Acquisition and excretion of Bartonella quintana by the cat flea,Ctenocephalides felis felis

Bartonella quintana is transmitted by the infected faeces of body lice. Recently, this bacterium was detected in cat fleas (Ctenocephalides felis) and in two humans with chronic adenopathy whose only risk factor was contact with cat fleas. In this study, a total of 960 C. felis were divided into 12 groups (2 control groups and 10 infected groups) each containing 80 fleas. The fleas were fed B. quintana‐inoculated human blood at different dilutions (≈3.6 × 10⁴ ? 8.4 × 10⁹ bacteria) for 4 days via an artificial membrane. Subsequently, all flea groups were fed uninfected blood until day 13 postinfection (dpi). On day 3 pi, B. quintana was detected with two specific genes by quantitative PCR in 60–100% of randomly chosen fleas per dilution: 52% (26/50) in the infected fleas in Trial 1 and 90% (45/50) of the fleas in Trial 2. B. quintana was also identified by molecular and culture assays in flea faeces. The average number of B. quintana as determined by qPCR decreased until the 11th dpi and was absent in both trials at the 13th dpi. Bacteria were localized only in the flea gastrointestinal gut by specific immunohistochemistry. Our results indicate that cat fleas can acquire B. quintana by feeding and release viable organisms into their faeces. Therefore, fleas may play a role as vectors of trench fever or other clinical manifestations that are caused by B. quintana. However, the biological role of C. felis in the transmission of B. quintana under natural conditions is yet to be defined.     

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐RNAi (VSRs). Interestingly, in spite of dominance of interferon‐based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double‐stranded RNA genome. Intriguingly, a time point‐dependent bimodal regulation of RNAi was observed in RV‐infected cells, where short interfering RNA (siRNA)‐based RNAi was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA‐mediated RNAi, via ubiquitin–proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.     

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.     

Core RNAi machinery and gene knockdown in the emerald ash borer (Agrilus planipennis)

The RNA interference (RNAi) technology has been widely used in insect functional genomics research and provides an alternative approach for insect pest management. To understand whether the emerald ash borer (Agrilus planipennis), an invasive and destructive coleopteran insect pest of ash tree (Fraxinus spp.), possesses a strong RNAi machinery that is capable of degrading target mRNA as a response to exogenous double-stranded RNA (dsRNA) induction, we identified three RNAi pathway core component genes, Dicer-2, Argonaute-2 and R2D2, from the A. planipennis genome sequence. Characterization of these core components revealed that they contain conserved domains essential for the proteins to function in the RNAi pathway. Phylogenetic analyses showed that they are closely related to homologs derived from other coleopteran species. We also delivered the dsRNA fragment of AplaScrB-2, a β-fructofuranosidase-encoding gene horizontally acquired by A. planipennis as we reported previously, into A. planipennis adults through microinjection. Quantitative real-time PCR analysis on the dsRNA-treated beetles demonstrated a significantly decreased gene expression level of AplaScrB-2 appearing on day 2 and lasting until at least day 6. This study is the first record of RNAi applied in A. planipennis.     

Large-scale monitoring of imidacloprid susceptibility in the cat flea, Ctenocephalides felis

Although on-animal topical treatment with compounds such as imidacloprid has revolutionized the control of the cat flea, Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae), the development of insecticide resistance is a continuing threat. As part of a highly co-ordinated and unprecedented resistance monitoring programme for C. felis, 1437 flea isolates were collected by veterinary clinics in Australia, Germany, France, the U.K. and 29 states in the U.S.A. from 2002 to 2009. About 65% of the collections were made from June to October each year and 71% of the collections were from cats. Collections of flea eggs were sent to one of five different laboratories, where they were tested with a diagnostic dose of imidacloprid (3 p.p.m.) applied to larval flea-rearing medium. Of the 1437 collections received, 1064 contained adequate numbers of eggs for testing. Of these isolates, untreated eggs failed to hatch in 22.7% and were not considered valid bioassays. Survival rates >5% and development of adult fleas (a threshold for further testing) occurred in only 22 isolates. They were re-tested with the same diagnostic dose and none produced >5% adult emergence. Complete dose-response bioassays were performed on three of the isolates that had triggered a second test and produced slopes, intercepts and LC(50) values similar to those for existing susceptible laboratory strains. Results confirmed sustained susceptibility of C. felis to imidacloprid, despite its widespread use for over a decade.     

Consumption of flea faeces and eggs by larvae of the cat flea,Ctenocephalides felis

The effects of the consumption of flea faeces and non-viable eggs on larval development in the cat flea Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) were investigated. Only 13.3% of larvae developed into adults when fed a diet of male or female flea faeces alone; however, 90% of larvae developed into adults when fed on flea faeces supplemented with non-viable flea eggs. When fed with non-viable eggs alone, larvae did not develop into adults. Nevertheless, non-viable eggs may provide critical supplemental nutrients, lacking in flea faeces and required for larval development. None of the larvae fed on flea faeces or non-viable eggs alone formed a cocoon. A diet of flea faeces alone significantly extended the second as well as third larval stadia compared to larvae fed on diets containing non-viable eggs. It is suggested that the cannibalism of fertile eggs may limit population growth in the cat flea.     

Indicators of sexual maturation and regression in the female cat flea, Ctenocephalides felis

Differences in the salivary glands, mesenteron epithelium and reproductive organs of female cat fleas, Ctenocephalides felis Bouché (Siphonaptera: Pulicidae), are related to the degree of reproductive maturation or regression. Contrary to previous ideas, blue bodies in the ovarioles are degenerate oocyte nuclei and their presence denotes failure of ripening oocytes to reach full maturity. A distinction between true corpora lutea and pseudo-corpora lutea is established, the presence of the former indicates successful oviposition, and of the latter, failure to complete maturation of eggs. Accurate indicators of sexual maturation and reproductive success are of potential value in assessing relative suitability of various hosts for a given flea species and therefore in assessing the degree of host specificity among fleas.     

Understanding attraction stimuli of the cat flea, Ctenocephalides felis, in non-chemical control methods

Comparisons were conducted of flea catches of four commercially available flea traps in the laboratory and under field conditions, in both rural and urban locations. The results clearly showed the My Flea Trap?, which utilizes an intermittent light to attract fleas, to be far superior in trapping ability to the three continuous light traps; it caught up to 23 times as many fleas as the other traps. Altering the lighting mechanism to provide continuous rather than intermittent light significantly decreased the number of fleas captured. In addition, the use of a green filter significantly increased trapping efficiency, whereas the addition of a heat source had no apparent effect.     

Identification,characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea,Ctenocephalides felis

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.     

Isolation, characterization, and recombinant expression of multiple serpins from the cat flea, Ctenocephalides felis

Several clones encoding serine protease inhibitors were isolated from larval and adult flea cDNA expression libraries by immunoscreening and PCR amplification. Each cDNA contained an open reading frame encoding a protein of approximately 45 kDa, which had significant sequence similarity with the serpin family of serine protease inhibitors. The thirteen cDNA clones isolated to date encode serpin proteins, which share a primary structure that includes a nearly identical constant region of about 360 amino acids, followed by a C-terminal variable region of about 40-60 amino acids. The variable C-terminal sequences encode most of the reactive site loop (RSL) and are generated by mutually exclusive alternative exon splicing, which may confer unique protease selectivity to each serpin. Utilization of an alternative exon splicing mechanism has been verified by sequence analysis of a flea serpin genomic clone and adjacent genomic sequences. RNA expression patterns of the cloned genes have been examined by Northern blot analysis using variable region-specific probes. Several putative serpins have been overexpressed using the cDNA clones in Escherichia coli and baculovirus expression systems. Two purified baculovirus-expressed recombinant proteins have N-terminal amino acid sequences identical to the respective purified native mature flea serpins indicating that appropriate N-terminal processing occurred in the virus-infected insect cells.     

Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects

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Dicing and slicing: the core machinery of the RNA interference pathway

RNA interference (RNAi) is broadly defined as a gene silencing pathway that is triggered by double-stranded RNA (dsRNA). Many variations have been described on this theme. The dsRNA trigger can be supplied exogenously, as an experimental tool, or can derive from the genome in the form of microRNAs. Gene silencing can be the result of nucleolytic degradation of the mRNA, or by translational suppression. At the heart of the pathway are two ribonuclease machines. The ribonuclease III enzyme Dicer initiates the RNAi pathway by generating the active short interfering RNA trigger. Silencing is effected by the RNA-induced silencing complex and its RNaseH core enzyme Argonaute. This review describes the discovery of these machines and discusses future lines of work on this amazing biochemical pathway.     

Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.     

MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.     

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

RNA interference mediated knockdown of juvenile hormone esterase gene in Bemisia tabaci (Gennadius): Effects on adults and their progeny

Juvenile hormone is responsible for regulating metamorphosis and reproduction in insects. Analysis of key elements of juvenile hormone regulation would enhance the understanding of this complex mechanism. Juvenile hormone esterase plays an important role in maintaining juvenile hormone titres in insects. In this study, effects of knockdown of juvenile hormone esterase gene (jhe) in Bemisia tabaci were studied using RNA interference (RNAi) technique. dsRNA corresponding to two conserved regions of jhe gene, substrate binding pocket site (jhe1), catalytic triad site (jhe2), green fluorescent protein gene (gfp) as control were synthesized. dsRNAs incorporated in artificial diet (20% sucrose solution) @ 2.5, 1.0, 0.5 and 0.1 μg/μl were fed to adult whiteflies for 48 h, followed by shifting whiteflies to live plants for next generation biology study. Based on qRT-PCR analyses, reduced jhe gene expression was observed in adult whiteflies after dsRNA feeding @ 2.5 and 1.0 μg/μl. jhe gene knockdown affects the survival and reproduction of whiteflies adversely in a dose-dependent manner. Moreover, oral feeding of dsRNA to adult whiteflies @ 2.5 and 1.0 μg/μl showed adverse effects on next generation of whitefly viz., lower egg hatchability and shortened egg incubation period. Minimum number of viable eggs (1.04 and 1.80 eggs/female) were observed when whiteflies were fed with highest concentration of dsjhe1 and dsjhe2 as compared to control (16.58 eggs/female). These data suggest that jhe gene acts as a major biological player in whitefly and its progeny and further indicate to be potential target for managing whitefly population.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

慢病毒介导的RNA干扰ppGalNAc-T2基因表达抑制Jurkat细胞增殖和迁移

在肿瘤中,黏蛋白O-糖基化有着重要的生物学功能.控制O-糖基化起始合成的是多肽∶N-乙酰氨基半乳糖转移酶家族,研究该酶家族对阐明O-糖基化在肿瘤中的作用机制有重要的意义.探讨了靶向干扰ppGalNAc-T2基因表达对白血病Jurkat细胞株增殖及迁移的影响.首先合成ppGalNAc-T2特异shRNA干扰及对照序列,将其连接至慢病毒干扰载体YH1;重组载体经双酶切、测序鉴定正确后与包装质粒共转染293T细胞,获得的病毒颗粒经过滤纯化后感染Jurkat细胞,流式细胞分选仪进行细胞分选以获得ppGalNAc-T2基因稳定干扰表达的Jurkat细胞,然后使用RT-PCR和Western blot方法对各组别细胞中ppGalNAc-T2基因表达情况进行分析,以确定ppGalNAc-T2基因表达被有效干扰;进一步利用MTT实验和Transwell实验分析ppGalNAc-T2基因干扰表达对Jurkat细胞增殖及迁移的影响.结果表明,成功构建了靶向干扰ppGalNAc-T2基因表达的慢病毒载体,感染Jurkat细胞后能稳定干扰ppGalNAc-T2基因表达.MTT和Transwell实验研究发现,下调ppGalNAc-T2基因表达对Jurkat细胞增殖和迁移有抑制作用.     

Suppression of bcl-2 gene by RNA interference increases chemosensitivity to cisplatin in nasopharyngeal carcinoma cell line CNE1

To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE1