Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

东亚飞蝗谷胱甘肽S-转移酶RNA干扰效率研究

东亚飞蝗Locusta migratoria manilensis(Meyen)是我国主要的农业害虫之一,已发现东亚飞蝗对某些农药产生了抗性,其抗性机制可能与谷胱甘肽硫转移酶(GST)代谢解毒相关.本研究利用特异性引物合成东亚飞蝗GST 4个不同家族基因的双链RNA(dsRNA),将dsRNA注射到东亚飞蝗幼虫体内,采...     

家蚕胚胎发育时期的RNA干涉研究

通过导入特定基因的dsRNA特异性地关闭该基因的功能,由此产生的现象为RNA干涉.为在家蚕胚胎发育时期建立有效的RNAi技术体系,在前人的基础上以家蚕第三白卵基因Bmwh3为材料,建立了有效的RNAi技术体系,结果表明,成功诱导第三白卵突变表型的有效注射时间为产卵后8 h内,wh3dsRNA的有效浓度须大于2.0 g/L.在发育胚胎的第三天注射wh3dsRNA,同样可诱导第三白卵突变的另一表型——半透明蚁蚕,根据实验结果初步推测,Bmwh3不仅参与眼色素和卵浆液膜色素前体的转运,还可能参与胚胎体壁色素前体的转运.     

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.     

Transport of orally delivered dsRNA in southern green stink bug,Nezara viridula

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24–72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. ³²P-labeled dsRNA injected into SGSB was processed into siRNA, but fed ³²P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.     

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm-TSP belongs to the trypsin-like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm-TSP leads to dramatic reductions in protease and cuticle-degrading activity of a molting fluid, which leads to molting defects from fourth-instar larvae (L4) to fifth-instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm-TSP plays a critical role in L. migratoria manilensis ecdysis.     

东亚飞蝗UBX结构域包含蛋白基因 LmUBX2 的克隆和表达分析

【目的】UBX结构域包含蛋白是p97/CDC48的辅助因子。p97在泛素化相关的多种细胞过程中起着重要的作用,如依赖泛素 蛋白酶体系统的蛋白质降解和同型膜融合等。本研究旨在克隆东亚飞蝗 Locusta migratoria manilensis (Meyen)的UBX结构域包含蛋白基因,分析其组织和发育表达格局,为进一步研究UBX结构域包含蛋白基因的功能奠定基础。【方法】通过分析东亚飞蝗的转录组数据克隆UBX结构域包含蛋白基因,采用实时定量PCR技术分析该基因在不同发育时期和成虫不同组织中的表达水平。【结果】克隆到东亚飞蝗的一个UBX结构域包含蛋白基因,命名为 LmUBX2。 LmUBX2 开放阅读框长1 020 bp,编码399个氨基酸,预测分子量和等电点分别为37.8 kDa和6.03,与其他UBX结构域包含蛋白的氨基酸一致性为37%~64%,N端和C端分别有一个保守的UBA结构域和UBX结构域。序列比较和系统发育分析发现 LmUBX2 属于SAKS1亚家族。定量分析发现,LmUBX2 在整个生命周期中都有表达,但成虫期的表达水平最高;在检测的所有组织中都有表达,但在精巢和卵巢中表达水平最高。【结论】研究结果说明 LmUBX2 可能参与东亚飞蝗多种生理过程,尤其可能与东亚飞蝗的生殖有关,但还需深入研究。     

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.     

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle,Leptinotarsa decemlineata

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.     

利用RNAi技术沉默小菜蛾类钙粘蛋白基因

RNA干扰（RNA interference, RNAi）是一种调控基因表达的方法, 其通过体外合成一段与内源靶基因同源的双链RNA（dsRNA）或siRNA, 导入生物体内, 使内源靶基因中同源mRNA降解, 从而达到阻抑基因表达的目的。类钙粘蛋白（cadherin-like protein）是位于昆虫中肠刷状缘膜囊(brush border membrane vesicles, BBMV)上与钙粘蛋白(cadherin)结构相似的物质, 是多种昆虫体内Bt杀虫蛋白的受体。本研究利用基因特异引物通过RT-PCR扩增了小菜蛾类钙粘蛋白基因的2个片段（CAD1和CAD2）, 合成相对应的双链RNA（double-stranded RNA, dsRNA）; 并将dsRNA通过显微注射导入小菜蛾3龄幼虫体内, 测定了不同靶位点、不同剂量、不同检测时间对目的基因mRNA表达量的影响。结果表明: 将70 nL CAD1对应的dsRNA注射到幼虫体内48 h后, 基因表达量显著下降, 72 h后恢复。免疫印迹检测结果表明, 类钙粘蛋白在注射dsRNA 48 h后幼虫BBMV中的含量明显下降。本实验成功实现了小菜蛾类钙粘蛋白基因的沉默, 该体系的成功建立为利用RNAi技术分析小菜蛾及其他鳞翅目昆虫基因的功能提供了参考。     

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae)

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.     

Scavenger receptor mediates systemic RNA interference in ticks

RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.     

A simple "soaking method" for RNA interference in the planarian<Emphasis Type="Italic"> Dugesia japonica</Emphasis>

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.