Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

RNA interference‐mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre‐adult life stages

In recent years, RNA interference (RNAi) has been validated as a viable approach for functional genetic studies in non‐model organisms. In this report we demonstrate the efficacy of RNAi in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). A L. lineolaris inhibitor of apoptosis gene (LlIAP) has been identified and cloned. The translated sequence encodes a 381 amino acid protein similar to other insect IAPs and contains two conserved baculovirus inhibitor of apoptosis protein repeat (BIR) domains. Microinjection of double stranded RNA (dsRNA) corresponding to two disparate portions of the gene resulted in decreased LlIAP mRNA quantities relative to controls. Both nymphs and adult specimens injected with IAP dsRNA exhibited significantly reduced lifespan compared with those injected with non‐insect dsRNA (eGFP). Thus, RNAi‐mediated knockdown of LlIAP expression has been correlated with a lethal phenotype in adults and nymphs.     

Systemic RNAi in western corn rootworm,Diabrotica virgifera virgifera,does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.     

GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER,Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE)

RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double‐strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase‐2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12–14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high‐efficiency determination of gene functions in this species.     

Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper,Nilaparvata lugens

RNA interference (RNAi) is a powerful strategy for gene function study in insects. Here, we described the development of a RNAi technique by microinjection of double-stranded RNA (dsRNA) in the brown planthopper Nilaparvata lugens. Based on the mortality and RNAi efficiency criteria, the conjunctive between prothorax and mesothorax was selected as the injection site and 50 nl as injection volume. Three genes with different expression patterns were selected to evaluate the RNAi efficiency. A comparable 40% decrease of gene expression was observed at the 4th day after injection for the ubiquitously expressed calreticulin and the gut specific cathepsin-B genes, but only 25% decrease at the 5th day for the central nervous system specific Nlβ2 gene. Double injection could increase the RNAi efficiency, such as from 25% to 53% for Nlβ2 gene. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in N. lugens.     

Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double‐stranded RNA

The induction of the naturally occurring phenomenon of RNA interference (RNAi) to study gene function in insects is now common practice. With appropriately chosen targets, the RNAi pathway has also been exploited for insect control, typically through oral delivery of dsRNA. Adapting current methods to deliver foreign compounds, such as amino acids and pesticides, to mosquitoes through sucrose solutions, we tested whether such an approach could be used in the yellow fever mosquito, Aedes aegypti. Using a non‐specific dsRNA construct, we found that adult Ae. aegypti ingested dsRNA through this method and that the ingested dsRNA can be recovered from the mosquitoes post‐feeding. Through the feeding of a species‐specific dsRNA construct against vacuolar ATPase, subunit A, we found that significant gene knockdown could be achieved at 12, 24 and 48 h post‐feeding.     

The regulation of lipid metabolism by a hypothetical P-loop NTPase and its impact on fecundity of the brown planthopper

BackgroundInsect fecundity can be regulated by multiple genes in several important signaling pathways which form an extremely complicated regulatory network. However, there are still many genes that have significant impact on insect fecundity but their action mode are still unknown.MethodsQuantitative real-time PCR (qRT-PCR), immunofluorescence and western blot were used to study the expression profile of Nl23867 in the brown planthopper, Nilaparvata lugens. RNA interference (RNAi), RNA-seq and isobaric tags for relative and absolute quantification (iTRAQ) were performed to investigate the action mode of Nl23867 in the regulation of fecundity. High performance liquid chromatography (HPLC) analysis was performed to detect the fatty acid contents.ResultsWe show that knockdown of Nl23867, a gene encoding a hypothetical P-loop NTPase, significantly decreased fecundity of N. lugens. Underdeveloped ovaries, fewer eggs laid and reduction in vitellogenin (Vg) protein expression were observed after RNAi knockdown of Nl23867, and most of the affected genes and pathways are fatty acid metabolism-related. We further determined that Nl23867 directly impacts the palmitic acid biosynthesis by regulating the expression of palmitoyl-protein thioesterase (PPT), subsequently affecting the content of total lipids in N. lugens.ConclusionsNl23867 regulates the fecundity of N. lugens by modulating the biosynthetic pathway of palmitic acid and affecting lipid metabolism during vitellogenesis and oocyte development.General significanceThe presented study pioneers the exploration into how a function-unknown gene takes part in the regulation of fecundity in an insect, and will contribute to the construction of gene regulatory network for insect fecundity.     

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated ³²P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.     

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm,Spodoptera frugiperda

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi–dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi–dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated ³²P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.     

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3‐dioxygenase (vermilion): Implications for insect farming

The gene vermilion encodes tryptophan 2,3‐dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA‐Seq was used to analyze the impact of vermilion‐specific RNA interference on gene expression. There was a 425‐fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G‐protein‐coupled receptor signaling, cell‐cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white‐eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.     

Double-stranded RNAs targeting inhibitor of apoptosis gene show no significant cross-species activity

RNA interference (RNAi) has become an integral part of mainstream research due to its versatility and ease of use. However, the potential nontarget effects associated with double-stranded RNAs (dsRNA) are poorly understood. To explore this, we used dsRNAs targeting the inhibitor of apoptosis (iap) gene from nine insect species and assayed their possible nontarget effects. For each assay, we used a control (dsRNA targeting the gene coding for green fluorescent protein, GFP) and a species-specific dsRNA targeting nine iap genes in insect species to evaluate target gene knockdown efficiency, apoptosis phenotype in cells and mortality in insects. Our results revealed that dsIAP efficiently knocks down iap gene expression and induces apoptosis phenotype and mortality in target insect species. In contrast, no significant knockdown of the iap gene expression, apoptosis phenotypes, or mortality were detected in cell lines developed from nontarget insects or nontarget insects treated with dsIAPs. Interestingly, even among closely related insects such as stink bugs, Nezara viridula, Halyomorpha halys, and Murgantia histrionica, with substantial sequence similarity among iap genes from these insects, no significant nontarget effects of dsIAP were observed under the conditions tested. These data demonstrate no significant nontarget effects for dsIAPs and suggest that the threat of nontarget effects of RNAi technology may not be substantial.