Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.


Introduction
The fruit fly (also known as the vinegar fly), D. melanogaster, has been extensively used as a model organism for infection and immunity against a variety of pathogens 1,2 . This work has offered fundamental insights into the physiological consequences of infection and was also pioneering in unraveling the molecular pathways underlying the host immune response against parasitoid, bacterial, fungal, and viral infections. This knowledge is not only useful to understand the innate immune response of insects and other invertebrates, but because many of the immune mechanisms are evolutionarily conserved between insects and mammals, Drosophila has also spurred the discovery of major immune mechanisms in mammals, including humans 3 .
Most work on Drosophila infection and immunity has focused on systemic infections, using inoculation methods that deliver pathogens directly into the body of the insect by pricking or injection 4,5,6 . The advantage of these methods in allowing the delivery of a controlled infectious dose is clear and supported by a large body of work on systemic infections. However, many naturally occurring bacterial pathogens of D. melanogaster are acquired through feeding on decomposing organic matter where gut immunocompetence plays a significant role in host defence 7,8,9,10,11,12,13,14,15 . Experiments that employ systemic infections bypass these defenses, and, therefore, provide an altogether different picture of how insects mount defenses against natural pathogens. This is especially relevant if the aim of the work is to test predictions about the ecology and evolution of infection, where the use of natural pathogens and routes of infection is important 16,17 . Recent work has highlighted how the route taken by pathogens significantly affects disease outcome 18,19 , elicits distinct immune pathways 20,21 , can determine the protective effect of inherited endosymbionts 16 , and may even play an important role in the evolution of host defenses 17 .
Another reason to employ oral routes of infection is that it allows the investigation of the variation in pathogen transmission by measuring bacterial shedding during fecal excretion following oral infection 22,23,24 . Understanding the sources of host heterogeneity in disease transmission is challenging in natural populations 25,26 , but measuring components of transmission, such as pathogen shedding, under controlled laboratory conditions offers a useful alternative approach 27 . By feeding flies bacteria and measuring bacterial shedding under a variety of genetic and environmental contexts in controlled experimental conditions, it is possible to identify sources of variation in transmission among hosts. expressed in colony forming units (CFUs). Finally, because gut immunocompetence results from a combination of epithelial barrier and humoral responses, we also measure the survival of fly lines where these defenses are disrupted. Specifically, Drosocrystallin (Dcy) mutants have been previously shown to be more susceptible to oral bacterial infection due to a depleted peritrophic matrix in the gut 29 . We also measure survival in a Relish (Rel) mutant which is impeded from producing antimicrobial peptides against Gram-negative bacteria via the IMD pathway 30 .

Maintain Flies
1. Maintain flies in 23 mL plastic vials containing 7 mL of freshly made Lewis medium (modified from reference 31 ; 1 L triple distilled H 2 O, 6.1 g agar, 93.6 g brown sugar, 68 g maize, 18.7 g instant yeast, 15 mL Tegosept anti-fungal agent) in incubators at 25

Prepare Experimental Flies
1. Collect the eggs of the parent generation in a population/embryo collection cage on a 75 mL apple-agar plate (1 L triple distilled H 2 O, 30 g agar, 33 g sucrose, 330 mL apple juice, 7 mL Tegosept anti-fungal agent) with a yeast paste spread (mix dry yeast with water to a peanut butter-like consistency). Add water-soaked cotton wool to the cage to provide moisture. NOTE: To avoid confounding effects caused by differences in larval rearing density, it is important that experimental flies in different vials are reared in similar densities. The above step is performed to avoid confounding effects. 2. Incubate for 24 h at 25 °C in a 12 h:12 h light:dark cycle until egg-laying has occurred. If there are too few eggs after 24 h, provide a longer habituation period. Replace apple-agar plates and allow egg-laying to occur for a further 24 h. 3. Take egg-laden apple-agar plates from the population cage. Remove the remaining yeast paste and any dead flies from the agar's surface. 4. Submerge the agar in 20 mL of 1x phosphate-buffered saline (PBS) and gently dislodge the eggs from the apple-agar with a fine paintbrush.
While suspended in PBS, transfer the eggs to a 50 mL centrifuge tube and leave for 5 min so the eggs sink to the bottom. NOTE: Most eggs are found on the outer edge of the agar. 5. Remove by cutting the bottom 4 mm of a p1000 filtered pipette tip and use the pipette tip to draw 1 mL of solution, taken from the bottom of the 50 mL centrifuge tube. Transfer this to a 1.5 mL microcentrifuge tube and allow it to settle. NOTE: When pipetting up eggs, snap-releasing the plunger is more efficient than a gentle release. 6. Remove by cutting the bottom 4 mm of a p20 filtered pipette tip. Set the pipette to a desired volume and draw from the bottom of the microcentrifuge tube. NOTE: With practice, a volume of 5 µL contains roughly 100 eggs. 7. Dispense the collected eggs onto the food and leave them to develop for the required amount of time.

Bacterial Culture
1. To grow P. entomophila and P. aeruginosa cultures, inoculate 10 mL of Luria-Bertani (LB) broth with 100 µL of a frozen bacterial stock at 30 °C (P. entomophila) and 37 °C (P. aeruginosa), respectively. Shake at 150 rpm overnight. Ensure that the bacterial culture reaches the saturation phase. 2. To ensure the bacteria used for inoculating the flies are in the exponential phase and rapidly replicating, inoculate the overnight culture into a new subculture, of a desired volume, the following morning. Ensure that the pre-inoculum is 10% of the total volume of the subculture culture. NOTE: Oral infection requires high . It is therefore necessary to grow a substantial volume of bacterial culture so that enough inoculation culture can be produced for the desired dose and experimental size. Calculate how much subculture is needed to produce the required infectious doses using the equation M s V s = M i V i , where M represents a culture's optical density measured at 600 nm (OD 600 ) value and V represents its volume. Subscript letters refer to whether the culture is used as a subculture (s) or an infectious dose (i). 3. Grow this subculture in a 2 L conical flask in a volume such that the subculture's surface falls (at most) just above the beginning of the flask's slope. Do not fill above this mark as it will stunt the growth of bacteria. 4. Ensure the bacteria in this subculture are in the exponential growth phase by measuring the OD every 30 min.
NOTE: This occurs after 3-5 h, where the subculture reaches an OD 600 between 0.6-0.8. 5. Pour equal volumes of this subculture across 50 mL centrifuge tubes and spin the subculture at 2,500 x g for 15 min at 4 °C to pellet the bacteria. Once pelleted, remove and then spin the supernatant again at the above conditions to confirm the removal of the vast majority of bacteria. NOTE: A pellet of negligible size (smaller than 1 mm in height) confirms this. 6. Combine the bacterial pellets of the separate tubes by re-suspending them in 5 mL of subculture supernatant and recombining these solutions in a single 50 mL tube. Spin this concentrated culture at 2,500 x g for 15 min at 4 °C to pellet the bacteria. 7. Remove the supernatant and re-suspend the final bacteria pellet in 5% sucrose water solution. Check the OD and adjust to the desired infectious dose (OD 600 = 100 for P. entomophila 8

Discussion
We present a protocol for reliably orally infecting D. melanogaster with bacterial pathogens. We focus on P. aeruginosa and P. entomophila, but this protocol can easily be adapted to enable infection of other bacterial species, e.g., Serratia marcescens 7 . Key aspects of this protocol will vary between bacterial species. Accordingly, the most efficient infectious dose, corresponding virulence, and host genotype susceptibility should all be considered and ideally tested in pilot studies. Exposing flies to bacterial cultures of a range of optical densities and measuring their infectious dose and survival is an appropriate starting point when working with new bacterial species or fly lines.
Protocol steps such as fly starvation prior to feeding and re-suspending bacterial pellets in 5% sucrose solution are commonplace in oral infection and increase the reliability of bacterial infection during exposure 7,8,9,10 . However, it is important to note that during exposure, flies essentially live on a surface of bacterial culture. In the process of walking on this culture, bacteria will become lodged on the fly's surface, especially on the cuticle or around the bristles 24 . These epicuticular bacteria, do not reflect a successful enteric infection but would still be detected by the fly homogenization and plating. To reduce the potential for false positives, it is essential to surface sterilize flies through immersion in 70% ethanol for up to 1 min.
When considering bacterial shedding rates, oral infection is essential. The number of pathogens a host releases into the environment is often difficult to measure and the internal load is often taken as a proxy for the severity of infection and therefore transmission 26,27 . Measuring bacterial load alongside bacterial shedding allows an examination of the relationship between these two important components of disease severity and spread 38 . One limitation of the method presented is that assaying the internal bacterial load of flies requires destructive sampling. This makes it difficult to investigate longitudinal trends of pathogen growth and clearance within the same individual. However, it is possible to overcome this limitation by destructively sampling cohorts of individuals at different stages of infection, under the assumption that the average microbe load in each cohort reflects the longitudinal pathogen dynamics within any given individual. Bacterial shedding does not suffer from the same limitations, and we offer examples of how shedding can be quantified in a cross-sectional sample, or longitudinally to investigate how shedding changes within an individual over time.
Many host and pathogen traits jointly determine an individual's propensity to transmit disease 25,26,39 . While the significance of these traits likely varies between host-pathogen systems, shedding is likely a major determinant of fecal-oral transmission. The ability to measure bacterial shedding opens the opportunity to test this assumption. Having characterized host-pathogen dynamics in a desired panel of fly lines,