Weeds often grow alongside crop plants. In addition to competing with crops for nutrients, water and space, weeds host insect vectors or act as reservoirs for viral diversity. However, little is known about viruses infecting rice weeds. In this work, we used metatranscriptomic deep sequencing to identify RNA viruses from 29 weed samples representing 23 weed species. A total of 224 RNA viruses were identified: 39 newly identified viruses are sufficiently divergent to comprise new families and genera. The newly identified RNA viruses clustered within 18 viral families. Of the identified viruses, 196 are positive-sense single-stranded RNA viruses, 24 are negative-sense single-stranded RNA viruses and 4 are double-stranded RNA viruses. We found that some novel RNA viruses clustered within the families or genera of several plant virus species and have the potential to infect plants. Collectively, these results expand our understanding of viral diversity in rice weeds. Our work will contribute to developing effective strategies with which to manage the spread and epidemiology of plant viruses.

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.

Superoxide dismutase (SOD) is an antioxidant metalloenzyme that catalyzes the dismutation of the superoxide anion O2− to O2 and H2O2. Many studies have focused on the role of SOD in response to abiotic stress, but its role during biotic stress, such as changes in organismal population density, has rarely been investigated. The oriental armyworm, Mythimna separata, is an economically important pest that exhibits phenotypic changes in response to population density. Solitary and gregarious phases occur at low and high population density, respectively. To examine the role of SODs in response to population density stress, we cloned two genes encoding SOD, MsCuZnSOD and MsMnSOD, and compared their expression in solitary and gregarious phases of M. separata. The MsCuZnSOD and MsMnSOD ORFs were 480 and 651 bp and encoded predicted protein products of 159 and 216 amino acids, respectively. The two SODs contained motifs that are typical of orthologous proteins. Real-time PCR indicated that the two SOD genes were expressed throughout developmental stages and were significantly upregulated in more mature stages of gregarious M. separata. Expression of the two SOD genes in various tissues of sixth-instar larvae was higher in gregarious versus solitary insects. Furthermore, expression of the SOD genes was significantly upregulated in response to crowding in solitary individuals, but suppressed in gregarious insects subjected to isolation. Collectively, these results suggest that population density may be key factor in the induction of SOD genes in M. separata.

Mythimna separata larvae exhibit both solitary and gregarious phases under low and high population density, respectively; furthermore, differences in morphology, behavior and physiology have been observed in the two phases. The integument plays an essential role in the fitness, general metabolism, communication, and survival of insects; however, differences in the integument ultrastructure and gene expression in the solitary and gregarious phases are largely unknown. In this study, the integument ultrastructure of larvae in the solitary and gregarious phases was compared, and transcriptome analysis was conducted to identify which genes were differentially expressed in the two phases. The results showed that the gregarious larvae had thicker integuments and more polygonal particles on the cuticle surface than solitary larvae. Using the Illumina HiSeq™ sequencing platform, 2774 differentially expressed genes (DEGs) were generated. Among these, many transcripts were identified with roles in the synthesis of fatty acids; structural components of the integument and the insecticide detoxification were differentially expressed in the integument of the two larval phases. qRT-PCR was used to validate expression patterns of the selected transcripts. This study provides a valuable resource for understanding the molecular basis of behavioral and physiological differences in the two phases of M. separata.

Mythimna separata, an important insect pest of grain crops, exhibits density-dependent prophylaxis (DDP). Most studies have focused on DDP from the perspective of immune system regulation. In this report, we show that two cuticular protein genes, MsCP1 and MsCP2, are involved in DDP of M. separata. MsCP1 and MsCP2 were highly expressed in 4th and 5th instar larvae of the M. separata gregarious high-density phase as compared to solitary, low-density individuals. The expression levels of MsCP1 and MsCP2 were significantly higher in the cuticle of gregarious larvae than in solitary individuals. When MsCP1 and MsCP2 were knocked down by star polycation (SPc) nanocarrier-mediated RNA interference (RNAi), the expression levels of the two genes were significantly downregulated in gregarious larvae as compared to dsGFP/SPc-treated gregarious larvae but were not significantly different from dsGFP/SPc-treated solitary larvae. Furthermore, silencing of MsCP1 and MsCP2 caused abnormal molting, alterations in ultrastructure, and reduced cuticle thickness in gregarious larvae. The mortality rates in dsMsCP1/SPc- and dsMsCP2/SPc-treated gregarious larvae were significantly higher after infection with the entomopathogenic fungus Beauveria bassiana as compared to the dsGFP/SPc-treated gregarious larva but were not significantly different from mortality rates in dsGFP/SPc-treated solitary larvae. Taken together, these findings suggest that upregulation of MsCP1 and MsCP2 are associated with resistance of gregarious M. separata to B. bassiana. These results broaden our understanding of insect DDP and are of great significance in the biological control of M. separata.

The oriental armyworm (OAW), Mythimna separata (Walker) (Lepidoptera: Noctuidae), is an important pest in China and causes serious economic losses in corn. The anthranilic diamide, chlorantraniliprole (CHL), has been widely used as a seed treatment to control corn pests; however, no information is available on the efficacy of this insecticide as a seed treatment for OAW. In this study, the efficacy of seed treatment with CHL alone and CHL combined with the neonicotinoid insecticide clothianidin (CHL+CLO) was evaluated for controlling OAW larvae in the laboratory and field conditions. Pot experiments demonstrated that seed treatment with CHL and CHL+CLO (both 240g a.i. 100kg⁻¹ seeds) resulted in >79 mortality of OAW larvae and a damage rate 70 within 14 DAE, which was significantly higher than CLO alone. These results suggest that CHL and CHL+CLO as seed treatments could effectively reduce OAW larval infestation in corn. This study validates the effectiveness of corn seed treatment for OAW as an alternative to conventional foliar applications.