Expression profile and function analysis of MsCSP17 and MsCSP18 in the larval development of Mythimna separata.

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.

Expressions and functions of RR-1 cuticular protein genes in the integument of Mythimna separata.

As the most outer layer between itself and the environment, integuments are necessary for insects with various important functions. Cuticular proteins (CPs) are the main components in integuments, while the functions of CP genes remain unknown in Mythimna separata (Walker), which is a devastating agricultural pest. In this study, 79 CP genes were identified from the transcriptomes of larval integuments, 57 of which were from the family containing conserved Rebers & Riddiford (R&R) consensus (CPR family). Amongst these CPRs, 44 genes belonged to the subfamily with RR-1 motif (RR-1 genes) and clustered into three clades, with the top 15 most abundant RR-1 genes identified based on fragments per kilobase per million mapped fragments (FPKM) values. RT-qPCR analysis showed that most of RR-1 genes such as MsCPR1-4 were highly expressed at larval stages and in their integuments. The expression levels of RR-1 genes were generally decreased at the beginning but increased at the late stage of molting process. RNAi was applied for six RR-1 genes, and MsCPR1-4 were knocked down significantly. Silence of MsCPR2 resulted in abnormal integument formed after molting, while knockdown of MsCPR3 and MsCPR4 led to failure of molting, respectively. No phenotype was obtained for the RNAi of MsCPR1. Therefore, the expression of RR-1 genes and their functions were analyzed in the development of integuments in M. separata, providing new insights of RR-1 genes and potential targets for the development of growth regulators and new insecticides for M. separata.

Deformed Mediated Larval Incisor Lobe Development Causes Differing Feeding Behavior between Oriental Armyworm and Fall Armyworm.

Mandibular incisor lobes are important for insect feeding behavior, living habits and niche. However, the molecular regulation of insect incisor lobe development remains unknown. In this study, we found that two maize pests, oriental armyworm Mythimna separata and fall armyworm Spodoptera frugiperda, have different feeding patterns in maize, which are closely associated with the different development patterns of their incisor lobes. Different from first to sixth instar S. frugiperda, which feed on leaf tissues and whorls with sharp incisor lobes, older instars of M. separata feed from leaf margins with no incisor lobes. Hox gene Deformed (Dfd) is important for head appendages, but its function in incisor lobe development is not clear. Here, Dfds were identified from two armyworm species, and both were expressed highly in heads and eggs. Interestingly, the expression levels of MsDfd were relatively high in larval mandibles and decreased dramatically from fourth-instar mandibles in M. separata. Knockdown of MsDfd resulted in malformed mandibles with no incisor lobe in M. separata, making the larvae unable to perform window-feeding. However, RNAi of SfDfd did not affect the mandibles and window-feeding pattern of S. frugiperda, indicating the different roles of Dfd in these two species. Moreover, the mortality of new first instar M. separata increased after feeding dsMsDfd but did not for S. frugiperda feeding dsSfDfd. These findings revealed that Dfd mediated the larval mandibular incisor lobe morphology, affecting its feeding pattern in M. separata, broadening the knowledge of Dfd functions in insect mandibles and feeding behavior.