Plexiglas

Behavioral testing apparatus are shown in Figure 1. Mice were randomly assigned to be tested exclusively on either the EZM or EPM, at either daily or weekly intervals for a total of five exposures to the apparatus. Experimental groups were represented in approximately equal numbers in several cohorts. The numbers of male and female mice tested in each maze and at each testing interval are shown in Table 1. EZM and EPM testing took place in the mornings and in the same room. The apparatus were obtained from Stoelting (Wood Dale, IL, USA) and both were elevated 50 cm above the floor. The EZM (Figure 1A) is an annular dark gray platform (60 cm in diameter) constructed of aluminum divided into four equal quadrants. Two opposite quadrants were “open”; the remaining two “closed” quadrants were surrounded by 16 cm high dark, opaque walls. Outer walls were constructed of dark gray plastic, inner walls were black Plexiglas. Quadrant lanes were 5 cm in width. The EPM (Figure 1B), also constructed of dark gray aluminum, consisted of two open arms (5 cm in width, 35 cm in length); perpendicular to the open arms were two closed arms of the same dimensions with opaque, dark gray plastic walls 16 cm high. The four arms met in a 5-cm center square region. Additional illumination for both mazes was provided by overhead fluorescent lamps; light levels in the open and closed regions of both mazes were approximately 1600 Lux and 200 Lux, respectively. The “open” regions of both mazes were surrounded by an edge approximately 1 cm high.
To start the EZM test, mice were placed at a randomly chosen boundary between an open and a closed zone, facing the inside of the closed zone. For EPM testing, mice were placed in the center of the maze, facing the inside of a closed arm. The tests were 5 min in duration. An overhead camera linked to a computer with Any-Maze software (Stoelting) tracked the position of the mouse and calculated the time spent in the open zones of the mazes, and the distance traveled during the test.

Black freshwater sediment was retrieved, in June 2019 (TS1) and October 2020 (TS2), from a pond at Aarhus University campus (Vennelyst Park), Denmark (56.164672, 10.207908) at a water depth of 0.5–1 m. The sediment was stored with overlying water at 15°C for 2 weeks.
Before inoculation, sediment was homogenized, sieved (pore size: 0.5 mm) and autoclaved in 2 L bottles for 20 min as described in Thorup et al. (2021) (link) and Marzocchi et al. (2022) (link). Cooled down sediment (15°C) was distributed into 20 and 10 ethanol-cleaned Plexiglas core liners that were closed with a rubber stopper at the bottom. After 24 h settling time, the stoppers were pushed upwards to align the sediment surface with the core liner edge. The cores were inoculated by transferring a clump of sediment from a two-week-old, pre-grown single-strain enrichment culture of Ca. Electronema aureum GS (Thorup et al., 2021 (link)) and submerged in an aquarium with autoclaved tap water. The aquarium was covered with aluminum foil to prevent algae formation, equipped with aeration and a lid to prevent excessive evaporation, and kept at 15°C. Overlying water was replenished and refreshed several times during the incubation periods.
During the first time series (TS1), O₂, pH, and EP profiles were measured combined with 16S rRNA sequencing and microscopy observations over 80 days, due to technical issues, the videos could not be used for flocking observations. The sequencing and microscopy observations were repeated in the second time series (TS2) with O₂ and EP measurements over 81 days, and additionally the pH was measured on day 81. Following microsensor measurements (of both TS1 and TS2), 1–2 of the measured cores were sliced based on geochemical zone: the oxic layer (determined by the oxygen penetration depth) and the anoxic layer (below the oxygen penetration depth). Samples for light microscopy and 16S rRNA amplicon sequencing were taken from the anoxic layer of each core, of which the latter were frozen at −80°C until RNA extraction.

The social choice test was carried out in a three-chambered apparatus, as previously described [33 (link),71 (link),72 (link)], that consisted of a center chamber and two end chambers. Before the start of the test and in a counter-balanced sequence, one end chamber was designated the social chamber, into which a stimulus mouse would be introduced, and the other end chamber was designed the nonsocial chamber. Two identical, clear Plexiglas cylinders with multiple holes to allow for air exchange were placed in each end chamber. In the habituation phase of the test (Phase 1), the experimental mouse freely explores the arena with empty cue cylinders in place for 10 min. In the social choice phase of the test (Phase 2), an age-matched stimulus mouse (M1) (adult, gonadectomized A/J mice) was placed in the cylinder in the social chamber while an inanimate object was simultaneously placed into the other cylinder in the nonsocial chamber. The social novelty phase immediately followed. In the social novelty phase (phase 3), the object used in phase 2 was replaced by a novel mouse (M2). The experimental mouse was tracked during a 10 min trial as it explores M2 and the familiarized mouse (M1) used in the choice phase. Image analysis software (ANY-maze) was used to determine the time of cue exploration and visits to each cue (snout of experimental mouse within 1 cm of cue cylinder) in all phases. The data was verified with manual video review and scoring.