Mosquito midgut harbors a complex community of microbes that affect the host metabolism, reproduction, fitness, and vet competence. With each gut microbe is a different effect. This video introduces the procedure to study the respective role of each bacteria strain or host physiology.
To acquire a culture of gut bacterial colonies, dissect the mosquito midgut under sterile condition. Ground the dissect midgut, serotologinate, and spread over the agar plate. Pick single colonies from the plate, and identify the bacteria species via their 16 SRNA gene sequence.
Feed the mosquitoes with COD Emboss containing antibiotics for consecutive of three days to remove gut microbiota. Effectiveness of antibiotic treatment can be confirmed by dissecting the midgut of the aseptic mosquito for colony forming unit assay prior to subsequent extremists. Then reintroduce specific bacteria species that previously isolated from mosquito gut via oral feeding assay.
The effect of bacteria on host physiology could be evaluated by comparing the untreated mosquitoes, antibiotic-treated mosquitoes, and the mosquitoes reintroduced specific restraint. Midgut dissection and the cultivatable bacteria isolation. To begin with, collect the mosquitoes in cage with an aspirator and cell the mosquitoes in the collection box.
Anesthetize the mosquitoes by subjecting to a temperature of 4 degree for three to five minutes. Keep the mosquitoes anesthetized by placing them in an ice cold Petri dish. Disinfect the bench, dissecting microscope, and forceps by spraying 75%ethanol to avoid contamination by bacteria from the environment.
Surface sterilize the mosquito by taping and shaking them in 75%ethanol for three minutes and rinse twice with PBS buffer. Bisect the mosquito separately on a sterile glass slide containing your drop of PBS. Carefully remove the legs, wings and head of the mosquito using forceps.
Clamp the mosquito's chest and the last section of abdomen was forceps and pull apart to isolate the digestive tract. Use forceps to remove the crop and the malpighian tube from the digestive tract. The crop is positioned at the pnterior of midgut and bulges after ingesting sugar water.
Malpighian tubes are group of slender excretory tubes on the junction of midgut and hindgut. Place the dissected makeup into EP tube with 200 microliters sterile PBS buffer and grind it with the sterile grinding pastor in the clean bench. Cereal to load the homogenate to three tempo delusions at 50 microliters of each delusion to the LB agar plate and desperate plate.
Incubate the plate as 37 degrees for one to two days until single colonies are feasible. Pick single colonies into a 150 milliliter conical flask containing 50 milliliter lb bras medium. shake the bacteria at 37 degree overnight.
Species identification. Extract TOTO DNA by bacterial genomic DNA construction kit and amplify 16 SRDNA by PCR. Recovery and the purification of DNA fragments from PCR products by Ambrose gel electrophoresis and the drove recovery kit.
Performed DNA sequencing on the purified DNA fragments to have a attained bacteria chain sequences. Run blast search to a query the 16 SRNA gene sequence of identify the bacteria against the bacteria and the archaea and database. Identification to the species level was defined as greater than or equal to 99%16 SRDNA sequence similarity to the closest team bank entry.
The isolate was assigned it to the corresponding, genus when is 16 SRDNA sequence, similarity was less than 99%and greater than or equal to 95%antibiotic treatment and bacterium re-introduction. weigh the required amount of sucrose penicillin and streptomycin to prepare us 10%sucrose solution, including trying 20 units of penicillin and the 20 microgram of streptomycin per milliliter. Infiltrate the cotton balls in the sucrose solution containing antibiotics and place it over the top of the paper cup containing mosquito.
Cover the cotton ball with a 10 centimeter Petri dish to prevent a waterfront from evaporating. Replace the cotton balls twice day and feed the mosquito for consecutive of three days. Mosquitoes are divided into two groups.
The first group was placed in a mosquito cup without any treatment, any cotton balls doctor with 10%So cross was given daily. The second group was treated with antibiotics and serve for three days. In each group, Sergei mosquitoes were dissected.
The Mika was extracted for total DNA extraction and the QPCR was performed using universal bacteria primers, figure one shows the expression of 16 SRNA in the control group and antibiotic treatment group. The results show that the cost or realization of penicillin and streptomycin was successful. prepare bacterial solution, measures a bacteria solution in the virus with a spectrophotometer, add one OT bacteria suspension into EP tube.
Centrifuge the suspension at 5, 000 RCA for five minutes at four degree Discard the supernatant, wash the bacteria pallet tries with sterile PBS buffer. Re-suspend the bacterial pallet with 200 microliters sterile PBS buffer Add 600 microliter, 10%sucrose solution, 200 microliter ATP and 200 microliter bacterial suspension into a new AP tube and mix well. Alternatively, the bacteria can be re-suspended in heat in activate your blood.
Preparation of heating activated blood. clot fresh blood with anticoagulant tube, take two to three milliliter of fresh blood. centrifuge at 1000 for 10 minutes at four degree to separate plasma and blood cells.
Collect the plasma into a new EP tube and herein activate activated at a 56 degree for one hour. Wash Blast house through your times with sterile PBS buffer. Rinse suspended blast house with heating activated plasma.
Assemble membrane and feeding system. Pull the film sickness to the maximum. fix with the plastic ring and workplace parafilm to avoid the leaking slowly add the prepared bacterial solution to the feeder unit, to the maximum wallet.
To avoid the bubble fell from one direction and seal the feeder unit. Connect the power supply. Wait until the temperature reaches 37 degree Installed the feeder unit with bacteria solution on the feeder.
Before feeding bacteria mosquitoes should be stopped for 24 hours to metabolize antibiotics. For the feed unit on the paper cup with mosquitoes, then allow feeding for 90 minutes. Fully encouraged mosquitoes could be picked out for further studies.
Representative results. Figure two show us the average accolade per mosquito. After plot feeding of antibiotic treated the mosquitoes we see meningosepticum.
Figure three shows the expression of a variant development related genes 24 hours after blood meal containing the C meningosepticum. The results show that there is no significant change in the egg production of the control group and the fading group. Thank you for your interest in bacteria or a feeding essay with antibiotic treated mosquitoes.
