Phenol-Oxidase Activity and Haemocytes Changes in Helicoverpa armigera Hübner Infected by Entomopathogenic Fungi, Beauveria bassiana and Metarhizium anisopliae

Document Type : Original Research

Authors
A. Hatami 1
R. Farshbaf Pour Abad 1
M. Saber 1
R. Motafakkerazad 2
1 Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Islamic Republic of Iran.
2 Department of Plant Sciences, Faculty of Natural Sciences, University of Tabriz, Tabriz, Islamic Republic of Iran.
10.22034/JAST.27.4.895
Abstract
Entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae are important and effective biocontrol agents against arthropod pests. Compared to chemical insecticides, insect pests do not easily develop resistance against these fungi. In this study, the lethal effects of exposure to B. bassiana and M. anisopliae, effects on phenol-oxidase activity, total haemocyte count, and changes in granulocytes and plasmatocytes were evaluated in 3rd instar larvae of Helicoverpa armigera. The LC50 values for B. bassiana and M. anisopliae were 0.795×106, and 5.972×107 spore mL-1, respectively. LC30 and LC10 of either entomopathogenic fungi were injected into the body of larvae, and 24 and 48 hours after injection, their hemolymph was extracted. After 24 hours, the highest and lowest phenol-oxidase activity was observed in LC30 of M. anisopliae, and LC10 of B. bassiana, respectively. After 48 hours of infection, phenol-oxidase activity increased in all treatments. At the LC30 of M. anisopliae, the highest phenol-oxidase activity was recorded, and other treatments also showed a significant difference compared to the control. Five types of hemocytes including prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes were identified in the hemolymph of larvae. The highest Total Hemocyte Count (THC) was recorded in LC30 M. anisopliae at 9 hours after initial infection. The highest number of granulocytes and plasmatocytes were recorded 9 hours after treatment in LC30 of M. anisopliae and LC30 of B. bassiana. Our results showed that both fungi had the ability to affect phenol-oxidase enzyme activity and haemocytes. These microbial insecticides exhibited high potential for controlling the pest.

Keywords

Subjects

Alizadeh, Z. 2014. Identification of entomopathogenic fungi from Khosrowshahr region based on phylogenetic analysis of ITS-rDNA sequence. M.Sc. thesis, Azarbaijan Shahid Madani University, 118 pp. (In Persian with English summary).
Bali, G.K., Kaur, S., Kour, B. 2013. Phenol-oxidase activity in haemolymph of Spodoptera litura (Fabricius) mediating immune responses challenge with entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillmin. J. Entomol. Zool. Stud., 1(6): 118-123.
Bogdan, C., Röllinghoff, M., Diefenbach, A. 2000. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol., 12(1): 64-76.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Cao, G. Jia, M., Zhao, X., Wang, L., Tu, X. Wang, G., Nong, N., Zhang, Z. 2016. Different effects of Metarhizium anisopliae strains IMI330189 and IBC200614 on enzymes activities and hemocytes of Locusta migratoria L. PLoS One, 11(5): 1-8.
Cerenius, L. and Söderhäll, K. 2021. Immune properties of invertebrate phenol-oxidases. Dev. Comp. Immunol., 122: 104098.
Deka, B., Baruah, C., Babu, A. 2021. Entomopathogenic microorganisms: their role in insect pest management. Egypt. J. Biol. Pest Control., 31(1): 1-8.
De Souza, T.D., Fernandes, F.O., Sanches, A.C., Polanczyk, R.A., 2020. Sublethal effects of different fungal isolates on Helicoverpa armigera (Lepidoptera: Noctuidae). Egypt J Biol Pest Control., 30(1): 1-12.
Essawy, M., Maleville, A., Brehelin, M. 1985. The Hemocytes of Heliothis armigera: Ultrastructure, Functions, and Evolution in the Course of Larval Developmen. J. Morphol., 186: 255-264.
Faraji, S., Mehrvar, A., Shadmehri, A.D. 2013. Studies on the virulence of different isolates of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metcsn.) Sorokin against Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Afr. J. Agric. Res., 8: 4157-4161.
Ferreira, J.M., and de Freitas Soares, F.E. 2023. Entomopathogenic fungi hydrolytic enzymes: A new approach to biocontrol?. J. Nat. Pestic Res., 3: 1-12.
Giglio, A., Battistella, S., Talarico, F.F., Brandmayr, T.Z. and Giulianini, P.G. 2008. Circulating hemocytes from larvae and adults of Carabus (Chaetocarabus) lefebvrei Dejean 1826 (Coleoptera, Carabidae): cell types and their role in phagocytosis after in vivo artificial non-self-challenge. Micron., 39: 552-558.
Gujar, G.T. and Kalia, V.K. 2005. Hemocyte Diversity of the American Bollworm Helicoverpa armigera. Phytoparasitica., 33(1): 17-27.

Hung, S. Y. and Boucias, D. G. 1992. Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua. J. Invertebr. Pathol., 60(2):152-158.
Istkhar, R. and Chaubey, A.K. 2018. Challenging the larvae of Helicoverpa armigera and assessing the immune responses to nematode-bacterium complex. Phytoparasitica., 46: 75-87.
Kalia, V., Chaudhari, S., Gujar, G. 2001. Changes in haemolymph constituents of American bollworm, Helicoverpa armigera Hübner infected with nucleopolyhedroviru. Indian. J. Exp. Biol., 39(11): 1123-1129.
Jones, J. C. 1962. Current concepts concerning insect hemocytes. Am. Zool, 2(2): 209-246.
Kalvnadi, E., Mirmoayedi, A., Alizadeh, M., Pourian, H.-R. 2018. Sub-lethal concentrations of the entomopathogenic fungus, Beauveria bassiana increase fitness costs of Helicoverpa armigera (Lepidoptera: Noctuidae). J. Invertebr. Pathol., 158: 32-42.
Kanost, M.R. and Gorman, M.J. 2008. Phenol-oxidases in insect immunity. Insect immunology, 1: 69-96.
Karim, S. 2000. Management of Helicoverpa armigera: a review and prospectus for Pakistan. Pak. J. Biol. Sci., 3(8): 1213-1222.
Kidanu, S. and Hagos, L. 2020. Research and application of entomopathogenic fungi as pest management option: a review. J. Environ. Earth Sci., 10(3): 31-39.
Khosravi, R., Sendi, J. J., Zibaee, A., Shokrgozar, M. A. 2014. Immune reactions of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) to the entomopathogenic fungus, Beauveria bassiana (Bals.-Criv.) Vuill and two developmental hormones. Invertebr surviv. j., 11: 11-21.

Lacey, L.A., Frutos, R., Kaya, H., Vail, P. 2001. Insect pathogens as biological control agents: do they have a future?. Biol. Control., 21(3): 230-248.
Lavine, M.D. and Strand, M.R. 2002. Insect hemocyte and their role in cellular immune responses. Insect Biochem. Mol. Biol.. 32: 1237-1242.
Leonard, C., Söderhäll, K., Ratcliffe, N.A. 1985. Studies on prophenol-oxidase and protease activity of Blaberus craniifer haemocytes. Insect Biochem., 15(6): 803-810.
Ling, E., Shirai, K., Kanekatsu, R.., Kiguchi, K. 2005. Hemocyte differentiation in the hematopoietic organs of the silkworm, Bombyx mori: prohemocytes have the function of phagocytosis. J. Cell Tissue Res., 320: 535-543.
Ling, E., Shirai, K., Kanekatsu, R., Kobayashi, Y., Tu, Z., Funayama, T. 2003. Why does hemocyte density rise at the wandering stage in the silkworm, Bombyx mori?. J. Insect Biotechnol. Sericol., 72: 101-10.
Liu, Z.-C., Zhou, L., Wang, J.-L., Liu, X.-S. 2021. Expression of a phenol-oxidase cascade inhibitor enhances the virulence of the fungus Beauveria bassiana against the insect Helicoverpa armigera. Dev. Comp. Immunol., 117: 1-7.
Mahmoud, D. Salem, D., Mo'men, S., Barakat, E., Salama, M. 2015. Purification and characterization of phenol-oxidase from immunized haemolymph of Schistocerca gregaria. Afr. J. Biotechnol., 14(44): 3027-3036.
Mingotti Dias, P., de Souza Loureiro, E., Amorim Pessoa, L.G., Mendes de Oliveira Neto, F., de Souza Tosta, RA., Teodoro, P.E. 2019. Interactions between fungal-infected Helicoverpa armigera and the predator Chrysoperla externa. Insects, 10(10): 1-11.
Mishra, G. and Omkar, 2021. Gram Pod Borer (Helicoverpa armigera). Polyphagous Pests of Crops: 311-348.
Mora, M.A.E., Castilho, A.M.C., Fraga, M.E. 2018. Classification and infection mechanism of entomopathogenic fungi. Arq. Inst. Biol., Sao Paulo., 8: 1-10.
Petlamul, W., Boukaew, S., Hauxwell, C., Prasertsan, P. 2019. Effects on detoxification enzymes of Helicoverpa armigera (Lepidoptera: Noctuidae) infected by Beauveria bassiana spores and detection of its infection by PCR. Sci. Asia., 45: 581–588.
Qu, S. and Wang, S. 2018. Interaction of entomopathogenic fungi with the host immune system. Dev Comp Immunol., 1-8.
Safavi, S., Kharrazi, A., Rasoulian, G.R., Bandani, A. 2010. Virulence of some isolates of entomopathogenic fungus, Beauveria bassiana on Ostrinia nubilalis (Lepidoptera: Pyralidae) larvae. J. Agric. Sci. Technol., 12(1): 13-21.
Shorey, H. and Hale, R. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol., 58(3): 522-524.
Singh, D., Raina, T.K., Singh, J. 2017. Entomopathogenic fungi: An effective biocontrol agent for management of insect populations naturally. J. Appl. Pharm. Sci. Res., 9(6): 833.
Söderhäll, K. and Cerenius, L. 1998. Role of the prophenol-oxidase-activating system in invertebrate immunity. Curr. Opin. Immunol., 10(1): 23-28.
Suzuki, A., Taguchi, H., Tamura, S. 1970. Isolation and structure elucidation of three new insecticidal cyclodepsipeptides, destruxins C and D and desmethyldestruxin B, produced by Metarrhizium anisopliae. Agric. Biol. Chem., 34(5): 813-816.
Tanaka, H., and Yamakawa, M. 2011. Regulation of the innate immune responses in the silkworm, Bombyx mori. Invertebrate surviv. j., 8: 59-69.
Tartar, A., Shapiro, A.M., Scharf, D.W., Boucias, D.G. 2005. Differential expression of chitin synthase (CHS) and glucan synthase (FKS) genes correlates with the formation of a modified, thinner cell wall in in vivo-produced Beauveria bassiana cells. Mycopathologia., 160: 303-314.
Wahlman, M. and Davidson, B.S. 1993. New destruxins from the entomopathogenic fungus Metarhizium anisopliae. J. Nat. Prod., 56(4): 643-647.

Zhong, K., Liu, Z.C., Wang, J.L., Liu, X.S. 2017. The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera. Arch. Insect Biochem. Physiol., 96(1): 1-10.
Zibaee, A., Bandani, A.R., Talaei-Hassanlouei, R., Malagoli, D. 2011. Cellular immune reactions of the sunn pest, Eurygaster integriceps, to the entomopathogenic fungus, Beauveria bassiana and its secondary metabolites. J. Insect Sci., 11(1): 1-16.
Zibaee, A. and Malagoli, D. (2014). Immune response of Chilo suppressalis Walker (Lepidoptera: Crambidae) larvae to different entomopathogenic fungi. Bull. Entomol. Res., 104(2): 155-163.
Journal of Agricultural Science and Technology
Volume 27, Issue 4
July and August 2025
Pages 895-908