This method can help answer key questions in the field of infection and immunity, such as how do individuals respond to different roots of infection? The main advantage of this technique is that it allows for the examination of gut immunocompetent which plays a significant role in host defense against infection, that are mainly acquired through feeding. This method has the potential to be used in species other than Drosophila, and can be used to study other pathogens as well, such as viruses.
Generally, individuals new to this method will struggle because of the required experimental rigor during these microbiology protocols. Maintain the flies in 23 milliliter plastic vials containing seven milliliters of freshly made Lewis medium. To plug the vials, use non-absorbent cotton wool.
Keep them at 25 degrees Celsius under a 12-12 light-dark cycle with a relative humidity of about 60%Every 14 days, transfer 20 to 30 adults to a new food vial, with a dash of dry yeast added to the surface. After two to three days, dispose of the adult flies if sufficient eggs have been laid. To collect eggs, prepare a population cage.
First, add water-soaked cotton wool to provide moisture. Then for a substrate, use a 75 milliliter plate filled with apple agar and a spread of yeast paste. Lastly, load the adults into a population cage.
On 24-hour intervals, replace the plate and check for the presence of eggs. Once sufficient eggs are laid, remove any dead flies from the plate and the remaining yeast paste from the collected plate. Then submerge the agar in 20 milliliters of PBS and gently dislodge the eggs, using a fine paint brush.
Transfer the eggs in the PBS into a 50 milliliter tube. Then let the eggs sink to the bottom over about five minutes. Next, shorten the end of a p1000 tip by about 4 millimeters, then use it to aspirate the eggs in solution.
A snap-release of the plunger is effective for drawing them up. Then transfer the eggs in PBS into a 1.5 milliliter tube and allow the eggs to settle. Now trim four millimeters off of a filtered p20 tip and then use it to transfer an aliquot of eggs onto a vial of food to develop.
To grow P.entomophila and P.aeruginosa cultures, inoculate 10 milliliter of LB broth with 100 microliters of a frozen stock and incubate it overnight with 150 rpm of shaking. Next, transfer the culture to a two liter conical flask and fill it with LB up to the beginning of the slope of the flask's walls. When the subculture reaches the exponential growth phase, transfer equal volumes to an even number of 50 milliliter tubes, then spin them down.
Then collect the supernatants and spin them down again. Now combine the bacterial pellets from all the tubes by resuspending each pallet in five milliliters of supernatant, and then recombine the suspensions in a single 50 milliliter tube. Then after another spin, resuspend the bacteria in 5%sucrose solution.
Now dilute a sample of this suspension 100-fold to reliably measure the OD of the suspension, and adjust the final concentration of the suspension accordingly to the desired infectious dose. Two to four hours before the oral infection, transfer the flies to standard agar vials containing no food. Meanwhile, for each fly, prepare an infection vial.
First, load the lid of a seven milliliter sample tube with 500 microliters of standard sugar agar and let it solidify. Next, stick a disk of filter paper in the agar, then pipette 100 microliters of bacterial culture directly onto the filter disc. For controls, use 5%sucrose water.
After the starvation period, transfer single flies to the prepared tubes. After incubating the flies for 18 to 24 hours, surface-sterilize the flies. Load individuals into a tube containing 100 microliters of 70%ethanol.
After 20 to 30 seconds, remove the ethanol and replace it with 100 microliters of triple-distilled water. After another 20 to 30 seconds, remove the water. Then add 100 microliters of PBS and homogenize the fly.
Transfer the homogenate to the top row of a 96-well plate and add 90 microliters of 1x PBS to every well below. Then using 10 microliter aliquots, serially dilute this sample to distinguish a range of CFU values. Next, plate the serial dilutions on an LB nutrient agar plate in five microliter droplets.
Position the droplets so they are discreetly located on the plate. Then incubate the plate overnight. The next day, calculate the number of CFUs from the dilution that produces 10 to 60 clearly visible colonies.
To measure bacterial load, transfer a single infected fly to a sterile 1.5 milliliter microcentrifuge tube. Then as previously described, surface-sterilize the flies, then homogenize them and make a CFU analysis. To measure bacterial shedding, transfer single flies to 1.5 milliliter tubes containing about 50 microliters of Lewis medium.
Change the tube every 24 hours. After three tubes have been populated for 24 hours, dispose of the flies. After each transfer, measure the bacteria contained in the tube.
Mix the tube content with 100 microliters of PBS using a vortex. Then measure the CFUs in the mixture by plating serial dilutions. Using the describe protocol, the effect of oral bacterial infection was investigated via mortality and bacterial shedding.
Shedding was measured longitudinally in the same flies and at single time points alongside an internal bacterial load. D.melanogaster was orally infected with P.aeruginosa or P.entomophila. Successful oral infection occurred after a 12 or 24 hour exposure period to bacterial cultures.
It was necessary to use more concentrated culture of P.entomophila. Male and female Oregon R flies cleared the P.aeruginosa infection at the same rate and shed the same number of P.aeruginosa CFUs. When infected with P.entomophila however, male and female Oregon R flies differed in the number of bacteria shed in a time-dependent manner.
Males and females died from P.aeruginosa and P.entomophila at different rates. By comparison, mutant flies that lack the protective peritrophic matrix in the gut epithelium die faster, as do mutants which lack a functional IMD immune pathway. After watching this video, you should have a good understanding of how to infect Drosophila orally with bacteria, and to measure key aspects of the resulting pathology, such as lifespan and bacterial shedding.
Once mastered, this technique can be done around six to eight hours, over a course of two days, if performed properly. Following this procedure, other methods such as qRT-PCR can be performed to answer additional questions, such as how infection changes immune gene expression. Don't forget, working with bacterial pathogens can be extremely hazardous, and precautions such as wearing the appropriate personal protective equipment and working within a laminar flow hood, should always be taken when performing these procedures.
