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The full-length cDNA of catalase (EfCAT) from Eisenia fetida was cloned (GenBank accession no. JN617999). Sequence characterization revealed that EfCAT protein sequence contained proximal heme-ligand signature sequence ((351)RLFSYSDTH(359)), two glycosylation sites (N(145) and N(436)), the proximal active site signature ((61)FDRERIPERVVHAKGAGA(78)), and 12 amino acids (N(145), H(191), F(195), S(198), R(200), N(210), Y(212), K(234), I(299), W(300), Q(302), and Y(355)), which were identified as putative residues involved in NADPH binding. These conserved motifs and catalase signature sequences were essential for the structure and function of EfCAT. The present study also investigated the effect of the veterinary food additive zinc oxide on antioxidant processes in E. fetida, at different concentrations and exposure durations. A significant increase (by 106.0 % compared to controls) in CAT activity at 500 mg/kg was registered at day 15. The superoxide dismutase (SOD) activity at 500 mg/kg increased to the maximum value (by 44.0 %) measured at day 15. There was a significant increase in glutathione peroxidase (GPx) activity for all concentrations after 5 days. The results showed that dietary Zn (500 mg/kg) causes oxidative damage to earthworms. At early stages of earthworms exposed to ZnO, GPx is the main enzyme to impair the oxidative status; while at later stages the enzymes CAT and SOD were the main indicators of oxidative stress. The antioxidant enzymatic variations may be an adaptive response of earthworms to survive in contaminated soils.

Avermectins are a series 16-membered macrocyclic lactone derivatives derived from the actinomycete Streptomyces avermectinius , which has been applied worldwide in veterinary, human medicine and agriculture. Abamectin is a mixture of avermectins containing ⩾80% avermectin B1a and ⩽20% avermectin B1b. In this paper, the apoptotic induction effect of abamectin on Spodoptera frugiperda (Sf9) cells was detected by using various techniques including cell proliferation assay, flow cytometry, morphology analysis with inverted phase contrast microscope, scanning electron microscope, transmission electron microscopy, enzyme activity assay and Western blot. The results revealed that within the concentrations of 5–15 μg/mL, abamectin inhibited the growth of the Sf9 cells and induced apoptotic cell death in a time- and dose-dependent manner. The proliferation inhibition rates of Sf9 cells were 6.45 ± 0.47%, 11.51 ± 0.20% and 12.97 ± 0.46% after 12 h of treatment with avermectin at the concentrations of 5, 10 and 15 μg/mL, which increased to 30.74 ± 0.57%, 36.14 ± 0.25% and 39.27 ± 0.63% after 72 h of the same treatments, respectively. The maximum apoptosis rate of each concentration was 24.13 ± 0.43%, 24.69 ± 0.38% and 27.285 ± 0.84%, respectively. Treated cells showed typical apoptosis morphological changes including cell swelling, chromatin condensation, apoptotic bodies, swollen mitochondria and high content of phagosome. Both mitochondrial events including mitochondrial membrane potential (Δψ m ) loss as well as cytochrome-c release into cytosol and significant activation of caspase-3 occurred during apoptosis. These data confirmed the apoptosis inducing effects of abamectin on S. frugiperda (Sf9) cell line, and provide preliminary studies on its mechanism.

Deregulated reactive oxygen species (ROS) production can lead to the disruption of structural and functional integrity of cells as a consequence of reactive interaction between ROS and various biological components. Catalase (CAT) is a common enzyme existing in nearly all organisms exposed to oxygen, which decomposes harmful hydrogen peroxide, into water and oxygen. In this study, the full length sequence that encodes CAT-like protein from Spodoptera litura named siltCAT (GenBank accession number: JQ_663444) was cloned and characterized. Amino acid sequence alignment showed siltCAT shared relatively high conservation with other insect, especially the conserved residues which defined heme and NADPH orientation. Expression pattern analysis showed that siltCAT mRNA was mainly expressed in the fat body, midgut, cuticle and malpighian tube, and as well as over last instar larvae, pupa and adult stages. RNA interference was used to silence CAT gene in SL-1 cells and the fourth-instar stage of S. litura larvae respectively. Our results provided evidence that CAT knockdown induced ROS generation, cell cycle arrest and apoptosis in SL-1 cells. It also confirmed the decrease in survival rate because of increased ROS production in experimental groups injected with double-stranded RNA of CAT (dsCAT). This study implied that ROS scavenging by CAT is important for S. litura survival.

The full length cDNA of copper/zinc superoxide dismutase (Cu/Zn-SOD) from Eisenia fetida (E. fetida) was cloned (GenBank accession no. JN579648). Sequence characterization revealed that the cDNA contained characteristic Cu/Zn-SOD family signatures (45GFHVHEFGDNT55 and 138GNAGGRLACGVI149), cysteines (Cys-58 and-146) predicted to form one disulphide bond, Cu-binding (His-47, -49, -64 and -120) and Zn-binding (His-64, -72, -81 and Asp-84). They were essential for the structure and function of Cu/Zn-SOD. Differential expression of stress-responsive genes like Cu/Zn-SOD, catalase (CAT), heat shock protein 70 (Hsp70) and metallothionein (MT) was applied as potential biomarkers to assess their efficacy for the ecotoxicological effects of dietary zinc oxide (ZnO) on E. fetida. The results showed that the expression of Cu/Zn-SOD and MT increased to reach the highest levels of 6.22 and 7.68 fold in a dose-dependent manner at day 10 respectively. The highest expression of 3.03 fold of CAT was registered at day 10. The transient expression of Hsp70 without consistent time- or/and dose-dependent was observed. It implied that the transcriptional patterns of Cu/Zn-SOD, CAT and MT could serve as early warning signals in ecotoxicological assessment of dietary ZnO on earthworms while the expression of Hsp70 was not well done, which is helpful to monitoring and regulation of ZnO in veterinary application.