında dört önemli ikincil endosimbiyont olan Wolbachia, Arsenophonus, Rickettsia ve Hamiltonella defensa'nın varlığını ve dağılımını bildiren ilk çalışmadır. Türlere özgü primerler ve tanımlayıcı bir Touchdown PCR protokolü kullanılarak, endosimbiyont enfeksiyonu örneklenen tüm bireylerde (n = 50) ayrı ayrı tespit edilmiştir. Arsenophonus en yaygın (%74), Wolbachia ise en az yaygın (%52) endosimbiyont olmuştur. Hiçbir yaprakbiti bireyi yalnızca Wolbachia veya Rickettsia ile enfekteli olmazken; çoğu birey ikili veya çoklu enfeksiyonlara konukçuluk yapmıştır. Sonuçlarımız, yaprakbitinin konukçuya uyumu, direnci ve potansiyel biyolojik mücadele stratejileri için çıkarımları olan karmaşık enfeksiyon örüntüleri olduğunu göstermektedir. Bu bulgular, A. punicae'nin mikrobiyal ekolojisinin anlaşılmasına katkıda bulunarak simbiyont aracılı zararlı mücadelesi konusunda gelecekte yapılacak araştırmalar için bir temel oluşturacaktır.

Anahtar Kelimeler

• Aphis punicae
• Arsenophonus
• Hamiltonella
• Rickettsia
• Wolbachia

Kaynakça

1. Augustinos, A.A, Santos-Garcia, D., Dionyssopoulou, E., Moreira, M., Papapanagiotou, A., Scarvelakis, M., & Bourtzis, K. (2011). Detection and characterization of Wolbachia infections in natural populations of aphids: is the latent variety fully resolved? PloS one, 6(12), e28695.
2. Ayoubi A, Talebi AA, Fathipour Y, & Mehrabadi M. (2020). Coinfection of the secondary symbionts, Hamiltonella defensa and Arsenophonus sp. contribute to the performance of the major aphid pest, Aphis gossypii (Hemiptera: Aphididae). Insect Science, 27, 86–98.
3. Bourtzis, K. (2008). Wolbachia-based technologies for insect pest population control. Transgenesis and the management of vector-borne disease, 104-113.
4. Brady C.M., & White, J.A. (2013). Cowpea aphid (Aphis craccivora) associated with different host plants has different facultative endosymbionts. Ecological Entomology, 38(4), 433-437.
5. Buchner, P. (1965). Endosymbiosis of animals with plant microorganisms. Interscience, New York. Chandler, S.M., Wilkinson, T.L., & Douglas, A.E. (2007). Impact of plant nutrients on the relationship between a herbivorous insect and its symbiotic bacteria. Proceedings of the Royal Society of London Series B: Biological Sciences, 275, 565–570.
6. Chen, D. Q., Campbell, B. C., & Purcell, A. H. (1996). A new Rickettsia from a herbivorous insect, the pea aphid Acyrthosiphon pisum (Harris). Current Microbiology, 33, 123-128.
7. Chen, D.Q., Montllor, C.B. & Purcell, A.H. (2000). Fitness effects of two facultative endosymbiotic bacteria on the pea aphid, Acyrthosiphon pisum, and the blue alfalfa aphid, A. kondoi. Entomololgia Experimentalis et Applicata, 95, 315–323.
8. Cocuzza, G. E. M., Mazzeo, G., Russo, A., Giudice, V. L., & Bella, S. (2016). Pomegranate arthropod pests and their management in the Mediterranean area. Phytoparasitica, 44, 393-409.

9. De Clerck, C., Tsuchida, T., Massart, S., Lepoivre, P., Francis, F., & Jijakli, M.H., (2014). Combination of genomic and proteomic approaches to characterize the symbiotic population of the banana aphid (Hemiptera: Aphididae). Environmental Entomolology, 43, 29–36.
10. De Clerck, C., Fujiwara, A., Joncour, P., Léonard, S., Félix, M.L., Francis, F., Jijakli, M.H., Tsuchida, T., & Massart, S. (2015). A metagenomic approach from aphid's hemolymph sheds light on the potential roles of co-existing endosymbionts. Microbiome, 3, 1–11.
11. Donner, S. H., Slingerland, M., Beekman, M. M., Comte, A., Dicke, M., Zwaan, B. J., Pannebakker, B. A., & Verhulst, E. C. (2024). Aphid populations are frequently infected with facultative endosymbionts. Environmental Microbiology, 26(3), e16599.
12. Douglas, A. E., Francois, C. L. M. J., & Minto, L. B. (2006). Facultative ‘secondary’bacterial symbionts and the nutrition of the pea aphid, Acyrthosiphon pisum. Physiological Entomology, 31(3), 262-269.
13. Elston, K. M. (2022). New Tools and Applications for Genetically Engineered Insect Symbionts. The University of Texas at Austin.
14. Feldhaar, H. (2011). Bacterial symbionts as mediators of ecologically important traits of insect hosts. Ecological Entomology, 36, 533–543.
15. Guo, J., Hatt, S., He, K., Chen, J., Francis, F., & Wang, Z. (2017). Nine facultative endosymbionts in aphids. A review. Journal of Asia-Pacific Entomology, 20(3), 794-801.
16. Gülmez, M., Çalışkan Keçe, A.F., Kaplan, M., & Ulusoy, M.R. (2022). Determination of Pest Insect Species at Pomegranate (Punica granatum L.) Orchards in Adıyaman and Siirt Provinces, Turkey. KSU Journal of Agriculture and Nature, 25(4), 677-686.
17. Higashi, C. H., Patel, V., Kamalaker, B., Inaganti, R., Bressan, A., Russell, J. A., & Oliver, K. M. (2024). Another tool in the toolbox: Aphid‐specific Wolbachia protect against fungal pathogens. Environmental Microbiology, 26(11), e70005.
18. Hurst, G. D., & Frost, C. L. (2015). Reproductive parasitism: maternally inherited symbionts in a biparental world. Cold Spring Harbor Perspectives in Biology, 7(5), a017699.
19. Hussain, M. D., Farooq, T., Kamran, A., Basit, A., Wang, Y., Smagghe, G., & Chen, X. (2025). Endosymbionts as hidden players in tripartite pathosystem of interactions and potential candidates for sustainable viral disease management. Critical Reviews in Biotechnology, 1-23.
20. Henry, L. M., Maiden, M. C., Ferrari, J. & Godfray, H.C. (2015). Insect life history and the evolution of bacterial mutualism. Ecology Letters, 18, 516–525.
21. Jeyaprakash, A., & Hoy, M. A. (2000). Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty‐three arthropod species. Insect Molecular Biology, 9(4), 393-405.
22. Jousselin, E., Cœur d'Acier, A., Vanlerberghe‐Masutti, F., & Duron, O. (2013). Evolution and diversity of A rsenophonus endosymbionts in aphids. Molecular Ecology, 22(1), 260-270.
23. Kahramanoglu, I., Usanmaz, S. (2016). Pomegranate production and marketing, CRC Press, Boca Raton.
24. Leonard, S. P., Powell, J. E., Perutka, J., Geng, P., Heckmann, L. C., Horak, R. D., Davies, B. W., Ellington, A. D., Barrick, J. E., & Moran, N. A. (2020). Engineered symbionts activate honey bee immunity and limit pathogens. Science, 367(6477), 573-576.
25. Liu, H., Zhao, D., Niu, H., Zhang, Z., Wang, N., Liu, X., & Guo, H. (2025). Arsenophonus and Wolbachia-mediated insecticide protection in Nilaparvata lugens. Journal of Pest Science, 98(1), 365-373.
26. Lukasik, P., van Asch, M., Guo, H., Ferrari, J., Godfray, H. C. J. (2013). Unrelated facultative endosymbionts protect aphids against a fungal pathogen. Ecology Letters, 16, 214–218.
27. Munson, M. A., Baumann, P., & Kinsey, M. G. (1991). Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. International Journal of Systematic and Evolutionary Microbiology, 41(4), 566-568.
28. Nadal‐Jimenez, P., Griffin, J. S., Davies, L., Frost, C. L., Marcello, M., & Hurst, G. D. (2019). Genetic manipulation allows in vivo tracking of the life cycle of the son‐killer symbiont, Arsenophonus nasoniae, and reveals patterns of host invasion, tropism and pathology. Environmental Microbiology, 21(8), 3172-3182.
29. Oliver, K. M., Russell, J. A., Moran, N. A., & Hunter, M. S. (2003). Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proceedings of the National Academy of Sciences, 100(4), 1803-1807.
30. Oliver, K. M., Moran, N. A., & Hunter, M. S. (2006). Costs and benefits of a superinfection of facultative symbionts in aphids. Proceedings of the Royal Society B: Biological Sciences, 273(1591), 1273-1280.
31. Perlman, S. J., Hunter, M. S., & Zchori-Fein, E. (2006). The emerging diversity of Rickettsia. Proceedings of the Royal Society B: Biological Sciences, 273(1598), 2097-2106.
32. Russell, J. A., Latorre, A., Sabater‐Muñoz, B., Moya, A., & Moran, N. A. (2003). Side‐stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea. Molecular Ecology, 12(4), 1061-1075.
33. Satar, G. (2020). Molecular comparison of some facultative endosymbiotic species obtained from aphids. Mediterranean Agricultural Sciences, 33(2), 189-194.
34. Sakurai, M., Koga, R., Tsuchida, T., Meng, X. Y., & Fukatsu, T. (2005). Rickettsia symbiont in the pea aphid Acyrthosiphon pisum: novel cellular tropism, effect on host fitness, and interaction with the essential symbiont Buchnera. Applied and Environmental Microbiology, 71(7): 4069-4075.
35. Slavenko, A., Ross, P. A., Mata, L., Hoffmann, A. A., & Umina, P. A. (2024). Modelling the spread of a novel endosymbiont infection in field populations of an aphid pest. Ecological Modelling, 497, 110851.
36. Thao, M. L., & Baumann, P. (2004). Evidence for multiple acquisition of Arsenophonus by whitefly species (Sternorrhyncha: Aleyrodidae). Current Microbiology, 48(2), 140-144.
37. Wagner, S. M., Martinez, A. J., Ruan, Y. M., Kim, K. L., Lenhart, P.A., Dehnel, A.C., Oliver, K. M. & White, J. A. (2015). Facultative endosymbionts mediate dietary breadth in a polyphagous herbivore. Functional Ecology. 29, 1402–1410.
38. Wang, Z., Su, X. M., Wen, J., Jiang, L. Y., & Qiao, G. X. (2014). Widespread infection and diverse infection patterns of Wolbachia in Chinese aphids. Insect Science, 21(3), 313-325.
39. Weldon, S., Strand, M., & Oliver, K. (2013). Phage loss and the breakdown of a defensive symbiosis in aphids. Proceedings of the Royal Society of London Series B: Biological Sciences, 280, 20122103.
40. Whitten, M. M., Facey, P. D., Del Sol, R., Fernández-Martínez, L. T., Evans, M. C., Mitchell, J. J., Bodger, O. G., & Dyson, P. J. (2016). Symbiont-mediated RNA interference in insects. Proceedings of the Royal Society B: Biological Sciences, 283(1825), 20160042.
41. Wilkes, T. E., Duron, O., Darby, A. C., Hypša, V., Nováková, E., & Hurst, G. D. (2011). 12 The Genus Arsenophonus. Manipulative tenants: bacteria associated with arthropods, 225.
42. Wulff, J. A. & White, J.A. (2015) The Endosymbiont Arsenophonus Provides a General Benefit to Soybean Aphid (Hemiptera: Aphididae) Regardless of Host Plant Resistance (Rag). Environmental Entomology, 44, 574–581.
43. Xu, S., Jiang, L., Qiao, G., & Chen, J. (2020). The bacterial flora associated with the polyphagous aphid Aphis gossypii Glover (Hemiptera: Aphididae) is strongly affected by host plants. Microbial Ecology, 79(4), 971-984.
44. Zhang, S., Luo, J., Wang, L., Zhang, L., Zhu, X., Jiang, W., & Cui, J. (2019) Bacterial communities in natural versus pesticide-treated Aphis gossypii populations in North China. Microbiology Open 8: e00652.
45. Zytynska, S. E., & Weisser, W. W. (2016). The natural occurrence of secondary bacterial symbionts in aphids. Ecological Entomology, 41(1), 13-26.