Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

Kazal-type serine protease inhibitors (KaSPI) play important roles in insect growth, development, digestion, metabolism and immune defence. In this study, based on the transcriptome of Mythimna separata, the cDNA sequence of MsKaSPI with Kazal domain was uploaded to GenBank (MN931651). Spatial and temporal expression analysis showed that MsKaSPI was expressed at different developmental stages and different tissues, and it was induced by 20-hydroxyecdysone in third-instar larvae of M. separata. After 24 h infection by Beauveria bassiana, the expression level of MsKaSPI and the corresponding MsKaSPI content were significantly up-regulated, being 6.42-fold and 1.91-fold to the control group, respectively, while the activities of serine protease, trypsin and chymotrypsin were inhibited. After RNA interference interfered with MsKaSPI for 6 h, the expression decreased by 73.44%, the corresponding content of MsKaSPI protein decreased by 55.66% after 12 h, and the activities of serine protease and trypsin were significantly enhanced. Meanwhile, both the larval and pupal stages of M. separata were prolonged, the weights were reduced and the number of eggs per female decreased by 181. Beauveria bassiana infection also increased the mortality of MsKaSPI-silenced M. separata by 18.96%. These prove MsKaSPI can not only result in slow growth and low fecundity of M. separata by regulating the activity of related protease, but also participate in the resistance to pathogenic fungi by regulating the serine protease inhibitor content and the activities of related serine protease.

Chemosensory proteins (CSPs) were necessary for insect sensory system to perform important processes such as feeding, mating, spawning, and avoiding natural enemies. However, their functions in non-olfactory organs have been poorly studied. To clarify the function of CSPs in the development of Mythimna separata (Walker) larvae, two CSP genes, MsCSP17 and MsCSP18, were identified from larval integument transcriptome dataset. Both of MsCSP17 and MsCSP18 contained four conserved cysteine sites (C × (6)-C × (18)-C × (2)-C), with a signal peptide at the N-terminal. RT-qPCR analysis showed that MsCSP17 and MsCSP18 have different expression patterns among different developmental stages and tissues. MsCSP17 was highly expressed in 1st–4th instar larvae, and MsCSP18 had high expression in adults. Both genes were expressed highly in larval head, thorax, integument and mandible. Moreover, both of MsCSP17 and MsCSP18 were lowly expressed in larval integuments when larvae molted for 6 h and 9 h from 3rd to 4th instar, but highly at the beginning and end phase during molting. After injection of dsMsCSP17 and dsMsCSP18, the expression levels of two genes decreased significantly, with the body weight of larvae decreased, the mortality increased, and the eclosion rate decreased. It was suggested that MsCSP17 and MsCSP18 contributed to the development of M. separata larvae.

Migratory insects display diverse behavioral strategies in response to external environmental shifts, via energy allocation of migration-reproduction trade-offs. However, how migratory insects distribute energy between migration and reproduction as an adaptive strategy to confront temporary low temperatures remains unclear. Here, we used Mythimna separata, a migratory cereal crop pest, to explore the effects of low temperature on reproductive performance, behavior, and energy allocation. We found that the influence of low temperatures on reproduction was not absolutely negative, but instead depended on the intensity, duration, and age of exposure to low temperature. Exposure to 6°C for 24 h significantly accelerated the onset of oviposition and ovarian development, and increased the synchrony of egg-laying and lifetime fecundity in 1-day-old adults compared to the control, while female's flight capacity decreased significantly on the first and second day after moths were exposed to 6°C. Furthermore, the abdominal and total triglycerides levels of females decreased significantly from exposure to low temperature, but their thoracic triglyceride content was significantly higher than the control on the third and fourth day. These results indicated that low temperatures induced M. separata to reduce energy investment for the development of flight system. This resulted in the shifting of moths from being migrants to residents during the environmental sensitive period (first day post-emergence). This expands our understanding of the adaptive strategy employed by migratory insects to deal with low temperatures and aids in the management of this pest species in China.

In recent decades, the oriental armyworm, Mythimna separata (Walker), has caused severe damage to staple grains in China. However, little is known about when M. separata begin their first migration and the role of males in reproduction and migration. Here, the migratory benefits and reproductive costs of flight frequency were examined in adults under laboratory conditions. We found that flying males had a positive effect on ovarian and reproductive development in females who flew for 1–2 nights by comparing two treatment groups (flying and nonflying male groups). Moreover, flying males decreased the flight capacity and flight propensity of females. In contrast, flight for more than two nights by males significantly inhibited ovarian and reproductive development in adult females. Compared with the controls (0 night), male flight for 1–2 nights significantly shortened the preoviposition period but significantly increased ovarian and reproductive development in females. However, male flight for more than three nights significantly inhibited female reproduction and flight capacity. These results indicate that M. separata begin their first migration within 2 days after emergence and fly for two nights. Prolonged flight times can result in significant reproductive costs. Females initiated their first migration earlier than males due to a stronger flight capacity. These observed findings will be useful for forecasting and monitoring population dynamics to prevent outbreaks of M. separata and reduce crop losses.

The oriental armyworm, Mythimna separata (Walker) is a serious pest of agriculture that does particular damage to Gramineae crops in Asia, Europe, and Oceania. Metamorphosis is a key developmental stage in insects, although the genes underlying the metamorphic transition in M. separata remain largely unknown. Here, we sequenced the transcriptomes of five stages; mature larvae (ML), wandering (W), and pupation (1, 5, and 10 days after pupation, designated P1, P5, and P10) to identify transition-associated genes. Four libraries were generated, with 22,884, 23,534, 26,643, and 33,238 differentially expressed genes (DEGs) for the ML-vs-W, W-vs-P1, P1-vs-P5, and P5-vs-P10, respectively. Gene ontology enrichment analysis of DEGs showed that genes regulating the biosynthesis of the membrane and integral components of the membrane, which includes the cuticular protein (CP), 20-hydroxyecdysone (20E), and juvenile hormone (JH) biosynthesis, were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs were enriched in the metabolic pathways. Of these DEGs, thirty CP, seventeen 20E, and seven JH genes were differentially expressed across the developmental stages. For transcriptome validation, ten CP, 20E, and JH-related genes were selected and verified by real-time PCR quantitative. Collectively, our results provided a basis for further studies of the molecular mechanism of metamorphosis in M. separata.

The oriental armyworm, Mythimna separata is an important crop pest in eastern Asia. Nocturnal insects, including nocturnal moths, have phototactic behavior to an artificial light source. Phototactic behavior in insects is species-specific in response to different wavelengths of light sources. Our previous study showed that green (520 nm) light emitting diode (LED) light resulted in a significantly higher phototactic behavior in M. separata moths compared to the other wavelength LED lights. The goal of the present study is to investigate the influence of green light illumination on biological characteristics of different developmental stages in M. separata. Our results revealed that when different developmental stages of M. separata were exposed to the green light illumination in a dark period, several biological characteristics in all developmental stages except for egg stage were positively changed, but those of F1 generation M. separata which are next generation of the adults exposed to the green light did not significantly change compared with the control level. These findings suggest that green light illumination at night (or dark period) has a positive effect on the development and longevity of M. separata.

The tachinid fly Drino inconspicuoides (Diptera: Tachinidae) is an ovolarviparous endoparasitoid whose larvae develop in the host haemocoel and avoids the host immune system. In this study, we investigated the immune evasion mechanisms of this species during infestation in the host Mythimna separata (Lepidoptera: Noctuidae). We discovered a unique ‘cloak’ that surrounded D. inconspicuoides larvae that penetrated into the host and determined through genomic polymerase chain reaction analysis that this structure originated from the host rather than the tachinid. The ‘cloak’ contained both haemocytes and fat body cells from the host, with the haemocytes assembling around the larvae first and the fat body cells then covering the haemocyte layer, following which the two mixed. Living D. inconspicuoides larvae that were wrapped in the ‘cloak’ were not melanized whereas encapsulated dead larvae were melanized, suggesting that this structure contributes to the avoidance of host immune reactions.

The period from March to mid April, when oriental armyworm Mythimna separata (Walker) moths migrate from South China to Central China over several nights, is an important window of time in annual armyworm population increase. The presence of nectar sources along the pathway of the migratory population is a prerequisite for moths to reach target habitats and lay eggs. Using flowering periods and geographic distributions, the major spring nectar plants suitable for M. separata moths were identified from among 102 species/varieties of apicultural nectar plants. The nectar plants proposed as important to M. separata include milk vetch Astragalus sinicus L., rape Brassica napa L. and six other species. Spearman's rank correlation analyses were conducted between the annual population size of M. separata and the acreages of milk vetch and rape in the daily stopover areas for migrating populations of M. separata during 1950–1979. The Spearman's coefficient between milk vetch and M. separata was 0.6259 and the correlation was highly significant (P<0.001). Further regression analysis with data from 1950–1979 and from 1980–1992 also revealed a close relationship between annual acreage of damaged crops/wheat and acreage of milk vetch. These results strongly suggest that the unprecedented enlargement in the geographic distribution of milk vetch from Central China into South China was the key factor in the frequent severe oriental armyworm outbreaks that occurred during 1966–1977. This is thought to be the first report in the world that reveals the key role of nectar sources in long distance, regional scale, migration of moths. The argument for the key role of milk vetch is supported by the simultaneous decline in the level of damage inflicted by M. separata and the acreage of milk vetch after 1980.