We experimentally examined the ontogeny of schooling and cranial neuromast development under contrasting CO2 conditions in Atlantic silverside larvae (Menidia menidia), an ecologically important forage fish in the Northwest Atlantic. Larvae were produced from wild spawners and reared them until 25 days post hatch (dph; Schooling Exp) or 34 dph (Neuromast Exp) at three contrasting pCO2 conditions (~400, ~2200, ~4200 µatm). The ontogeny of schooling was quantified by recording daily larval positions in their replicate rearing containers using burst-photography, and the distance between all individuals was calculated to derive relative larval distance distributions (rLDD). rLDD captured the transition from non-aggregated (4-6 dph) to increasingly aggregated larval distributions (>7 dph) with a modal peak in rLDDs at ~20 mm. The appearance of the rLLD mode was delayed under high CO2 conditions, suggesting a 1-5 day delay in the onset of schooling relative to CO2 controls

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Curation Notes
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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

This section documents curation actions performed prior to publication review with the submitter, and additional information relevant to understanding and reusing this dataset. It distinguishes changes made to the submitted (meta)data from unresolved issues and/or enhancements that improve future reuse and interoperability.

CURATION ACTIONS PERFORMED ON DATA

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CURATION ACTIONS PERFORMED ON METADATA

- BCO-DMO's standard metadata entry and text formatting steps were performed. See: https://www.bco-dmo.org/how-to/standard-curation-edits

ISSUES POTENTIALLY IMPACTING REUSE

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Coverage: Eastern Long Island Sound, CT, USA

Description from NSF award abstract:
Coastal marine ecosystems provide a number of important services and resources for humans, and at the same time, coastal waters are subject to environmental stressors such as increases in ocean acidification and reductions in dissolved oxygen. The effects of these stressors on coastal marine organisms remain poorly understood because most research to date has examined the sensitivity of species to one factor, but not to more than one in combination. This project will determine how a model fish species, the Atlantic silverside, will respond to observed and predicted levels of dissolved carbon dioxide (CO2) and oxygen (O2). Shorter-term experiments will measure embryo and larval survival, growth, and metabolism, and determine whether parents experiencing stressful conditions produce more robust offspring. Longer-term experiments will study the consequences of ocean acidification over the entire life span by quantifying the effects of high-CO2 conditions on the ratio of males to females, lifetime growth, and reproductive investment. These studies will provide a more comprehensive view of how multiple stressors may impact populations of Atlantic silversides and potentially other important forage fish species. This collaborative project will support and train three graduate students at the University of Connecticut and the Stony Brook University (NY), two institutions that attract students from minority groups. It will also provide a variety of opportunities for undergraduates to participate in research and the public to learn about the study, through summer research projects, incorporation in the "Women in Science and Engineering" program, and interactive displays of environmental data from monitoring buoys. The two early-career investigators are committed to increasing ocean literacy and awareness of NSF-funded research through public talks and presentations.

This project responds to the recognized need for multi-stressor assessments of species sensitivities to anthropogenic environmental change. It will combine environmental monitoring with advanced experimental approaches to characterize early and whole life consequences of acidification and hypoxia in the Atlantic silverside (Menidia menidia), a valued model species and important forage fish along most of the US east coast. Experiments will employ a newly constructed, computer-controlled fish rearing system to allow independent and combined manipulation of seawater pCO2 and dissolved oxygen (DO) content and the application of static and fluctuating pCO2 and DO levels that were chosen to represent contemporary and potential future scenarios in productive coastal habitats. First CO2, DO, and CO2 × DO dependent reaction norms will be quantified for fitness-relevant early life history (ELH) traits including pre- and post-hatch survival, time to hatch, post-hatch growth, by rearing offspring collected from wild adults from fertilization to 20 days post hatch (dph) using a full factorial design of 3 CO2 × 3 DO levels. Second, the effects of tidal and diel CO2 × DO fluctuations of different amplitudes on silverside ELH traits will be quantified. To address knowledge gaps regarding the CO2-sensitivity in this species, laboratory manipulations of adult spawner environments and reciprocal offspring exposure experiments will elucidate the role of transgenerational plasticity as a potential short-term mechanism to cope with changing environments. To better understand the mechanisms of fish early life CO2-sensitivity, the effects of temperature × CO2 on pre- and post-hatch metabolism will be robustly quantified. The final objective is to rear silversides from fertilization to maturity under different CO2 levels and assess potential CO2-effects on sex ratio and whole life growth and fecundity.

Related references:
Gobler, C.J. and Baumann, H. (2016) Hypoxia and acidification in ocean ecosystems: Coupled dynamics and effects on marine life. Biology Letters 12:20150976. doi:10.1098/rsbl.2015.0976

Baumann, H. (2016) Combined effects of ocean acidification, warming, and hypoxia on marine organisms. Limnology and Oceanography e-Lectures 6:1-43. doi:10.1002/loe2.10002

Depasquale, E., Baumann, H., and Gobler, C.J. (2015) Variation in early life stage vulnerability among Northwest Atlantic estuarine forage fish to ocean acidification and low oxygen Marine Ecology Progress Series 523: 145–156.doi:10.3354/meps11142