<meta charset="utf8"></head><body>마이크로바이옴 분석을 위한 고정밀 모기 장기 분리

<ol>
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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. 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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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<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>
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dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level

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.

<meta charset="utf8"></head><body>저자 스포트라이트: 공생체 및 벡터 마이크로바이옴 연구를 위한 모기 장기 절개 최적화

장기 수준의 마이크로바이옴 분석을 위한 모기 난소, 중장 및 타액선 해부

Our research focuses on microbiome community in mosquito at the scale of individual organs using metagenomic analysis we study symbionts in ovaries, midgut and salivary glands. By preventing cross contamination during dissection we aim to study how these microbes affect mosquito biology at front scale. and adding targeted control methods.Working with individual organs it's really complex because we need to work on them intact without causing any damage or contamination between organs. Plus we only have a small amount of genetic material available. Research on vector mosquitoes typically focuses on the world organism.However, interactions between viruses, bacteria and mosquitoes can differ depending mosquito specific organ. By carefully dissecting and isolating mosquito organs we can study each compartment individually and gain a better understanding of how these interactions function.

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

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

829 조회수.  University of Montpellier, INRAe, CNRS, IRD. 우리의 연구는 메타게놈 분석을 사용하여 난소, 중장 및 타액선의 공생체를 연구하는 개별 장기 규모의 모기의 마이크로바이옴 군집에 중점을 둡니다. 해부 중 교차 오염을 방지함으로써 우리는 이러한 미생물이 전면 규모에서 모기 생물학에 어떤 영향을 미치는지 연구하는 것을 목표로 합니다. 표적 제어 방법을 추가합니다.개별 장기를 다루?...

비디오: 저자 스포트라이트: 공생체 및 벡터 마이크로바이옴 연구를 위한 모기 장기 절개 최적화

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.

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.

연구

생물학

Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

To begin, obtain surface-sterilized mosquitoes in a 1.5-milliliter tube containing PBS. Transfer a cleaned mosquito onto a drop of sterile 1X PBS placed on the microscope slide. Using tweezers, hold the mosquito's thorax to prevent it from moving.Then place a needle at the neck just below the head and gently pull on the mosquito's head to separate it from the thorax. Using a needle, cut the salivary glands at the level of the salivary duct. Retrieve the glands with the needle and place them into a new drop of PBS to rinse.Next, employing a needle, retrieve the salivary glands. Place the mosquito on its back to begin dissecting the ovaries. Using forceps, hold the mosquito by the thorax to prevent movement and puncture the third abdominal segment, starting from the bottom, with a needle.While piercing the third abdominal segment, pull down the abdomen to open it and reveal the internal organs. Remove any remaining pieces of the mosquito exoskeleton to facilitate organ release. Retrieve both ovaries with sterilized forceps and place them into a new drop of sterile PBS on the microscope slide to rinse.Transfer the rinsed ovaries into a 1.5-milliliter tube containing 100 microliters of preservation buffer. Next, using a sterile needle, separate the midgut from the foregut and hindgut. Remove the Malpighian tubules from the midgut and place the midgut into a new drop of sterile PBS to rinse it and prevent contamination.Then transfer the midgut to another drop of sterile PBS. With forceps, open the midgut to release bacteria. Pipette all the PBS-containing bacteria and the midgut tissue into a new sterilized 1.5-milliliter tube containing 100 microliters of preservation buffer.Add five microliters of PBS onto the same area of the microscope slide where the midgut was previously placed. Pipette the PBS back into the same 1.5-milliliter tube to retrieve any bacteria that may have stuck to the slide. Finally, use sterile forceps to grasp the mosquito carcass and store it in a sterilized 1.5-milliliter tube containing 100 microliters of preservation buffer.