Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

1. Collect and Prepare Flies

1. Rear D. melanogaster under the desired experimental conditions. Take care not to overcrowd the flies during rearing and make sure that larval densities are consistent across treatments, since environmental conditions during the larval stage can profoundly affect immune defense phenotypes during adult stage.¹⁵
2. Collect experimental flies 0 - 3 days after eclosion from the pupal case and transfer them onto fresh medium.
3. House the collected flies at a desired temperature (temperatures between 22 °C and 28 °C are generally suitable) until they are aged 5 – 7 days post-eclosion.
   NOTE: This allows sufficient time for the flies to complete metamorphosis and become mature adults, but is well before senescence begins.
4. Sort the desired number of flies into a separate vial prior to infection, again taking care to avoid overcrowding.
   NOTE: Only males are infected in this demonstration but is equally possible to infect females using the procedure described.

2. Culture and Prepare Bacteria

1. Prepare a master plate of the chosen bacteria at least 2 days prior to infection. Streak the bacteria from a 15% glycerol stock stored indefinitely at -80 °C. Store the master plate at 4 °C for up to 2 weeks. Always streak the master plates directly from the frozen glycerol stock. Avoid serial passage of bacteria from plate to plate, as repeated passage in culture can cause bacteria to evolve attenuated virulence.
   NOTE: This example makes use of Providencia rettgeri and Escherichia coli.
2. Use the following procedure to prepare a bacterial suspension for injection.
   1. Grow a 2 ml culture of bacteria by inoculating sterile medium with a single colony isolated from the master plate. Grow the bacteria to stationary phase (e.g. O/N growth at 37 °C).
      NOTE: Both P. rettgeri and E. coli grow well in Luria Broth at temperatures from 20 – 37 °C with gentle shaking.
   2. Once the culture has reached stationary phase, gently pellet cells from approx. 600 µl of the culture in a tabletop centrifuge (3 min at 5,000 x g), discard the supernatant, and resuspend the bacteria in approx. 1,000 µl of sterile phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂PO₄, 1.8 mM KH₂PO₄, pH = 7.4).
   3. Dilute the resuspended cells in sterile PBS to achieve an optical density (OD) appropriate for the bacterium being used, measured with a spectrophotometer as the absorbance at 600 nm. Use this suspension to infect the flies.
      NOTE: A₆₀₀ = 0.1 or 1.0 corresponds respectively to approximately 10⁸ and 10⁹ Providencia rettgeri or E. coli per ml. Because both live and dead bacteria contribute to optical density, it is important to harvest cultures early in stationary phase before bacterial corpses accumulate and distort the relationship between optical density and the number of viable bacteria introduced during infection.

3. Infect the Flies

NOTE: As Drosophila immunity is influenced by circadian rhythm, it is important to perform infections at a similar time of day across experimental replicates.¹⁶

3.1) Using a Nanoinjector

1. Prepare the glass needles for infection.
   1. Prepare a glass needle by pulling a borosilicate glass capillary using a micropipette puller.
   2. Using forceps, break off the tip of the needle to create an opening of approximately 50 µm diameter to allow ejection of liquid.
2. Assemble the injector.
   1. Place the sealing O-ring and then the white spacer (large dimple facing outwards) onto the metal plunger.
   2. Fill the glass needle with mineral oil using a syringe with a 30 G needle.
   3. Put the filled glass needle through the collet and then place the larger O-ring around its base, about 1mm from the blunt end of the needle.
   4. Slide the needle onto the metal plunger and gently screw on the collet until secure.
   5. Eject most of the mineral oil from the injector, but make sure that there is still 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 or bacterial suspension, or between the two liquids.
   6. Set the injector to the desired volume for injection (between 9 nl and 50 nl).
3. Generate wounding controls.
   1. Fill the injector needle with sterile PBS by carefully inserting the tip of the capillary needle in a tube of the media and pressing the “fill” button on the injector.
      NOTE: Sterile bacterial growth media can also be used a wounding control if the experiment requires injection of bacteria suspended in their growth medium. However, because bacterial growth media contains components that may stimulate the immune system or have other effects on the host, an inert carrier such as PBS is preferable.
   2. Anaesthetize the desired number of flies under a light flow of CO₂.
   3. Inject the flies in the anterior abdomen on the ventrolateral surface with the sterile PBS.
      NOTE: It is also possible to inject flies at other sites, such as the sternopleural plate of the thorax, but it is important to keep the injection site consistent within each experiment.¹⁷
   4. Place injected flies into fresh vials with new medium, laying the vials on their side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.
      NOTE: It is recommended to inject the PBS control flies before injecting bacteria to experimental flies so the same needle can be used for both treatments. It will not always be possible to use the same needle for an entire experiment. In that case, it may be desirable to record which needle was used with which flies and to include needle identity as an experimental factor in statistical analysis.
4. Inject the bacterial pathogen.
   1. Eject the remaining sterile media from the injector and refill the same needle with the bacterial suspension.
   2. Repeat the above procedure (3.1.3), now injecting the flies with the bacterial suspension prepared in procedure 2.2.

3.2) With Septic Pinprick

1. Prepare the needle for pricking.
   1. Melt the end of a 200 µl micropipette tip and insert a 0.15 mm insect minutien pin into the molten plastic. Allow the plastic to solidify such that the pin is held in place with approximately 0.5 cm of pin extending from the plastic.
2. Generate wounding controls.
   1. Anaesthetize the desired number of flies under a light flow of CO₂.
   2. Prick the flies in the sternopleural plate of the thorax with the needle, avoiding the attachment sites of the wings and the legs. If necessary, gently remove flies from the minutien pin using soft forceps.
      NOTE: It is also possible to prick the flies at other sites, such as the anterior abdomen on the ventrolateral surface, but it is important to keep the injection site consistent within each experiment.¹⁷ Pricking through the cuticle of the abdomen tends to be more difficult than pricking of the thorax and is therefore less common.
   3. 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 to prevent the flies from becoming stuck to the food.
3. Introduce the bacterial infection.
   1. Anaesthetize the desired number of flies under a light flow of CO₂.
   2. Prick each fly in the same location as the media controls, dipping the tip of the pin into the bacterial suspension prepared in procedure 2.2 before pricking each fly.
   3. 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 to prevent the flies from becoming stuck to the food.

3.3) Assess the Infectious Dose Delivered

1. Infect a set of flies as described above in either procedure 3.1 or 3.2, only instead of returning the flies to food vial to recover from anesthesia, place each fly into a microcentrifuge tube on ice.
2. Add 250 µl PBS to each tube and homogenize the flies either using a pestle or a bead beater.
3. Plate the homogenate on an LB agar plate, either using a spiral plater or a serial dilution.
   1. To plate using serial dilution, transfer each fly homogenate to the first row of a 96 well plate.
   2. Fill each well of the remaining rows with 90 µl of PBS.
   3. Using a multichannel pipette, take 10 µl from the first row containing fly homogenate and dispense into the second row.
   4. Pipette up and down at least 10 times to thoroughly mix, and then take 10 µl and transfer to the third row. Repeat this procedure using the remaining rows.
   5. Starting from the bottom row (most dilute bacteria suspension), use the multichannel pipette to take 10 µl from each well and deposit on an LB plate, taking care that the samples are dispensed as discrete spots that do not touch each other. Repeat until all wells have been sampled from each row, dispensing them in descending order of dilution on the LB plate. 
   6. Leave the plate at RT until the spots have completely soaked into the LB plate.
      NOTE: Drying the LB plate for a few days at RT prior to use is recommended to ensure that the liquid is readily absorbed, minimizing the chances of accidental contact between sample spots.
4. Incubate the plates O/N, taking care not to overgrow the plates so colonies remain small and discrete.
   NOTE: Depending on the bacterial growth medium and incubation temperature used, colonies derived from the fly’s endogenous gut microbiota may eventually appear on the plate. However, most bacteria used for pathogenic infection grow much faster than the gut microbiota on LB agar at 37 °C.
5. Remove plates from the incubator when the experimental bacteria have grown visible colonies (typically 8 – 12 hr), but before Drosophila gut microbiota colonies appear (approximately 36 hr).  For slow growing experimental bacteria, use a selective antibiotic in the LB agar to remove any colonies from the gut microbiota.
6. Count the number of colonies for each homogenate.
   1. For spiral plates, count the colonies that grow using an automated colony counter that can estimate the bacterial load per ml of homogenate based on the number and position of the colonies on the plate.
      NOTE: Spiral counts are most accurate when the bacterial concentration is in the range of 5 x 10² to 5 x 10⁴ bacteria per ml.
   2. For spot plates, manually count the colonies for each fly from whichever dilution contains 30 – 300 colonies and calculate the number of bacteria per ml of original homogenate.

4. Characterize Survivorship of Infection

1. Assay mortality post-injection.
   1. Place infected flies and wounded controls in fresh vials and maintain in an incubator at a desired temperature (25 °C with a 12:12 light:dark cycle is recommended). Do not put more than 15 flies into a single vial as it becomes difficult to count more than 15 living flies.
   2. Count the number of living and dead flies each day, or more frequently if appropriate.
   3. In order to maintain food quality, flip the flies to new food on a regular schedule.
      1. If the vial contains only males, transfer them to fresh vials every three days. If the vial contains females, transfer to fresh vials every two days because larval progeny will liquefy the food, increasing the risk that adult flies may get stuck and resulting in overestimated rates of mortality due to infection.
2. Statistically analyze the data.
   1. Plot survival as a Kaplan-Meier curve, which indicates the probability that an individual will survive to a measured time-point post injection.¹⁸ For survival curves that do not cross, perform pair-wise comparisons using a Log-Rank Test (also called Mantel Cox Test) or perform more complex analyses using a Cox Proportional Hazards Model, which can incorporate several factors and their interactions. For survival curves that cross, perform a stratified Cox analysis.¹⁷

5. Assay Bacterial Load Post-infection

1. Measure bacterial load.
   1. At a prescribed time post-injection, repeat the procedure used to assess infectious dose to determine bacterial load over the course of the infection (see protocol 3.3).
      NOTE: If using the spiral plater, dilute homogenates so that the bacterial suspension falls within a range of 1,000 – 100,000 bacterial per ml.
2. Analyze the data.
   1. If bacterial load is non-normal, either transform the data to better approximate a normal distribution or analyze the data using non-parametric statistical analysis (e.g. Mann-Whitney U test). Use a Box-Cox analysis to find the optimal transformation; natural log or base -10 log transformations are often effective.¹⁹
   2. If the data are sufficiently normally distributed and there are only two treatments being contrasted, perform a t-test to determine whether the two treatments result in different mean bacterial loads. If there are multiple experimental variables or other potentially predictive factors, use Analysis of Variance (ANOVA) to determine which factors are significant predictors of bacterial load.¹⁹

6. Assay Transcriptional Activation of Immune System Genes

1. To coarsely visualize immune activation after infection, infect transgenic flies that express green fluorescent protein (GFP) under the control of a promoter from an antimicrobial peptide gene as previously described.¹⁸
2. View the infected flies under a fluorescence-capable microscope; GFP induction should become visible in the fat body within the first 6 – 10 hr after infection.
3. For more precise quantification of immune activation, use quantitative PCR on cDNA template (qRT-PCR) to measure the abundance of antimicrobial peptide gene transcripts.
   NOTE: The expression of immune genes can be measured at any time after (or before) infection. Generally speaking, induction of immune gene expression begins approximately 4 hr after injection and increases over the next 24 hr.
   1. Anesthetize flies using CO₂.
   2. Place 15 flies into a microfuge tube for each RNA extraction.
      NOTE: It is recommended to collect at least three replicate samples for each treatment condition.
   3. Extract RNA from the flies and generate first-strand cDNA using any of a number of standard procedures or commercial kits. Store RNA at -80 °C. Store cDNA at -20 °C for periods of a few weeks and at -80 °C for long term storage. Prior to RNA extraction, store flies at -80 °C.
   4. Perform quantitative PCR (qPCR) on target genes of interest using the cDNA as a template. Refer to Table 1 for primer sequences to amplify the antibacterial peptide genes Diptericin A, Drosomycin, Defensin, Attacin A, and Metchnikowin as well as the housekeeping control gene rp49 (also known as rpL32).
      NOTE: The genes encoding the antimicrobial peptides Diptericin A and Drosomycin provide very good readouts of D. melanogaster immune activity induced mainly through the Imd and Toll pathways, respectively. In order to control for sample-to-sample variation in RNA yield and the efficiency of reverse transcription, standardize the recorded expression of target genes against a reference housekeeping gene whose expression is not expected to change with infection such as rp49 (also known as rpL32).
   5. Analyze the data.
      1. For rough approximation of expression differences, calculate the ratio of the starting quantity (SQ) value obtained from the test gene (e.g., Diptericin A) relative to the SQ value obtained from the reference gene (e.g., rp49) for each sample (calculated as SQ_–DptA/SQ_–rp49). Scale these ratios to a baseline control treatment such as an uninfected handling control. Arbitrarily set the control expression level equal to 1.0 and visualize experimental treatments as relative fold changes. Estimate the SQ value for each gene against a standard curve generated from a dilution series of known-quantity cDNA.
         NOTE: Statistical analysis of ratios can be complicated, so this approach is better for quick approximation than for rigorous analysis. If PCR efficiency is low, design new primers to improve PCR kinetics if possible. For qPCR reactions with near-perfect efficiency (a doubling of PCR product every cycle), the threshold cycle (C_T) can be substituted for the SQ value.
      2. For simple experiments with optimally efficient amplification, data may be analyzed using the ΔΔC_T method.¹⁹ For each sample, subtract the C_T value of the reference gene (e.g., rp49) from the C_T of the test gene (e.g., Diptericin A) to yield a ΔC_T. Then subtract the ΔC_T of the baseline control from the ΔC_T for each experimental sample to yield a ΔΔC_T value for each sample; this ΔΔC_T is indicative of relative differences in gene expression levels between treatments, where 2^(-ΔΔCT) approximates the fold change in expression.
         NOTE: This analysis can badly misestimate fold-change in expression of the target gene if the PCR of either the test gene or the reference gene has low efficiency.
      3. For the most rigorous analysis, conduct analysis of variance (ANOVA) using the estimated expression of the test gene (e.g. Diptericin A) as the response variable and estimated expression of the control gene (e.g. rp49) and other experimental factors as explanatory variables.
         NOTE: This approach is relatively robust to inefficiency in PCR amplification and allows analysis of more complicated experimental designs.