Rice leaf folder Cnaphalocrocis medinalis is one of the most serious pests of rice in rice-planting regions worldwide. DsRNA-degrading nucleases (dsRNases) are important factors in reducing the efficiency of RNA interference (RNAi) in different insects. In this study, a dsRNase gene from C. medinalis (CmdsRNase) was cloned and characterized. The CmdsRNase cDNA was 1395 bp in length, encoding 464 amino acids. The CmdsRNase zymoprotein contains a signal peptide and an endonuclease NS domain that comprises six active sites, three substrate-binding sites, and one Mg²⁺-binding site. The mature CmdsRNase forms a homodimer with a total of 16 α-helices and 20 β-pleated sheets. Homology and phylogenetic analyses revealed that CmdsRNase is closely related to dsRNase2 in Ostrinia nubilalis. Expression pattern analysis by droplet digital PCR indicated that the expression levels of CmdsRNase varied throughout the developmental stages of C. medinalis and in different adult tissues, with the highest expression levels in the fourth-instar larvae and the hemolymph. CmdsRNase can degrade dsRNA to reduce the efficiency of RNAi in C. medinalis. Co-silencing of CmCHS (chitin synthase from C. medinalis) and CmdsRNase affected significantly the growth and development of C. medinalis and thus improved RNAi efficacy, which increased by 27.17%. These findings will be helpful for green control of C. medinalis and other lepidopteran pests by RNAi.

Coridius chinensis belongs to Dinidoridae, Hemiptera. Previous studies have indicated that C. chinensis contains abundant polypeptides with antibacterial and anticancer activities. Antimicrobial peptides (AMPs), as endogenous peptides with immune function, play an indispensable role in the process of biological development and immunity. AMPs have become one of the most potential substitutes for antibiotics due to their small molecular weight and broad-spectrum antimicrobial activity. In this study, a defensin CcDef2 from C. chinensis was characterized based on bioinformatics and functional analyses. The mature peptide of CcDef2 is a typical cationic peptide composed of 43 amino acid residues with five cations, and contains three intramolecular disulfide bonds and a typical cysteine-stabilized αβ motif in defensins. Phylogenetic analysis showed that CcDef2 belongs to the insect defensin family. Analysis of gene expression patterns showed that CcDef2 was expressed throughout developmental stages of C. chinensis with high levels at the nymphal stage and in adult tissues tested with the highest level in the fat body. In addition, the CcDef2 expression was significantly upregulated in adults infected by bacteria. After expressed in Escherichia coli BL21(DE3) and renatured, the recombinant CcDef2 showed a significant antibacterial effect on three kinds of Gram-positive bacteria. These results indicate that CcDef2 is an excellent antibacterial peptide and a highly effective immune effector in the innate immunity of C. chinensis. This study provides a foundation for further understanding the function of CcDef2 and developing new antimicrobial drugs.

Coridius chinensis is a valuable medicinal insect resource in China. Previous studies have indicated that the antibacterial and anticancer effects of the C. chinensis extract mainly come from the active polypeptides. Lysozyme is an effective immune effector in insect innate immunity and usually has excellent bactericidal effects. There are two kinds of lysozymes in insects, c-type and i-type, which play an important role in innate immunity and intestinal digestion. Studying lysozyme in C. chinensis will be helpful to further explore the evolutionary relationship and functional differences among lysozymes of various species and to determine whether they have biological activity and medicinal value. In this study, a lysozyme CcLys2 was identified from C. chinensis. CcLys2 contains 223 amino acid residues, and possesses a typical domain of the c-type lysozyme and a putative catalytic site formed by two conserved residues Glu32 and Asp50. Phylogenetic analysis showed that CcLys2 belongs to the H-branch of the c-type lysozyme. The analysis of spatiotemporal expression patterns indicated that CcLys2 was mainly expressed in the fat body of C. chinensis adults and was highly expressed in the second- and fifth-instar nymphs. In addition, CcLys2 was significantly up-regulated after injecting and feeding bacteria. In the bacterial inhibition assay, it was found that CcLys2 had antibacterial activity against Gram-positive bacteria at a low pH. These results indicate that CcLys2 has muramidase activity, involves in the innate immunity of C. chinensis, and is also closely related to the bacterial immune defense or digestive function of the intestine.

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Akt (also known as protein kinase B) belongs to the multifunctional serine/threonine kinase family and is an important component of the insulin signaling pathway that plays a key role in many biological processes such as cell growth, proliferation, and survival. However, few studies have reported the effect of Akt on reproduction in Hemiptera. In this study, we cloned and characterized the Akt gene from Coridius chinensis (CcAkt). The open reading frame of CcAkt has a length of 1,563 bp and encodes 520 amino acids. It has a conserved pleckstrin homology domain (PH), catalytic domain of serine/threonine protein kinases (S_TKc), and extension of Ser/Thr-type protein kinases (S_TK_X). Phylogenetic analysis showed that CcAkt and HhAkt of Halyomorpha halys had the highest similarity. Analysis of temporal and spatial expression patterns revealed that CcAkt is expressed throughout development and in various tissues of C. chinensis adults. CcAkt was highly expressed in the female adult and the fourth-instar nymph, as well as in the testis and ovary of C. chinensis. Injection of bovine insulin and methoprene induced the CcAkt expression, whereas that of 20-hydroxyecdysone significantly reduced the CcAkt expression. These three hormones, however, induced the expression of vitellogenin (Vg) and vitellogenin receptor (VgR). In unmated females, knockdown of CcAkt resulted in decreased expression of CcVg and CcVgR, stunted the development of the ovarioles, decreased the number of eggs and hatching rate. These findings from RNA interference experiment suggested that CcAkt may be involved in regulating the reproduction of C. chinensis.