This work was supported by the National Natural Science Foundation of China (Grant No. 81902094, 81600497), and the Science and Technology Plan Project of Hunan Province (2019RS1036).

1. Midgut dissection and cultivable bacteria isolation
<ol>
	<li>Prepare the mosquito for dissection.
	<ol>
		<li>Collect the mosquitoes 7&#8211;9 days after emergence with an aspirator. Anesthetize the collected mosquitoes by subjecting them to a temperature of 4 &#176;C for 3&#8211;5 min and keep the mosquitoes anesthetized in an ice-cold Petri dish until dissection.</li>
	</ol>
	</li>
	<li>Sterilize laboratory instruments and the mosquito surface.
	<ol>
		<li>Sterilize the experimen...

<ol>
	<li>Tolle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito-borne+diseases.">Mosquito-borne diseases.</a> Current Problems in Pediatric and Adolescent Health Care. 39 (4), 97-140 (2009).</li><li>Wu, P., Yu, X., Wang, P., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbovirus+lifecycle+in+mosquito:+acquisition,+propagation+and+transmission.">Arbovirus lifecycle in mosquito: acquisition, propagation and transmission.</a> Expert Reviews in Molecular Medicine. 21, 1 (2019).</li><li>Jayakrishnan, L., Sudhikumar, A. V., Aneesh, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+gut+inhabitants+on+vectorial+capacity+of+mosquitoes.">Role of gut inhabitants on vectorial capacity of mosquitoes.</a> Journal of Vector Borne Diseases. 55 (2), 69 (2018).</li><li>Jupatanakul, N., Sim, S., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+microbiome+modulates+vector+competence+for+arboviruses.">The insect microbiome modulates vector competence for arboviruses.</a> Viruses. 6 (11), 4294-4313 (2014).</li><li>Moro, C. V., Tran, F. H., Raharimalala, F. N., Ravelonandro, P., Mavingui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+culturable+bacteria+including+Pantoea+in+wild+mosquito+Aedes+albopictus.">Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus.</a> BMC Microbiology. 13 (1), 70 (2013).</li><li>Chouaia, B., 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=Molecular+evidence+for+multiple+infections+as+revealed+by+typing+of+Asaia+bacterial+symbionts+of+four+mosquito+species.">Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species.</a> Applied and Environmental Microbiology. 76 (22), 7444-7450 (2010).</li><li>Terenius, O., 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=Midgut+bacterial+dynamics+in+Aedes+aegypti.">Midgut bacterial dynamics in Aedes aegypti.</a> FEMS Microbiology Ecology. 80 (3), 556-565 (2012).</li><li>Bando, 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=Intra-specific+diversity+of+Serratia+marcescens+in+Anopheles+mosquito+midgut+defines+Plasmodium+transmission+capacity.">Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity.</a> Scientific Reports. 3, 1641 (2013).</li><li>Telang, A., Skinner, J., Nemitz, R. Z., McClure, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome+and+culture-based+methods+reveal+candidate+bacterial+mutualists+in+the+Southern+house+mosquito+(Diptera:+Culicidae).">Metagenome and culture-based methods reveal candidate bacterial mutualists in the Southern house mosquito (Diptera: Culicidae).</a> Journal of Medical Entomology. 55 (5), 1170-1181 (2018).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PloS One. 6 (9), (2011).</li><li>Xiao, X., 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+Mesh-Duox+pathway+regulates+homeostasis+in+the+insect+gut.">A Mesh-Duox pathway regulates homeostasis in the insect gut.</a> Nature Microbiology. 2 (5), 17020 (2017).</li><li>Gu&#233;gan, 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=Short-term+impacts+of+anthropogenic+stressors+on+Aedes+albopictus+mosquito+vector+microbiota.">Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.</a> FEMS Microbiology Ecology. 94 (12), 188 (2018).</li><li>Valzania, L., Coon, K. L., Vogel, K. J., 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=Hypoxia-induced+transcription+factor+signaling+is+essential+for+larval+growth+of+the+mosquito+Aedes+aegypti.">Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), 457-465 (2018).</li><li>Coon, K. L., Vogel, K. J., 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+rely+on+their+gut+microbiota+for+development.">Mosquitoes rely on their gut microbiota for development.</a> Molecular Ecology. 23 (11), 2727-2739 (2014).</li><li>de O Gaio, 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=Contribution+of+midgut+bacteria+to+blood+digestion+and+egg+production+in+Aedes+aegypti+(diptera:+culicidae)(L).">Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera: culicidae)(L).</a> Parasites &amp; Vectors. 4 (1), 105 (2011).</li><li>Ramirez, J. 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=Reciprocal+tripartite+interactions+between+the+Aedes+aegypti+midgut+microbiota,+innate+immune+system+and+dengue+virus+influences+vector+competence.">Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence.</a> PLoS Neglected Tropical Diseases. 6 (3), 1561 (2012).</li><li>Dong, Y., Morton, J. C., Ramirez, J. L., Souza-Neto, J. A., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entomopathogenic+fungus+Beauveria+bassiana+activate+toll+and+JAK-STAT+pathway-controlled+effector+genes+and+anti-dengue+activity+in+Aedes+aegypti.">The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti.</a> Insect Biochemistry and Molecular Biology. 42 (2), 126-132 (2012).</li><li>Anglero-Rodriguez, Y. I., 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=An+Aedes+aegypti-associated+fungus+increases+susceptibility+to+dengue+virus+by+modulating+gut+trypsin+activity.">An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity.</a> Elife. 6, 28844 (2017).</li><li>Wu, 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=A+gut+commensal+bacterium+promotes+mosquito+permissiveness+to+arboviruses.">A gut commensal bacterium promotes mosquito permissiveness to arboviruses.</a> Cell Host &amp; Microbe. 25 (1), 101-112 (2019).</li><li>M&#246;hlmann, T. W., 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=Impact+of+gut+bacteria+on+the+infection+and+transmission+of+pathogenic+arboviruses+by+biting+midges+and+mosquitoes.">Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes.</a> Microbial Ecology. , (2020).</li><li>Llorca, M., Gros, M., Rodr&#237;guez-Mozaz, S., Barcel&#243;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preservation+for+the+analysis+of+antibiotics+in+water.">Sample preservation for the analysis of antibiotics in water.</a> Journal of Chromatography. A. 1369, 43-51 (2014).</li><li>Berendsen, B., Elbers, I., Stolker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+stability+of+antibiotics+in+matrix+and+reference+solutions+using+a+straightforward+procedure+applying+mass+spectrometric+detection.">Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection.</a> Food Additives &amp; Contaminants: Part A, Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 28 (12), 1657-1666 (2011).</li><li>Hill, C. L., Sharma, A., Shouche, Y., Severson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+midgut+microflora+and+dengue+virus+impact+on+life+history+traits+in+Aedes+aegypti.">Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti.</a> Acta Tropica. 140, 151-157 (2014).</li><li>Eng, M. W., 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=Multifaceted+functional+implications+of+an+endogenously+expressed+tRNA+fragment+in+the+vector+mosquito+Aedes+aegypti.">Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti.</a> PLoS Neglected Tropical Diseases. 12 (1), 0006186 (2018).</li><li>Kajla, M. K., Barrett-Wilt, G. A., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria:+A+novel+source+for+potent+mosquito+feeding-deterrents.">Bacteria: A novel source for potent mosquito feeding-deterrents.</a> Science Advances. 5 (1), 6141 (2019).</li><li>Gon&#231;alves, G. G. 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=Use+of+MALDI-TOF+MS+to+identify+the+culturable+midgut+microbiota+of+laboratory+and+wild+mosquitoes.">Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes.</a> Acta Tropica. 200, 105174 (2019).</li><li>Kuss, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+microbiota+promote+enteric+virus+replication+and+systemic+pathogenesis.">Intestinal microbiota promote enteric virus replication and systemic pathogenesis.</a> Science. 334 (6053), 249-252 (2011).</li><li>Rani, A., Sharma, A., Rajagopal, R., Adak, T., Bhatnagar, R. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serratia+odorifera+a+midgut+inhabitant+of+Aedes+aegypti+mosquito+enhances+its+susceptibility+to+dengue-2+virus.">Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.</a> PLoS One. 7 (7), 40401 (2012).</li><li>Behura, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+microbiota+and+metagenomics,+and+its+relevance+to+disease+transmission.">Mosquito microbiota and metagenomics, and its relevance to disease transmission.</a> Nature. 436, 257-260 (2013).</li><li>Dickson, L. 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Research on host-microbe interactions have found that different gut microbes affect their host physiology via divergent mechanisms. This article introduces the method to investigate the respective role of mosquito gut microbe, including dissecting mosquito midgut, culturing cultivable gut bacteria, antibiotic treatment, and reintroducing the bacteria of interest.
For successful antibiotic treatment, the following details must be considered in conducting the experiment. In this protocol, mosqui...

Mosquitoes are considered to be the most important vectors of human pathogenic diseases, transmitting over a hundred pathogens including Zika virus, Dengue virus, and Plasmodium parasites1. When mosquitoes take a blood meal to acquire nutrients for oviposition, they can accidentally ingest pathogens from an infected host via the digestive tract2. Importantly, the mosquito midgut, which plays a pivotal role in both blood meal digestion and pathogen entrance, harbors a highly dynamic microbiome3.
Several studies have characterized lab-reared and field-collected mosquito microbiota using either a culture-dependent method or a bacteria sequencing assay4,5,6. Species including Pantoea, Serratia, Klebsiella, Elizabethkingia, and Enterococcus are commonly isolated from mosquitoes in various studies5,7,8,9. Interestingly, mosquito gut microbiota fluctuates dynamically in both the community diversity and the amount of bacteria species, affected by the development stage, species, geographical origin, and feeding behavior4. Studies show that blood feeding dramatically increases the total bacterial load with rapid expansion of species from Enterobacteriaceae and a reduction in overall diversity10,11. In addition, mosquito gut microbiota of the larval stage is usually eradicated when the insect undergoes metamorphosis during pupation and eclosion; thus, newly emerged adult mosquitoes need to repopulate their microbiota4.
Gut microbiota modulates insect physiology in various aspects, including nutrient absorption, immunity, development, reproduction, and vector competence12. Axenic mosquito larvae fail to develop beyond the first instar while a bacteria oral supply rescues development, indicating that the mosquito gut microbe is essential for larval development13,14. Besides, depletion of gut bacteria retards blood meal digestion and nutrient absorption, affects oocyte maturation, and decreases oviposition15. In addition, mosquitoes with gut microflora elicit higher immune responses compared to antibiotic-treated mosquitoes, with constantly elevated antimicrobial peptide expression against other pathogens to infect16. Antibiotics are usually orally administered to remove pan gut bacteria in these studies, and then experiments are conducted to compare the difference between axenic mosquitoes and mosquitoes with commensal microbes. However, the mosquito midgut harbors a diverse community of microbes, and each bacteria species could exert a distinct effect toward the host physiology.
Mosquito microbiota regulates vector competence with divergent effects. Colonization by Proteus isolated from field-derived mosquitoes of dengue-endemic areas confers upregulated antimicrobial peptide expression and resistance against dengue virus infection16. The entomopathogenic fungus Beauveria bassiana activates the Toll and JAK-STAT immune pathway against arbovirus infection17. By contrast, the fungus Talaromyces isolated from Aedes aegypti midgut facilitates dengue virus infection by modulating gut trypsin activity18. In addition, Serratia marcescens promotes arbovirus transmission through a secretory protein called SmEnhancin, which digests the mucin layer on the intestinal epithelium of mosquitoes19.
This procedure provides a systematic and intuitive method for dissection of the mosquito midgut, isolation of cultivable bacteria colonies, identification of the bacteria species, and reintroduction via oral feeding. It provides representative results of blood feeding with a commensal bacterium, Chryseobacterium meningosepticum, on mosquito ovary development and oviposition.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td >Sigma</td>
			<td >A2383</td>
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions</td>
		</tr>
		<tr >
			<td >Aedes aegypti</td>
			<td></td>
			<td></td>
			<td>Female mosquitoes</td>
		</tr>
		<tr >
			<td >Anticoagulant tube</td>
			<td>BD Vacutainer</td>
			<td>363095</td>
			<td>Collect fresh blood</td>
		</tr>
		<tr >
			<td >Centrifuge tube</td>
			<td>Sangon Biotech</td>
			<td>F601620-0010</td>
			<td>1.5 ml, Natural, Graduated, Sterile</td>
		</tr>
		<tr >
			<td >Cotton balls</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposable Tissue Grinding Pestle</td>
			<td>Sangon Biotech</td>
			<td>F619072-0001</td>
			<td>70 mm Long, Conical, Blue, Sterile</td>
		</tr>
		<tr >
			<td >Ethanol absolute</td>
			<td>Paini</td>
			<td></td>
			<td>Dilute it to 75% ethanol</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td>RWD</td>
			<td>F11029</td>
			<td>Dissection</td>
		</tr>
		<tr >
			<td >Hemotek Membrane Feeding System</td>
			<td>Hemotek</td>
			<td></td>
			<td>Components of the feeding system, including&#160; Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.</td>
		</tr>
		<tr >
			<td >Incubator shaker</td>
			<td></td>
			<td>ZQZY-78AN</td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculation Loops</td>
			<td>Sangon Biotech</td>
			<td>F619312-0001</td>
			<td>10 &#956;l, Yellow</td>
		</tr>
		<tr >
			<td >LB Agar Powder</td>
			<td>Sangon Biotech</td>
			<td>A507003</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g&#65307; Agar 15.0 g.</td>
		</tr>
		<tr >
			<td >LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g.</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Zeiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper cup</td>
			<td></td>
			<td></td>
			<td>Place mosquito</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td>Sangon Biotech</td>
			<td>F104002</td>
			<td>4 inx 125 ft</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Sangon Biotech</td>
			<td>F611203</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin G procaine salt hydrate</td>
			<td>Sangon Biotech</td>
			<td>A606248</td>
			<td>White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol</td>
		</tr>
		<tr >
			<td >Single Channal Pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Streptomycin sulfate</td>
			<td>Sangon Biotech</td>
			<td>A610494</td>
			<td>Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins.</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td>Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine.</td>
		</tr>
		<tr >
			<td >TIANamp Bacteria DNA Kit</td>
			<td>TIANGEN</td>
			<td>DP302</td>
			<td>Extract DNA&#160;</td>
		</tr>
		<tr >
			<td >Utility Fabric-Mosquito Netting White</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td>Scintic Industries</td>
			<td>S1-0246</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5ml EP tube</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr >
			<td >10X PBS buffer</td>
			<td>Sangon Biotech</td>
			<td>E607016</td>
			<td>This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.</td>
		</tr>
	</tbody>
</table>

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.

The midguts of mosquitoes treated with antibiotics and without antibiotics were taken out for DNA extraction, and qPCR was performed with universal bacterial primers. Figure 1 shows the expression of bacterial 16S rRNA in the control group and antibiotic treatment group. The results show that about 98% of the gut bacteria have been removed, and the gut sterilization of penicillin and streptomycin was successful.
With the methods described, bacteria strains were isolated and id...

Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

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 ...

Mosquito midgut harbors a complex community of microbes that affect the host metabolism, reproduction, fitness, and vet competence. With each gut microbe is a different effect. This video introduces the procedure to study the respective role of each bacteria strain or host physiology.To acquire a culture of gut bacterial colonies, dissect the mosquito midgut under sterile condition. Ground the dissect midgut, serotologinate, and spread over the agar plate. Pick single colonies from the plate, and identify the bacteria species via their 16 SRNA gene sequence.Feed the mosquitoes with COD Emboss containing antibiotics for consecutive of three days to remove gut microbiota. Effectiveness of antibiotic treatment can be confirmed by dissecting the midgut of the aseptic mosquito for colony forming unit assay prior to subsequent extremists. Then reintroduce specific bacteria species that previously isolated from mosquito gut via oral feeding assay.The effect of bacteria on host physiology could be evaluated by comparing the untreated mosquitoes, antibiotic-treated mosquitoes, and the mosquitoes reintroduced specific restraint. Midgut dissection and the cultivatable bacteria isolation. To begin with, collect the mosquitoes in cage with an aspirator and cell the mosquitoes in the collection box.Anesthetize the mosquitoes by subjecting to a temperature of 4 degree for three to five minutes. Keep the mosquitoes anesthetized by placing them in an ice cold Petri dish. Disinfect the bench, dissecting microscope, and forceps by spraying 75%ethanol to avoid contamination by bacteria from the environment.Surface sterilize the mosquito by taping and shaking them in 75%ethanol for three minutes and rinse twice with PBS buffer. Bisect the mosquito separately on a sterile glass slide containing your drop of PBS. Carefully remove the legs, wings and head of the mosquito using forceps.Clamp the mosquito's chest and the last section of abdomen was forceps and pull apart to isolate the digestive tract. Use forceps to remove the crop and the malpighian tube from the digestive tract. The crop is positioned at the pnterior of midgut and bulges after ingesting sugar water.Malpighian tubes are group of slender excretory tubes on the junction of midgut and hindgut. Place the dissected makeup into EP tube with 200 microliters sterile PBS buffer and grind it with the sterile grinding pastor in the clean bench. Cereal to load the homogenate to three tempo delusions at 50 microliters of each delusion to the LB agar plate and desperate plate.Incubate the plate as 37 degrees for one to two days until single colonies are feasible. Pick single colonies into a 150 milliliter conical flask containing 50 milliliter lb bras medium. shake the bacteria at 37 degree overnight.Species identification. Extract TOTO DNA by bacterial genomic DNA construction kit and amplify 16 SRDNA by PCR. Recovery and the purification of DNA fragments from PCR products by Ambrose gel electrophoresis and the drove recovery kit.Performed DNA sequencing on the purified DNA fragments to have a attained bacteria chain sequences. Run blast search to a query the 16 SRNA gene sequence of identify the bacteria against the bacteria and the archaea and database. Identification to the species level was defined as greater than or equal to 99%16 SRDNA sequence similarity to the closest team bank entry.The isolate was assigned it to the corresponding, genus when is 16 SRDNA sequence, similarity was less than 99%and greater than or equal to 95%antibiotic treatment and bacterium re-introduction. weigh the required amount of sucrose penicillin and streptomycin to prepare us 10%sucrose solution, including trying 20 units of penicillin and the 20 microgram of streptomycin per milliliter. Infiltrate the cotton balls in the sucrose solution containing antibiotics and place it over the top of the paper cup containing mosquito.Cover the cotton ball with a 10 centimeter Petri dish to prevent a waterfront from evaporating. Replace the cotton balls twice day and feed the mosquito for consecutive of three days. Mosquitoes are divided into two groups.The first group was placed in a mosquito cup without any treatment, any cotton balls doctor with 10%So cross was given daily. The second group was treated with antibiotics and serve for three days. In each group, Sergei mosquitoes were dissected.The Mika was extracted for total DNA extraction and the QPCR was performed using universal bacteria primers, figure one shows the expression of 16 SRNA in the control group and antibiotic treatment group. The results show that the cost or realization of penicillin and streptomycin was successful. prepare bacterial solution, measures a bacteria solution in the virus with a spectrophotometer, add one OT bacteria suspension into EP tube.Centrifuge the suspension at 5, 000 RCA for five minutes at four degree Discard the supernatant, wash the bacteria pallet tries with sterile PBS buffer. Re-suspend the bacterial pallet with 200 microliters sterile PBS buffer Add 600 microliter, 10%sucrose solution, 200 microliter ATP and 200 microliter bacterial suspension into a new AP tube and mix well. Alternatively, the bacteria can be re-suspended in heat in activate your blood.Preparation of heating activated blood. clot fresh blood with anticoagulant tube, take two to three milliliter of fresh blood. centrifuge at 1000 for 10 minutes at four degree to separate plasma and blood cells.Collect the plasma into a new EP tube and herein activate activated at a 56 degree for one hour. Wash Blast house through your times with sterile PBS buffer. Rinse suspended blast house with heating activated plasma.Assemble membrane and feeding system. Pull the film sickness to the maximum. fix with the plastic ring and workplace parafilm to avoid the leaking slowly add the prepared bacterial solution to the feeder unit, to the maximum wallet.To avoid the bubble fell from one direction and seal the feeder unit. Connect the power supply. Wait until the temperature reaches 37 degree Installed the feeder unit with bacteria solution on the feeder.Before feeding bacteria mosquitoes should be stopped for 24 hours to metabolize antibiotics. For the feed unit on the paper cup with mosquitoes, then allow feeding for 90 minutes. Fully encouraged mosquitoes could be picked out for further studies.Representative results. Figure two show us the average accolade per mosquito. After plot feeding of antibiotic treated the mosquitoes we see meningosepticum.Figure three shows the expression of a variant development related genes 24 hours after blood meal containing the C meningosepticum. The results show that there is no significant change in the egg production of the control group and the fading group. Thank you for your interest in bacteria or a feeding essay with antibiotic treated mosquitoes.

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JoVE Journal

Biology

6.1K visualizações.  Hunan Normal University. Mosquito midgut abriga uma comunidade complexa de micróbios que afetam o metabolismo hospedeiro, reprodução, aptidão e competência veterinária. Com cada micróbio intestinal é um efeito diferente. Este vídeo introduz o procedimento para estudar o respectivo papel de cada cepa de bactérias ou fisiologia hospedeira.Para adquirir uma cultura de colônias bacterianas intestinais, disseque o mosquito midgut sob condição estéril. Aterrar o meio-boca dissecado, serotologinado, e e...