In vitro screening of 65 mycotoxins for insecticidal potential

Abstract

The economic losses and threats to human and animal health caused by insects and the pathogens transmitted by them require effective and environmentally-friendly methods of controlling them. One such group of natural biocontrol agents which may be used as biopesticides is that of the entomopathogenic fungi and their toxic secondary metabolites (mycotoxins). The present in vitro work examined the insecticidal potential of 65 commercially-available mycotoxins against the insect Sf-9 cell line. Mammalian Caco-2 and THP-1 cell lines served as reference controls to select insecticidal mycotoxins harmless to mammalian cells. All tested mycotoxins significantly reduced the in vitro proliferation of the Sf-9 cells and evoked morphological changes. Ten of the mycotoxins found to strongly inhibit Sf-9 proliferation also had moderate or no effect on Caco-2 cells. The THP-1 cells were highly resistant to the tested mycotoxins: doses 103 times higher were needed to affect viability and morphology (1 μg/ml for THP-1 versus 1 ng/ml for Sf-9 and Caco-2). Nine mycotoxins significantly decreased Sf-9 cell proliferation with minor effects on mammalian cells: cyclosporins B and D, cytochalasin E, gliotoxin, HC toxin, paxilline, penitrem A, stachybotrylactam and verruculogen. These may be good candidates for future biopesticide formulations.

Fig 1. Dose-response effects of four mycotoxins on the viability of THP-1 cells in vitro.

The statistical significance of the differences observed between treated cultures and controls at all concentrations of each tested mycotoxin were tested using Dunnett test. Each test was performed in 3 independent replications; control C2 (solvent treatment) in 20 replications. Statistical significance: * p < 0.05, ** p <0.001, *** p <0.0001.

Fig 2. Dose-response effects of aflatoxin M1 and T2-toxin on the mortality of THP-1 cells in vitro.

The statistical significance of the differences observed between treated cultures and controls at all concentrations of each tested mycotoxin were tested using Dunnett test. Each test was performed in 3 independent replications; controls: C1 (no treatment) and C2 (solvent treatment), each in 3 replications. Statistical significance: * p < 0.05, ** p <0.001, *** p <0.0001.

Fig 3. Examples of characteristic mycotoxin-induced morphological changes in Caco-2 and Sf-9 cells.

(1) untreated Caco-2 control—C1, (2) Caco-2 control treated with solvent—C2, (3) Caco-2 treated aflatoxicol, (4) Caco-2 treated 3-acetyldeoxynivalenol, (5) Caco-2 treated aflatoxin B1, (6) Caco-2 treated aflatoxin G2, (7) Caco-2 treated α-amanitin, (8) Caco-2 treated antibiotic PF 1052, (9) Caco-2 treated cyclosporin H, (10) Caco-2 treated cytochalasin B, (11) Caco-2 treated gliotoxin, (12) Caco-2 treated HT-2 toxin, (13) Caco-2 treated roquefortine C, (14) Caco-2 treated stachybotrylactam, (15) Caco-2 treated T2 tetraol, (16) Caco-2 treated wortmannin, (17) Caco-2 treated zearalenone, (18) untreated Sf-9—C1, (19) Sf-9 treated with solvent—C2, (20) Sf-9 treated 3-acetyldeoxynivalenol, (21) Sf-9 treated aflatoxin B2, (22) Sf-9 treated alternariol, (23) Sf-9 treated alternariol-9-methyl ether, (24) Sf-9 treated fumonisin B2, (25) Sf-9 treated neosolaniol, (26) Sf-9 treated ochratoxin A, (27) Sf-9 treated patulin, (28) Sf-9 treated stachybotrylactam, (29) Sf-9 treated T2 tetraol, (30) Sf-9 treated verruculogen. White arrow—mitotic cells, yellow arrow—spindle cell, green arrow—vacuoles, blue arrow—malformed cell, orange arrow—monolayer surface protrusions, purple arrow—cells detached from the monolayer, black arrow—fragments of disintegrated cells. Scale bars: 50 μm.

Fig 4. Examples of characteristic mycotoxin-induced morphological changes in THP-1 cells.

(1) untreated THP-1 control—C1, (2) THP-1 control treated with solvent—C2, (3) aflatoxicol, (4) aflatoxin B2, (5) aflatoxin G2, (6) cyclosporin C, (7) cyclosporin H, (8) cytochalasin C, (9) diacetoxyscripenol, (10) fumigaclavine A, (11) moniliformin, (12) ochratoxin A, (13) patulin, (14) paxilline, (15) sterigmatocystin. White arrow—mitotic cells, red arrow—swelled and deformed cell, green arrow—vacuoles, black arrow—fragments of disintegrated cells. Scale bars: 50 μm.