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.     

Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [³H]imidacloprid binding sites with different affinities (K_d = 3.5 ± 0.6 pM and 1.5 ± 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlα1, Nlα2 and Nlβ1 subunits into one receptor complex and of Nlα3, Nlα8 and Nlβ1 into another. Immunodepletion of Nlα1 or Nlα2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlα3 or Nlα8 caused the selective loss of the high-affinity site. Immunodepletion of Nlβ1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlα4, Nlα6, Nlα7 and Nlβ2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlα1, Nlα2 and Nlβ1 constitute the lower affinity binding site, whereas nAChRs containing Nlα3, Nlα8 and Nlβ1 constitute the higher affinity binding site for imidacloprid in N. lugens.     

Insulin receptors regulate the fecundity of Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

Two InR (insulin receptor) genes have been identified in the Nilaparvata lugens. In this study, we used RNA interference (RNAi) to investigate the role of InR genes in the fecundity of N. lugens. The expression of NLInR1 and NLInR2 genes was simultaneously silenced with mixture of dsInR1 and dsInR2 (dsInRs) injection. Our results showed that larvae RNAi against both NLInR1 and NLInR2 reduced the number of eggs laid by N. lugens and some eggs as well as ovaries were abnormal. In addition, the relative expression of Vg (vitellogenin) and VgR (vitellogenin receptor) was significantly reduced on the 4th and 6th days after insects treated with larvae RNAi reached the adult stage. We also determined the relative expression levels of insulin/insulin-like signaling (IIS) related genes in RNAi-treated larvae and found that the expression levels of Chico (homologous receptor substrate), Akt (protein kinase B), PI3K (phosphoinositide 3-kinase), and PTEN (phosphatase and tensin homolog) genes decreased whereas FOXO (forkhead box O) and GSK3 (glycogen synthase kinase-3) levels increased on the 4th and 6th days after insects reached the adult stage. These results indicate that silencing of NLInR1 and NLInR2 reduces the fecundity of N. lugens through the IIS pathway.     

NlCDK1 gene is required for the ovarian development and survival of the brown planthopper, Nilaparvata lugens Stål,as revealed by RNA interference

CDK1 is a kind of cyclin-dependent kinases (CDKs) involved in diverse biological processes besides cell cycle regulation in various species. However, little is known about CDK1 in Nilaparvata lugens Stål (brown planthopper, BPH). In this study, the full-length cDNA of NlCDK1, a CDK1 gene in BPH, was obtained using rapid-amplification of cDNA ends (RACE) technique. RT-qPCR revealed that the expression of NlCDK1 was relatively low and stable at nymph stages from 1st star to 5th star, but increased to higher levels in gravid females 3?day to 5?day post-eclosion. Feeding-based RNA interference (RNAi) with 0.5?μg/μL dsNlCDK1 achieved an efficient knockdown of target gene expression in females after being fed with dsRNA for 6?days, and the mRNA level of NlCDK1 decreased to 19.9% in comparison with that of the dsGFP control. As a result, RNA inerference with dsNlCDK1 caused a significant decrease of the BPH survival rate to 28.0% of that for the control. RNAi of NlCDK1 also suppressed the ovarian development and significantly affected the fecundity of the females, leading to a 70.2% reduction to the number of the offspring. These data suggest that NlCDK1 is required for the ovarian development and survival of the brown planthopper.     

褐飞虱Nl15基因的克隆及功能分析

【目的】植食性刺吸式口器昆虫的唾液蛋白参与调控植物抗虫防御反应,影响其对寄主植物的适应性。本研究旨在通过克隆褐飞虱Nilaparvata lugens重要唾液蛋白基因Nl15,调查其时空表达模式,明确其在褐飞虱致害性中的作用。【方法】基于褐飞虱IR56种群转录组数据,用RT-PCR克隆褐飞虱基因Nl15 cDNA序列,并进行生物信息学分析。利用qPCR检测其在褐飞虱TN1和IR56种群不同发育阶段(卵、1-5龄若虫和雌雄成虫)和雌成虫不同组织(头、胸、腹和足)中的表达模式。通过显微注射dsRNA对褐飞虱TN1和IR56种群的4龄若虫进行Nl15的RNAi,利用qPCR检测Nl15 RNAi后褐飞虱若虫中Nl15的相对表达量以及Nl15 RNAi后褐飞虱若虫取食3 d时水稻植株中防御相关基因(OsLecRK4, OsMPK10, OsWRKY24, OsLox, OsNPR1和OsGns5)的相对表达量,并生物测定Nl15 RNAi后褐飞虱的存活率以及成虫蜜露量和体质量增量。【结果】克隆了褐飞虱Nl15 cDNA序列(GenBank登录号：OK181113),其开放阅读框长1 008 bp;预测编码335个氨基酸,理论等电点为7.54,分子量为38.7 kD,含有23个氨基酸的信号肽序列和一个糖基化修饰位点,不存在跨膜结构域和其他已知的功能域;Nl15与灰飞虱Laodelphax striatellus同源蛋白氨基酸序列一致性为45%。发育表达谱结果表明,Nl15在褐飞虱各个发育阶段均表达,在3-4龄若虫中的表达量最高;组织表达谱结果表明,Nl15在褐飞虱雌成虫头部中的表达量最高,且在IR56种群头部中的表达量高于在TN1种群头部中的。RNAi实验结果表明,与注射dsGFP的对照组相比,注射dsNl15的处理组中Nl15的表达量显著降低了89.5%,褐飞虱的存活率以及成虫蜜露量和体质量增量均显著降低,上述6个水稻防御相关基因的表达量显著上调。【结论】褐飞虱IR56种群中的Nl15参与褐飞虱与水稻的防御与反防御分子互作。本研究为进一步阐述褐飞虱克服抗虫基因的机制及揭示昆虫与植物互作的分子网络提供了思路。     

The Insect Ecdysone Receptor is a Good Potential Target for RNAi-based Pest Control

RNA interference (RNAi) has great potential for use in insect pest control. However, some significant challenges must be overcome before RNAi-based pest control can become a reality. One challenge is the proper selection of a good target gene for RNAi. Here, we report that the insect ecdysone receptor (EcR) is a good potential target for RNAi-based pest control in the brown planthopper Nilaparvata lugens, a serious insect pest of rice plants. We demonstrated that the use of a 360 bp fragment (NlEcR-c) that is common between NlEcR-A and NlEcR-B for feeding RNAi experiments significantly decreased the relative mRNA expression levels of NlEcR compared with those in the dsGFP control. Feeding RNAi also resulted in a significant reduction in the number of offspring per pair of N. lugens. Consequently, a transgenic rice line expressing NlEcR dsRNA was constructed by Agrobacterium- mediated transformation. The results of qRT-PCR showed that the total copy number of the target gene in all transgenic rice lines was 2. Northern blot analysis showed that the small RNA of the hairpin dsNlEcR-c was successfully expressed in the transgenic rice lines. After newly hatched nymphs of N. lugens fed on the transgenic rice lines, effective RNAi was observed. The NlEcR expression levels in all lines examined were decreased significantly compared with the control. In all lines, the survival rate of the nymphs was nearly 90%, and the average number of offspring per pair in the treated groups was significantly less than that observed in the control, with a decrease of 44.18-66.27%. These findings support an RNAi-based pest control strategy and are also important for the management of rice insect pests.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.

Results

Here we present an optimized feeding method that results in phenotypes at least as strong as those produced by direct injection of dsRNA for embryonic lethal genes, and stronger for genes with post-embryonic phenotypes. In addition, the interference effect generated by feeding can be titrated to uncover a series of hypomorphic phenotypes informative about the functions of a given gene. Using this method, we screened 86 random genes on consecutive cosmids and identified functions for 13 new genes. These included two genes producing an uncoordinated phenotype (a previously uncharacterized POU homeodomain gene, ceh-6, and a gene encoding a MADS-box protein) and one gene encoding a novel protein that results in a high-incidence-of-males phenotype.

Conclusions

RNAi by feeding can provide significant information about the functions of an individual gene beyond that provided by injection. Moreover, it can be used for special applications for which injection or the use of mutants is sometimes impracticable (for example, titration, biochemistry and large-scale screening). Thus, RNAi by feeding should make possible new experimental approaches for the use of genomic sequence information.     

Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans

Background  

In Caenorhabditis elegans, injection of double-stranded RNA (dsRNA) results in the specific inactivation of genes containing homologous sequences, a technique termed RNA-mediated interference (RNAi). It has previously been shown that RNAi can also be achieved by feeding worms Escherichia coli expressing dsRNA corresponding to a specific gene; this mode of dsRNA introduction is conventionally considered to be less efficient than direct injection, however, and has therefore seen limited use, even though it is considerably less labor-intensive.     

Stable and heritable gene silencing in the malaria vector Anopheles stephensi

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.     

Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo

RNA interference (RNAi) has become an essential technique in entomology research. However, RNAi efficiency appears to vary significantly among insect species. Here, the sensitivity of four insect species from different orders to RNAi was compared to understand the reason for this variation. A previously reported method was modified to monitor trace amounts of double-stranded RNA (dsRNA). After the administration of dsRNA, the dynamics of its content was determined in the hemolymph, in addition to the capability of its degradation in both the hemolymph and the midgut juice. The results showed that injection of dsRNA targeting the homologous chitinase gene in Periplaneta americana, Zophobas atratus, Locusta migratoria, and Spodoptera litura, with doses (1.0, 2.3, 11.5, and 33.0 μg, respectively) resulting in the same initial hemolymph concentration, caused 82%, 78%, 76%, and 20% depletion, respectively, whereas feeding doses based on body weight (24, 24, 36, and 30 μg) accounted for 47%, 28%, 5%, and 1% depletion. The sensitivity of insects to RNAi was observed to be as follows: P. americana > Z. atratus >> L. migratoria >> S. litura. In vivo monitoring revealed that RNAi effects among these insect species were highly correlated with the hemolymph dsRNA contents. Furthermore, in vitro experiments demonstrated that the hemolymph contents after dsRNA injection were dependent on hemolymph degradation capacities, and on the degradation capabilities in the midgut juice, when dsRNA was fed. In conclusion, the RNAi efficacy in different insect species was observed to depend on the enzymatic degradation of dsRNA, which functions as the key factor determining the inner target exposure dosages. Thus, enzymatic degradation in vivo should be taken into consideration for efficient use of RNAi in insects.     

Juvenile hormone regulation of vitellogenin synthesis in the red flour beetle,Tribolium castaneum

To elucidate the endocrine regulation of vitellogenin (Vg) synthesis in the red flour beetle, Tribolium castaneum, the titers of juvenile hormone (JH) and ecdysteroids in the whole body of female beetles were measured and compared with Vg mRNA levels. Juvenile hormone levels remained high while the ecdysteroid levels declined steadily during 1–5 days post adult emergence (PAE). The Vg mRNA levels began to increase by the end of 3rd day PAE and peaked by the 4th–5th day PAE. Gene expression profiling by microarray and quantitative real-time PCR analyses of RNA isolated from 1 to 5 days PAE beetles revealed that the genes coding for proteins involved in JH biosynthesis and action, but not those involved in 20-hydroxyecdysone (20E) biosynthesis and action had similar expression patterns as the genes coding for Vg. RNA interference (RNAi)-aided knock-down in the expression of these genes showed that both JH and 20E were required for Vg gene expression. However, Vg mRNA was induced by the application of JH III but not by the injection of 20E into the previtellogenic females. These data suggest that JH is required for Vg synthesis in the fat body and 20E influences Vg synthesis through its action on oocyte maturation.     

Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi.     

Molecular characterization and gene functional analysis of Dicer‐2 gene from Nilaparvata lugens (Hemiptera: Geometroidea)

Abstract Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer‐2 (Dcr‐2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full‐length complementary DNA (cDNA) of N. lugens Dcr‐2 (NlDcr‐2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr‐2 gene. The complete Dcr‐2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr‐2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr‐2 was significantly reduced by NlDcr‐2 double‐stranded RNA (dsRNA) (dsDcr‐2). Fifty‐five per cent decrease of NlDcr‐2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion.     

Application of RNAi technology to the inhibition of zebrafish GtHalpha, FSHbeta, and LHbeta expression and to functional analyses

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.