Assessment of Sesamia nonagrioides (Lepidoptera: Noctuidae) EcR and USP Genes as Targets for Exogenous Non-Persistent RNAi

RNA interference (RNAi) is a transforming technology with high potential for practical applications in biology, including specific and safe insect pest control. For developing RNAi-based pest-control products no general recommendations exist and the best strategy needs to be determined for each insect pest separately on a case-by-case basis. In this research, the potential of silencing the genes encoding the subunits of the ecdysone receptor complex, EcR and Ultraspiracle (USP) by RNAi was evaluated in the corn borer, Sesamia nonagrioides, using different delivery approaches and targeting different developmental stages. In conjunction with our previous research it is demonstrated that prepupae are sensitive to RNAi triggered by dsRNA injection and that feeding of dsRNA-expressing bacteria throughout S. nonagrioides’ larval life can lead to limited developmental malformations with no potent insecticidal results. Our results, consistent with previous studies, indicated a great fluctuation of exogenous RNAi effectiveness in the Lepidopteran species, suggesting that further factors should be taken into consideration in order to expand this very promising field into the ‘’RNAi-resistant’’ insect species.

Cytochrome P450s Are Essential for Insecticide Tolerance in the Endoparasitoid Wasp Meteorus pulchricornis (Hymenoptera: Braconidae)

With the widespread application of insecticides, parasitoid wasps may also be under risk when exposed to insecticides directly at their free-living stages. The endoparasitoid wasp Meteorus pulchricornis is the predominant natural enemy of many lepidopteran pests, such as Spodoptera litura and Helicoverpa armigera. The cytochrome P450 monooxygenases constitute a ubiquitous and complex superfamily of hydrophobic, haem-containing enzymes. P450s are involved in the detoxification of many xenobiotics. However, their exact roles in the tolerance mechanism in parasitoids toward insecticides has received less attention. Here, 28 P450 genes in M. pulchricornis were identified from a previously constructed transcriptome dataset. These P450 genes belonged to CYP2, -3, and -4, and mitochondrial clans. Subsequently, eight candidate MpulCYPs were selected from four CYP clans to validate their expression patterns under phoxim, cypermethrin, and chlorfenapyr exposure by qRT-PCR. The results showed that all three insecticides had significant effects on the expression of MpulCYPs. To further study the function of P450s, CYP369B3 was silenced, and its expression levels of CYP369B3 were significantly decreased. Survival analysis indicated that after dsRNA injection, the mortality rate of wasps was significantly increased when M. pulchricornis females were exposed to insecticides compared to control groups. Our findings provide a theoretical base for elucidating the mechanism of insecticide tolerance and promote functional research on P450 genes in parasitoid wasps.

Identification and Characterization of the Succinate Dehydrogenase Complex Iron Sulfur Subunit B Gene in the Oriental River Prawn, Macrobrachium nipponense

Previous studies have revealed that SDHB has potential functions in the male sexual differentiation and development in M. nipponense through providing ATP. In this study, the functions of Mn-SDHB were further analyzed in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, western-blot, and RNA interference (RNAi), combined with the histological observations. The full-genome sequence of Mn-SDHB was 54,608 bp at Chromosome 34, including 7 introns and 6 exons. The full-length cDNA sequence of Mn-SDHB was 1,268 base pairs (bp) long with an open reading frame of 807 bp, encoding for 268 amino acids. The highest expression level of Mn-SDHB in different tissues was observed in the testis, and male prawns at post-larval developmental stage 25 during different developmental stages, indicating that SDHB was potentially involved in the male sexual development in M. nipponense. In situ hybridization and western-blot analysis indicated that SDHB plays essential roles in the testis development. The in situ hybridization analysis also implies the potential roles of Mn-SDHB in ovarian development. The expressions of Mn-IAG were decreased after Mn-SDHB dsRNA injection, indicating SDHB has the positive regulatory effects on IAG in M. nipponese. Thus, SDHB was involved in the mechanism of the male sexual development. The testis development was inhibited, and sperms were rarely observed after 10 days of Mn-SDHB dsRNA injection, indicating SDHB has positive effects on the male sexual development in M. nipponense. This study highlights the functions of SDHB in M. nipponense, which provide new insights for the future studies of the male sexual development in other crustacean species.

RNAi-Mediated Silencing of the Chitinase 5 Gene for Fall Webworm (Hyphantria cunea) Can Inhibit Larval Molting Depending on the Timing of dsRNA Injection

Chitinases, which are crucial enzymes required for chitin degradation and reconstruction, are often selectively considered to be effective molecular targets for pest control due to their critical roles in insect development. Although the Hyphantria cunea chitinase gene has been reported previously, its sequence characteristics, gene function, and feasibility as a potential target for pest management were absent. In the present study, we characterized the H. cunea chitinase gene and designated it HcCht5. Phylogenic and domain structure analysis suggested that HcCht5 contained the typical chitinase features and was clustered into chitinase group I. Tissue-specific and developmental expression pattern analysis with Real-Time Quantitative PCR (RT-qPCR) showed that HcCht5 was mainly expressed in the integument tissues and that the transcript levels peaked during molting. RNA interference (RNAi)-mediated silencing of HcCht5 caused 33.3% (2 ug) and 66.7% (4 ug) mortality rates after double-stranded RNA (dsRNA) injection. Importantly, the interference efficiency of HcCht5 depended on the injection time of double-stranded RNA (dsRNA), as the pre-molting treatment achieved molt arrest more effectively. In addition, transcriptome sequencing (RNA-seq) analysis of RNAi samples demonstrated silencing of the down-regulated HcCht5 genes related to chitin metabolism and molting hormone signaling, as well as genes related to detoxification metabolism. Our results indicate the essential role of HcCht5 in H. cunea development and detail the involvement of its gene function in the larval molting process.

Characterization, Knockdown and Parental Effect of Hexokinase Gene of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) Revealed by RNA Interference

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.

Transcriptome Analysis of Sogatella furcifera (Homoptera: Delphacidae) in Response to Sulfoxaflor and Functional Verification of Resistance-Related P450 Genes

The white-back planthopper (WBPH), Sogatella furcifera, is a major rice pest in China and in some other rice-growing countries of Asia. The extensive use of pesticides has resulted in severe resistance of S. furcifera to variety of chemical insecticides. Sulfoxaflor is a new diamide insecticide that acts on nicotinic acetylcholine receptors (nAChRs) in insects. The aim of this study was to explore the key genes related to the development of resistance to sulfoxaflor in S. furcifera and to verify their functions. Transcriptomes were compared between white-back planthoppers from a susceptible laboratory strain (Sus-Lab) and Sus-Lab screened with the sublethal LC25 dose of sulfoxaflor for six generations (SF-Sel). Two P450 genes (CYP6FD1 and CYP4FD2) and three transcription factors (NlE78sf, C2H2ZF1 and C2H2ZF3) with upregulated expression verified by qRT-PCR were detected in the Sus-Lab and SF-Sel strains. The functions of CYP6FD1 and CYP4FD2 were analyzed by RNA interference, and the relative normalized expressions of CYP6FD1 and CYP4FD2 in the SF-Sel population were lower than under dsGFP treatment after dsRNA injection. Moreover, the mortality rates of SF-Sel population treated with the LC50 concentration of sulfoxaflor after the injecting of dsRNA targeting CYP6FD1 and CYP4FD2 were significantly higher than in the dsGFP group from 72 h to 96 h (p < 0.05), and mortality in the CYP6FD1 knockdown group was clearly higher than that of the CYP4FD2 knockdown group. The interaction between the tertiary structures of CYP6FD1 and CYP4FD2 and sulfoxaflor was also predicted, and CYP6FD1 showed a stronger metabolic ability to process sulfoxaflor. Therefore, overexpression of CYP6FD1 and CYP4FD2 may be one of the primary factors in the development of sulfoxaflor resistance in S. furcifera.

Molecular Characterization of Primary Juvenile Hormone Responders Methoprene-Tolerant (Met) and Krüppel Homolog 1 (Kr-h1) in Grapholita molesta (Lepidoptera: Tortricidae) with Clarification of Their Roles in Metamorphosis and Reproduction

Methoprene-tolerant (Met) is a putative JH intracellular receptor that transduces JH signal by activation of the inducible Krüppel homolog 1 (Kr-h1). We analyzed the gene sequences of Met and Kr-h1 and their patterns of expression in Grapholita molesta (Busck) immature and adult stages in order to better understand the roles of these primary JH responders in regulating the metamorphosis and reproduction of this global pest of fruit crops. The deduced amino acid sequences of both GmMet and GmKr-h1 were highly homologous to those of other Lepidoptera, especially the cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Peak expression of GmMet occurred during the last 3 to 5 d of the final instar, followed by that of GmKr-h1, in the last 3 d of final instar. Similar patterns of GmMet and GmKr-h1 expression were detected across various tissue types in the fifth-instar larvae, with the highest expression observed in the head, followed by the epidermis, and the fat body. When expression of GmMet and GmKr-h1 was knocked down via dsRNA injection in the fifth instar, the results were increased larval mortality, abnormal pupation, delayed pupal duration, reduced adult emergence, extended preoviposition period, and reduced fecundity. We infer that both GmMet and GmKr-h1 participated in regulation of metamorphosis and reproduction in G. molesta, the former acting upstream of the latter, and could present biorational targets for novel pest control compounds.

Oxytocin/vasopressin-like peptide inotocin regulates cuticular hydrocarbon synthesis and water balancing in ants

Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the antCamponotus fellah. Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed thatinotocin receptoris specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targetinginotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression ofcytochrome P450 4G1(CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.