RNA interference (RNAi) is a promising method for pest control; however, some studies have showed that the degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) is one of the factors that may reduce RNAi efficiency in lepidopteran insects. In this study, we cloned two dsRNase genes named CmdsRNase5 and CmdsRNase6 from rice leaffolder Cnaphalocrocis medinalis, a notorious insect pest of rice. Open reading frames (ORFs) of CmdsRNase5 and CmdsRNase6 are 1317 and 1185 bp in length, encoding 438 and 394 amino acids, respectively. These two genes were expressed at the highest level in the third-instar larvae throughout developmental stages and highly expressed in the midgut and hemolymph of C. medinalis. RNAi efficiencies of CmdsRNase5 and CmdsRNase6 were 57.44 % and 63.94 % on day 3, respectively. The RNAi efficiency of a target gene CmCHS (chitin synthase from C. medinalis) was 58.70% on day 5 and this efficiency was 87.63 % after co-silencing of CmCHS + CmdsRNase5 + CmdsRNase6. The findings suggested that co-silencing of CmdsRNases alongside CmCHS mitigated the degradation of dsCmCHS and enhanced the RNAi efficiency in C. medinalis, leading to phenotypic deformities, increased mortality, and a significant reduction in both egg production and hatching rate. Transcriptome analysis indicated CmdsRNase5 or CmdsRNase6 knockdown affected the expression of many important functional genes, thereby hindering the growth and development of C. medinalis. The concurrent silencing of both CHS and dsRNases provides a novel strategy for RNAi-mediated green control of C. medinalis and other lepidopteran pests.

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.

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, a serine/threonine protein kinase catalytic domain, and a serine/threonine protein kinase domain. 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 expression was high in female adult and the fourth instar nymph stage of C. chinensis . In C. chinensis adult, CcAkt expression was highest in the testis and ovary. Injection of bovine insulin induced CcAkt expression, whereas that of 20-hydroxyecdysone significantly reduced CcAkt expression. Both hormones, however, induced the expression of vitellogenin ( Vg ) and vitellogenin receptor ( VgR ). In unmated females, CcAkt knockout resulted in decreased expression of CcVg and CcVgR , stunted the development of the ovarioles, decreased the number of eggs and hatching rate. These findings suggest that CcAkt may be involved in regulating the reproduction of C. chinensis .

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.

Parental RNAi (pRNAi) is a response of RNA interference in which treated insect pests progenies showed a gene silencing phenotypes. pRNAi of CmGNA gene has been studied in Cnaphalocrocis medinalis via injection. Our results showed significant reduction in ovulation per female that was 26% and 35.26% in G1 and G2 generations, respectively. Significant reduction of hatched eggs per female were observed 23.53% and 45.26% as compared to control in G1-G2 generations, respectively. We also observed the significant variation in the sex ratio between female (40% and 53%) in G1-G2 generations, and in male (65%) in G1 generation as compared to control. Our results also demonstrated the significant larval mortality (63% and 55%) and pupal mortality (55% and 41%), and significant reduction of mRNA expression level in G1 and G2 generations. Our findings have confirmed that effectiveness of pRNAi induced silencing on the CmGNA target gene in G1-G2 generations of C. medinalis. These results suggested the potential role of pRNAi in insect pest resistance management strategies.

The rice leaf folder (RLF), Cnaphalocrocis medinalis, is one of the major pests of rice, and chitin synthase is a key enzyme for the chitin synthesis pathway in insects. In this study, the chitin synthase B gene from C. medinalis (CmCHSB) was cloned and characterized. The cDNA of CmCHSB is 4824 bp in length, containing an open reading frame of 4578 nucleotides that encodes 1525 amino acids. The CmCHSB zymoprotein consists of 10 transmembrane domains (TMDs) in the N-terminus, a middle conserved catalytic domain, and 7 TMDs in the C-terminus. Homology and phylogenetic analyses revealed that CmCHSB possesses the closest relationship with its homolog in Ostrinia furnacalis. CmCHSB was expressed throughout development and in all of the adult tissues tested, with the highest expression level in the adult and in the midgut. Silencing of CmCHSB through RNA interference (RNAi) severely affected RLF larval growth and caused larval lethality. Our results revealed that CmCHSB is essential for RLF growth and development, which sheds new light on the characteristics and functions of this gene. These findings will be helpful for green control of RLF, by targeting the CmCHSB gene using RNAi technology.

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.

The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect.

Hexokinase (HK) is a key enzyme in chitin biosynthesis in insects and plays an important role in development and energy regulation. It also performs a crucial role in the synthesis of Glucose-6-phosphate and its putative functions are studied via injection of dsRNA corresponding to the hexokinase gene from Cnaphalocrocis medinalis (CmHK). This study was designed to analyze the characteristics and expression patterns of HK-related genes in various tissues of C. medinalis at different developmental stages. The CmHK ORF is a 1359 bp in length, encoding a protein of 452 amino acids, with homology and cluster analysis showing that CmHK shares an 85.11% sequence similarity with hexokinase from Ostrinia furnacalis.CmHK was highly expressed in the ovary and in the fifth instar larvae. Injection of dsCmHK significantly suppressed mRNA expression (73.6%) 120 h post-dsRNA injection as compared to a control group. The results demonstrated an increased incidence of larval and pupal mortality of 80% and 78%, respectively, with significant variation in the sex ratio between males (68.33%) and females (35%), overt larval deformities, and a reduction in average weight gain observed 120 h post-dsRNA injection. In addition, dsCmHK-injected C. medinalis showed a significant reduction in ovulation per female and larval hatching rate, along with increased larval and pupal mortality and variation in male and female emergence over three generations (G1, G2, and G3). Taken together, the outcomes of the study provide a foundation to study gene function and a new dimension to control C. medinalis by transgenic RNAi technology.

The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel, is one of the most destructive pests causing heavy rice yield loss worldwide. In the present study, amplified fragment length polymorphism (AFLP) was used to analyze the genetic differentiation and transmission routes of 26 RWW geographical populations from Guizhou and 6 other provinces in China. The genetic distance between the 26 populations ranged from 0 to 0.693; the genetic distance between Guizhou and 6 other provinces populations varied from 0.043 to 0.693, with an average of 0.382. There was higher genetic variation among the populations from Guizhou and 6 other provinces compared with populations within Guizhou. The Mantel test showed no correlation between the genetic and geographical distances of the 26 populations. According to genetic distance, clustering, and time that RWW was found in these regions, we deduced that the pest entered Guizhou and spread in this province most likely via three routes: (1) from Hebei to Zunyi and southward to the south-central regions; (2) from Sichuan to Bijie and eastward to the central regions; and (3) from Yunnan through Anshun to the central regions and Qiandongnan prefecture. RWW spread in a natural manner, including flight, crawling, swimming, and by human-associated mechanisms, comprising dissemination through paddies and hay and hitchhiking on human transportations.

To provide strains for biological control of rice pests,the insect-resistant gene Cry 1C from GenBank was optimized,added BamH I and Spe I restriction enzyme cutting sites at both sides,and artificially synthesized.The modified Bt gene was named Cry 1C * ,which was inserted into pTCK303 after double restriction-enzyme digestion to construct recombinant expression vector pTCK303-Cry 1C * ,and transferred into A.tumefaciens LBA4404.The PCR,double digestion,and sequencing results showed that the recombinant vector was successfully constructed(2 342 bp).The PCR result showed that the expression vector pTCK303-Cry 1C * had been transformed into A.tumefaciens LBA4404,successfully constructing a genetic engineering bacteria.

BACKGROUND:The rice leaf folder (RLF), Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae), is one of the most destructive pests affecting rice in Asia. Although several studies have been performed on the ecological and physiological aspects of this species, the molecular mechanisms underlying its developmental regulation, behavior, and insecticide resistance remain largely unknown. Presently, there is a lack of genomic information for RLF; therefore, studies aimed at profiling the RLF transcriptome expression would provide a better understanding of its biological function at the molecular level.PRINCIPAL FINDINGS:De novo assembly of the RLF transcriptome was performed via the short read sequencing technology (Illumina). In a single run, we produced more than 23 million sequencing reads that were assembled into 44,941 unigenes (mean size = 474 bp) by Trinity. Through a similarity search, 25,281 (56.82%) unigenes matched known proteins in the NCBI Nr protein database. The transcriptome sequences were annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). Additionally, we profiled gene expression during RLF development using a tag-based digital gene expression (DGE) system. Five DGE libraries were constructed, and variations in gene expression were compared between collected samples: eggs vs. 3(rd) instar larvae, 3(rd) instar larvae vs. pupae, pupae vs. adults. The results demonstrated that thousands of genes were significantly differentially expressed during various developmental stages. A number of the differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR).CONCLUSIONS:The RLF transcriptome and DGE data provide a comprehensive and global gene expression profile that would further promote our understanding of the molecular mechanisms underlying various biological characteristics, including development, elevated fecundity, flight, sex differentiation, olfactory behavior, and insecticide resistance in RLF. Therefore, these findings could help elucidate the intrinsic factors involved in the RLF-mediated destruction of rice and offer sustainable insect pest management.

This paper summarizes the progress in the research of chemical compositions,pharmacological actions,and insecticidal activities of R.arisaematis and R.pinellia to explore their medicinal value and development potential and make full use of their variety resources.

The genetic diversity of M.persicae populations in tobacco and Chinese cabbage fields was analyzed by microsatellite marker technique to study genetic differentiation characteristics of M.persicae with different body color on different hosts.The results showed that the genetic relationship between two populations with different body color on tobacco was similar to that between two populations with different body color on Chinese cabbage and the genetic relationship between red populations was similar to that between green populations.The tested M.persicae sample belonged to the incomplete cycloid type.

The red-spotted grouper, Epinephelus akaara, is a protogynous hermaphroditic fish that shows the characteristic of natural sex change. In this study, 2-year-old female groupers were successfully reversed to functional males by oral administration of 17α-methyltestosterone (MT) for 42 days. The protein inhibitor of the neuronal nitric oxide synthase (PIN) gene was cloned from sex-reversed male gonads using modern suppression subtractive hybridization (SSH), cDNA synthesis and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA of PIN is 499bp containing a 270bp open reading frame (ORF) that encodes 89 amino acids. Virtual Northern blotting and reverse transcription-PCR (RT-PCR) analysis revealed that PIN was specifically transcribed in sex-reversed male gonads. Tissue-specific expression analysis showed that PIN gene was expressed in the brain, heart, liver, spleen, and kidney but not in the muscle tissue. Analyses of the expression pattern by RT-PCR and Western blotting indicated that transcription and the level of expression of PIN in the gonads increased gradually during the transformation from female to male. The results showed that PIN is strongly expressed in the sex-reversed male gonad but scarcely in the female gonad, and that its expression is upregulated as the change of sex proceeds. Taken together, these findings demonstrate that PIN is associated with the MT-induced sex transition of the red-spotted grouper, but the precise role of the gene in this process remains to be further investigated.