Development of densovirus-based bioinsecticide (Tenebriokiller) for use in the control of mealworm, Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae)

Abstract

Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae) is one of the major storage pests affecting a large proportion of pasta, flour, starch, and rice. Densoviruses are biocontrol agents with a single-stranded DNA genome that is enveloped and very small in size. In this study, crude TmDNV-TR was formulated using bovine gelatin, and the formulation called Tenebriokiller was prepared in Türkiye. The dose of virus in the formulation was determined to be 2.8x108 copies/mL-1. Five different doses were used (2.8x108, 2.8x107, 2.8x106, 2.8x105, and 2.8x104 copies/mL-1) against 10th instar T. molitor larvae. The mortality was detected 100% (LD50: 4 x106 copies/mL-1, LT50: 6 day, LD95: 2.2 x108 copies/mL-1 and LT95: 9 day) by day 14. The stability of the crude virus and Tenebriokiller was tested at different storage temperatures (4 and 25°C) over various periods (3 and 6 months). Accordingly, the mortality rate of the fresh formulation was 100% as in the previous step, while at 4°C the mortality rate at the end of 3 and 6 months remained 100%, and at 25°C, the mortality rate at the end of 3 and 6 months was 98% and 95% respectively, at the end of day 14. Preliminary pathogenicity tests in the laboratory showed that the virus was highly lethal to the target organism. The virus was therefore formulated to maintain its stability during storage, and a prototype formulation was developed.

Keywords

• Biocontrol
• densovirus
• Tenebrio molitor
• virus formulation
• virus stabilization

Un kurdu, Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae)'un kontrolünde kullanılmak üzere densovirüs bazlı biyoinsektisitin (Tenebriokiller) geliştirilmesi

Öz

Tenebrio molitor L., 1758 (Coleoptera: Tenebrionidae), makarna, un, nişasta ve pirincin büyük bir kısmını etkileyen önemli depo zararlılarından biridir. Densovirüsler, zarflı ve çok küçük boyutlu tek zincirli bir DNA genomuna sahip biyolojik kontrol ajanlarıdır. Bu çalışmada, sığır jelatini kullanarak ham TmDNV-TR formüle edilmiş ve Tenebriokiller adı verilen formülasyon Türkiye’de hazırlanmıştır. Formülasyondaki virüs dozunun 2.8x108 kopya/mL-1 olduğu belirlenmiştir. Beş farklı doz (2.8x108, 2.8x107, 2.8x106, 2.8x105 ve 2.8x104 kopya/mL-1) 10. dönem T. molitor larvalarına uygulanmıştır. Ölüm oranı 14. günün sonunda %100 (LD50: 4 x106 kopya/mL-1, LT50: 6 gün, LD95: 2.2 x108 kopya/mL-1 ve LT95: 9 gün) olarak belirlenmiştir. Ham virüsün ve Tenebriokiller'in stabilitesi farklı depolama sıcaklıklarında (4 ve 25°C) çeşitli sürelerde (3 ve 6 ay) test edilmiştir. Buna göre, taze formülasyonun ölüm oranı bir önceki adımda olduğu gibi %100 iken, 4°C'de 3 ve 6 ayın sonunda ölüm oranı %100, 25°C'de ise 3 ve 6 ayın sonunda ölüm oranı sırasıyla %98 ve %95 olarak belirlenmiştir. Laboratuvardaki ön patojenite testleri virüsün hedef organizma için oldukça öldürücü olduğunu göstermiştir. Bu nedenle virüs, depolama sırasında stabilitesini koruyacak şekilde formüle edilerek, bir prototip formülasyon üretilmiştir.

Anahtar Kelimeler

• Biyolojik mücadele
• densovirüs
• Tenebrio molitor
• virüs formülasyonu
• virüs kararlılığı

Kaynakça

1. Abbott, W. S., 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18 (2): 265-267.
2. Armien, A. G., R. Polon, D. Rejmanek, R. B. Moeller & B. M. Crossley, 2023. Outbreak of densovirus with high mortality in a commercial mealworm (Tenebrio molitor) farm: A molecular, bright-field, and electron microscopic characterization. Veterinary Pathology, 60 (5): 689-703.
3. Aş, Y. & G. B. Eroğlu, 2025. Densovirinae: An Eco-Friendly Alternative in Biological Control. Eurasian Journal of Molecular and Biochemical Science, 4 (1): 45-55.
4. Aş, Y., Z. Selvitopi & G. B. Eroğlu, 2025. Two novel densoviruses from storage pests insects (Zophobas morio and Tenebrio molitor) in Türkiye: Genomic and ultrastructural comparison. Journal of Stored Products Research, 111: 102549 (1-8).
5. Batool, K., J. Xiao, Y. Xu, T. Yang, P. Tao, S. Zhao, J. Chen, I. Alam, Y. Xie, J. Gu & X. Chen, 2022. Densovirus oil suspension significantly improves the efficacy and duration of larvicidal activity against Aedes albopictus. Viruses, 14 (3): 475 (1-18).
6. Bayramoglu, Z., D. Gencer & I. Demir, 2023. Development of novel betabaculovirus (HycuGV-Hc1) as a biopesticide (HycuGV-TR61) and its efficacy on the fall webworm, Hyphantria cunea Drury (Lepidoptera: Erebidae) larvae. Egyptian Journal of Biological Pest Control, 33 (1): 21 (1-7).
7. Black, P. N., C. D. Blair, A. Butcher, J. L. Capinera & G. M. Happ, 1981. Biochemistry and ultrastructure of iridescent virus type 29. Journal of Invertebrate Pathology, 38 (1): 12-21.
8. Brendell, M. J. D., 1975. Handbooks for the identification of British insects. Coleoptera, Tenebrionidae. Volume 10. Royal Entomological Society, London, UK, 22 pp.

9. Burges, H. D. & K. A. Jones, 1998. “Formulation of Bacteria, Viruses and Protozoa to Control Insects, 33-127”. In: Formulation of Microbial Biopesticides: Beneficial Microorganisms, Nematodes and Seed Treatments (eds. H. D. Burges). Springer Dordrecht, Netherlands, 412 pp.
10. Cory, J. S., 2003. “Ecological Impacts of Virus Insecticides: Host Range and Non-Target Organisms, 73-91”. In: Environmental Impacts of Microbial Insecticides: Need and Methods for Risk Assessment (Eds. H. M. T. Hokkanen & A. E. Hajek). Dordrecht: Springer Netherlands, 269 pp.
11. Cotmore, S. F., M. Agbandje-McKenna, M. Canuti, J. A. Chiorini, A. M. Eis-Hubinger & J. Hughes. 2019. ICTV Report Consortium. ICTV virus taxonomy profile: Parvoviridae. Journal of General Virology, 100 (3): 367-368.
12. Deka, B., C. Baruah & A. Babu, 2021. Entomopathogenic microorganisms: Their role in insect pest management. Egyptian Journal of Biological Pest Control, 31 (1): 1-8.
13. Dong, W., W. YongMing, W. ChunXiu, Z. Zhen & X. Zheng, 2012. Review of environmental-friendly public health insecticides. 23 (5): 485-488.
14. Eroglu, G. B. & Z. Demirbag, 2022. An environmentally safe and tolerant microbial insecticide utilizing Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR). Egyptian Journal of Biological Pest Control, 32 (1): 53 (1-7).
15. Garofalo, C., V. Milanović, F. Cardinali, L. Aquilanti, F. Clementi & A. Osimani, 2019. Current knowledge on the microbiota of edible insects intended for human consumption: A state-of-the-art review. Food Research International, 125 (2019): 108527 (1-32).
16. Gencer, D., A. Yesilyurt, M. Güllü, İ. Demir & R. Nalcacıoglu, 2020. Insecticidal activities of wild type and recombinant invertebrate iridescent viruses on five common pests. Turkish Journal of Entomology, 44 (3): 365-373.
17. Guo, H., J. Zhang & Y. Hu, 2000. Complete nucleotide sequence and genomic organization of Periplaneta fuliginosa densonucleosis virus. Chinese Science Bulletin, 45 (19): 1782-1786.
18. Hernandez-Pelegrin, L., V. I. Ros, S. Herrero & C. Savio, 2025. Novel RNA viruses in a commercial colony of Tenebrio molitor. Journal of Invertebrate Pathology, 211: 108351 (1-8).
19. Huger, A. M., 1969. Virose bei larven des mehlkäfers: Tenebrio molitor. Naturwissenschaften, 56 (9): 466-467.
20. Jakob, N. J., K. Müller, U. Bahr & G. Darai, 2001. Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus. Virology, 286 (1): 182-196.
21. Johnson, R. M. & J. L. Rasgon, 2018. Densonucleosis viruses (‘densoviruses’) for mosquito and pathogen control. Current Opinion in Insect Science, 28: 90-97.
22. Kadji, F. M. N., K. Kotani, H. Tsukamoto, Y. Hiraoka & K. Hagiwara, 2022. Stability of enveloped and nonenveloped viruses in hydrolyzed gelatin liquid formulation. Virology Journal, 19 (1): 94 (1-8).
23. Kavallieratos, N. G., E. J. Michail, M. C. Boukouvala, E. P. Nika & A. Skourti, 2019. Efficacy of pirimiphos-methyl, deltamethrin, spinosad and silicoSec against adults and larvae of Tenebrio molitor L. on wheat, barley and maize. Journal of Stored Products Research, 83: 161-167.
24. Kelly, D. C., M. D. Ayres, T. Lescott, J. S. Robertson & G. M. Happ, 1979. A small iridescent virus (type 29) isolated from Tenebrio molitor: a comparison of its proteins and antigens with six other iridescent viruses. Journal of General Virology, 42 (1): 95-105.
25. Kittayapong, P., K. J. Baisley & S. L. O'Neill, 2001. A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand. The American Journal of Tropical Medicine and Hygiene, 61 (4): 612-617.
26. La Fauce, K. A., R. Layton & L. Owens, 2007. TaqMan real-time PCR for detection of hepatopancreatic parvovirus from Australia. Journal of Virological Methods, 140 (1-2): 10-16.
27. Luo, M., D. Zhu, J. Lin, X. Zhou, C. Zheng & X. Pu, 2021. Preparation and performance of insect virus microcapsules. Egyptian Journal of Biological Pest Control, 31 (1): 104 (1-12).
28. Nalcacioglu, R., H. Muratoglu, A. Yesilyurt, M. M. Van Oers, J. M. Vlak & Z. Demirbag, 2016. Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin. Journal of Invertebrate Pathology, 138: 104-111.
29. Ozgen, A., H. Muratoglu, Z. Demirbag, J. M. Vlak, M. M. Van Oers & R. Nalcacioglu, 2014. Construction and characterization of a recombinant invertebrate iridovirus. Virus Research, 189: 286-292.
30. Penzes, J. J., M. Holm, S. A. Yost & J. T. Kaelber, 2024. Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles. Cell, 187 (20): 5604-5619.
31. Pigeyre, L., M. Schatz, M. Ravallec, L. Gasmi, N. Negre, C. Clouet, M. Seveno, K. El Koulali, M. Decourcelle, Y. Guerardel, D. Cot, T. Dupressoir, A.-S. Gosselin-Grenet & M. Ogliastro, 2019. Interaction of a densovirus with glycans of the peritrophic matrix mediates oral infection of the lepidopteran pest Spodoptera frugiperda. Viruses, 11 (9): 870 (1-21).
32. Plata-Rueda, A., L. Martinez & M. Santos, 2017. Insecticidal activity of garlic essential oil and their constituents against the mealworm beetle, Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Scientific Report, 7 (1): 46406 (1-11).
33. Pöllinger-Zierler, B., A. Lienhard, C. Mayer, S. Berner, R. Rehorska, A. Schöpfer & M. Grasser, 2023. Tenebrio molitor (Linnaeus, 1758): Microbiological screening of feed for a safe food choice. Foods, 12 (11): 2139 (1-10).
34. Ribeiro, N., M. Abelho & R. Costa, 2018. A review of the scientific literature for optimal conditions for mass rearing Tenebrio molitor (Coleoptera: Tenebrionidae). Journal of Entomological Science, 53 (4): 434-454. https://doi.org/10.18474/JES17-67.1.
35. Rwegoshora, R. T. & P. Kittayapong, 2004. Pathogenicity and infectivity of the Thai-strain densovirus (AThDNV) in Anopheles minimus SL. Southeast Asian Journal of Tropical Medicine and Public Health, 35 (3): 630-634.
36. Stoops, J., S. Crauwels, M. Waud, J. Claes, B. Lievens & L. Van Campenhout, 2016. Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption. Food Microbiology,53 (Part B): 122-127.
37. Su, K. & C. Wang, 2015. Recent advances in the use of gelatin in biomedical research. Biotechnology letters, 37 (11): 2139-2145.
38. Szelei, J., J. Woodring, M. S. Goettel, G. Duke, F. X. Jousset, K. Y. Liu, Z. Zadori, Y. Li, E. Styer, D. G. Boucias, R. G. Kleespies, M. Bergoin & P. Tijssen, 2011. Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics. Journal of Invertebrate Pathology, 106 (3): 394-399.
39. Talukder, F. 2009. Pesticide resistance in stored-product insects and alternative biorational management: a brief review. Journal of Agricultural and Marine Sciences, 14 (1): 9-15.
40. Thapa, A., J. Tamang & M. Rai, 2020. “Viral Insecticides, 329-351”. In: Precision Agriculture and Sustainable Crop Production (Eds. H. K. Chourasia, K. Acharya & V. K. Singh). Today & Tomorrow’s Printers and Publishers, New Delhi, India, 631 pp.
41. Tokarev, Y.S., S. M. Malysh, Y. V. Volodartseva, A. V. Gerus & M. V. Berezin, 2020. Molecular identification of a densovirus in healthy and diseased Zophobas morio (Coleoptera, Tenebrionidae), Intervirology, 62 (5-6): 222-226.
42. Topuz, F. C. & G. Boran, 2018. Jelatin bazlı yenilebilir film ve kaplamalar. Akademik Gıda, 16 (3): 332-339 (in Turkish with abstract in English).
43. Wang, Y. J., Q. Yao, K. P. Chen, Y. Wang, J. Lu & X. Han, 2007. Characterization of the genome structure of Bombyx mori densovirus (China isolate). Virus Genes, 35 (1): 103-108.
44. Williams, T., V. Barbosa‐Solomieu & V. G. Chinchar, 2005. A decade of advances in iridovirus research. Advances in Virus Research, 65: 173-248.