抗生素处理蚊子的细菌口服喂养测定
1Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, College of Life Science,Hunan Normal University, 2State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory for Microbial Molecular Biology, College of Life Science,Hunan Normal University, 3Department of Infectious Diseases, the Second Xiangya Hospital,Central South University
Summary
本文提出了一个协议，研究单个蚊子肠道细菌的影响，包括隔离和识别蚊子中生可培养微生物，抗生素消耗蚊子肠道细菌，并重新引入一个特定的细菌物种。
Abstract
蚊子中游人具有高度动态的微生物群，影响宿主的新陈代谢、繁殖、健身和病媒能力。已开展研究，以调查肠道微生物作为一个整体的影响;然而，不同的微生物会对宿主产生不同的影响。本文提供了研究每种特定蚊帐微生物作用和潜在机理的方法。
该协议包含两个部分。第一部分介绍如何解剖蚊子中，分离可培养的细菌菌落，并识别细菌物种。第二部分提供了产生抗生素处理的蚊子的程序，并重新引入一种特定的细菌物种。
Introduction
蚊子被认为是人类致病疾病最重要的媒介，传播超过一百种病原体，包括寨卡病毒、登革热病毒和疟原虫1。当蚊子采取血餐来获得营养细胞，他们可以意外地从受感染的宿主通过消化道2摄入病原体。重要的是，在血粉消化和病原体入口中起着关键作用的蚊子中游，它蕴藏着高度动态的微生物群3。
几项研究已经确定实验室饲养和现场收集的蚊子微生物群使用培养依赖方法或细菌测序测定4，5，6。,5,6在各种研究中，包括潘5,7,8,托亚、塞拉蒂亚、克莱布西拉、Elizabethkingia伊丽莎白金氏菌和Enterococcus肠球菌在内的物种通常与蚊子分离。有趣的是，受发育阶段、物种、地理来源和喂养行为的影响，蚊子肠道微生物群在社区多样性和细菌种类中波动剧烈。研究表明，随着肠杆菌物种的迅速扩张和整体多样性的减少，供血Enterobacteriaceae量显著增加,了细菌总负荷。此外，幼虫阶段的蚊子肠道微生物群通常在昆虫在繁殖和闭合过程中发生变质时被根除;因此，新出现的成年蚊子需要重新填充其微生物群4。
古特微生物群在营养吸收、免疫、发育、繁殖和载体能力等各个方面调节昆虫生理学。斧头蚊子幼虫未能发育超过第一星，而细菌口服供应抢救发展，表明蚊子肠道微生物是幼虫发育的关键13，14。,14此外，肠道细菌的耗竭会延缓血餐消化和营养吸收，影响卵母细胞成熟，减少卵巢15。此外，与抗生素治疗的蚊子相比，具有肠道微生物群的蚊子具有更高的免疫反应，与其他病原体的抗菌肽表达不断升高，感染16。在这些研究中，通常口服抗生素来去除泛肠道细菌，然后进行实验，以比较斧头蚊子和蚊子与共生微生物的区别。然而，蚊子中古人蕴藏着各种各样的微生物群落，每种细菌物种对宿主生理机能产生明显的影响。
蚊子微生物群调节病媒能力，具有不同的效应。从登革热流行地区的外地蚊子中分离出的Proteus的殖民化，可产生对登革热病毒感染的增强抗菌肽表达和抵抗力。致病真菌博韦里亚巴西亚纳激活收费和JAK-STAT免疫途径对阿尔博病毒感染17。相比之下，从伊蚊中分离出的真菌Talaromyces通过调节肠道尝试素活性18促进登革热病毒感染。 Aedes aegypti此外，Serratia Marcescens通过一种叫做Sm Enhancin的分Sm泌蛋白促进arbo病毒的传播，这种蛋白质消化蚊子肠道上皮上的粘液层。
该程序为解剖蚊子中口、分离可培养细菌菌落、鉴定细菌种类和通过口服喂养重新引入提供了系统、直观的方法。它提供了具有代表性的用共体细菌、菊花 菌、蚊子卵巢发育和卵巢的供血结果。
Protocol
1. 中古解剖和可培养细菌分离 准备蚊子解剖。 用吸气器在出现7~9天后收集蚊子。麻醉收集的蚊子，让它们在4°C的温度下3~5分钟，并保持蚊子在冰冷的培养皿中麻醉，直到解剖。 对实验室仪器和蚊子表面进行消毒。 通过喷洒 75% 乙醇对实验台进行消毒，解剖显微镜、钳子和玻璃幻灯片，以避免被环境中的细菌污染。 准备一个?…
Representative Results
用抗生素治疗和没有抗生素治疗的蚊子中游被提取出来进行DNA提取，用通用细菌底剂进行qPCR。 图1 显示了对照组和抗生素治疗组中细菌16S rRNA的表达。结果表明，约98%的肠道细菌已被去除，青霉素和链霉素的肠道灭菌工作成功。 通过所述方法，分离和鉴定细菌菌株。 C. 性消化是 一种非感染性、氧化酶阳性的克阴性有氧杆菌 ，属于菊花菌。 图…
Discussion
对宿主-微生物相互作用的研究发现，不同的肠道微生物通过不同的机制影响宿主生理。本文介绍了研究蚊子肠道微生物各自作用的方法，包括解剖蚊虫、栽培肠道细菌、抗生素治疗和重新引入感兴趣的细菌。
要成功进行抗生素治疗，在进行实验时必须考虑以下细节。在该协议中，蚊子使用湿润的棉球进行治疗，用10%蔗糖溶液，包括20单位青霉素和20μg每mL链霉素3天<sup class="xre…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（第81902094号，81600497）和湖南省科技计划项目（2019RS1036）的支持。
Materials
| Adenosine 5′-triphosphate disodium salt hydrate | Sigma | A2383 | Adenosine 5′-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions |
| Aedes aegypti | Female mosquitoes | ||
| Anticoagulant tube | BD Vacutainer | 363095 | Collect fresh blood |
| Centrifuge tube | Sangon Biotech | F601620-0010 | 1.5 ml, Natural, Graduated, Sterile |
| Cotton balls | |||
| Disposable Tissue Grinding Pestle | Sangon Biotech | F619072-0001 | 70 mm Long, Conical, Blue, Sterile |
| Ethanol absolute | Paini | Dilute it to 75% ethanol | |
| Forceps | RWD | F11029 | Dissection |
| Hemotek Membrane Feeding System | Hemotek | Components of the feeding system, including Hemotek temperature controller, feeder-housing assembly, metal feeder assembled. | |
| Incubator shaker | ZQZY-78AN | ||
| Inoculation Loops | Sangon Biotech | F619312-0001 | 10 μl, Yellow |
| LB Agar Powder | Sangon Biotech | A507003 | Tryptone 10.0 g； Yeast Extract 5.0 g； NaCl 10.0 g； Agar 15.0 g. |
| LB Broth Powder | Sangon Biotech | A507002 | Tryptone 10.0 g； Yeast Extract 5.0 g； NaCl 10.0 g. |
| Microscope | Zeiss | Stemi508 | |
| Paper cup | Place mosquito | ||
| Parafilm | Sangon Biotech | F104002 | 4 inx 125 ft |
| Petri dish | Sangon Biotech | F611203 | |
| Penicillin G procaine salt hydrate | Sangon Biotech | A606248 | White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol |
| Single Channal Pipettor | Gilson | ||
| Streptomycin sulfate | Sangon Biotech | A610494 | Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins. |
| Sucrose | Sangon Biotech | A502792 | Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine. |
| TIANamp Bacteria DNA Kit | TIANGEN | DP302 | Extract DNA |
| Utility Fabric-Mosquito Netting White | |||
| Vortex mixer | Scintic Industries | S1-0246 | |
| 1.5ml EP tube | Sangon Biotech | F600620 | |
| 10X PBS buffer | Sangon Biotech | E607016 | This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4. |
References
- Tolle, M. A. Mosquito-borne diseases. Current Problems in Pediatric and Adolescent Health Care. 39 (4), 97-140 (2009).
- Wu, P., Yu, X., Wang, P., Cheng, G. Arbovirus lifecycle in mosquito: acquisition, propagation and transmission. Expert Reviews in Molecular Medicine. 21, 1 (2019).
- Jayakrishnan, L., Sudhikumar, A. V., Aneesh, E. M. Role of gut inhabitants on vectorial capacity of mosquitoes. Journal of Vector Borne Diseases. 55 (2), 69 (2018).
- Jupatanakul, N., Sim, S., Dimopoulos, G. The insect microbiome modulates vector competence for arboviruses. Viruses. 6 (11), 4294-4313 (2014).
- Moro, C. V., Tran, F. H., Raharimalala, F. N., Ravelonandro, P., Mavingui, P. Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus. BMC Microbiology. 13 (1), 70 (2013).
- Chouaia, B., et al. Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species. Applied and Environmental Microbiology. 76 (22), 7444-7450 (2010).
- Terenius, O., et al. Midgut bacterial dynamics in Aedes aegypti. FEMS Microbiology Ecology. 80 (3), 556-565 (2012).
- Bando, H., et al. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity. Scientific Reports. 3, 1641 (2013).
- Telang, A., Skinner, J., Nemitz, R. Z., McClure, A. M. Metagenome and culture-based methods reveal candidate bacterial mutualists in the Southern house mosquito (Diptera: Culicidae). Journal of Medical Entomology. 55 (5), 1170-1181 (2018).
- Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya. PloS One. 6 (9), (2011).
- Xiao, X., et al. A Mesh-Duox pathway regulates homeostasis in the insect gut. Nature Microbiology. 2 (5), 17020 (2017).
- Guégan, M., et al. Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota. FEMS Microbiology Ecology. 94 (12), 188 (2018).
- Valzania, L., Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America. 115 (3), 457-465 (2018).
- Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. Mosquitoes rely on their gut microbiota for development. Molecular Ecology. 23 (11), 2727-2739 (2014).
- de O Gaio, A., et al. Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera: culicidae)(L). Parasites & Vectors. 4 (1), 105 (2011).
- Ramirez, J. L., et al. Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence. PLoS Neglected Tropical Diseases. 6 (3), 1561 (2012).
- Dong, Y., Morton, J. C., Ramirez, J. L., Souza-Neto, J. A., Dimopoulos, G. The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti. Insect Biochemistry and Molecular Biology. 42 (2), 126-132 (2012).
- Anglero-Rodriguez, Y. I., et al. An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity. Elife. 6, 28844 (2017).
- Wu, P., et al. A gut commensal bacterium promotes mosquito permissiveness to arboviruses. Cell Host & Microbe. 25 (1), 101-112 (2019).
- Möhlmann, T. W., et al. Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes. Microbial Ecology. , (2020).
- Llorca, M., Gros, M., Rodríguez-Mozaz, S., Barceló, D. Sample preservation for the analysis of antibiotics in water. Journal of Chromatography. A. 1369, 43-51 (2014).
- Berendsen, B., Elbers, I., Stolker, A. Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection. Food Additives & Contaminants: Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment. 28 (12), 1657-1666 (2011).
- Hill, C. L., Sharma, A., Shouche, Y., Severson, D. W. Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti. Acta Tropica. 140, 151-157 (2014).
- Eng, M. W., et al. Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti. PLoS Neglected Tropical Diseases. 12 (1), 0006186 (2018).
- Kajla, M. K., Barrett-Wilt, G. A., Paskewitz, S. M. Bacteria: A novel source for potent mosquito feeding-deterrents. Science Advances. 5 (1), 6141 (2019).
- Gonçalves, G. G. A., et al. Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes. Acta Tropica. 200, 105174 (2019).
- Kuss, S. K., et al. Intestinal microbiota promote enteric virus replication and systemic pathogenesis. Science. 334 (6053), 249-252 (2011).
- Rani, A., Sharma, A., Rajagopal, R., Adak, T., Bhatnagar, R. K. Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector. BMC Microbiology. 9 (1), (2009).
- Apte-Deshpande, A., Paingankar, M., Gokhale, M. D., Deobagkar, D. N. Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus. PLoS One. 7 (7), 40401 (2012).
- Behura, S. K. Mosquito microbiota and metagenomics, and its relevance to disease transmission. Nature. 436, 257-260 (2013).
- Dickson, L. B., et al. Diverse laboratory colonies of Aedes aegypti harbor the same adult midgut bacterial microbiome. Parasites & Vectors. 11 (1), 1-8 (2018).
Cite This Article
Liu, X., Wu, S., Li, W., Zhang, M., Wu, Y., Zhou, N., Wu, P. A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. J. Vis. Exp. (163), e61341, doi:10.3791/61341 (2020).