Fly Strains
Fruit flies were cultured in incubators at a temperature of 25℃, with humidity ranging between 40% ~ 60%, under a 12-hour light-dark cycle. The lights were on from 8:00 am to 8:00 pm, and off from 8:00 pm to 8:00 am. The wild-type strain w1118 flies, obtained from the Vienna Drosophila Resource Center (VDRC), were used as a standard wild-type strain, with the FlyBase ID: FBgn0003996 CG2759.
The Tracking System
To track the locomotion levels of fruit flies under different test conditions, we captured their movement trajectories using our custom-developed system. We employed the mrobo D3 night vision HD sports camera to record the movement of the fruit fly, with videos output in AVI format, resolution of 1920*1080P, and a frame rate of 24 fps. The video setup included eight evenly spaced test tubes, each containing a single fly, separated by dividers to prevent interference. For efficient tracking, videos were edited eight separate clips, each featuring one test tube, converted to 1920i resolution in MP4 format, and muted. Subsequently, movement tracking was conducted using a Python 3.5 script that utilized optical flow techniques to track and record the trajectory of the fruit fly. This was achieved by analyzing the visual displacement of objects between two consecutive frames, capturing the position changes and accurately plotting their movement paths as x and y coordinates in pixels.
The experimental design
To explore the impact of various mating systems on fruit fly locomotion, we carefully selected healthy virgin male and female fruit flies from the w1118 white-eyed strain, known for its standard genetic background, and placed them in conical flasks filled with fresh culture medium for breeding. After the initial batch of pupae emerged, adult flies were removed, and a thorough process was initiated, wherein newly hatched virgin flies were sorted into fresh test tube cultures every six hours. Each fly was placed in its own tube and labeled. This process continued until there were no further observations of emerging virgin flies.
In the preliminary phase of our experiment, we selected 4 virgin male and 4 female flies. The pre-experiment phase started one day after the flies had emerged, with each group being carefully transferred to individual test tubes. These tubes were then placed in an observation device designed to simulate natural environmental conditions with a lighting schedule that mirrored the natural day cycle: lights on from 8:00 am to 8:00 pm, and lights off from 8:00 pm to 8:00 am. We tracked the flight paths of the flies within these tubes over a 24-hour period. Subsequently, we utilized a Python 3.5 script to quantify their activity levels and movement speeds, specifically identifying the 1-hour window where fly activity was at its peak for closer observation in subsequent experiments.
For the main experimental phase, the flies were grouped according to their mating system into six conditions: male virgin (males housed in pairs without females), female virgin (females housed in pairs without males), monogamy (one male to one female), serial monogamy (males and females rotated among partners), male polygamy (one male with multiple females), and female polygamy (one female with multiple males) (Fig. 1). Each group was placed in same environmental conditions and cultured for seven days, with their test tubes being changed daily. Specific interventions were then applied to each group: for the serial monogamy group, female flies were rotated daily to a new test tube, effectively changing their male partners. In the polygamy groups, new unmated flies of the opposite sex were introduced daily, and the previous day’s mates were removed.
Group 1, the male virgin group, had two male virgin flies per tube. Group 2, the female virgin group, contained two female virgin flies per tube. Group 3, the monogamy group, contained one male and one female fly per tube. Group 4, the serial monogamy group, started with one unmated male and female per tube, with female flies rotated to the next tube daily. Group 5, the male polygamy group, started with one unmated male and one unmated female per tube. Each day, a new unmated female was introduced, while the female from the previous day was removed. Group 6, the female polygamy group, followed a similar protocol with unmated males introduced daily. All groups were cultured under identical conditions for 7 days with daily tube changes.
After the seven-day culture period, each fruit fly was isolated in individual test tubes and cultured for an additional six days, with the culture medium being refreshed every 48 hours. During this period, we recorded the daily activity levels and average movement speeds of each fly.
Data Analysis
Our study aims to explore the effects of different mating systems on the locomotion of fruit flies. Therefore, based on the previous researches (Chi et al. 2020; Han et al. 2024, 2021a, b), we designed behavioral measurement and analysis methods to quantify indices of locomotion. We first recorded the activity of virgin male and female flies in test tubes over a day using our custom-developed system and employed a Python 3.5 script to capture the position of fruit flies in every frame of the video. The positions were recorded as x and y coordinates in pixels to track their movement trajectories, with periods of inactivity lasting over five minutes being noted as sleep duration. Further, we documented the total sleep duration of each virgin fly every 30 minutes for statistical analysis.
To analyze the locomotion level of fruit flies, we defined activity level and average movement speed, which quantify the ability of fruit flies to move over time. The activity level is defined as the percentage of points at which the fruit fly moves within a unit of time relative to all captured points of the fruit fly, while the average movement speed is calculated as the total distance moved divided by the unit of time.
Statistical Analysis
In the analysis of data across all groups, activity levels and average movement speed that fell more than two standard deviations below the group's mean were considered outliers and were excluded from the analysis. Statistical analyses were performed using Statistical Product and Service Solutions version 22.0 (SPSS 22.0). Descriptive statistics were utilized to examine the sleep duration of fruit flies at various times throughout the day. Independent sample T-test were employed to assess the activity levels and average movement speeds of virgin male fruit flies and virgin female fruit flies during the initial measurement. Mixed variance analysis was applied to investigate the differences in locomotion levels at different times under various mating systems among fruit flies.