We thank Gilbert Legoff for teaching Jordan Tutagata how to dissect salivary glands from mosquitoes and Giuliano Mucci for helping with photos of the mosquito organs. We thank Anna-Bella Failloux and Nonito Pages for helpful discussion on the protocol.&#160;This work was supported by the ERC RosaLind Starting Grant &#8220;948135&#8221; to JR.&#160;We thank the Vectopole platform (IRD, Montpellier) for providing technical support and for the rearing and maintenance of the mosquito populations.

NOTE: Figure 1 shows a schematic of the method summarizing the different steps of the protocol.
1. Material preparation
<ol>
	<li>Before starting the experiment, prepare a bleach solution with 0.5% active chlorine (recommended by the World Health Organization) to disinfect the equipment and the workbench.
	<ol>
		<li>In 900 mL of water, dissolve three soluble tablets, each containing 1.5 g of active chlorine. Wait 15 min for the tablets to fully dissolve, resulting in a 0.5% bleach solution.</li>
		<li>Place the 0.5% bleach solution in a spray bottle. Then, spray the solution onto all dissection equipment, including microscopic slides and workbench. 
		NOTE: The equipment and workbench should be left to dry for ~10 min before use.</li>
	</ol>
	</li>
	<li>To rinse the mosquitoes before each dissection to prevent contamination of the samples with external bacteria, prepare four 1.5 mL newly sterilized tubes: two containing 96% ethanol and two others containing 1x PBS buffer (phosphate-buffered saline).</li>
	<li>Prepare the tubes to store the samples after the dissection of each mosquito: four 1.5 mL sterile tubes, each containing 100 &#181;L of preservation buffer. 
	NOTE: Prepare one tube for the salivary glands, one for the ovaries, one for the midgut, and one for the mosquito carcass.</li>
</ol>
2. Mosquito collection
<ol>
	<li>Collect and keep adult female individuals alive (from field or laboratory, 4-7 days) until surface sterilization.</li>
</ol>
3. Surface sterilization of mosquitoes
NOTE: Clean forceps and needles and use new, sterilized tubes between each organ and individual. If bleach is unavailable, ensure to thoroughly flame-sterilize the dissecting forceps and needles between the dissection of each individual and organ to prevent cross-contamination of biological material. Dissection instruments should be dry, without any bleach/ethanol residue, before the next use.
<ol>
	<li>To avoid females emptying their gut content when exposed to alcohol, cool-anesthetize them on ice or in a freezer at -20 &#176;C (for 4 min so that the tissues and organs do not freeze).</li>
	<li>Transfer individual mosquitoes into a new sterilized 1.5 mL tube with 1 mL of 96% ethanol. Vortex gently (manually) for 30 s (shake the mosquito well in the tube to detach the scales and potential bacterial contaminants on the surface of the insects). Rinse twice using tubes containing ethanol.</li>
	<li>Transfer the mosquito into a new sterilized 1.5 mL tube with 1 mL of sterile 1x PBS solution and vortex gently (manually) for 30 s (to avoid DNA precipitation with ethanol). Rinse twice using tubes containing sterile 1x PBS.</li>
</ol>
4. Dissection of mosquitoes
<ol>
	<li>Place several small drops of sterile 1x PBS onto the slide to facilitate the placement of the mosquito and rinsing of dissected organs. 
	NOTE: The drops of sterile PBS should be large enough to accommodate the mosquito organs.</li>
	<li>Take the cleaned mosquito and place it onto a drop of sterile 1x PBS on the microscope slide.</li>
	<li>Dissect the salivary glands with sterilized forceps and needle as described below.
	<ol>
		<li>Hold the mosquito&#39;s thorax with tweezers to prevent it from moving. Then, place a needle at the neck, below the head, and gently pull on the mosquito&#39;s head to separate it from the thorax. 
		NOTE: The salivary glands are located in the upper thorax and are connected to the head by the salivary duct. During dissection, it is crucial not to break this duct as the salivary glands will not come with the head when you pull it from the thorax. This is why it is important to have a fresh mosquito, not a frozen one.</li>
		<li>When the head separates from the thorax, observe the two salivary glands emerge, each composed of three lobes. Cut the salivary glands at the level of the salivary duct, retrieve them with a needle, and place them into a new drop of sterile 1x PBS to rinse it and prevent inter-organ contaminations.</li>
		<li>Retrieve the salivary glands using a needle and place them into a new sterilized tube containing 100 &#181;L of preservation buffer.</li>
	</ol>
	</li>
	<li>Dissect the ovaries with sterilized forceps and needle as follows.
	<ol>
		<li>Place the mosquito on its back. Using forceps to hold the mosquito by the thorax to prevent movement during dissection, puncture the third abdominal segment (starting from the bottom) with a needle. 
		NOTE: Optionally, remove the legs and wings of the mosquito to facilitate dissection.</li>
		<li>While piercing the third abdominal segment, pull down the abdomen to open it and reveal the internal organs. Remove any remaining pieces of mosquito exoskeleton to facilitate the release of the organs. 
		NOTE: Organs in the abdomen such as the gut, Malpighian tubules, and ovaries are visible.</li>
		<li>Retrieve both ovaries with sterilized forceps and place them in a new drop of sterile 1x PBS on the microscope slide to rinse them. Grab the ovaries and place them in the 1.5 mL tube with the 100 &#181;L of preservation buffer.</li>
	</ol>
	</li>
	<li>Now that the abdomen is open and the organs are exposed, retrieve the midgut with new sterilized forceps and/or needle.
	<ol>
		<li>Separate the midgut from the foregut and hindgut; then, remove the Malpighian tubules from the midgut.Grab the midgut using a sterile needle and place it on a new sterile 1x PBS drop to rinse it and prevent inter-organ contaminations.</li>
		<li>Transfer to a new sterile 1x PBS drop. Open the midgut with forceps to free and enrich for bacteria as described in Supplementary File 1. Grab the midgut with forceps and pipette all the PBS containing bacteria to store the entire material (liquid and tissue) in a new sterilized 1.5 mL tube with 100 &#181;L of preservation buffer.</li>
		<li>Add again 5 &#181;L of 1x PBS on the previously used place in the microscope slide and directly pipette back PBS to the same 1.5 mL tube to retrieve bacteria that may have stuck to the microscope slide.</li>
	</ol>
	</li>
	<li>Use sterile forceps to grasp the carcass (remaining body) and store it in a new sterilized 1.5 mL tube with 100 &#181;L of preservation buffer.</li>
	<li>Storage of material until further processing
	<ol>
		<li>Label tubes with &#39;Species, Organ, Date, Researcher&#39;s name&#39;.</li>
		<li>Store dissected ovaries, midgut, salivary glands, and carcass at -20 &#176;C until processing.</li>
	</ol>
	</li>
	<li>Keep track in a spreadsheet of all associated metadata for the samples, including Species (or genus level only), Collection date, Country, Province, Latitude, Longitude, Name of the person who did the dissection, Gravid female or not, Blood meal or not.</li>
</ol>

High-Precision Mosquito Organ Isolation for Microbiome Analyses

<ol>
	<li>Achee, N. L., 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=Alternative+strategies+for+mosquito-borne+arbovirus+control.">Alternative strategies for mosquito-borne arbovirus control.</a> PLOS Negl Trop Dis. 13 (1), e0006822 (2019).</li><li>Hoffmann, A. A., 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=Successful+establishment+of+Wolbachia+in+Aedes+populations+to+suppress+dengue+transmission.">Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.</a> Nature. 476 (7361), 454-457 (2011).</li><li>Porter, J., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+lives+of+Wolbachia.">The cellular lives of Wolbachia.</a> Nat Rev Microbiol. 21 (11), 750-766 (2023).</li><li>Coon, K. L., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquitoes+host+communities+of+bacteria+that+are+essential+for+development+but+vary+greatly+between+local+habitats.">Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats.</a> Mol Ecol. 25 (22), 5806-5826 (2016).</li><li>Didion, E. 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=Microbiome+reduction+prevents+lipid+accumulation+during+early+diapause+in+the+northern+house+mosquito,+Culex+pipiens+pipiens.">Microbiome reduction prevents lipid accumulation during early diapause in the northern house mosquito, Culex pipiens pipiens.</a> J Insect Physiol. 134, 104295 (2021).</li><li>Gabrieli, P., 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=Mosquito+trilogy:+Microbiota,+immunity+and+pathogens,+and+their+implications+for+the+control+of+disease+transmission.">Mosquito trilogy: Microbiota, immunity and pathogens, and their implications for the control of disease transmission.</a> Front Microbiol. 12, 630438 (2021).</li><li>Garrig&#243;s, M., Garrido, M., Panisse, G., Veiga, J., Mart&#237;nez-de La Puente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+West+Nile+Virus+and+the+microbiota+of+Culex+pipiens+vectors:+A+literature+review.">Interactions between West Nile Virus and the microbiota of Culex pipiens vectors: A literature review.</a> Pathogens. 12 (11), 1287 (2023).</li><li>Zheng, R., 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=Holobiont+perspectives+on+tripartite+interactions+among+microbiota,+mosquitoes,+and+pathogens.">Holobiont perspectives on tripartite interactions among microbiota, mosquitoes, and pathogens.</a> ISME J. 17 (8), 1143-1152 (2023).</li><li>Flores, G. A. 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=Wolbachia+dominance+influences+the+Culex+quinquefasciatus+microbiota.">Wolbachia dominance influences the Culex quinquefasciatus microbiota.</a> Sci Rep. 13 (1), 18980 (2023).</li><li>Hegde, 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=Interkingdom+interactions+shape+the+fungal+microbiome+of+mosquitoes.">Interkingdom interactions shape the fungal microbiome of mosquitoes.</a> Anim Microbiome. 6 (1), 11 (2024).</li><li>Suo, P., 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=Seasonal+variation+of+midgut+bacterial+diversity+in+Culex+quinquefasciatus+populations+in+Haikou+city,+Hainan+province,+China.">Seasonal variation of midgut bacterial diversity in Culex quinquefasciatus populations in Haikou city, Hainan province, China.</a> Biology. 11 (8), 1166 (2022).</li><li>Gruber-Vodicka, H. R., Seah, B. K. B., Pruesse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=phyloFlash:+Rapid+small-subunit+rRNA+profiling+and+targeted+assembly+from+metagenomes.">phyloFlash: Rapid small-subunit rRNA profiling and targeted assembly from metagenomes.</a> mSystems. 5 (5), e00920-e00920 (2020).</li><li>Gra&#241;a-Miraglia, L., 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=Spirochetes+isolated+from+arthropods+constitute+a+novel+genus+Entomospira+genus+novum+within+the+order+Spirochaetales.">Spirochetes isolated from arthropods constitute a novel genus Entomospira genus novum within the order Spirochaetales.</a> Sci Rep. 10 (1), 17053 (2020).</li><li>Reveillaud, J., 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=The+Wolbachia+mobilome+in+Culex+pipiens+includes+a+putative+plasmid.">The Wolbachia mobilome in Culex pipiens includes a putative plasmid.</a> Nat Commun. 10 (1), 1051 (2019).</li><li>Ghousein, A., 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=pWCP+is+a+widely+distributed+and+highly+conserved+Wolbachia+plasmid+in+Culex+pipiens+and+Culex+quinquefasciatus+mosquitoes+worldwide.">pWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex pipiens and Culex quinquefasciatus mosquitoes worldwide.</a> ISME Commun. 3 (1), 40 (2023).</li><li>Juma, E. O., Kim, C. -. H., Dunlap, C., Allan, B. F., Stone, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culex+pipiens+and+Culex+restuans+egg+rafts+harbor+diverse+bacterial+communities+compared+to+their+midgut+tissues.">Culex pipiens and Culex restuans egg rafts harbor diverse bacterial communities compared to their midgut tissues.</a> Parasit Vectors. 13 (1), 532 (2020).</li><li>Lee, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GToTree:+a+user-friendly+workflow+for+phylogenomics.">GToTree: a user-friendly workflow for phylogenomics.</a> Bioinformatics. 35 (20), 4162-4164 (2019).</li><li>Parks, D. H., 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=A+complete+domain-to-species+taxonomy+for+bacteria+and+Archaea.">A complete domain-to-species taxonomy for bacteria and Archaea.</a> Nat Biotechnology. 38 (9), 1079-1086 (2020).</li></ol>

The authors declare that they have no conflicts of interest.

We recommend paying particular attention to the sequence of organ dissection, starting with the salivary glands. Indeed, we observed that they were more easily extracted from the thorax of Culex specimens if the mosquito&#39;s integrity was preserved. Damage to the abdomen or thorax could reduce pressure in the mosquito&#39;s body, impeding the procedure. It is, however, also possible to cut between the thorax and the abdomen and then pull out the salivary glands from the head and thorax (A. B. Failloux, personal communication). Additionally, acquiring proficient dissecting skills can be challenging, so we suggest practicing on an adequate number of specimens prior to the experiment.
Isolating mosquito organs while avoiding cross-contamination in a systematic manner is crucial for a wide range of downstream mosquito microbiota analyses. A genome-resolved metagenomic study following the dissection of single ovaries from Culex pipiens specimens in Southern France allowed the discovery of the first plasmid of Wolbachia in Culex pipiens (pWCP14). Using a similar approach, we investigated the distribution and frequency of pWCP in Culex pipiens and Culex quinquefasciatus specimens&#160;from both continental and island areas worldwide, across various environmental and laboratory conditions. Overall, the data revealed a remarkably conserved Wolbachia plasmid element in Culex mosquitoes, suggesting a crucial role for this mobile element in endosymbiont biology15, warranting further analysis.
Here, we provide additional examples of mosquito microbiome analyses on midgut and ovary samples obtained using this systematic procedure. We observed a clear difference in microbiota between tissues (Figure 5), with both shared and organ-specific bacterial taxa. As expected, the presence of Wolbachia was detected in both organs, with higher relative abundance (based on unassembled short reads) and mean coverage of MAGs in the mosquito ovaries compared to the midguts, consistent with the observation that this endosymbiont is transmitted through the ovaries and subsequently spreads to the somatic tissues. Although this study was restricted to samples from New Caledonia, this protocol may facilitate the investigation of Wolbachia&#39;s genomic variability on a global scale, as well as its role in various phenotypes, including density regulation of itself and viral protection. Moreover, this work exemplifies how the dissection procedure presented here permits the examination of the taxonomic diversity and potential functional capabilities of mosquito symbionts within midgut samples.
We obtained two enterobacterial draft genomes that were only present in two midgut samples, confirming the lack of contamination between organs for these two specimens. Regarding spirochetes, detected both in the midgut and the ovaries of individual 3, Juma and colleagues, 2020 observed the presence of these bacteria on the surface of egg rafts. The authors suggested that the bacterial communities found in the egg rafts might primarily be maternally inherited from the ovaries, given that the egg rafts were kept in deionized and bacteria-free water. However, they could not rule out the possibility of bacterial colonization occurring right after oviposition and recommended further study on the ovary microbiome16.
While initially designed for specimens of the Culex pipiens species complex, we foresee the applicability of this protocol to a broader community of medical entomologists studying other vectors such as Anopheles or Aedes. By operating at the level of individual organs, this method may enable both intra- and inter-individual genomic comparisons, offering insights into symbiont genomic variability at a fine scale, with the potential of advancing vector control strategies. This method of dissection and isolation of the salivary glands, ovaries, and midgut, preventing microbial cross-contamination, could also be a useful protocol for viral infection dynamics studies within these three organs.

Mosquitoes spread a wide range of pathogens causing diseases and are a serious threat to public health. Due to an increased prevalence of insecticide resistance among mosquito populations and in the absence of effective vaccines against these pathogens, new biocontrol methods that focus on the mosquito microbiome are emerging. In particular, the intracellular bacterium Wolbachia, which can interfere with pathogen transmission and manipulate host reproduction, stands out1,2,3. In addition, other mosquito symbionts are central to the survival, development, or immune system of their host, as well as in infection and transmission of pathogens, and show great promise for their exploitation to fight vector-borne diseases4,5,6,7,8.
Microorganisms associated with mosquitoes span all domains of microbial life (including bacteria, eukaryotes, and fungi) that interact intimately with their host but also with each other in the different body compartments9,10. Therefore, a better understanding of the microbiota, its potential seasonal variation11, and the mechanisms by which its members interact directly in distinct mosquito tissues can help develop new targeted biocontrol methods or improve existing ones. However, the majority of symbionts remain resistant to cultivation, rendering their characterization impossible.
The advent of second- and third-generation sequencing methods, together with state-of-the-art assembly and binning approaches, have enabled the reconstruction of microbial genomes and accessing the diversity and functional potential of non-cultivable microbes. Here, we present a method for the dissection of the ovaries, midgut, and salivary glands of female mosquitoes while preventing cross-contamination. This protocol can be followed by genomic DNA extraction and subsequent metabarcoding or shotgun metagenomic sequencing to explore the diversity and function of mosquito microbiota at the organ level. We provide an example of mosquito dissection and microbiome data for Culex spp. specimens although this protocol can be extended to vectors of other genera&#160;such as Anopheles or Aedes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol 96&#176;</td>
			<td>Fisher scientific</td>
			<td>10332562</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular magnifier&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>RAJA</td>
			<td>145517</td>
			<td>150 tablets of 1.5 g</td>
		</tr>
		<tr>
			<td>DNA prep kit</td>
			<td>Illumina</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Previously known as Nextera DNA Flex</td>
		</tr>
		<tr>
			<td>DNA-RNA Shield (50 mL)</td>
			<td>Zymo research</td>
			<td>ZR1100-50</td>
			<td>Preservation buffer</td>
		</tr>
		<tr>
			<td>DNeasy Blood and Tissue Kit&#160;</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>DNA extraction kit</td>
		</tr>
		<tr>
			<td>Filter tips 20 &#181;L</td>
			<td>Starlab</td>
			<td>S1120-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 200 &#181;L</td>
			<td>Starlab</td>
			<td>S1120-8710</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 1000 &#181;L</td>
			<td>Starlab</td>
			<td>S1122-1730-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST (Fine Science Tools)</td>
			<td>11252-20</td>
			<td>Dumont Forceps #5</td>
		</tr>
		<tr>
			<td>Library quantification kit</td>
			<td>Roche</td>
			<td>Provided by MGX sequencing platform</td>
			<td>KAPA Library Quanitification Kits</td>
		</tr>
		<tr>
			<td>Micropipettes 2-20 &#181;L</td>
			<td>Eppendorf</td>
			<td>6.291704</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 20-200 &#181;L</td>
			<td>Eppendorf</td>
			<td>6.291703</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 100-1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>7.648488</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides&#160;</td>
			<td>Epredia</td>
			<td>J1800BMNZ</td>
			<td>dimension : 75 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Terumo</td>
			<td>AN*2719R1</td>
			<td></td>
		</tr>
		<tr>
			<td>NGS kit&#160;</td>
			<td>Agilent</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Fragment Analyzer Systems HS Genomic DNA 50kb Kit</td>
		</tr>
		<tr>
			<td>NovaSeq 6000</td>
			<td>Illumina</td>
			<td>Provided by MGX sequencing platform</td>
			<td>Sequencer</td>
		</tr>
		<tr>
			<td>PBS Phosphate Buffered Saline (Sterile)</td>
			<td>Fisher scientific</td>
			<td>10212990</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent black marker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Eppendorf&#160;</td>
			<td>Dutscher</td>
			<td>33871</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Support for needles</td>
			<td>FST (Fine Science Tools)</td>
			<td>26016-12</td>
			<td>Moria MC1 Pin Holder 12 cm</td>
		</tr>
	</tbody>
</table>

dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a significant public health threat. With the rise of insecticide resistance and the absence of effective vaccines, new strategies are emerging that focus on the mosquito's microbiota. Nevertheless, the majority of symbionts remain resistant to cultivation. Characterizing the diversity and function of bacterial genomes in mosquito specimens, therefore, relies on metagenomics and subsequent assembly and binning strategies. The obtention and analysis of Metagenome-Assembled Genomes (MAGs) from separated organs can notably provide key information about the specific role of mosquito-associated microbes in the ovaries (the reproductive organs), the midgut (key for food digestion and immunity), or the salivary glands (essential for the transmission of vector-borne diseases as pathogens must colonize them to enter the saliva and reach the bloodstream during a blood meal). These newly reconstructed genomes can then pave the way for the development of novel vector biocontrol strategies. To this aim, it is required to isolate mosquito organs while avoiding cross-contamination between them or with microorganisms present in other mosquito organs. Here, we describe an optimized and contamination-free dissection protocol for studying mosquito microbiome at the organ level.

x`Mosquito dissections 
Following the protocol, we collected and isolated the ovaries, midgut, salivary glands, and the carcass from two Culex pipiens molestus specimens (including a gravid female) from a laboratory colony. We confirmed clean dissections following the observation of entire (that is, unbroken) and well-isolated organs with no remaining debris under the binocular. The whole body, head, and thorax, dissected salivary glands, ovaries, and midgut of a Culex pipiens molestus specimen are shown in Figure 2. As expected, the midgut and salivary glands were an order of magnitude smaller than the mosquito ovaries. The eggs of a gravid Culex specimen, together with its midgut and Malpighian tubules, are presented in Figure 3. Of note, dissections are more prone to fail when the material is not fresh and tissue is likely to break (Figure 4B,C). We therefore suggest dissecting material right after collection (within less than 12 h) when possible as tissues are still elastic (Figure 4A). Similarly, dissection of frozen material can be performed but there is a much higher risk of failure and cross-contamination between organs due to fragile tissues (Figure 4).
Microbiome data 
In addition, we collected and separated the midguts and ovaries from three individual Culex quinquefasciatus specimens from Noumea, New Caledonia, following the same procedure. We extracted DNA from each organ, prepared samples for whole genome sequencing, and performed microbiome data analyses as detailed in Supplemental File 1. A bacterial taxonomic diversity analysis on unassembled quality-filtered short reads using PhyloFlash12 showed distinct dominant taxa in midguts compared to ovaries (Figure 5A). Notably, ovarian bacterial communities were dominated by Wolbachia, with the additional presence of a Spirochaetaceae phylotype in the ovaries of individual 3, while midgut communities exhibited a wider diversity, including Gammaproteobacteria, Spirochaetaceae, and Firmicutes. From the same sequencing data, we reconstructed four Metagenome-Assembled Genomes (MAGs) with completion &#62; 80% and redundancy &#60; 5%, belonging to classes Spirochaetia, Alpha-, and Gammaproteobacteria (Table 1).
As expected, MAGs reconstructed herein did not cover the full taxonomic diversity predicted by the PhyloFlash results due to the specific shortcomings of genome reconstruction using metagenomic short-reads. The MAG assigned to Wolbachia (Ref. MAG 3 in Table 1) was detected in all ovaries and two of the midguts and had higher coverage in the ovaries (Figure 5B). We also reconstructed two MAGs belonging to the Enterobacteriaceae family, including genus Pantoea (Ref. MAG 1 and 2 in Table 1), in the midguts obtained from Culex individuals 2 and 3, that were not detected in the corresponding ovaries (Figure 5B). Finally, we also reconstructed one Spirochaetaceae bacterial genome, Ref. MAG 4 (Table 1), assigned to genus BR149 that was successfully isolated from Culex pipiens midguts by Gra&#241;a-Miraglia and colleagues13. Interestingly, this MAG was detected in the midguts of individuals 1 and 3, as well as in the ovaries of individual 3 (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67128/67218fig01v2.jpg" /> 
Figure 1: Schematic of the method summarizing the different steps. Material preparation, mosquito collection, mosquito cleaning, mosquito dissections, and storage. <a href="https://www.jove.com/files/ftp_upload/67128/67218fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67128/67218fig02.jpg" /> 
Figure 2: Culex pipiens molestus female. (A) Whole body. (B) Head and thorax. (C) Dissected salivary glands. (D) Dissected ovaries. (E) Dissected gut with Malpighi tubules. Scale bars = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67128/67218fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67128/67218fig03.jpg" /> 
Figure 3: Dissected abdomen of a gravid Culex pipiens molestus female. The foregut, midgut, hindgut, Malpighian tubules, and eggs of the specimen are shown. Red dashes indicate where to cut to separate the midgut from the foregut and hindgut. Scale bar = 500 &#181;m. Abbreviations: F = foregut; M = midgut; H = hindgut; E = eggs; MT = Malpighian tubules. <a href="https://www.jove.com/files/ftp_upload/67128/67218fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67128/67218fig04.jpg" /> 
Figure 4: Dissection of fresh and frozen Culex mosquito specimens. White rectangles illustrate&#160;(A)&#160;mosquito tissues with intact organs from a freshly dissected specimen and (B,C) broken biological material from specimens that have been frozen before dissection. Scale bars = 720 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67128/67218fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67128/67218fig05.jpg" /> 
Figure 5: Example microbiome analysis on three Culex quinquefasciatus individuals. (A)&#160;Visualization of bacterial diversity estimated through the extraction of SSU rRNA reads with PhyloFlash12 in midguts and ovaries of the three specimens. (B) Mean coverage of the reconstructed MAGs over the samples. Abbreviations: MAG = Metagenome-Assembled Genome; Ind = Individual. <a href="https://www.jove.com/files/ftp_upload/67128/67218fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MAG</td>
			<td>Ref. MAG 4</td>
			<td>Ref. MAG 2</td>
			<td>Ref. MAG 1</td>
			<td>Ref. MAG 3</td>
		</tr>
		<tr>
			<td>Length (bp)</td>
			<td>1,287,790</td>
			<td>4,910,866</td>
			<td>4,751,276</td>
			<td>1,298,266</td>
		</tr>
		<tr>
			<td>Number of contigs</td>
			<td>9</td>
			<td>162</td>
			<td>149</td>
			<td>123</td>
		</tr>
		<tr>
			<td>GC %</td>
			<td>34.05</td>
			<td>55.45</td>
			<td>54.05</td>
			<td>34.15</td>
		</tr>
		<tr>
			<td>Completion (%)</td>
			<td>84.5</td>
			<td>97.18</td>
			<td>98.59</td>
			<td>91.55</td>
		</tr>
		<tr>
			<td>Redundancy (%)</td>
			<td>0</td>
			<td>2.82</td>
			<td>4.22</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Domain</td>
			<td>Bacteria</td>
			<td>Bacteria</td>
			<td>Bacteria</td>
			<td>Bacteria</td>
		</tr>
		<tr>
			<td>Phylum</td>
			<td>Spirochaetota</td>
			<td>Proteobacteria</td>
			<td>Proteobacteria</td>
			<td>Proteobacteria</td>
		</tr>
		<tr>
			<td>Class</td>
			<td>Spirochaetia</td>
			<td>Gammaproteobacteria</td>
			<td>Gammaproteobacteria</td>
			<td>Alphaproteobacteria</td>
		</tr>
		<tr>
			<td>Order</td>
			<td>WRBN01</td>
			<td>Enterobacterales</td>
			<td>Enterobacterales</td>
			<td>Rickettsiales</td>
		</tr>
		<tr>
			<td>Family</td>
			<td>WRBN01</td>
			<td>Enterobacteriaceae</td>
			<td>Enterobacteriaceae</td>
			<td>Anaplasmataceae</td>
		</tr>
		<tr>
			<td>Genus</td>
			<td>BR149</td>
			<td>Pantoea</td>
			<td>-</td>
			<td>Wolbachia</td>
		</tr>
	</tbody>
</table>
Table 1: Reconstructed MAGs from the three Culex quinquefasciatus individuals. Genome size, number of contigs, proportion of GC, estimates of completion, and redundancy based on the single-copy core gene collection available in Anvi&#39;o17 and taxonomy obtained using GTDB18.
Supplemental File 1: Detailed example procedure for microbiome data analysis starting from sample collection, DNA extraction, and whole genome sequencing followed by the bioinformatic workflow for genome reconstruction and estimation of prokaryotic and eukaryotic read proportion <a href="https://www.jove.com/files/ftp_upload/67128/67128_SupplementaryMaterial_15.07.2024_JR_fin.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Optimizing Mosquito Organ Dissection for Studying Symbionts and Vector Microbiomes

Microbial communities in mosquitoes hold great promise for vector biocontrol strategies. Most symbionts are uncultivable, requiring metagenomic analyses. We describe a method to dissect female mosquitoes and separate ovaries, midgut, and salivary glands preventing cross-contamination, facilitating microbiome studies at the organ level, and enhancing understanding of microorganisms' roles in mosquito biology.

Dissection of Mosquito Ovaries, Midgut, and Salivary Glands for Microbiome Analyses at the Organ Level

The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a ...

dissection-mosquito-ovaries-midgut-salivary-glands-for-microbiome

Research

JoVE Journal

Biology

1.4K Views.  University of Montpellier, INRAe, CNRS, IRD. Microbial communities in mosquitoes hold great promise for vector biocontrol strategies. Most symbionts are uncultivable, requiring metagenomic analyses. We describe a method to dissect female mosquitoes and separate ovaries, midgut, and salivary glands preventing cross-contamination, facilitating microbiome studies at the organ level, and enhancing understanding of microorganisms' roles in mosquito biology.

Video: Author Spotlight: Optimizing Mosquito Organ Dissection for Studying Symbionts and Vector Microbiomes

Dissection of Drosophila Ovaries

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...