Establishing a Defined Gut Bacterial Population in Adult Mosquitoes

Abstract

Source: Liu, X., et al. A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. J. Vis. Exp. (2020)

This video demonstrates the depletion of native gut microbiota in mosquitoes using an antibiotic-containing sucrose solution. This is followed by controlled recolonization with a bacterial suspension delivered through a membrane feeding system to establish a defined bacterial population in the mosquito gut.

Protocol

1. Antibiotic treatment and bacterium reintroduction

1. Prepare the antibiotic solution.
   1. Weigh the required amount of sucrose, penicillin, and streptomycin to prepare a 10% sucrose solution including 20 units of penicillin and 20 µg of streptomycin per mL.
2. Feed mosquitoes with the antibiotic solution.
   1. Anesthetize the mosquitoes in 4 °C for 3–5 min. Transfer the mosquitoes to a paper cup. Cover the top with gauze and enclose the gauze with tape to prevent mosquitoes from escaping.
   2. Dip sterile cotton balls into the antibiotic solution and place them carefully on the mosquito cup. Squeeze the cotton balls before use to prevent mosquitoes from drowning in the dripping cotton balls.
   3. Cover the cotton balls with a 10 cm Petri dish to prevent moisture evaporation. Replace the cotton balls twice a day for three consecutive days.
3. Preparation of bacterial suspension
4. Measure the bacterial solution with a spectrophotometer. Add 1 OD (OD₆₀₀) bacterial suspension into a 1 mL tube. Centrifuge the suspension at 5,000 x g for 5 min at 4 °C.
5. Discard the supernatant and add 1 mL of sterile 1x PBS buffer to a 1.5 mL tube for suspension precipitation.
6. Centrifuge at 5,000 x g for 5 min at 4 °C; discard the supernatant.
7. Repeat steps 2.4.2 and 2.4.3.
8. Add 200 µL of sterile 1x phosphate-buffered saline (PBS) buffer to the bacterial precipitation.
9. Add 600 µL of 10% sucrose solution, 200 µL of 10 mm adenosine 5′-triphosphate (ATP), which acts as a phagostimulant, and 200 µL of bacterial suspension into a sterile 1.5 mL tube. Mix by vortexing.
10. Feed the mosquito with the bacteria suspension.
    1. Starve the mosquitoes for 24 h, allowing antibiotics to metabolize before feeding bacteria.
    2. Assemble the membrane-feeding system.
    3. Seal the sterilized feeder unit with parafilm or alternatively, a collagen membrane.
    4. Put on a plastic ring and fix it with parafilm to avoid breaking due to friction during feeding.
    5. Slowly add the prepared bacterial solution to the feeder unit. 
       Note: Only one well of the two-well feeder unit can be filled with the solution, and cover the feeder unit only from the reagent-dropping side.
    6. Connect the feeder unit to a feeding system preheated to 37 °C, which simulates human temperature to attract mosquitoes. Place the feeding device on a paper cup filled with mosquitoes and feed for 90 min.
    7. After feeding, anesthetize the 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.
    8. Pick the fully engorged mosquitoes for further studies.

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
Hemotek membrane feeding system	Hemotek		Components of the feeding system, including Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.
Parafilm	Sangon Biotech	F104002	4 in x 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
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.
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.