Infections
The pathogen used in our experiments, P. entomophila , is a naturally-occurring gram-negative bacteria isolated from D. melanogaster in Guadeloupe (Vodovar et al. , 2005; Liehl et al. , 2006). It is acquired during feeding and at high doses kills about 60% of D. melanogaster adults within 72 hours and almost 70% of larvae in 48 hours (Liehl et al. , 2006). It has been found to elicit both local and systemic immune responses involving a range of host responses including the secretion of specific anti-microbial peptides, repair and regeneration of epithelial cells in the gut as a result of damage caused by the pathogen (Vodovar et al. , 2005; Liehl et al. , 2006), and leads to large scale changes in gene expression in response to this pathogen (Chakrabarti et al. , 2012). This system has been used to study the genetic basis of immunity (Chakrabarti et al. , 2012; Neyen et al. , 2014; Bou Sleimanet al. , 2015) as well as in an evolutionary context in work looking at life history trade-offs (Vijendravarma et al. , 2015) and sexual selection (Joye & Kawecki, 2019).
We obtained an isolate of P. entomophila from Bruno Lemaitre (EPFL). Bacteria were plated from glycerol stocks 3 days prior to infection on standard LB-agar plates supplemented with 1% milk and grown for two days at room temperature. On the day before the infection, a single colony was transferred to a 50ml Erlenmeyer pre-culture flask with 12.5ml LB and incubated for 8 hours in a shaking incubator at 29°C and 180rpm. The pre-culture flask was then transferred to a 2L Erlenmeyer flask with 400ml LB (or 1L Erlenmeyer with 200ml LB) and the culture was incubated overnight in the same shaking incubator at 29°C and 180rpm. On the next day, the bacterial culture was centrifuged at 2500 g at 4°C for 20 min. The pellet was re-suspended and mixed with sucrose and water to obtain a final infection cocktail with an OD of 300. The sham treatment was performed with a 2.5% sucrose solution.
Oral infection was performed as previously described (Neyen et al. , 2014). Flies were first starved for 4 hours and then transferred to a vial with a filter paper layered over food and soaked with 150µl of the bacterial cocktail. Males were left in these vials for 24-26 hours after which they were transferred to interaction vials with females. Dead flies were counted at 2, 4, 20 and 24 hours after pathogen exposure.