scholarly journals RNA Interference Is Enhanced by Knockdown of Double-Stranded RNases in the Yellow Fever Mosquito Aedes aegypti

Insects ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 327 ◽  
Author(s):  
David Giesbrecht ◽  
Daniel Heschuk ◽  
Ian Wiens ◽  
David Boguski ◽  
Parker LaChance ◽  
...  
Keyword(s):  
Rna Interference ◽  
Aedes Aegypti ◽  
Fluorescent Protein ◽  
Mosquito Control ◽  
Ex Vivo ◽  
Double Stranded Rna ◽  
Industry Standards ◽  
Rna Targeting ◽  
Aegypti Genome ◽  
Control Technologies

RNA interference (RNAi) techniques are being developed for a range of pest insect control technologies, including the sterile insect technique (SIT) and double-stranded RNA (dsRNA)-based insecticides. In SIT applications, where >99% of the released males should be sterile to meet industry standards, the efficiency of RNAi will need to be improved for many insect species if this technology is to be adopted. Endogenous dsRNases can impede dsRNA delivery in some insects, and, here, we investigated whether dsRNases in the midgut could limit RNAi efficacy in the mosquito Aedes aegypti. Ten putative dsRNases were identified in the Ae. aegypti genome, with two highly expressed in the midguts of larvae. Using an ex vivo assay, we observed that dsRNA was rapidly degraded within the mosquito larva’s gut. Double-stranded RNA targeting these two dsRNases, when fed to the larvae, effectively reduced gut dsRNase activity. When these dsRNase-specific dsRNAs were co-delivered with dsRNA targeting a cyan fluorescent protein (CFP) reporter gene, greater knockdown of CFP fluorescence was observed. These results suggest that inhibiting dsRNase activity could enable the implementation of RNAi-based mosquito control methods.

scholarly journals Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

Insects ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 90-103 ◽  
Author(s):  
Francis Nunes ◽  
Aline Aleixo ◽  
Angel Barchuk ◽  
Ana Bomtorin ◽  
Christina Grozinger ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Interference ◽  
Honey Bee ◽  
Fluorescent Protein ◽  
Double Stranded Rna ◽  
Green Fluorescent ◽  
Target Effects

scholarly journals Double-Stranded RNA-Degrading Enzymes Reduce the Efficiency of RNA Interference in Plutella xylostella

Insects ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 712
Author(s):  
Jin-Zhi Chen ◽  
Ying-Xia Jiang ◽  
Miao-Wen Li ◽  
Jian-Wen Li ◽  
Ben-Hu Zha ◽  
...  
Keyword(s):  
Rna Interference ◽  
Plutella Xylostella ◽  
Intestinal Tract ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Double Stranded Rna ◽  
Degrading Enzymes ◽  
Rnai Efficiency ◽  
Green Fluorescent

DsRNA-degrading enzymes (dsRNases) have been recognized as important factors in reducing RNA interference (RNAi) efficiency in different insect species. However, dsRNases in Plutella xylostella are still unknown. We identified the full-length cDNAs of PxdsRNase1, PxdsRNase2, PxdsRNase3, and PxdsRNase4. Gene expression profile showed that PxdsRNase1 was mainly expressed in the hemolymph; and that PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. The expression of PxCht (Chitinase of P. xylostella) in P. xylostella larvae injected with the mixture of dsPxCht (dsRNA of PxCht) and dsPxdsRNase1 (dsRNA of PxdsRNase1), dsPxdsRNase2 (dsRNA of PxdsRNase2), or dsPxdsRNase3 (dsRNA of PxdsRNase3) was significantly higher than that in the larvae injected with the mixture of dsGFP (dsRNA of green fluorescent protein gene, GFP) and dsPxCht; the transcription level of PxCht in the larvae feeding on the mixture of dsPxCht and dsPxdsRNase1, dsPxdsRNase2, or dsPxdsRNase3 was significantly higher than that in the larvae feeding on the mixture of dsPxCht and dsGFP. The recombinant protein of PxdsRNase1 degraded dsRNA rapidly, PxdsRNase3 cleaved dsRNA without complete degradation, and PxdsRNase2 could not degrade dsRNA in vitro. These results suggested that PxdsRNases1, PxdsRNases2, and PxdsRNases3 were involved in the dsRNA degradation to reduce RNAi efficiency with different mechanisms.

scholarly journals RNAi by Soaking Aedes aegypti Pupae in dsRNA

Insects ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 634
Author(s):  
Fiza Arshad ◽  
Arvind Sharma ◽  
Charleen Lu ◽  
Monika Gulia-Nuss
Keyword(s):  
Functional Analysis ◽  
Rna Interference ◽  
Aedes Aegypti ◽  
Simple Procedure ◽  
Standard Technique ◽  
Malaria Mosquito ◽  
Double Stranded Rna ◽  
Carrier Molecule ◽  
Mosquito Anopheles Gambiae ◽  
Osmotic Challenge

RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.

scholarly journals A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽  
2021 ◽  
Vol 2 (3) ◽  
Author(s):  
Nelly Redolfi ◽  
Elisa Greotti ◽  
Giulia Zanetti ◽  
Tino Hochepied ◽  
Cristina Fasolato ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Brain Slices ◽  
Resonance Energy ◽  
Mouse Line ◽  
Isolated Cells ◽  
Genomic Locus ◽  
Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

scholarly journals Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1187
Author(s):  
Michael Wassenegger ◽  
Athanasios Dalakouras
Keyword(s):  
Dna Methylation ◽  
Rna Interference ◽  
Gene Silencing ◽  
Plant Cells ◽  
Transcriptional Gene Silencing ◽  
Rnai Machinery ◽  
Double Stranded Rna ◽  
Post Transcriptional Gene Silencing ◽  
Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

scholarly journals Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Teresa D Shippy ◽  
Jianhua Guo ◽  
Susan J Brown ◽  
Richard W Beeman ◽  
Robin E Denell
Keyword(s):  
Rna Interference ◽  
Expression Pattern ◽  
Tribolium Castaneum ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Homeotic Gene ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Similar Expression ◽  
Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

Detection of Flaviviral-Like DNA Sequences in Aedes aegypti (Diptera: Culicidae) Collected From Argentina

2021 ◽  
Author(s):  
Melisa B Bonica ◽  
Dario E Balcazar ◽  
Ailen Chuchuy ◽  
Jorge A Barneche ◽  
Carolina Torres ◽  
...  
Keyword(s):  
Public Health ◽  
Aedes Aegypti ◽  
Dna Sequences ◽  
Immature Stages ◽  
Public Health Burden ◽  
The Past ◽  
Health Burden ◽  
The World ◽  
Aegypti Genome ◽  
Dengue Epidemics

Abstract Diseases caused by flaviviruses are a major public health burden across the world. In the past decades, South America has suffered dengue epidemics, the re-emergence of yellow fever and St. Louis encephalitis viruses, and the introduction of West Nile and Zika viruses. Many insect-specific flaviviruses (ISFs) that cannot replicate in vertebrate cells have recently been described. In this study, we analyzed field-collected mosquito samples from six different ecoregions of Argentina to detect flaviviruses. We did not find any RNA belonging to pathogenic flaviviruses or ISFs in adults or immature stages. However, flaviviral-like DNA similar to flavivirus NS5 region was detected in 83–100% of Aedes aegypti (L.). Despite being previously described as an ancient element in the Ae. aegypti genome, the flaviviral-like DNA sequence was not detected in all Ae. aegypti samples and sequences obtained did not form a monophyletic group, possibly reflecting the genetic diversity of mosquito populations in Argentina.

scholarly journals Permethrin Resistance in Aedes aegypti Affects Aspects of Vectorial Capacity

Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 71
Author(s):  
Tse-Yu Chen ◽  
Chelsea T. Smartt ◽  
Dongyoung Shin
Keyword(s):  
Aedes Aegypti ◽  
Sodium Channel ◽  
Mosquito Control ◽  
Vector Competence ◽  
Vectorial Capacity ◽  
Parental Population ◽  
Control Programs ◽  
Channel Gene ◽  
Sodium Channel Gene ◽  
Specific Pcr

Aedes aegypti, as one of the vectors transmitting several arboviruses, is the main target in mosquito control programs. Permethrin is used to control mosquitoes and Aedes aegypti get exposed due to its overuse and are now resistant. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world and the potential influence on vectorial capacity needs to be studied. Here we selected a permethrin resistant (p-s) Aedes aegypti population from a wild Florida population and confirmed the resistance ratio to its parental population. We used allele-specific PCR genotyping of the V1016I and F1534C sites in the sodium channel gene to map mutations responsible for the resistance. Two important factors, survival rate and vector competence, that impact vectorial capacity were checked. Results indicated the p-s population had 20 times more resistance to permethrin based on LD50 compared to the parental population. In the genotyping study, the p-s population had more homozygous mutations in both mutant sites of the sodium channel gene. The p-s adults survived longer and had a higher dissemination rate for dengue virus than the parental population. These results suggest that highly permethrin resistant Aedes aegypti populations might affect the vectorial capacity, moreover, resistance increased the survival time and vector competence, which should be of concern in areas where permethrin is applied.

scholarly journals RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 41
Author(s):  
Ya-Wen Chang ◽  
Yu-Cheng Wang ◽  
Xiao-Xiang Zhang ◽  
Junaid Iqbal ◽  
Yu-Zhou Du
Keyword(s):  
Rna Interference ◽  
Expression Analysis ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Control Strategies ◽  
Vacuolar Atpase ◽  
Invasive Pest ◽  
Liriomyza Trifolii ◽  
Horticultural Crops ◽  
Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

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