Characterisation of a copper/zinc superoxide dismutase from Pieris rapae and its role in protecting against oxidative stress induced by chlorantraniliprole.

Insecticide exposure typically leads to abnormally high levels of reactive oxygen species (ROS) and oxidative damage in insects. Superoxide dismutases (SODs) are potent antioxidant enzymes for ROS scavenging that are essential to protect insects against insecticide-induced oxidative injury. The small white butterfly, Pieris rapae, is an economically important lepidopteran pest of cruciferous crops, and the anthranilic diamide insecticide chlorantraniliprole is widely used to control this organism. However, whether chlorantraniliprole causes oxidative stress, and whether SODs are involved in ROS scavenging, remains unclear in P. rapae. In this study, an intracellular copper/zinc SOD (designated PrSOD1) gene was identified and characterised in P. rapae. The gene consists of four exons and three introns, and the PrSOD1 protein encoded by the gene has typical highly conserved features of CuZnSODs, including two signature motifs and seven Cu/Zn-interacting residues. Transcription of PrSOD1 was highest in the larval fat body and at the fifth-instar larval stage. Recombinant PrSOD1 protein expressed in Escherichia coli displayed antioxidant activity and high thermal and pH stability, confirming that PrSOD1 encodes a functional enzyme. Exposure to three sublethal doses of chlorantraniliprole for 6, 12 or 24 h resulted in significantly increased malondialdehyde concentration in P. rapae larvae, indicating insecticide-induced oxidative stress. Furthermore, both PrSOD1 transcription levels and CuZnSOD activity were quickly (6 and 12 h, respectively) upregulated in larvae subjected to chlorantraniliprole, strongly suggesting that PrSOD1 plays an important role in protecting against oxidative damage and possibly chlorantraniliprole tolerance in P. rapae.

Molecular characterization of a typical 2-Cys thioredoxin peroxidase from the Asiatic rice borer Chilo suppressalis and its role in oxidative stress.

In insects, thioredoxin peroxidase (TPX) plays an important role in protecting against oxidative damage. However, studies on the molecular characteristics of TPXs in the Asiatic rice borer, Chilo suppressalis, are limited. In this work, a cDNA sequence (CsTpx3) encoding a TPX was identified from C. suppressalis. The deduced CsTPX3 protein shares high sequence identity and two positionally conserved cysteines with orthologs from other insect species, and was classified as a typical 2-Cys TPX. CsTpx3 was expressed most highly during the fifth-instar larval stage, and transcripts were most abundant in the midgut. Recombinant CsTPX3 protein expressed in Escherichia coli displayed the expected peroxidase activity by removing H2 O2 . Furthermore, CsTPX3 protected DNA from oxidative damage, and E. coli cells overexpressing CsTPX3 exhibited long-term resistance to oxidative stress. Exposure to various oxidative stressors, such as cold (8°C), heat (35°C), bacteria (E. coli), and two insecticides (chlorpyrifos and lambda-cyhalothrin), significantly upregulated transcription of CsTpx3. However, exposure to abamectin had no such effect. Our results provide valuable information for future studies on the antioxidant mechanism in this insect species.

Molecular Characterization of a Mitochondrial Manganese Superoxide Dismutase From Chilo suppressalis (Lepidoptera: Crambidae).

In insects, superoxide dismutases (SODs) play a critical role in the scavenging of harmful reactive oxygen species (ROS) and protecting against oxidative stress induced by various environmental stresses. The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is an economically important insect pest of rice crops. In this study, a mitochondrial manganese SOD (Cs-mMnSOD) gene was characterized in C. suppressalis. The deduced Cs-mMnSOD protein has typical highly conserved features of mitochondrial manganese SODs, including four manganese binding residues, the signature DVWEHAYY peptide, and a mitochondrial-targeting sequence at the N-terminus. Transcription of Cs-mMnSOD was detectable at all developmental stages, but highest in pupae. Furthermore, the mRNA level of Cs-mMnSOD was strongly upregulated (more than twofold increase) following exposure to low and high temperatures (4, 30 and 35°C), insecticides (chlorpyrifos and chlorantraniliprole), and chemical reagents (cumene hydroperoxide, paraquat, H2O2 and CdCl2), but slightly elevated (less than twofold increase) in response to 8°C, abamectin and CuSO4. Additionally, the Cs-mMnSOD transcription results were consistent with the enzymatic activity data of the protein product. Purified recombinant Cs-mMnSOD protein expressed in Escherichia coli displayed SOD activity and thermostability. Furthermore, E. coli cells overexpressing Cs-mMnSOD exhibited long-term resistance to the oxidative inducers cumene hydroperoxide and paraquat. Our findings indicate that Cs-mMnSOD plays an important role in protecting C. suppressalis against oxidative damage.