The Regulation Of Calcineurin To Phenoloxidase And Other Biological Enzymes Involved In Immunity Of Locusta Migratoria L.

As one of the most important pest in migratory locusts, Locusta migratoria L. can cause huge losses in agriculture in China. The immune-related enzymes, sueh as phenoloxidase, play a pivotal role in innate immune system of insects against pathogenic fungi Metarrhizium anisopliae. Recent studies has found that calcineurin (CaN), as a serine/threonine phosphatases under control of Ca2+ and calmodulin (CaM), is involved in the regulation of the antimicrobial peptides (AMPs) production in humoral immunity of Drosophila melanogaster. And yet, little research has addressed about the relationship between CaN and immune-related enzymes. Therefore, the activities of CaN and immune-related enzymes were assayed after locusts were treated with inhibitors of phenoloxidase, CaN and Ca2+ channels. These indicated that CaN might play a critical role in immunity of L. migratoria and the study might provide the basis of the mechanism that Metarrhizium anisopliae suppress host immunity via the inhibition of CaN activity.1. The effects of a phenoloxidase inhibitor Kojic acid on biological enzymes activities of L. migratoriaTo investigate the role of phenoloxidase (PO) in immune system of insects, the activities of PO, acetylcholinesterase (AChE), chitinase (CHI), general esterases (ESTs), glutathione-S-transferases (GSTs), aryl acylamidase (AA), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) were assayed after L. migratoria were treated with Kojic acid. PO, AChE, CHI, ESTs, GSTs, AA activities decreased in the Kojic acid-treated locusts decreased in comparison with control after 3 d. POD, SOD and CAT activities increased after 3 d and decreased after 5 d in comparison with control. The results indicated that Kojic acid showed inhibitory effects on PO and other biochemical enzymes of locusts.2. The effects of Ruthenium Red which is a ryanodine receptor (RYR) antagonist on biological enzymes in L. migratoriaTo investigate the role of RYR in immune system in insects, the activities of PO, AChE, ESTs, GSTs, POD, SOD and CAT were assayed after L. migratoria were treated with Ruthenium Red (RR). The results showed that there was no obvious toxicity of RR to locusts. The ESTs, AChE and GSTs activities increased early and only GSTs and AChE activities decreased finally after locusts were treated with RR. RR showed a inhibitory effect on PO early. In conclusion, the activities of biological enzymes of locusts were affected when the RYR was inhibited, the decreased of PO and AChE activities might be caused the inhibition of the release of Ca2+ from RYR. These provide theoretical references of the target and synergist of insecticides about RYR.3. The activation of calcineurin in L. migratoriaTo investigate the activation of calcineurin (CaN) in insects, the CaN activity was assayed after L. migratoria were treated with 2-APB, Trifluoperazine (TFP) and Cyclosporine A (CsA) which were inhibitors of inositol 1,4,5-trisphosphate receptor (InsP3R), calmodulin (CaM) and CaN respectively. The results showed that the activity of CaN was decreased when locusts were treated with CsA after 1 d,2-APB after 3 d and TFP after 3,5,7 d in vivo while it was not decreased by CsA,2-APB and TFP in vitro (unpublished), which indicated that CaN might not be activated when InsPsR and CaM were inhibited by 2-APB and TFP in L. migratoria. We showed that, the InsP3R and CaM were required for the activation of CaN in L. migratoria.4. The relationship between CaN and other biological enzymesIn this study, a potential relationship between CaN and other biological enzymes was investigated by assaying CaN and other biological enzymes activities after L. migratoria were treated with pharmacological inhibitors (2-APB, TFP and CsA). To our surprise, locusts were fed with 2-APB, TFP and CsA resulting the decreased of CaN and biological enzymes activities, such as PO, AChE, detoxification enzymes (GSTs and ESTs) and antioxidant enzymes (POD, SOD and CAT). This result suggested that the decrease of biological enzymes might be related with the inhibition of CaN in L. migratoria.5. The difference of survival rate after locusts were treated with CsA and Metarrhizium anisopliae.To investigate the role of CaN in locusts against M. anisopliae, the survival rate difference was tested after locusts were treated with CsA and M. anisopliae. The results showed that the survival rate of locusts fed with CsA and treated with M. anisopliae (2.5E+07 spores/mL) decreased in comparison with no CsA-treated locusts. But there was no difference survival rate of locusts treated with M. anisopliae (2.5E+08 spores/mL). These indicate that CaN play a critical role in locusts immune responses against M. anisopliae and the M. anisopliae might affect the CaN of locusts. In conclusion, calcineurin might be important in immune system of locusts and might be investigated as a new target for insecticides. These results provide the theoretical basis of the damage of M. anisopliae to immune system of insects related to CaN.