Whole-Mount In Situ Hybridization for Analysis of Gene Expression during <i>Aedes aegypti</i> Development: Figure 1.

Haugen, Morgan; Tomchaney, Michael; Kast, Kristopher; Flannery, Ellen; Clemons, Anthony; Jacowski, Caitlin; Holland, Wendy Simanton; Le, Christy; Severson, David W.; Duman-Scheel, Molly

doi:10.1101/pdb.prot5509

Cited by 34 publications

(37 citation statements)

References 7 publications

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“…Furthermore, while this technique clearly generates knockdown beyond the gut, we are only beginning to assess the tissues for which this technique is effective, and this may vary among species. It will therefore be useful to measure knockdown in particular tissues, which can be achieved by dissecting tissues of interest from whole animals for qRT-PCR assays or through in situ hybridization (Figures 4–6) 20,21 and immunohistochemistry (Figure 4) 20 assays (see Haugen et al 27 and Clemons et al 28 , respectively, for troubleshooting these protocols). Since knockdown varies from individual to individual, it is helpful to perform double-labeling assays to assess phenotypes in combination with knockdown levels (Figure 4) 20 , which greatly facilitates phenotype characterization.…”

Section: Discussionmentioning

confidence: 99%

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Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Zhang

Mysore

Flannery

et al. 2015

JoVE

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SHORT ABSTRACT Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae. LONG ABSTRACT Vector mosquitoes inflict more human suffering than any other organism—and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

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Section: Discussionmentioning

confidence: 99%

“…27 protocol for confirmation of knockdown as described previously 11,20 and discussed in the representative results section below.…”

Section: Protocolmentioning

confidence: 99%

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Zhang

Mysore

Flannery

et al. 2015

JoVE

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“…Whole-mount in situ hybridization experiments were performed as previously described [35]. Stained tissue preparations were imaged on a Zeiss Axioimager equipped with a Spot Flex camera.…”

Section: Methodsmentioning

confidence: 99%

Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti

Mysore

Andrews

et al. 2014

BMC Dev Biol

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BackgroundEssentially nothing is known about the genetic regulation of olfactory system development in vector mosquitoes, which use olfactory cues to detect blood meal hosts. Studies in Drosophila melanogaster have identified a regulatory matrix of transcription factors that controls pupal/adult odorant receptor (OR) gene expression in olfactory receptor neurons (ORNs). However, it is unclear if transcription factors that function in the D. melanogaster regulatory matrix are required for OR expression in mosquitoes. Furthermore, the regulation of OR expression during development of the larval olfactory system, which is far less complex than that of pupae/adults, is not well understood in any insect, including D. melanogaster. Here, we examine the regulation of OR expression in the developing larval olfactory system of Aedes aegypti, the dengue vector mosquito.ResultsA. aegypti bears orthologs of eight transcription factors that regulate OR expression in D. melanogaster pupae/adults. These transcription factors are expressed in A. aegypti larval antennal sensory neurons, and consensus binding sites for these transcription factors reside in the 5’ flanking regions of A. aegypti OR genes. Consensus binding sites for Single-minded (Sim) are located adjacent to over half the A. aegypti OR genes, suggesting that this transcription factor functions as a major regulator of mosquito OR expression. To functionally test this hypothesis, chitosan/siRNA nanoparticles were used to target sim during larval olfactory development. These experiments demonstrated that Sim positively regulates expression of a large subset of OR genes, including orco, the obligate co-receptor in the assembly and function of heteromeric OR/Orco complexes. Decreased innervation of the antennal lobe was also noted in sim knockdown larvae. These OR expression and antennal lobe defects correlated with a larval odorant tracking behavioral defect. OR expression and antennal lobe defects were also observed in sim knockdown pupae. ConclusionsThe results of this investigation indicate that Sim has multiple functions during larval and pupal olfactory system development in A. aegypti.

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“…Magnusson et al (2011) assessed sex-specific transcriptomes throughout Anopheles gambiae development and characterized the functions of several testis- and ovary-specific genes during gonad development. Functional genetic analysis of nervous system development has been performed in A. aegypti (Clemons et al, 2011; Haugen et al, 2011; Sarro et al, 2013; Mysore et al, 2013, 2014a, 2014b), an emerging model for vector mosquito development studies (Clemons et al, 2010a). A recent functional genetic study explored the development of sexual dimorphism in the A. aegypti pupal nervous system (Tomchaney et al, 2014).…”

Section: Sexual Dimorphism a Critical Aspect Of Pathogen Transmissiomentioning

confidence: 99%

“…These investigations were facilitated by the work of Mysore et al (2011), who used cross-reactive Drosophila antibodies to establish the first set of molecular markers for the developing mosquito brain. Many of the antibodies work well in conjunction with a combined whole mount in situ hybridization/protein localization protocol (Haugen et al, 2010), which employs a detergent-treatment permeabilization step that has facilitated mRNA localization in many arthropod species (Patel et al, 2001; Duman-Scheel et al, 2002). The results obtained validated the microarray data and laid a foundation for future studies.…”

Section: Global and Spatial Analysis Of Sexually Dimorphic Gene Exprementioning

confidence: 99%

Developmental neurogenetics of sexual dimorphism in Aedes aegypti

Duman-Scheel

Syed

2015

Front. Ecol. Evol.

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Sexual dimorphism, a poorly understood but crucial aspect of vector mosquito biology, encompasses sex-specific physical, physiological, and behavioral traits related to mosquito reproduction. The study of mosquito sexual dimorphism has largely focused on analysis of the differences between adult female and male mosquitoes, particularly with respect to sex-specific behaviors related to disease transmission. However, sexually dimorphic behaviors are the products of differential gene expression that initiates during development and therefore must also be studied during development. Recent technical advancements are facilitating functional genetic studies in the dengue vector Aedes aegypti, an emerging model for mosquito development. These methodologies, many of which could be extended to other non-model insect species, are facilitating analysis of the development of sexual dimorphism in neural tissues, particularly the olfactory system. These studies are providing insight into the neurodevelopmental genetic basis for sexual dimorphism in vector mosquitoes.

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Whole-Mount In Situ Hybridization for Analysis of Gene Expression during Aedes aegypti Development: Figure 1.

Cited by 34 publications

References 7 publications

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti

Developmental neurogenetics of sexual dimorphism in Aedes aegypti

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