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An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2).

ABSTRACT: Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host-pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL^-1) was better than other two M. anisopliae (134.59 µg mL^-1) and B. bassiana (128.65 µg mL^-1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL^-1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL^-1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL^-1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

SUBMITTER: Ghosh SK

PROVIDER: S-EPMC8346544 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Microfungal applications are increasing daily in the medical science. Several species of Trichoderma are widely used in agricultural fields as biological control and plant growth promoting agents. The application of Trichoderma asperellum as an entomopathogenic fungus against the Anopheles mosquito, a vector of malaria, is a novel control approach. Controlling malaria with eco-friendly management practices is an urgent need. We isolated three T. asperellum from different natural sources using serial dilution and mosquito baiting techniques. The fungi were identified on the basis of phenotypical and molecular characteristics. The fungi were grown in different natural media to examine spore production ability and the fungal spore suspensions were applied to the anopheline larvae to determine their larvicidal activity in vitro. We investigated the efficacy of crude ME (methanolic extract) and different methanolic fractions (MFs) of the fungal extracts against anopheline larvae. Methanolic Fraction 8 (MF8) exhibited the strongest larvicidal activity. A GC-MS analysis of MF8 and a Chemolibrary search were performed to identify the active agents in the fungal extracts. Among the three isolates of T. asperellum, the TaspSKGN2 isolate showed the lowest LD50 (2.68 × 107 conidia/mL) and LT50 values (12.33 h). The crude ME exhibited LD50 values of 0.073 mg/mL and LT50 values of 11.33 h. MF8 showed LD50 values of 0.059 mg/mL and LT50 values of 8.57 h. In GC-MS study of MF8, 49 compounds were found. Among these, seven compounds (2,3-di hydro thiopene, p-cymene, alpha-pinene, hexadecanoic acid, 8-methyl quinoline, (Z,Z)-9,12-octa decadienoic acid, methyl ester, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-Pyran-4-one-) with high abundance were found to have insecticidal efficacy by a literature survey. We detected a reduction in the phenoloxidase content inside the cuticle and hemolymph of the anopheline larvae after a few hours of interaction with ME (0.073 mg/mL). Thus Trichoderma asperellum has new applications for the control of Anopheles spp. malaria vectors.

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An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2).

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