Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The overall goal of this procedure is to deliver robust and repeatable bacterial infections to drosophila, melanogaster, then to subsequently measure infection severity and quantify the host immune response. This is accomplished by first delivering the bacterial infection into the flies using a nano injector. Post mortality can be tracked post-infection over time, and the systemic bacterial load can be determined at any point by homogenizing infected flies, and measuring the number of bacteria present.

The host immune defense can also be quantified by measuring the expression level of antibacterial peptide genes, ultimately quantification and characterization of bacterial infection. In drosophila, melanogaster can be used to test how genetic and environmental factors alter immune competence of the fly. This method can be used to address key questions in the field of insect immunity, such as how host genetic and environmental factors influence immune capability or how distinct bacteria vary in their infection Kinetics, Although we are using this method to measure immune defense of drosophila melanogaster against bacterial infection, it can also be applied to other insects and can make use of other types of pathogens like viruses or fungi.

A common problem people experience when they're first learning this method is that they may cause too much injury to flies, which can result in excessively high mortality, even in controls. Visual demonstration of this method is critical for learning how to do infection with minimum damage to the fly. Since our host species oph melanogaster is so small, infection must be done under the microscope, which adds to the initial complexity of the technique.

However, we believe that a small demonstration of the technique will render it accessible even to scientists who have only limited experience with flies. To begin the experiment, select a single colony from a previously prepared master plate and inoculate a flask of sterile medium. Grow the bacteria overnight at 37 degrees Celsius to stationary phase.

Once the culture has reached stationary phase, gently pellet the cells from 600 microliters of the culture in a tabletop centrifuge for three minutes at 5, 000 times. G, then discard the Rena and Resus. Suspend the bacteria in 1000 microliters of sterile phosphate buffered saline or PBS.

Next, dilute the resuspended cells in sterile PBS to achieve the desired 600 nanometer absorbance and use this suspension to infect the flies lies. Prepare a glass needle by pulling a borrow silicate glass capillary and use forceps to break off the tip of the needle to create a 50 micrometer diameter opening. Next place the ceiling O-ring and the white spacer with the large dimple facing outward onto the metal plunger of the injector.

Fill the glass needle with mineral oil using a syringe with a 30 gauge needle. Then place the filled glass needle through the collet and place the larger O-ring around its base one millimeter from the blunt end of the needle. Slide the needle onto the metal plunger and gently screw on the collet until it is secure.

Dispel the mineral oil from the injector, leaving a small volume of oil in the needle to act as a barrier between the injector and the bacterial suspension. Make sure that there are no air bubbles within the mineral oil, bacterial suspension, or between the two liquids. Then set the injector to the desired volume for injection.

Carefully insert the tip of the capillary needle in a tube of sterile PBS and press the fill button on the injector to fill the injector needle to generate wounding controls. Inject anesthetized flies with sterile PBS in the anterior abdomen on the ventral lateral surface place the injected flies into fresh vials with new media and lay the vials on their side to prevent the flies from becoming stuck to the food before they recover from their anesthesia. Next, eject the remaining sterile media from the injector and refill the same needle with the bacterial suspension.

Repeat the procedure and inject the flies with the bacterial suspension. Alternatively, the infection can be delivered by a septic pin prick. To prepare the infecting pin, melt the end of a 200 microliter micro pipette tip and insert a 0.15 millimeter insect mnuchin pin into the molten plastic.

Allow the plastic to solidify so that the pin is held in place with 0.5 centimeters of pin extending from the plastic anesthetize flies with carbon dioxide and prick the flies in the sternal pleural plate of the thorax with the needle, avoiding the attachment sites of the wings and the legs. Remove the flies from the minutia pin using soft forceps. Make sure to dip the pin into the bacterial suspension between pricking each fly.

Place the pricked flies into a fresh vial with new fly medium laying the vial on its side until all of the flies have recovered from the anesthesia. Place a subset of the infected flies into individual micro centrifuge tubes on ice. Add 250 microliters of PBS to each tube and use a pestle to homogenize the flies.

Next plate the homogenate on an LB aerate using a spiral plater. Alternatively to plate the homogenate by serial dilution. Transfer each fly homogenate to the first row of a 96.

Well plate and fill each well of the remaining rows with 90 microliters of PBS using a multi-channel pipette. Take 10 microliters from the first row containing fly homogenate and dispense the homogenate into the second row. Pipe it up and down at least 10 times to thoroughly mix the homogenate.

Then take 10 microliters of the homogenate and transfer it to the next row. Repeat this procedure using the remaining rows starting from the bottom row. Use the multi-channel pipette to take 10 microliters from each well and deposit it on an LB plate.

Ensure that the samples are dispensed as discrete spots that do not touch each other. Repeat this step until all wells have been sampled from each row, dispensing them in descending order of dilution on the LB plate. Whichever plating method is used, take care not to overgrow the plates so colonies remain small and discreet.

Remove the plate from the incubator. Once the experimental bacteria have grown visible colonies, but before colonies from the drosophila, gut microbiota become visible approximately 24 hours later for spiral plates. Count the colonies that grow using an automated colony counter that can estimate the bacterial load per milliliter of homogenate based on the number and position of the colonies on the plate.

For spot plates, manually count the colonies for each fly from whichever dilution contains 30 to 300 colonies and calculate the number of bacteria per milliliter of original homogenate. Flies were injected with 50 nanoliters of bacterial suspensions covering a range of optical densities and immediately homogenized and plated infection dose varies with the optical density of the bacterial suspension used for injection. Initial bacterial loads strongly correlates with initial OD injected.

This protocol enables the observation of different types of infections. Flies were infected with providencia rei, which can cause a chronic sub lethal infection that persists for 20 days or longer. Flies infected with coli will clear the infection within six hours.Post-infection.

Induction of the immune system can be estimated through RNA isolation and subsequent Q rtpcr R of antibacterial peptide transcript analogously, but less quantitatively flies expressing GFP under the control of antimicrobial peptide. Gene promoters can be used to visualize induction of the immune system Once mastered. This technique can allow for the infection of hundreds of flies per hour.

Any number of phenotypes could conceivably be measured in the flies after they're infected, including fity learning ability, metabolic status, or virtually any other trait that can be imagined. After watching this video, you should have a good understanding of how to deliver robust and repeatable bacterial infections to drosophila, melanogaster, and how to subsequently measure infection severity and quantify the host immune response. Don't forget that certain bacteria can post human health risks.

Make sure that proper biosafety procedures are followed and that adequate containment measures are in place to keep infected flies from escaping. Thanks for watching and good luck with your experiments.