A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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).
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. |
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