Dataset: Atlantic silverside HypOA experiments: Larval morphometric measurements

Two experimental treatment levels were used for this experiment: a control treatment [100% DO (8.9 mg L-1) and 650 µatm pCO2 (7.83 pHTotal Scale] and a treatment combining hypoxia and ocean acidification [hereafter referred to as HypOA; 40% DO (3.6 mg L-1) and 2,300 µatm pCO2 (7.33 pHT)]. Following the initial five-day holding period, adult fish were exposed to their respective experimental treatment conditions for 10 days. Parental conditioning to HypOA involved exposing adult fish to a fluctuating DO and pCO2 regime, mimicking the diurnal fluctuations seen in nearshore marine environments. After the conditioning phase, adult silversides were spawned, and their offspring were reared under both conditions in a fully-factorial experimental design. Subsamples for hatch morphometrics (N = 10) were made on the first morning when a growth replicate had at least 20 new hatchlings. The larvae were euthanized by an overdose of MS-222 and were fixed in 4% buffered formalin for 24 h and transferred to 75% ethanol for storage. The remaining hatchlings from growth replicates were moved to a new 20-L container for continued rearing. Larvae were provided daily rations of newly hatched brine shrimp nauplii (San Francisco Bay strain) at 2-3 nauplii ml-1. Replicates were siphoned daily for uneaten food and waste. The continuous seawater flow through ensured that ammonia levels remained near 0 ppm, verified daily via API® Ammonia Test Kit. At 19 dpf (which corresponds to either 9 or 10 days dph based on the treatment and hatch timing) the survival replicates were recounted to determine the final survival rate. Larvae were subsampled for morphometric measurements (N = 12) and fixed using the same protocol as described above. 

Dates and Locations:

Wild Atlantic silverside adults were collected from Mumford Cove, CT (41°19'18.1"N 72°01'02.8"W) a shallow embayment adjacent to the eastern end of the Long Island Sound on May 31, 2022. Adults were transported to the Environmental Systems Laboratory at Woods Hole Oceanographic Institution where we have developed at large experimental seawater system to facilitate multistressor research on fish.   Parental conditioning of wild adults took place between May 31 to June 17, 2022. Embryo and larval rearing took place between June 17 to July 7, 2022.

Taxnomic Identifier, LifeSciences Identifier (LSID):
Menidia menidia, urn:lsid:marinespecies.org:taxname:159228

Instrument summary:

The experiment was conducted at the Environmental Systems Laboratory at Woods Hole Oceanographic Institution (Falmouth, MA, USA) in a custom designed automated seawater system comprised of eight 900-L circle main tanks (four tanks per treatment level) and one 600-L mixing tank. A schematic of the experimental setup is provided in Fig. S1.  Each main tank was outfitted with multiple 20-L rearing containers (white buckets fitted with six 3-cm flowthrough holes covered in 300-µm screening) to accommodate groups of embryos and larvae. Four main tanks were allocated to the control treatment received temperature-controlled seawater sourced from Vineyard Sound. This water was sand-filtered, UV-sterilized, and constantly maintained at the control treatment's DO and pCO2 conditions. The HypOA treatment conditions were automatically maintained within the mixing tank using custom software developed in Arduino IDE and operated by a Teensy® 4.1 Development Board. In short, the mixing tank received a continuous flow of ambient seawater, with DO, pH, and temperature measurements taken every 10 seconds by Pyroscience® Ultra Compact (PICO) DO and pH meters, fitted with fiberoptic DO or pH sensors and PT100 temperature sensors. Calibration of the probes was performed weekly according to the manufacturer's guidelines using oxygen and pHT calibration solutions provided by Pyroscience®. HypOA levels were regulated through a simple feedback mechanism: when the current DO or pH exceeded the treatment set point, the software opened a solenoid valve to introduce compressed gas (N2 or CO2) into ultrafine gas-diffusers positioned at the bottom of the mixing tank until the setpoint was reached. The software was optimized to implement brief and precisely controlled gas additions, ensuring highly stable DO and pH conditions in the mixing tank. Adjusted HypOA seawater was pumped to the main tanks and individual rearing containers as required.

DO levels in individual rearing units were cross-verified using a handheld Pyroscience Firesting®-GO2 meter fitted with an optical DO sensor. A two-point calibration (100% and 0% DO) was made daily using air-saturated seawater and a concentrated NaSO3 seawater solution. pH conditions were verified via a Hach HQ11D pH meter fitted with a Hach PHC28101 IntelliCal pH probe. The probe was calibrated daily using pHT buffers from PyroScience®. To validate target pCO2 conditions, seawater was sampled every 1 – 2 days from one of the replicate rearing containers per offspring treatment group, resulting in 12 seawater samples per offspring treatment group during the 19-day experiment. Seawater samples were poisoned with mercuric chloride and stored in sealed bottles for later analysis of carbon chemistry parameters. Temperature and salinity (via refractometer) were recorded at the time of sampling. Samples were measured in triplicate for dissolved inorganic carbon (DIC) using an Apollo AS-D1 analyzer connected to a Picarro G-2121i cavity ringdown system. Total alkalinity (TA) was measured using an open-system Gran titration on 5-mL samples in triplicate, using a Metrohm 805 Dosimat and a robotic Titrosampler. Both systems were calibrated against seawater Certified Reference Materials (Andrew Dickson, University of California San Diego, Scripps Institution of Oceanography, https://www.nodc.noaa.gov/ocads/oceans/Dickson_CRM/batches.html). The remaining in situ carbon chemistry parameters of pHT, pCO2, bicarbonate, and carbonate ions were calculated using the R package seacarb (Gattuso et al., 2020) based on direct measurements of DIC and TA and the salinity and temperature at the time of water sampling. Equilibrium constants for the dissociation of carbonic acid in seawater (K1 and K2) followed Mehrbach et al. (1973) and refitted by Dickson and Millero (1987). The constant for KHSO4 followed Dickson (1990).