The oriental armyworm, Mythimna separata, is a major insect pest in China and other Asian countries. Unfortunately, suitable reference genes for quantitative real-time PCR (qRT-PCR) have not been previously identified in M. separata for normalizing target gene expression. In this study, we evaluated the expression stability of eight candidate genes (18S, ACT, EF1-α, GAPDH, RPS7, RPS13, RPL32 and TUB) in M. separata using the comparative ΔCt method, BestKeeper, Normfinder geNorm and ReFinder, a comprehensive software platform. The results indicated that the appropriate reference gene varied depending on the experimental conditions. We found that ACTIN, EF1-α and TUB were optimal for different developmental stages; TUB, RPS13 and EF1-α showed the most stable expresssion in different tissues; RPS13 and 18S were the best reference genes for monitoring expression under high temperature conditions; TUB, RPS13 and RPS7 exhibited the most stable expression under larval-crowding conditions; RPS7, EF1-α, RPL32 and GAPDH were the best for pesticide exposure experiments. This study provides tools for reliable normalization of qRT-PCR data and forms a foundation for functional studies of target gene expression in M. separata.

The development and reproduction of the fall armyworm (FAW), Spodoptera frugiperda, which were reared on artificial diets containing nano-graphene oxide (GO), were determined based on age-stage, two-sex life table analysis. The results showed that GO had adverse effects on FAWs. Compared with the control, the duration of the egg stage and first, second, and sixth instar larval stages increased with increasing GO concentrations; however, the lifespan of male and female adults decreased with increasing GO concentrations. Weights of FAW pupae that were supplied with GO-amended diets increased by 0.17–15.20% compared to the control. Intrinsic growth, limited growth, and net reproductive rates of FAWs feeding on GO supplemented diets were significantly lower than the control, while mean generational periods (0.5 mg/g: 38.47; 1 mg/g: 40.38; 2 mg/g: 38.42) were significantly longer than the control. The expression of genes encoding vitellogenin (Vg) and vitellogenin receptor (VgR) expression was abnormal in female FAW adults feeding on GO-amended diets; the number of eggs laid decreased relative to the control, but Vg expression increased. In conclusion, GO prolonged the developmental period of FAWs, decreased fecundity, and led to a decline in the population size. The study provides a basis for the rational use of GO as a pesticide synergist for FAW control.

High population density (crowding) becomes a stress factor in insects. The oriental armyworm, Mythimna separata (Walker), displays gregarious and solitary phases at high and low population densities, respectively. In this study, we compared life history, disease resistance, and induction of antioxidant enzymes and heat shock protein (HSPs) in two phases of M. separata larvae. Results showed that gregarious larvae had a faster growth rate and lower pupal weight compared to solitary larvae. Furthermore, gregarious individuals exhibited higher survival rates than solitary individuals after Beauveria bassiana infection. The gregarious larvae had higher malondialdehyde content compared to solitary ones, but no differences in total antioxidant capacity were observed between the two larval phases before or after infection. Superoxide dismutase and glutathione peroxidase activities were significantly lower in gregarious M. separata larvae than solitary individuals before infection, but no difference was detected in two phases after infection. However, peroxidase and catalase activities in the two phases showed no difference either before or after infection. Hsp19.8 and Hsp90 expression in gregarious larvae were up-regulated when compared to solitary individuals before or after infection. CuZnSOD expression was not different between the two phases before infection, but it was up-regulated in gregarious ones compared to solitary ones after infection. However, expression of other stress-related genes in gregarious larvae was either repressed or unchanged when compared to solitary individuals before or after infection. Thus, larval crowding changed life history, improved disease resistance of M. separata larvae, and induced variable response of antioxidant enzymes and HSPs to fungal infection.

The diamides, chlorantraniliprole (CHL) and cyantraniliprole (CYA), have been used as seed treatment agents against the fall armyworm (FAW), Spodoptera frugiperda in China. However, large-scale application of these two insecticides is prohibited because of their high cost. The neonicotinoid insecticides, clothianidin (CLO) and thiamethoxam (THI), are cheaper and widely used. In this study, we tested the efficacy of CHL + CLO and CYA + THI as seed treatment agents against FAW larvae both in laboratory and field conditions. Laboratory experiments showed that the two binary mixtures (both 240 g.a.i.100 kg⁻¹ corn seeds) caused FAW mortality exceeded 84.00% at 14 days after seedling emergence (DAE). The mortality of the binary mixtures were similar to either CHL (300 g.a.i.100 kg⁻¹corn seeds) or CYA (144 g a.i.100 kg⁻¹corn seeds), but higher than CLO (120 g.a.i.100 Kg⁻¹corn seeds) or THI (180 g a.i.100 kg⁻¹corn seeds). Two independent field experiments showed that both binary mixtures resulted in above 68.00% control efficacy at 14 DAE, suggesting that these insecticidal combinations could effectively control FAW over a relative long period. In addition, both binary mixtures showed no negative effects on the growth and development of corn seedlings. The residues of binary mixtures in corn leave were also lower at 28 DAE as compared to residues in CHL or CYA alone. Most importantly, the costs of CHL + CLO were reduced up to 50% and CYA + THI up to 20% when compared to singly used chemical. Totally, our results indicated that CHL + CLO and CYA + THI had the same control efficacy as CHL or CYA alone, but with much lower cost.