Establishing a Defined Gut Bacterial Population in Adult Mosquitoes

Begin with a paper cup containing adult mosquitoes, sealed with gauze to prevent their escape.

Place cotton balls soaked in an antibiotic-containing sucrose solution on the gauze, and cover them with a Petri dish to maintain humidity.

As the mosquitoes feed, they ingest the antibiotics, eliminating their endogenous gut microbiota.

Remove the cotton and initiate a starvation period to clear residual antibiotics from the mosquitoes' gut.

Take a tube containing bacteria suspended in a sucrose solution supplemented with a phagostimulant to stimulate feeding.

Take a sealed feeder unit, load it with the bacterial suspension, and secure it.

Connect the unit to a preheated feeding device and place it over the cup containing the pre-starved mosquitoes.

As the mosquitoes feed, bacteria travel into the gut and colonize it, forming a defined bacterial population.

The fed mosquitoes carrying this bacterial population are ready for host-pathogen interaction studies.

Infiltrate the sterile cotton balls in the sucrose solution containing antibiotics and place it over the top of the paper cup containing mosquito.

Cover the cotton balls with a 10-centimeter Petri dish to prevent the water from evaporating. Replace the cotton balls twice a day and feed the mosquito for a consecutive of three days. Prepare bacterial solution. Measure the bacterial solution in the UV-visible spectrophotometer. Add one OD bacteria suspension into EP tube.

Centrifuge the suspension at 5,000 RCF for 5 minutes at 4 degree.

Discard the supernatant. Wash the bacterial pellet thrice with sterile PBS buffer. Re-suspend the bacterial pellet with 200 microliters sterile PBS buffer. Add 600 microliter 10% sucrose solution, 200 microliter ATP, and 200 microliter bacterial suspension into a new EP tube and mix well. Slowly add the prepared bacterial solution to the feeder unit, to the maximum volume.

To avoid the bubbles fell from one direction and seal the feeder unit. Connect the power supply. Wait until the temperature reaches 37 degree. Install the feeder unit with bacterial solution on the feeder. Before feeding bacteria, mosquitoes should be stopped for 24 hours to metabolize antibiotics. Put the feeder unit on the paper cup with mosquitoes, then allow feeding for 90 minutes. Fully engorged mosquitoes could be picked out for further studies.