Name: dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level
Uploaded: 2024-10-04T14:25:00.000+00:00
Duration: 3 min 58 s
Description: The global burden of mosquito-transmitted diseases, including malaria, dengue, West Nile, Zika, Usutu, and yellow fever, continues to increase, posing a ...

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

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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. 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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><tbody><tr><td>Alcohol 96&amp;deg;</td><td>Fisher scientific</td><td>10332562</td><td></td></tr><tr><td>Binocular magnifier&amp;nbsp;</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&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>DNA extraction kit</td></tr><tr><td>Filter tips 20 &amp;micro;L</td><td>Starlab</td><td>S1120-3810</td><td></td></tr><tr><td>Filter tips 200 &amp;micro;L</td><td>Starlab</td><td>S1120-8710</td><td></td></tr><tr><td>Filter tips 1000 &amp;micro;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 &amp;micro;L</td><td>Eppendorf</td><td>6.291704</td><td></td></tr><tr><td>Micropipettes 20-200 &amp;micro;L</td><td>Eppendorf</td><td>6.291703</td><td></td></tr><tr><td>Micropipettes 100-1000 &amp;micro;L</td><td>Eppendorf</td><td>7.648488</td><td></td></tr><tr><td>Microscope slides&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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

1.5K 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 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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Immunology and Infection

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

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

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Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes