The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. Here, we exploit the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive behaviour. Contrary to expectations, we found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, we show that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection.

Bacterial infection: The bacterial strains used in this study include Serratia marcescens (DB11), Staphylococcus aureus (SH1000), Listeria monocytogenes (EGD-e), Escherichia coli (DH5α), Pectinobacterium carotovorum carotovorum 15 (ECC15) and Micrococcus luteus (clinical isolate, gift from Prof William Wade, King's College London). The bacterial strains were cultured overnight (see extended methods) at and cultures were pelleted by centrifugation at 4500g for 2 minutes. The pellet was diluted in filter-sterilised PBS (phosphate buffered saline) to a defined concentration. 50nl of diluted bacterial solution was injected employing a nano-injector (MPP1-3 Pressure Injector, Applied Scientific Instrumentation) into the abdomen of anaesthetised flies.

Survival assay:Infected flies and controls were placed in groups of 10-15 in vials at 29°C. The number of live flies infected with pathogenic strains was counted at regular intervals until all the infected flies were dead. Flies injected with non-pathogenic strains were counted at regular intervals for 72 hours.

Behavioural assays and parameters: All behavioural experiments were done in between zeitgeber time (ZT) 01 and ZT10 at 25°C. Mating assays were carried out in courtship chambers (20mm in diameter, 5mm in height), which have built-in dividers that allow separation of the flies before the experiment. For single pair mating assay, flies were injected with bacteria or vehicle solution (PBS) and immediately placed in the courtship chamber with food. Before the behavioural measurement began, the uninfected flies of the opposite sex were introduced using a fly aspirator. The dividers were opened before the assay and behaviours were recorded for one hour. For mate choice assays, a focal fly was given a choice between an infected and a healthy (PBS) mate. The infected and healthy flies were marked with acrylic paint 48 hours before experimentation. After injection, both infected and PBS flies were transferred to a courtship chamber with food. The focal fly was aspirated into the chamber before behavioural experimentation and behaviours were recorded for one hour. Courtship index (in experiments with infected males and controls) was measured as the proportion of time the male spends courting from the beginning of courtship until 10 minutes or end of copulation. Mating success was measured as the percentage of flies that mated within one hour. Copulation latency (in experiments with infected females and controls) was measured as the time taken to copulate from the start of courtship. For competitive mating assays, the focal fly’s first mate choice was recorded (i.e., if the fly chose to mate with a healthy or infected mate).

Locomotion Assay: A single fly was aspirated into a circular chamber (2 cm in diameter, 0.5cm in height), and locomotion was recorded for 5 minutes at 60 frames per second using Blackfly S U3-13Y3M camera. Individual trajectories were computed using custom-written Bonsai software. From the trajectories, two parameters were computed: walking speed and total distance travelled. A fly was considered to be walking if the instantaneous speed was greater than 4mm/s. The parameter “walking speed” refers to the average instantaneous speed over 5 minutes. The parameter “total distance” refers to the total Euclidean distance travelled by the fly in 5 minutes. 

Raw Data_Rose_et_al.xlsx file contains all the raw data files for Rose et al. 2022. Summary.R was used for statistical analysis and Figure1.Rmd for figure creation.