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 m....

Protocol

1. Midgut dissection and cultivable bacteria isolation

1. Prepare the mosquito for dissection.
   1. Collect the mosquitoes 7–9 days after emergence with an aspirator. Anesthetize the collected mosquitoes by subjecting them to a temperature of 4 °C for 3–5 min and keep the mosquitoes anesthetized in an ice-cold Petri dish until dissection.
2. Sterilize laboratory instruments and the mosquito surface.
   1. Sterilize the experiment bench, dissecting microscope, forceps, and glass slide by spraying 75% ethanol to avoid contamination by bacteria from the environment.
   2. P....

Results

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.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.