Effect of Metarhizium anisopliae IPPM202 Extracellular Proteinases on Midgut of Locusta migratoria manilensis

Metarhizium anisopliae, an entomopathogenic fungus, can produce four extracellular proteases, subtilisin (Pr1), trypsin (Pr2), metalloproteases (Pr3), and cysteine proteases (Pr4), which are important for pathogenicity of M. anisopliae in target hosts. In order to understand their function in M. anisopliae pathogenicity, third-instar nymphs of Locusta migratoria were fed with a diet containing either conidia of M. anisopliae strain IPPM202 or in combination with one of the four inhibitors (TPCK: tosyl-phenylalanine chloromethyl-ketone, inhibitor of Pr1: EDTA: ethylenediaminetetraacetic acid, inhibitor of Pr3: APMSF: 4-amidinophenyl methanesulfonyl fluoride, inhibitor of Pr2: CI1: cathepsin inhibitor 1, inhibitor of Pr4). The effects on mortality, midgut integrity, and the gut enzymes peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and phenol oxidase (PO) were examined. The results indicated that exposure to IPPM202/TPCK and IPPM202/CI1 caused decreased mortality to L. migratoria with no loss of midgut epithelial cellular integrity. On the other hand, exposure to IPPM202/APMSF or IPPM202/EDTA mixtures resulted in higher mortality similar to PPM202, with severely damaged epithelial gut cells with fragmented microvilli, broken endoplasmic reticulum, and disrupted nucleus membrane. The activity of the protective enzymes POD, SOD, CAT, and PO all increased significantly when L. migratoria was treated with IPPM202 only, but decreased when any one of the inhibitors was added. We further concluded that TPCK, a subtilisin (Pr1) inhibitor, and CI1, a cysteine protease (Pr4) inhibitor, played important roles in the pathogenicity of the M. anisopliae strain IPPM202. Conversely, trypsin (Pr2) and metalloproteases (Pr3) did not have a role in the given process. We further concluded that trypsin (Pr2) and metalloproteases (Pr3) do not contribute to the fungal infection process, while the subtilisin (Pr1) inhibitor TPCK and cysteine protease (Pr4) inhibitor CI1 play critical roles in the pathogenicity of Metarhizium anisopliae strain IPPM202, thus providing a foundation for targeted biocontrol strategies.