The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

Differential responses of migratory locusts to systemic RNA interference via double‐stranded RNA injection and feeding

The migratory locust, Locusta migratoria, is one of the most destructive agricultural pests and has been widely used as a model system for insect physiology, neurobiology and behavioural research. In the present study, we investigated the effects of RNA interference (RNAi) using two delivery methods for double‐stranded RNA (dsRNA) molecules, namely, injection and feeding, to develop a potential new pest control strategy. Our results showed that locusts have a sensitive and systemic response to the injection of dsRNAs in a dose‐dependent manner, but do not respond to the feeding of dsRNAs. Further experiments suggested that the ineffectiveness of dsRNA feeding was attributable to the rapid degradation of dsRNA, which was probably induced by nuclease enzymes in the locust midgut. Moreover, we identified almost all the homologous genes involved in the endocytosis‐mediated dsRNA uptake from the locust genome, which provided possible clues regarding the dsRNA uptake mechanisms from the intestine to the midgut epithelium. These findings reveal the differential response models of fourth instar locust nymphs to dsRNA delivery methods, contribute to the current understanding of insect RNAi mechanisms and provide important information for the further application of RNAi as a genetic tool and pest control strategy.     

Key factors determining variations in RNA interference efficacy mediated by different double‐stranded RNA lengths in Tribolium castaneum

Double‐stranded RNA (dsRNA) length may affect RNA interference (RNAi) efficacy. Herein, variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum. Through in vitro experiments with T. castaneum midgut, dsRNA accumulation in the midgut, degradation by midgut homogenates and persistence in haemolymph after injection were tested to determine the causes of RNAi efficacy variation. The comparative efficacies of dsRNAs were 480 bp ≈ 240 bp  >  120 bp > 60 bp >> 21 bp. The combined midgut dsRNA accumulation and midgut homogenate‐induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning, but was likely not the main determinant of the variation in longer dsRNAs’ (≥60 bp) bioactivity. In vitro RNAi experiment with T. castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes, suggesting little variation in intracellular RNAi machinery’s affinity for different dsRNA lengths. In vivo haemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. In addition, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of haemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length.     

Knockdown of genes in the Toll pathway reveals new lethal RNA interference targets for insect pest control

RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well‐established model, with a strong and robust RNAi response, which can be used as a high‐throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large‐scale screening, to encompass others identified using targeted knowledge‐based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double‐stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal‐related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies.     

A MC motif in silkworm Argonaute 1 is indispensible for translation repression

Small RNA‐mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA‐induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double‐stranded RNA (dsRNA)‐induced RNAi, whereas Ago3 and Siwi are involved in the Piwi‐interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)‐mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA‐decapping enzyme 2 (Dcp2) protein in the Drosophila processing‐bodies (P‐bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P‐bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.     

dsRNA uptake and persistence account for tissue‐dependent susceptibility to RNA interference in the migratory locust,Locusta migratoria

RNA interference (RNAi) by introducing double‐stranded RNA (dsRNA) is a powerful approach to the analysis of gene function in insects; however, RNAi responses vary dramatically in different insect species and tissues, and the underlying mechanisms remain poorly understood. The migratory locust, a destructive insect pest and a hemimetabolic insect with panoistic ovaries, is considered to be a highly susceptible species to RNAi via dsRNA injection, but its ovary appears to be completely insensitive. In the present study, we showed that dsRNA persisted only briefly in locust haemolymph. The ovariole sheath was permeable to dsRNA, but injected dsRNA was not present in the follicle cells and oocytes. The lack of dsRNA uptake into the follicle cells and oocytes is likely to be the primary factor that contributes to the ineffective RNAi response in locust ovaries. These observations provide insights into tissue‐dependent variability of RNAi and help in achieving successful gene silencing in insensitive tissues.     

Molecular characterization of insulin‐like peptides in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

Insulin‐like peptides (ILPs) including insulin, insulin‐like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin‐specific G protein‐coupled receptor‐binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)‐mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1–4 dramatically impaired female fecundity. These findings indicate that NlILP1–4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.     

RNA interference and dietary inhibitors induce a similar compensation response in Tribolium castaneum larvae

Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double‐stranded RNA (dsRNA) constructs of different regions of the gene (3′, middle, 5′ and entire coding regions). RNA sequencing and quantitation (RNA‐seq) was used to evaluate knockdown and specificity amongst the treatments. Overall, target gene expression decreased in all treatment groups, but was more severe and specific in dsRNA targeting the 3′ and entire coding regions, encoding the proteolytic active site in the enzyme. Additional cysteine cathepsin genes also were down‐regulated (off‐target effects), but some were up‐regulated in response to RNAi treatment. Notably, some serine peptidase genes were increased in expression, especially in dsRNA targeting 5′ and middle regions, and the response was similar to the effects of dietary cysteine protease inhibitors. We manually annotated these serine peptidase genes to gain insight into function and relevance to the RNAi study. The data indicate that T. castaneum larvae compensate for the loss of digestive peptidase activity in the larval gut, regardless of the mechanism of disruption.     

Delivery of RNA interference triggers to sensory neurons in vivo using herpes simplex virus

Importance of the field: Pain is a hugely important area of research attracting considerable academic and commercial interest. However, the application of RNA interference (RNAi) to the study of nociceptive processes and the development of new analgesics has been limited by the specific challenges associated with the delivery of RNAi triggers to the cell bodies of sensory neurons in the dorsal root ganglia (DRG).Areas covered in this review: In the past five years, delivery of small-interfering RNA (siRNA) to the DRG and spinal cord has achieved effective and specific silencing of targeted genes in various animal models of pain. However, delivery of short-hairpin RNA (shRNA) or artificial microRNA (miRNA) to sensory neurons in vivo has not been feasible using most delivery systems currently available. What the reader will gain: Replication-defective vectors based on herpes simplex virus (HSV), which are particularly efficient at targeting DRG neurons, have been recently engineered to express shRNA and artificial miRNA. Whilst silencing induced by siRNA is transient and requires relatively high doses of silencing triggers, HSV-mediated expression of shRNA/miRNA in sensory neurons allows silencing of targeted genes for at least one week following a single injection.Take home message: The potential to use inducible or tissue-specific promoters and to simultaneously silence multiple gene targets, in addition to recent studies suggesting that artificial miRNAs may have improved safety profiles, hold clear advantages for the use of miRNA-based vectors for gene silencing in sensory neurons.     

Feeding‐based RNA interference of a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is the most devastating rice insect pest to have given rise to an outbreak in recent years. RNA interference (RNAi) is a technological breakthrough that has been developed as a powerful tool for studying gene function and for the highly targeted control of insect pests. Here, we examined the effects of using a feeding‐based RNAi technique to target the gene trehalose phosphate synthase (TPS) in N. lugens. The full‐length cDNA of N. lugens TPS (NlTPS) is 3235 bp and has an open reading frame of 2424 bp, encoding a protein of 807 amino acids. NlTPS was expressed in the fat body, midgut and ovary. Quantitative real‐time PCR (qRT‐PCR) analysis revealed that NlTPS mRNA is expressed continuously with little change during the life of the insect. Efficient silencing of the TPS gene through double‐stranded RNA (dsRNA) feeding led to rapid and significant reduction levels of TPS mRNA and enzymatic activity. Additionally, the development of N. lugens larvae that had been fed with the dsRNA was disturbed, resulting in lethality, and the cumulative survival rates dropped to 75.56, 64.44, 55.56 and 40.00% after continuous ingestion of 0.5 µg/µl dsRNA for 2, 4, 7 and 10 days, respectively. These values were significantly lower than those of the insects in the control group, suggesting that NlTPS dsRNA may be useful as a means of insect pest control.     

Scavenger receptor‐mediated endocytosis facilitates RNA interference in the desert locust,Schistocerca gregaria

RNA interference (RNAi) has become a widely used loss‐of‐function tool in eukaryotes; however, the delivery of double‐stranded (ds)RNA) to the target cells remains a major challenge when exploiting the RNAi‐technology. In insects, the efficiency of RNAi is highly species‐dependent. Yet, the mechanism of cell entry in insects has only been characterized in a cell line of the fruit fly, Drosophila melanogaster, a species that is well known to be poorly amenable to environmental RNAi. In the present paper, we demonstrate that silencing vacuolar H‐ATPase 16 (vha16) and clathrin heavy chain (clath), two components of the Clathrin‐dependent endocytosis pathway, together with pharmacological inhibition of scavenger receptors with polyinosine and dextran sulphate, can significantly attenuate the highly robust RNAi response in the desert locust, Schistocerca gregaria.     

小RNA在血液肿瘤研究中应用的新进展

小RNA包括siRNA和miRNA两种，前者是伴随着RNAi现象发现的，是对外源基因剪切加工形成的，是生物维持自身基因组稳定性的一种机制，而miRNA是基因组中固有的，是在转录后水平调节基因表达的重要机制。小RNA的作用方式有两种：介导目标RNA降解和抑制蛋白质翻译。前者要求小RNA与目标RNA精确互补，而后者只要求部分互补，采取何种机制取决于互补程度而不是其来源。RNAi作为下调基因表达的手段，在功能基因组学中已有广泛的应用。对血液肿瘤发病相关基因，尤其对染色体易位造成的相关融合基因、凋亡相关基因及多药耐药基因的干扰研究表明，该技术不但是研究机制的有力手段，而且具有临床应用前景。对microRNA的研究发现，它在多种血液肿瘤如多种淋巴瘤和白血病中存在表达的变化，并与多种癌基因相关，提示它广泛参与血液肿瘤的发病机制。本文就RNA干扰和小RNA的发现和作用，以及小RNA在血液肿瘤研究中的应用作一综述。     

RNA interference in Pardosa pseudoannulata,an important predatory enemy against several insect pests,through ingestion of dsRNA-expressing Escherichia coli

RNA interference is an important technology for gene functional research in many organisms. The pond wolf spider (Pardosa pseudoannulata) is an important natural enemy of rice field pests. To facilitate large-scale gene functional research in this spider species and others, we developed an RNA interference (RNAi) method via ingestion of bacteria expressing dsRNA. The dsRNA targeting a cytochrome P450 monooxygenase (cyp41g2) was expressed in Escherichia coli HT115 (DE3). And then the bacterial suspension was fed to 14–20 days old spiderlings. The mRNA abundance of the target gene was significantly reduced after 3-day's ingestion of bacteria expressing dsRNA, and between day 5 and 7, RNAi efficiency remained stable. Thus, we selected 5 days as the optimum interference time. Furthermore, the bacteria resuspension containing 20 ng/μl dsRNA was selected as the optimum concentration. To evaluate the applicability of this method, three other genes with different tissue expression pattern were also selected as targets. And the mRNA abundance of all the four target genes was significantly reduced with RNAi efficiency between 66.0% and up to 86.9%. The results demonstrated that the oral delivery of bacteria expressing dsRNA would be an effective RNAi method for the gene functional study in P. pseudoannulata.