The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae)

We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.     

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.     

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.     

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.     

Reduction in deformed wing virus infection in larval and adult honey bees (Apis mellifera L.) by double-stranded RNA ingestion

Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.     

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.     

Identification,characterization and functional analysis of a chitin synthase gene in the brown citrus aphid,Toxoptera citricida (Hemiptera,Aphididae)

Chitin synthase (CHS) is a crucial enzyme involved in the final step of the insect chitin biosynthetic pathway. In this study, we cloned the full‐length cDNA sequence of a chitin synthase gene (TCiCHS) from the brown citrus aphid, Toxoptera citricida, an important citrus pest and the main vector of citrus tristeza virus worldwide. TCiCHS was expressed during the entire lifecycle and in all insect tissues examined. Expression was highest in first–second‐instar nymphs, nymph–adult transitions and in the abdomen (6.7‐fold higher than head). Embryos had a higher expression level than the integument. Fourth‐instar nymphs were exposed to 5 and 500 mg/l concentrations of the chitin synthesis inhibitor diflubenzuron (DFB) for 48 h and had the highest mortality at the 500 mg/l concentration. The mRNA expression levels of TCiCHS were significantly enhanced upon the exposure of nymphs to both low and high DFB concentrations. Silencing of TCiCHS occurred through plant‐mediated double‐stranded RNA (dsRNA) feeding. Most dsRNA‐fed nymphs were unable to moult to the next stage, and the expression of TCiCHS decreased 48% compared with controls. These results demonstrate that TCiCHS plays an important role in nymph to adult development, is possibly help identify molecular targets for To. citricida control.     

RNA干扰技术抑制急性白血病细胞THP-1中SALL4基因表达的研究

目的 抑制SALL4基因在急性白血病细胞THP-1中的表达,探讨其在白血病发病机制中的作用.方法 将表达SALL4干扰序列的质粒载体转染至急性白血病细胞THP-1中,分为4组:(1)空白对照组:不加处理;(2)转染对照组:加入空pRS质粒载体;(3)转染一组:加入SALL4-shRNA-pRS-1干扰质粒的转染复合体;(4)转染二组:加入SALL4-shRNA-pRS-2干扰质粒的转染复合体.采用实时荧光定量PCR和WB技术观察THP-1细胞中SALL4基因mRNA及蛋白表达的变化,建立抑制SALL4表达的体系.实时荧光定量PCR检测SALL4受抑后Wnt/β-catenin信号通路中C-myc、Cyclin D1、β-catenin基因的表达改变,采用流式细胞术分析THP-1细胞凋亡情况.结果 实时荧光定量PCR结果 显示,转染一组、转染二组、转染对照组、空白对照组SALL4基因的表达水平分别为(36.0±4.3)%、(32.0±2.4)%、(102.0±6.5)%、(100.0±2.6)%,差异有统计学意义(F=226.3,P<0.05);转染一组、转染二组的SALL4基因表达水平显著低于空白对照组,差异有统计学意义(t值分别为19.7、19.1,P<0.05);转染对照组SALL4基因表达水平与空白对照组比较差异无统计学意义(t=1.1,P＞0.05).WB检测结果 显示,转染一组、转染二组SALL4蛋白表达与空白对照组和转染对照组相比明显下降.以上基因和蛋白表达水平均提示SALL4干扰效率良好.SALL4基因抑制表达后,转染一组C-myc基因、β-catenin基因、Cyclin D1基因的表达分别为(44.0±6.2)%、(44.0±5.1)%和(107.0±13.6)%,转染二组为(22.0±4.5)%、(25.0±3.5)%和(48.0±7.6)%,转染对照组为(42.0±3.5)%、(59.0±3.7)%及(79.0±5.6)%.转染一组、转染二组C-myc基因表达水平显著低于空白对照组的(103.0±7.5)%,差异有统计学意义(t值分别为10.1、9.5,P均<0.05);转染一组、转染二组β-catenin基因的表达显著低于空白对照组的(100.0±4.1)%,差异有统计学意义(t值分别为23.3、22.9,P均<0.05);转染一组、转染二组Cyclin D1基因的表达显著低于空白对照组的(102.0±6.0)%,差异有统计学意义(t值分别为17.4、12.4,P均<0.05).SALL4基因被抑制后,转染一组、转染二组、转染对照组及空白对照组的细胞凋亡率分别为(57.2±9.1)%、(34.4±8.6)%、(14.4±3.6)%及(14.8±4.8)%,差异有统计学意义(F=42.5,P<0.05).转染一组、转染二组细胞凋亡率显著高于空白对照组(t值分别为9.7、4.5,P均<0.05).结论 抑制急性白血病细胞THP-1中SALL4基因表达后,Wnt/β-catenin信号通路的C-myc、Cyclin D1及β-catenin基因的表达随之下调,并促进了细胞凋亡.     

ER type I signal peptidase subunit (LmSPC1) is essential for the survival of Locusta migratoria manilensis and affects moulting,feeding, reproduction and embryonic development

The endoplasmic reticulum type I signal peptidase complex (ER SPC) is a conserved enzyme that cleaves the signal peptides of secretory or membrane preproteins. The deletion of this enzyme leads to the accumulation of uncleaved proteins in biomembranes and cell death. However, the physiological functions of ER SPC in insects are not fully understood. Here, a catalytic subunit gene of ER SPC, LmSPC1, was cloned from Locusta migratoria manilensis and its physiological functions were analysed by RNA interference (RNAi). The LmSPC1 open reading frame encoded a protein of 178 amino acids with all five conserved regions of signal peptidases. RNAi‐mediated knockdown of LmSPC1 resulted in high mortality. Sixty‐nine per cent of dead nymphs died of abnormal moulting, corresponding to decreased activity of moulting fluid protease. Moreover, insects in the RNAi group experienced a decline in food intake, and a decrease in the secretion of total protein and digestive enzymes from midgut tissues to the midgut lumen. Furthermore, the females produced fewer eggs and eggs with disrupted embryogenesis. These results indicate that LmSPC1 is required for the secretion of secretory proteins, affects physiological functions, including moulting, feeding, reproduction and embryonic development, and is essential for survival. Therefore, LmSPC1 may be a potential target for locust control.     

RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach,Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha‐amylase Blattella germanica Tergal Gland protein‐1 (BGTG‐1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG‐1 protein. BGTG‐1 gene expression profiles were determined by using quantitative real‐time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG‐1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG‐1 gene expression by injecting BGTG‐1 homologous double‐stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG‐1 dsRNA showed ～90% lower BGTG‐1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.     

RNA interference-mediated knock-down of Bla g 1 in the German cockroach, Blattella germanica L., implicates this allergen-encoding gene in digestion and nutrient absorption

We used RNA interference (RNAi) to silence the expression of a gene encoding Bla g 1, a human allergen produced by the German cockroach, Blattella germanica L., to study its function in cockroach physiology. Females injected with 1 µg of double-stranded RNA contained 64% less Bla g 1 protein and Bla g 1 mRNA abundance was reduced by 91.4% compared to sham-injected females. Bla g 1 knockdown slowed the pace of weight gain, midgut growth, and colleterial gland and basal oocyte maturation, resulting in delayed egg case formation and lower fecundity. Exogenous juvenile hormone treatments rescued reproduction in RNAi-treated females, suggesting that Bla g 1 silencing lowered endogenous juvenile hormone, probably by reducing food intake and nutrient absorption.