Evaluation of Anti-bacterial Activity Induced by Penicillium mallochii in the Hemolymph of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)

Abstract

Anti-microbial peptides (AMPs) exhibit anti-bacterial, anti-fungal and anti-parasite activity and are essential effectors for the immune response of insects. Insect hemolymph contains AMPs, which are one of the sources of antibiotics effective on drug-resistant microorganisms. This study was conducted to induce antimicrobial activity in hemolymph by topical application of different doses of Penicillium mallochii conidia and its metabolite to Ephestia kuehniella larvae. Tetracycline antibiotic disks (TE-10 µg, Sigma), Sulfametaxozole trimethoprim (SXT-25 µg, Sigma), PBS, sterile water, and non-induced hemolymphs of larvae were used as control groups. In total hemolymph induced with metabolite extract, 24-h application was determined to be more effective on test bacteria than 48-h application. The largest zone diameter was observed against Escherichia coli (20mm) in hemolymph collected 24 h after metabolite application. Antimicrobial activity was highly increased (24h and 48h) when larvae were induced with P. mallochii conidial suspension. The largest zone diameter was observed against Proteus vulgaris and Klebsiella pneumonia (20 and 24 mm) in hemolymph collected 24 h after conidial suspension application. When larvae were induced with fungus metabolite and conidia, the zone of inhibition was 1.5-2.5-fold larger than that of the control hemolymph, indicating a higher antimicrobial activity after application. In general, this study provides a novel contribution to the knowledge regarding enhancement of antimicrobial activity in response to fungal infections in larvae.

Keywords

• Anti-bacterial activity
• Penicillium mallochii
• Ephestia kuehniella
• Hemolymph
• Fungi

References

1. Abdel-Moneim, A. M. E., El-Saadony, M. T., Shehata, A. M., Saad, A. M., Aldhumri, S. A., Ouda, S. M., & Mesalam, N. M. (2021). Antioxidant and antimicrobial activities of Spirulina platensis extracts and biogenic selenium nanoparticles against selected pathogenic bacteria and fungi. Saudi Journal of Biological Sciences, 29 (2), 1197-1209. https://doi.org/10.1016/j.sjbs.2021.09.046
2. Al-Marzoqi, A. H., Imad, H. H., & Salah, A. I. (2015). Analysis of bioactive chemical components of two medicinal plants (Coriandrum sativum and Melia azedarach) leaves using gas chromatography-mass spectrometry (GC-MS). African Journal of Biotechnology, 14, 2812-2830. https://doi:10.5897/AJB2015.14956
3. Andra, J., Berninghausen, O., & Leippe, M. (2001). Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans. Medical Microbiology and Immunology, 189 (3), 169-173. https://doi.org/10.1007/s430-001-8025-x
4. Bland, M. L. (2023). Regulating metabolism to shape immune function: Lessons from Drosophila. Seminars in Cell and Developmental Biology, 138, 128-141. https://doi.org/10.1016/j.semcdb.2022.04.002
5. Boderick, N. A., Welchman, D. P., & Lemaitre, B. (2009). Recognition and Response to Microbial Infection in Drosophila. (Rolff, J., & Reynolds, S. E.) (Eds.). Insect Infection and Immunity, Evolution, Ecology, and Mechanisms. Oxford University Press.
6. Bouhri, Y., Aşkun, T., Tunca, B., Deniz, G., Aksoy, S. A., & Mutlu, M. (2020). The orange-red pigment from Penicillium mallochii: Pigment production, optimization, and pigment efficacy against Glioblastoma cell lines. Biocatalysis and Agricultural Biotechnology, 23, 101451. https://doi.org/10.1016/j.bcab.2019.101451
7. Browne, N., Heelan, M., & Kavanagh, K. (2013). An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes. Virulence, 4, 597-603. https://doi.org/10.4161/viru.25906
8. Casanova-Torres, A. M., & Goodrich-Blair, H. (2013). Immune signaling and antimicrobial peptide expression in Lepidoptera. Insects, 4 (3), 320-38. https://doi.org/10.3390/insects4030320.

9. Coggins, S. A., Estévez-Lao, T. Y., & Hillyer, J. F. (2012). Increased survivorship following bacterial infection by the mosquito Aedes aegypti as compared to Anopheles gambiae correlates with increased transcriptional induction of antimicrobial peptides. Developmental & Comparative Immunology, 37, 390–401. https://doi.org/10.1016/j.dci.2012.01.005
10. Eleftherianos, I., Heryanto, C., Bassal, T., Zhang, W., Tettamanti, G., & Mohamed, A. (2021). Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites. Immunology, 164, 401-432. https://doi.org/10.1111/imm.13390
11. El-Saadony, M. T., Elsadek, M. F., Mohamed, A.S., Taha, A. E., Ahmed, B. M., & Saad, A. M. (2020). Effects of chemical and natural additives on cucumber juice’s quality, shelf life, and safety. Foods, 9 (5), 639. https://doi.org/10.3390/foods9050639
12. El-Saadony, M. T., Sitohy, M. Z., Ramadan, M. F., & Saad, A. M. (2021). Green nanotechnology for preserving and enriching yogurt with biologically available iron (II). Innovative Food Science & Emerging Technologies, 69, 102645. https://doi.org/10.1016/j.ifset.2021.102645
13. Fancelli, M., Dias, A. B., Delalibera, I. J., Cerqueira de Jesus, S., Souza do Nascimento, A., & Oliveira e Silva, S. (2013). Beauveria bassiana Strains for Biological Control of Cosmopolites sordidus (Germ.) (Coleoptera: Curculionidae) in Plantain. BioMed Research International, 184756. https://doi.org/10.1155/2013/184756
14. Haine, E. R., Moret, Y., Siva-Jothy, M. T., & Rolff, J. (2008). Antimicrobial defense and persistent infection in insects. Science, 322, 1257-1259. https://doi.org/10.1126/science.1165265
15. Hultmark, D., Steiner, H., Rasmuson, T., & Boman, H. G. (1980). Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. European Journal of Biochemistry, 106, 7-16. https://doi.org/10.1111/j.1432-1033.1980.tb05991.x
16. Junqueira, J. C., & Mylonakis, E. (2019). Current status and trends in alternative models to study fungal pathogens. Journal of Fungi, 5 (1), 12. https://doi.org/10.3390/jof5010012
17. Korner, P., & Schmid-Hempel, P. (2004). In vivo dynamics of an immune response in the bumble bee Bombus terrestris. Journal of Invertebrate Pathology, 87, 59-66. https://doi.org/10.1016/ j.jip.2004.07.004
18. Kurtuluş, A., Pehlivan, S., Achiri, T. D., & Atakan, E. (2020). Influence of different diets on some biological parameters of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Journal of Stored Products Research, 85, 101554. https://doi.org/10.1016/j.jspr.2019.101554
19. Latifi, M., Alikhani, M. Y., Salehzadeh, A., Nazari, M., Bandani, A. R., & Zahirnia, A. H. (2015). The antibacterial effect of American cockroach hemolymph on the nosocomial pathogenic bacteria. Avicenna Journal of Clinical Microbiology and Infection, 2, e23017. https://doi.org/10.17795/ajcmi-23017
20. Lazzaro, B. P., & Rolff, J. (2011). Immunology. Danger, microbes, and homeostasis. Science, 332, 43-44. https://doi.org/10.1126/science.1200486
21. League, G. P., Estevez-Lao, T. Y., Yan, Y., Garcia-Lopez, V. A., & Hillyer, J. F. (2017). Anopheles gambiae larvae mount stronger immune responses against bacterial infection than adults: evidence of adaptive decoupling in mosquitoes. Parasite Vector, 10, 367. https://doi.org/10.1186/s13071-017-2302-6
22. Lemaitre, B., & Hoffmann, J. (2007). The host defense of Drosophila melanogaster. Annual Review of Immunology, 25, 697-743. https://doi.org/10.1146/annurev.immunol.25.022106.141615
23. Lu, H. L. & St. Leger, R. J. (2016). Insect Immunity to entomopathogenic fungi. Advances in Genetics, 94, 251-285. https://doi.org/10.1016/bs.adgen.2015.11.002
24. Manniello, M. D., Moretta, A., Salvia, R., Scieuzo, C., Lucchetti, D., Vogel, H., Sgambato, A., & Falabella, P. (2021). Insect antimicrobial peptides: Potential weapons to counteract the antibiotic resistance. Cellular and Molecular Life Sciences, 78, 4259-4282. https://doi.org/10.1007/s00018-021-03784-z
25. Marshall, S. H., & Arenas, G. (2003). Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology. Electronic Journal of Biotechnology, 6, 271-284.
26. Morejon, B., & Michel, K. (2023). A zone-of inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph. Frontiers in Cellular and Infection Microbiology, 13, 891577. https://doi.org/10.3389/fcimb.2023.891577
27. Moreno-Garcia, M., Córdoba-Aguilar, A., Condé, R., & Lanz-Mendoza, H. (2013). Current immunity markers in insect ecological immunology: assumed trade-offs and methodological issues. Bulletin of Entomological Research, 103(2), 127–139. https://doi.org/10.1017/S000748531200048X
28. Pasupuleti, M., Schmidtchen, A., & Malmsten, M. (2012) Antimicrobial peptides: key components of the innate immune system. Critical Reviews in Biotechnology, 32 (2), 143-71. https://doi.org/10.3109/07388551.2011.594423
29. Radwan, M. H, Alaidaroos, B. A., Jastaniah, S.D., Abu El-Naga, M. N., El-Gohary, E. E., Barakat, E. M. S., El Shafie, A. M., Abdou, M. A., Mostafa, N. G., El-Saadony, M. T., & Momen, S. A. A. (2022). Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis. Saudi Journal of Biological Sciences, 29 (4), 2892-2903. https://doi.org/10.1016/j.sjbs.2022.01.025
30. Rhodes, V. L., Thomas, M. B., & Michel, K. (2018). The interplay between dose and immune system activation determines fungal infection outcome in the African malaria mosquito, Anopheles gambiae. Developmental & Comparative Immunology, 85, 125-133. https://doi.org/10.1016/j.dci.2018.04.008
31. Rivera, K. G., Díaz, J., Chavarría-díaz, F., Garcia, M., Urb, M., Thorn, R. G., Louis-seize, G., & Janzen, D. H. (2012). Penicillium mallochii and P. guanacastense, two new species isolated from Costa Rican caterpillars. Mycotaxon, 119, 315-328. https://doi.org/10.5248/119.315
32. Robertson, M., & Postlethwait, J. H. (1986). The humoral antibacterial response of Drosophila adults. Developmental & Comparative Immunology, 10, 167-179. https://doi.org/10.1016/0145-305x(86)90001-7
33. Saad, A. M., El-Saadony, M. T., Mohamed, A.S., Ahmed, A. I., & Sitohy, M. Z. (2021). Impact of cucumber pomace fortification on the nutritional, sensorial and technological quality of soft wheat flour-based noodles. International Journal of Food Science & Technology, 56 (7), 3255-3268. https://doi.org/10.1111/ijfs.14970
34. Seraj, U., Hoq, M., Anwar, M., & Chowdhury, S. (2003). A 61 kDa antibacterial protein isolated and purified from the hemolymph of the american cockroach Periplaneta americana. Pakistan Journal of Biological Sciences, 6 (7), 715-720. https://doi.org/10.3923/pjbs.2003.715.720
35. Shafaghat, A. (2012). Phytochemical and antimicrobial activities of Lavandula officinalis leaves and stems against some pathogenic microorganisms. Journal of Medicinal Plants Research, 6, 455-460. https://doi.org/10.5897/JMPR11.1166
36. Swelum, A. A., Shafi, M. E., Albaqami, N. M., El-Saadony, M. T., Elsify, A., Abdo, M., & Mohamed, E. (2020). COVID-19 in human, animal, and environment: a review. Frontiers in Veterinary Science. 7, 578. https://doi.org/10.3389/fvets.2020.00578
37. Teixeira, M. F. N. P., Souza, C. R., & Morais, P. B. (2022). Diversity and enzymatic capabilities of fungi associated with the digestive tract of larval stages of a shredder insect in cerrado and Amazon Forest, Brazil. Brazilian Journal of Biology, 82, e260039. https://doi.org/10.1590/1519-6984.265681
38. Ursic-Bedoya, R., Buchhop, J., Joy, J. B., Durvasula, R., & Lowenberger, C. (2011). Prolixicin: a novel antimicrobial peptide isolated from Rhodnius prolixus with diferential activity against bacteria and Trypanosoma cruzi. Insect Molecular Biology, 20, 775-786. https://doi.org/10.1111/j.1365-2583.2011.01107
39. Uvell, H., & Engström, Y. (2007). A multilayered defense against infection: combinatorial control of insect immune genes. Trends in Genetics, 23, 342-349. https://doi.org/10.1016/j.tig.2007.05.003
40. Wojda, I., Cytryńska, M., Zdybicka-Barabas, A., & Kordaczuk, J. (2020). Insect defense proteins and peptides. Sub-Cellular Biochemistry, 94, 81-121. https://doi.org/10.1007/978-3-030-41769-7-4
41. Yoon, B., Jackman, J., Valle-González, E., & Cho, N. J. (2018). Antibacterial free fatty acids and monoglycerides: biological activities, experimental testing, and therapeutic applications. International Journal of Molecular Sciences, 19 (4), 1114. https://doi.org/10.3390/ijms19041114
42. Zahirnia, A., Seifi-Kar, M., & Nasirian, H. (2023). Evaluation of antifungal activity of hemolymph of American cockroaches against three human invasive fungal species: Aspergillus niger, Candida albicans and Penicillium oxalicum. International Journal of Tropical Insect Science. https://doi.org/10.1007/s42690-023-01056-w
43. Zhang, Z. M., Wu, W. W., & Li, G. K. (2009). Study of the alarming volatile characteristics of Tessaratoma papillosa using SPME-GC-MS. Journal of Chromatographic Science, 47, 291-295. https://doi.org/10.1093/chromsci/47.4.291