Perspex

Forced alternation tests were conducted using a symmetrical Y-maze made of a grey steel bottom plate with grey Perspex^® walls (Stoelting Co, Wood Dale, IL). Each arm of the Y-maze was 35 cm long, 5 cm wide, and 10 cm high, and the wall at the end of each arm was marked with a different black and white pattern. To reduce anxiety in the animals, light in the testing area was dimmed to 30 ± 5 lux.
The protocol for the forced alternation test was modified from Melnikova et al. [21 (link)]. Mice were handled for three days before testing. The test consisted of a 5 min sample trial (T1) followed by a 5 min retrieval trial (T2). For the scopolamine experiment, mice were dosed with scopolamine or vehicle 30 min before T1. In T1, the mouse was placed into the end of the start arm, facing the wall and away from the center. The mouse was then allowed to explore two arms of the Y-maze, while entry into the third arm was blocked. After the sample trial, the mouse was returned to its home cage for a 30 min inter-trial interval. In T2, the block in arm 3 was removed, the mouse was again placed into the start arm, and then allowed to access all three arms of the maze. If a mouse climbed on the maze wall, it was immediately returned into the abandoned maze arm. After each animal and between T1 and T2, the maze was wiped with a Quatricide^® dilution to prevent odor cues. An arm entry was recorded when 85% of a mouse’s body entered the arm. Time in Novel Arm [%] was defined as the time spent in the novel arm divided by the time spent in all arms during the first minute of the retrieval trial T2. Forced Alternation [%] was defined as the percent of mice entering first the novel arm during T2 [22 (link)]. Mice with less than three arm entries in the first minute of T2 were excluded from the analysis.

After habituation to the laboratory (the animals were not handled during this time), each cage of two mice were randomly assigned (via random number generator) to one of two treatment groups, tail or tunnel handled (n = 16 per group). Mice were then only handled by their designated method (tail or tunnel handled) by the same handler wearing nitrile gloves, which were were rubbed in soiling bedding before each handling session (from mice of the same sex and strain) and a laboratory coat that was contaminated with mouse scent^{13 ,14 (link)}. Tail handling involved grasping a mouse at the base of its tail using the thumb and forefinger, and then lifting onto the sleeve of the laboratory coat for 30 seconds before being returned to its home cage. For tunnel handling, the mouse was guided into the Perspex tunnel, and lifted above the cage and held for 30 seconds. For the first two days, the handler’s hands were loosely cupped over the ends of the tunnel to prevent escape. Mice were handled twice daily for 30 seconds, 60 seconds apart, for the first nine days. Prior to handling, the nesting material (care was taken not to disrupt the structure) and home cage tunnel were removed. This procedure was also conducted once weekly to coincide with the drinking experiments (days 17, 24 and 31). For routine husbandry practices, such as cage cleaning, mice were captured and transferred using their designated handling method either on the sleeve for tail handled mice, or in the tunnel for tunnel handled mice. The same protocol was used when transferring mice to behavioural tests, i.e. the elevated plus maze, open field test and sucrose drinking chambers.

All training and experimental procedures were conducted in eight rodent touchscreen operant chambers (Bussey-Saksida Rat Touchscreen Chamber, Lafayette Instruments) and ABET II software for touchscreens was used for experimental control and data recording (Lafayette Instruments). Each touchscreen chamber (21.6 × 17.8 × 12.7 cm) was housed within a sound-attenuating box (Med Associates Inc.) and consisted of a touchscreen monitor (15.0-inch, screen resolution 1024 × 768), fan (providing ventilation and white noise), stainless steel grid floor, tone generator, house light (LED), magazine unit (with light and infrared beam to detect entries), and reward dispenser. Liquid reward (Ensure Light Vanilla) was used. The inner chambers were composed of three black plastic walls, trapezoidal in shape to help guide the focus of the animal toward the touchscreen and reward delivery area. A black Perspex “mask” (h 38 × 28 cm) was placed over the touchscreen monitor with three response windows (h 15 × 6 cm) cut into it. Response windows were 1.5 cm apart, while the outer response windows were 4.5 cm from the edge of the mask. Attached at a 90° angle 16 cm above the stainless-steel grid floor was a spring-hinged “shelf” (d 6 cm, w 20.5 cm). The positioning of the shelf required rats to stop and rear up toward the screen to explicitly facilitate attention toward touchscreen stimuli. All touchscreen procedures including presentation of stimuli and data collection were performed using the ABET II Touchscreen software (Lafayette Instrument Co) and WhiskerServer interface (Cambridge University Technical Services Ltd.) installed on a Windows PC.

For D. longicaudata and P. cosyrae parasitization assays, B. dorsalis larvae were used. Briefly, B. dorsalis embryos were dechorionated and raised as described above for each experimental group until the larvae reached the 2nd instar stage. Using soft forceps, a set of a hundred 2nd instar B. dorsalis larvae were randomly selected from each fly line and individually transferred to larval oviposition units containing autoclaved carrot diets [56 (link)] (modified by excluding nipagen). The carrot diets for the BMALs and axenic and symbiotic lines were supplemented with 500 μL (1 × 10⁸) of the respective bacterial species, distilled water, or a liquid diet previously fed on by larvae with an intact microbiome, respectively. The oviposition units were transferred to a Perspex cage (12 × 12 × 12 cm) holding 10 D. longicaudata or P. cosyrae mated female parasitoids for parasitization. In a recent study, Gwokyalya et al. [22 (link)] demonstrated that D. longicaudata achieved oviposition above 90%, whereas P. cosyrae achieved similar oviposition rates when host exposure was carried out for six hours. Consequently, in this study, exposure to D. longicaudata was implemented for two hours, while exposure to P. cosyrae was carried out for six hours. Post-parasitization, the host larvae were retrieved from the oviposition units and transferred to the autoclaved carrot diets. The carrot diets were supplemented with 500 µL of the respective bacteria inoculum for the BMALs, a regular liquid larval diet for the symbiotic lines, or distilled water for the axenic lines. The host larvae were left to feed and subsequently pupariate. The puparia were transferred to four-liter transparent lunch boxes (18 × 11 × 15 cm) covered with a cotton mesh and monitored until fly and/or parasitoid emergence.
Fopius arisanus parasitization assays were conducted using B. dorsalis eggs. Briefly, 150 dechorionated embryos were transferred to a filter paper laid on an agar plate and moistened with a 1000 µL (1 × 10⁸) inoculum of the respective bacteria species for each BMAL. For the axenic lines, distilled water was used to moisten the filter paper, whereas, for the symbiotic lines, 1000 µL of the liquid diet used to raise normal laboratory flies was used. The agar plates were incubated for one hour (to allow for efficient bacterial infection in mono-associated and symbiotic lines), after which the oviposition units were transferred to individual Perspex cages (12 × 12 × 12 cm) containing 10 mated F. arisanus females for parasitization, for six hours. Post-parasitization, B. dorsalis eggs were retrieved from the oviposition units and transferred to autoclaved liquid larval diets [55 (link)] (excluding streptomycin and nipagen) supplemented with 500 µL of the respective bacteria inoculum for the BMALs, a normal larval liquid diet for the symbiotic lines, or distilled water for the axenic lines and left to feed and subsequently pupariate. Consequently, the puparia were transferred to transparent 4-liter lunch boxes (18 × 11 × 15 cm) covered with a cotton mesh and monitored for fly and/or parasitoid emergence.
The parasitization experiments for each fly line and parasitoid were conducted individually in sterile conditions. All the wasps used in these assays underwent parasitization by exposing them to bacteria-free B. dorsalis larvae or eggs prior to parasitization assays. The wasps that did not exhibit oviposition behavior within 15 min were removed and replaced with new ones. All fly lines and parasitoids were kept at 25–27 °C, 60–70% humidity, and 12:12 day:light photoperiod.