We thank Margo Herre and the Leslie Vosshall lab for sharing their in-situ hybridization protocol for Aedes aegypti olfactory appendages. This work was supported by grants from the National Institutes of Health to C.J.P. (NIAID R01Al137078), a HHMI Hanna Gray fellowship to J.I.R, a Johns Hopkins Postdoctoral Accelerator Award to J.I.R, and a Johns Hopkins Malaria Research Institute Postdoctoral Fellowship to J.I.R. We thank the Johns Hopkins Malaria Research Institute and Bloomberg Philanthropies for their support.

1. Considerations and preparation of materials
<ol>
	<li>Decide if whole-mount or cryo-section of tissue will be appropriate. This protocol is optimized for whole-mount in situ imaging of RNA in the Anopheles mosquito antenna and maxillary palp without cryo-sectioning. If samples are thicker than 5 mm, cryo-sectioning is recommended to enable probe penetration.</li>
	<li>Identify the genes of interest and copy the sequences including introns and exons from a suitable database. Transcr...

Chemosensory Gene Expression Mapping in Mosquito Olfactory Appendages

<ol>
	<li>Konopka, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+in+Anopheles+mosquitoes.">Olfaction in Anopheles mosquitoes.</a> Chem Senses. 46, (2021).</li><li>Raji, J. I., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+ionotropic+receptors+in+human+host-seeking+mosquitoes.">Chemosensory ionotropic receptors in human host-seeking mosquitoes.</a> Curr Opin Insect Sci. 54, 100967 (2022).</li><li>Young, A. P., Jackson, D. J., Wyeth, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technical+review+and+guide+to+RNA+fluorescence+in+situ+hybridization.">A technical review and guide to RNA fluorescence in situ hybridization.</a> PeerJ. 8, e8806 (2020).</li><li>Herre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-canonical+odor+coding+in+the+mosquito.">Non-canonical odor coding in the mosquito.</a> Cell. 185 (17), 3104-3123.e28 (2022).</li><li>Raji, J. I., Konopka, J. K., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+antennal-expressed+ionotropic+receptors+in+the+malaria+mosquito.">A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito.</a> Cell Rep. 42 (2), 112101 (2023).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+in+situ+amplification+for+multiplexed+imaging+of+mRNA+expression.">Programmable in situ amplification for multiplexed imaging of mRNA expression.</a> Nat Biotechnol. 28 (11), 1208-1212 (2010).</li><li>Choi, H. M. T., Beck, V. A., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+in+situ+hybridization+chain+reaction:+higher+gain,+lower+cost,+greater+durability.">Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.</a> ACS Nano. 8 (5), 4284-4294 (2014).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145 (12), dev165753 (2018).</li><li>Schwarzkopf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridization+chain+reaction+enables+a+unified+approach+to+multiplexed,+quantitative,+high-resolution+immunohistochemistry+and+in+situ+hybridization.">Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.</a> Development. 148 (22), dev199847 (2021).</li><li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+a+multiplexed+zoo+of+mRNA+expression.">Mapping a multiplexed zoo of mRNA expression.</a> Development. 143 (19), 3632-3637 (2016).</li><li>Pitts, R. J., Derryberry, S. L., Zhang, Z., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+receptors+in+the+malaria+vector+mosquito+Anopheles+gambiae+tuned+to+amines+and+carboxylic+acids.">Variant ionotropic receptors in the malaria vector mosquito Anopheles gambiae tuned to amines and carboxylic acids.</a> Sci Rep. 7, 40297 (2017).</li><li>Rinker, D. C., Zhou, X., Pitts, R. J., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+transcriptome+profiles+of+anopheline+mosquitoes+reveal+human+host+olfactory+specialization+in+Anopheles+gambiae.">Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae.</a> BMC Genomics. 14, 749 (2013).</li><li>Maguire, S. E., Afify, A., Goff, L. A., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-receptor-mediated+regulation+of+chemosensory+gene+expression+in+the+malaria+mosquito+Anopheles+gambiae.">Odorant-receptor-mediated regulation of chemosensory gene expression in the malaria mosquito Anopheles gambiae.</a> Cell Rep. 38 (10), 110494 (2022).</li><li>Athrey, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+gene+expression+in+olfactory+organs+of+the+anthropophilic+Anopheles+coluzzii+and+zoophilic+Anopheles+quadriannulatus.">Chemosensory gene expression in olfactory organs of the anthropophilic Anopheles coluzzii and zoophilic Anopheles quadriannulatus.</a> BMC Genomics. 18 (1), 751 (2017).</li><li>Task, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoreceptor+co-expression+in+Drosophila+melanogaster+olfactory+neurons.">Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons.</a> eLife. 11, e72599 (2022).</li><li>Shah, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+RNA+detection+at+depth+by+hybridization+chain+reaction+and+tissue+hydrogel+embedding+and+clearing.">Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.</a> Development. 143 (15), 2862-2867 (2016).</li><li>Trivedi, V., Choi, H. M. T., Fraser, S. E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+quantitative+analysis+of+mRNA+expression+within+intact+vertebrate+embryos.">Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.</a> Development. 145 (1), dev156869 (2018).</li><li>Herre, M., Greppi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+in+situ+hybridization+and+immunohistochemistry+to+visualize+gene+expression+in+peripheral+chemosensory+tissues+of+mosquitoes.">RNA in situ hybridization and immunohistochemistry to visualize gene expression in peripheral chemosensory tissues of mosquitoes.</a> Cold Spring Harb Protoc. 2023 (1), 48-54 (2023).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+the+Year:+spatially+resolved+transcriptomics.">Method of the Year: spatially resolved transcriptomics.</a> Nat Methods. 18 (1), 9-14 (2021).</li></ol>

The third generation of hybridization chain reaction (HCR) is remarkable for its sensitivity and robustness to visualize several RNA targets8. HCR WM-FISH has been successfully used on the embryos of Drosophila, chicken, mice, and zebrafish as well as the larvae of nematodes and zebrafish10,16,17. Mosquito antennae and maxillary palps are typically prone to high autofluorescence and weak probe pe...

Mosquito vectors such as Anopheles gambiae rely on a rich repertoire of chemosensory genes expressed in their peripheral olfactory appendages to thrive in a complex chemical world and identify behaviorally relevant odors emanating from human hosts, detect nectar sources, and locate oviposition sites1. The mosquito antenna and the maxillary palp are enriched with chemosensory genes that drive odor detection in these olfactory appendages. Three main classes of ligand-gated ion channels drive odor detection in mosquitoes&#39; olfactory appendages: the Odorant receptors (ORs), which function with an obligate Odorant receptor co-receptor (Orco); the Ionotropic receptors (IRs), which interact with one or more IR coreceptors (IR8a, IR25a, and IR76b); the chemosensory Gustatory receptors (GRs), which function as a complex of three proteins to detect carbon dioxide (CO2)1,2.
RNA fluorescence in situ hybridization is a powerful tool for detecting the expression of endogenous mRNA3. In general, this method utilizes a fluorophore-tagged single stranded nucleic acid probe with sequence complementary to a target mRNA. Binding of the fluorescent RNA probe to the target RNA allows identification of cells expressing a transcript of interest. Recent advancements now enable the detection of transcripts in whole-mount mosquito tissues4,5. The first generation of hybridization chain reaction (HCR) technology used an RNA-based HCR amplifier; this was improved upon in a second-generation method that instead used engineered DNA for the HCR amplifier6,7. This upgrade resulted in a 10x increase in signal, a dramatic decrease in production cost, and significant improvement in the durability of reagents6,7.
In the protocol, we describe the utilization of a third generation HCR whole-mount RNA fluorescence in situ hybridization (HCR RNA WM-FISH) method designed for detecting the spatial localization and expression of any gene8,9. This two-step method first utilizes nucleic acid probes specific for the mRNA of interest, but which also contain an initiator recognition sequence; the second step utilizes fluorophore-tagged hairpins which bind to the initiator sequence to amplify the fluorescent signal (Figure 1). This method also allows for the multiplexing of two or more RNA probes and amplifying probe signals to facilitate RNA detection and quantification8. Visualizing the transcript abundance and RNA localization patterns of chemosensory genes expressed in the olfactory appendages offers the first line of insight into chemosensory gene functions and odor coding.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplification buffer</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2) 1M&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-50UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Chymotrypsin</td>
			<td>Sigma-Aldrich</td>
			<td>CHY5S-10VL&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>VWR</td>
			<td>20901-551</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251</td>
			<td>Number 5</td>
		</tr>
		<tr>
			<td>Gel loading tip</td>
			<td>Costar</td>
			<td>4853</td>
			<td>1-200 &#181;L tip</td>
		</tr>
		<tr>
			<td>Hairpins&#160;</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>h1 and h2 initiator splits</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>IR25a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK149&#160;</td>
			<td>AGAP010272</td>
		</tr>
		<tr>
			<td>IR41t.1 probe</td>
			<td>Molecular Instruments</td>
			<td>&#160;Probe Set ID: PRK978</td>
			<td>AGAP004432</td>
		</tr>
		<tr>
			<td>IR64a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK700&#160;</td>
			<td>AGAP004923</td>
		</tr>
		<tr>
			<td>IR75d probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK976</td>
			<td>AGAP004969</td>
		</tr>
		<tr>
			<td>IR76b probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRI998</td>
			<td>AGAP011968</td>
		</tr>
		<tr>
			<td>IR7t probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRL355</td>
			<td>AGAP002763</td>
		</tr>
		<tr>
			<td>IR8a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK150</td>
			<td>AGAP010411</td>
		</tr>
		<tr>
			<td>LoBind Tubes</td>
			<td>VWR</td>
			<td>80077-236</td>
			<td>0.5 mL DNA/RNA LoBind Tubes</td>
		</tr>
		<tr>
			<td>Magnessium Chloride (MgCl2) 1M</td>
			<td>Thermo Fisher</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher&#160;</td>
			<td>A412-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Thermo Fisher</td>
			<td>43-879-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Denville Scientific</td>
			<td>Model 135</td>
			<td>3-D Mini rocker</td>
		</tr>
		<tr>
			<td>Orco probe</td>
			<td>Molecular Instruments</td>
			<td>Probe set ID PRD954</td>
			<td>AGAP002560</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (20% )</td>
			<td>Electron Microscopy Services&#160;</td>
			<td>15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X PBS)</td>
			<td>Thermo Fisher</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe hybridization buffer</td>
			<td>Molecular Instruments</td>
			<td>https://www.molecularinstruments.com/</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Probe wash buffer</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Proteinase-K</td>
			<td>Thermo Fisher</td>
			<td>AM2548</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline-Sodium Citrate (SSC) 20x&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-557-044</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Diamond</td>
			<td>Thermo Fisher&#160;</td>
			<td>S36972</td>
			<td>mounting solution</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl) 5M</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160; (10%)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (10% )</td>
			<td>Teknova</td>
			<td>T0027</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass</td>
			<td>Carolina</td>
			<td>742300</td>
			<td>&#160;1 5/8&#34; square; transparent</td>
		</tr>
	</tbody>
</table>

hybridization chain reaction rna whole-mount fluorescence in situ hybridization of chemosensory genes in mosquito olfactory appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes 

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Our group is interested in the chemo-sensory system of mosquitoes. We hope that by targeting a mosquito sense of taste and smell, we can stop it from finding us and biting us. Hybridization chain reaction fluorescent in situ hybridization is a recent development.This technology is highly sensitive, robust, and capable of multiplexing several gene targets such that we can visualize the expression and localization of these genes simultaneously within their native environment. Special transcriptomics is a cutting edge method used in the field of spatial biology. This technology is capable of multiplexing several thousands of genes, and you can visualize the expression of these genes simultaneously.Our protocol allows us to directly visualize which neurons in the antenna express any of the 50 or so possible ionotropic receptors. This provides us with a foundational roadmap to guide our understanding of how this classical factor receptors might mediate behaviors. Olfactory neurons within olfactory organs of an insect like the antenna, are typically buried inside a protective chitinous shell.This makes visualizing olfactory receptor expression in those neurons very challenging. Our protocol uses a combination of chitinase treatments and a state-of-the-art fluorescence and see-through protocols to overcome these challenges.

Robust detection of chemosensory genes in Anopheles antenna 
We investigated the sensitivity of the HCR FISH method (Figure 1) to detect the expression of chemosensory receptors in mosquito olfactory tissues. Guided by the RNA transcript data reported earlier on the female Anopheles mosquito antenna, we generated probes to target a variety of IRs. The average transcript values from four independent antennal transcriptome studies revealed that Ir41t.1 (...

Watch this Scientific Journal Video about Visualizing Olfactory Receptor Expression in Mosquitoes at JoVE.com

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

hybridization-chain-reaction-rna-whole-mount-fluorescence-situ