DOI: 10.5061/dryad.76hdr7szw
title: Data from: Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria

description: 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.
description: <em>Bacterial infection: </em>The bacterial strains used in this study include
 <em>Serratia marcescens (DB11)</em>, <em>Staphylococcus
 aureus (SH1000)</em>, <em>Listeria monocytogenes
 </em>(EGD-e), <em>Escherichia</em> <em>coli
 (DH5α)</em>, <em>Pectinobacterium carotovorum carotovorum 15
 (ECC15) </em>and <em>Micrococcus luteus </em>(clinical
 isolate, gift from Prof William Wade, King's College
 London)<em>. </em>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.<br><br><em>Survival assay:</em>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.<br><br><em>Behavioural assays and parameters:
 </em>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<em>, </em>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<em>, </em>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).<br><br><em>Locomotion Assay:</em> 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. 
description: 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.