Dataset: Schooling behavior in Atlantic silverside (Menidia menidia) larvae reared at contasting pCO2 conditions in a laboratory experiment conducted with embryos from adults collected in Long Island Sound in 2022

Spawning Atlantic silversides (Menidia menidia) were caught on May 15th, 2022 in Mumford Cove (41°19’25”N 72°01’07”W). Fish were captured using a beach seine (30 × 2m) and were transported to the University of Connecticut’s Rankin Seawater Laboratory, separated by sex, and held at 20°C in 80-l aerated tanks. The following day, fish were strip-spawned (following Murray et al., 2014, 2017; Malvezzi et al., 2015), and within one hour embryos were randomly distributed into 20-l replicate rearing containers. 

Experimental Design:

Each container was equipped with 4 mesh-covered holes (7 cm diameter, 150 µm mesh) to allow water exchange with the individual surrounding 600-l tanks in the Automatic Larval Fish Rearing System (ALFiRiS). This custom-built system consists of nine such recirculation units that are independently controlled for pH and temperature via a customized LabView program (Baumann et al., 2022; Zavell and Baumann, 2024). ALFiRiS measures conditions once per hour via sampling pumps connected to a central pH sensor (Hach pHD digital electrode) that is calibrated weekly using NIST 2-point pH references. As needed, the system automatically adjusts pH /pCO_² levels by triggering solenoid valves to inject standardized amounts of 100% bone dry CO₂ (to decrease pH) or CO₂-stripped air (to increase pH), while maintaining desired temperatures via computer-controlled submersible heaters (Finnex Deluxe Titanium, 800W) or in-line chillers (DeltaStar ⅓ horsepower). All trials experiments were conducted at 22°C to resemble late spring conditions at Mumford Cove normally experienced by spawning silversides and their larvae (Murray and Baumann, 2018).

Two experiments were done to quantify schooling and cranial neuromast development. In our schooling ontogeny experiment we reared silversides from fertilization to 25 days post hatch (dph) at three CO₂ levels, while repeatedly conducting standardized burst photography to identify larval positions – and subsequently larval distance distributions - in each rearing container. This simple but robust, quantitative, and observer-unbiased metric showed ontogenetic changes that clearly differed between CO₂ treatments. Three CO₂ treatments were used, referred to hereafter as control (8.15 pH units, ~400 µatm CO₂), elevated (7.45pH units, ~2200 µatm CO₂), or extreme (7.20 pH units, ~4200 µatm CO₂) CO₂ conditions. The control CO₂ treatment is characteristic of the average local surface ocean and of coastal systems at the start of the silverside spawning season in spring (Murray et al., 2014). The elevated CO₂ level represents the maximum predicted open ocean CO₂ for the next 300 years (Caldeira and Wickett, 2005), and it is both a common benchmark in ocean acidification studies as well as a level already observed in productive coastal systems during summer (Baumann et al., 2015). The extreme CO₂ level is rarely reached yet in contemporary systems (Baumann et al., 2015), but may become more common in productive nearshore habitats under future climate and eutrophication scenarios (Wallace et al., 2014). 

Actual CO₂ levels were determined biweekly by measuring total alkalinity (A_T; Murray and Baumann, 2018) in 300 mL samples of filtered seawater (10 µm) taken from each tank. Salinity was measured concurrently using a handheld refractometer. Endpoint titrations (G20 Potentiometric Titrator, Mettler Toledo) were calibrated with Dr. Andrew Dickson’s certified reference material for AT in seawater (UCSD Scripps Institution of Marine Science; Dickson et al., 2003) and titration accuracy was (±1%). Measured temperature, salinity, pH, and AT were then used to calculate CO2 (CO2SYS V2.1; https://cdiac.ess-dive.lbl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/) using K1, K2 constants from Mehrbach et al. (1973), refitted by Dickson & Millero (1987), and Dickson (1990) for potassium bisulfate (KHSO₄). 

Upon hatch, larvae were fed both a powdered weaning diet (Otohime Marine Fish Diet, size A1, Reed Mariculture, Campbell, CA, USA) and newly hatched brine shrimp nauplii ad libitum (Artemia salinia, San Francisco Strain, brineshrimpdirect.com, Ogdon, UT, USA). During the remainder of each experiment larvae were fed daily ad libitum rations of newly hatched nauplii. Ammonia was measured daily (API Saltwater Test Kit) to assure that levels remained < 0.25 ppm. Rearing containers were siphoned daily to remove uneaten food and detritus, and 25% partial water changes were conducted weekly. Salinity (27 – 31 psu) and light conditions (15 h light: 9 h dark) reflected optimal levels for growth for this species (Middaugh et al., 1987). Throughout both experiments, sampled larvae were euthanized and fixed in cold (2-4°C) 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). 48 hours following fixation, larvae were transferred to 70% ethanol in deionized water, which was changed twice to remove excess PFA.

Schooling Ontogeny Experiment: 

To address the effects of ocean acidification on schooling, a schooling experiment quantified larval behavior under the three different CO₂ conditions. Approximately 500 embryos were reared to hatch in one 20-l rearing container for each of 3 ALFiRiS units tanks per CO₂ treatment (except for the extreme CO₂ treatment, where a fungal outbreak rendered one unit unusable). Hatching occurred 8 days post fertilization (May 23rd, 2022). At 1 day post hatch (dph), 50 larvae were randomly distributed into each of two ‘schooling’ containers per ALFiRiS unit. Schooling containers were the same as all other containers, but had one additional, mesh-covered hole located 2 cm above the container bottom to drain excess water during schooling trials. Therefore, the control and elevated CO₂ treatments each had 6 schooling containers (2 per ALFiRiS unit) and 3 rearing containers (1 per ALFiRiS unit). In the extreme treatment, we added a third schooling container per ALFiRiS unit (total of 6 schooling and 2 rearing containers).

Daily schooling trials were conducted via burst photography daily from 4 to 14 dph and then 3× a week until the completion of the experiment at 25 dph (16, 18, 21, 23, 25 dph). Each day, each schooling container was carefully lifted and placed on top of a pedestal in the tank to slowly lower water level to a depth of only 2 cm. A custom-built fiberglass plate with an aperture for a camera lens (GoPro Hero 4) was placed 28 cm from the water surface, ensuring consistent resolution and light levels (PAR: 3.61x1012 µE × m^-2 × s^-1  ) while removing any glare on the water’s surface. The entire area of the container was visible in field of view. After a 3-minute acclimation period, photographs were recorded for every 2 seconds for 1 minute  for a total of 30 pictures). At the end of each trial, schooling containers were gently refilled with water. One (1/6) and two (2/6) replicate schooling containers at extreme and elevated CO₂ treatments, respectively, exhibited high larval mortality limiting recordings only through 7 dph. A full set of schooling data was obtained  from 6 (6/6), 4 (4/6), and 5 (5/6) replicate containers in the control, elevated, and extreme CO₂ treatments, respectively. 

Then 15 of the 30 images per replicate schooling container were randomly selected for analysis by a technician blind to the treatments. In each image they manually located the head of every larva using ImagePro Premier® (v. 9.1, Media Cybernetics) and then the x and y coordinates of the detected larvae were used to calculate the distance between all individuals [in pixels; N_Distances = N_larvae*((N_larvae-1)/2)), thus for instance, 50 larvae yielded 1225 distance measurements] (Fig.1). Distance was converted from pixels to mm ( 0.148 mm pixel-1 given the 300 mm diameter of the rearing container. For each image, distances were then binned into 1 mm intervals and used to calculate relative larval distance distributions for each replicate schooling container (i.e., across 15 images) per dph for each CO₂ treatment. Images with fewer than 10 detected larvae were excluded from analysis. In total, 2,196 images containing 41,407 detected larvae were analyzed yielding 417,569 distances for further analysis. At the end of the experiment, larvae were euthanized and fixed. After being euthanized and fixed, larvae were photographed using calibrated images (Nikon SMZ1000; Image-Pro Premier V.9.3.3, Media Cybernetics, Rockville, MD) and measured for standard length (SL, in mm), eye diameter (ED; horizontal diameter of the orbit,, in mm), and lens diameter (LD, in mm), and the ratio of ED:SL was calculated as a proxy for relative visual capacity (Caves et al. 2017, Lisney et al 2020). 

Organism Identifier (LSID, Life Sciences Identifier):

Menidia menidia, urn:lsid:marinespecies.org:taxname:159228