Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Study description:
*see "Related Datasets" section for access to related datasets from this experiment

These data were generated during a cross-generational laboratory experiment testing how parental exposure to a combined hypoxia and acidification treatment affected offspring responses and HypOA tolerance in an important coastal forage fish species, Atlantic silverside Menidia menidia. The experiment was conducted during June and July 2022 at Woods Hole Oceanographic Institution’s Environmental Systems Laboratory.   Wild adult Atlantic silversides were conditioned to two treatment levels: control (100% dissolved oxygen [DO] / 650 µatm pCO2) or HypOA conditions (40% DO / 2300 µatm pCO2) for 10 days. After artificial fertilization, their offspring were reared in a factorial experimental design to test how parental conditioning to the stressors affected the survival, development, and growth of embryos and larvae when reared under control or HypOA conditions. The larvae were sampled for morphometric measurements at hatch (9 or 10 days post-fertilization) and after 10 days of larval rearing (19 days post-fertilization). We evaluated survival to hatch (fertilization to 10 days post-fertilization), during larval development (hatch to 10 days post hatch), and over the entire rearing interval (fertilization to 19 days post-fertilization). We found that direct offspring exposure to HypOA lowered survival and growth; however, embryos fertilized by HypOA conditioned parents showed better survival under HypOA conditions. By contrast, we found that larvae from HypOA-conditioned parents exhibited, on average, slower post-hatch growth and slightly lower larval survival in offspring groups reared under both treatment conditions. The data demonstrate that parental conditioning does affect offspring responses, with evidence for both positive and negative effects on offspring viability and tolerance to HypOA. 

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).

Hatch counts: Larvae were counted from replicate rearing containers and moved to new replicates for continued rearing or sampled for morphometric measurements. 

Survival statistics: Larvae were counted at hatch (9 - 10 days post fertilization) and after 9 or 10 days of larval rearing (19 days post fertilization). Survival was summarized over three intervals: 1) embryo survival (fertilization to hatch), 2) larval survival (hatch to 9 or 10 days post hatch), 3) overall survival (fertilization to 9 or 10 days post hatch). Survival is reported by individual rearing replicate (N = 4 per treatment). 

* Sheet "data" of submitted file "Dataset2_Murray et al_HatchingData.xlsx" was imported into the BCO-DMO data system for this dataset.
** Missing data values are displayed differently based on the file format you download from BCO-DMO.  They are blank in csv files, "NaN" in MatLab files, etc.
* Dates converted to ISO 8601 format
* Sheet "data" of submitted file "Dataset3_Murray et al_SurvivalStatistics.xlsx" was added as a supplemental file.

NSF Award Abstract:

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).

Coastal marine ecosystems face multiple anthropogenic stressors including increasingly severe events of co-occurring acidification and hypoxia. These periodic but acute environmental stressors can directly impact the abundance, diversity, and commercial value of coastal fish stocks. Rapid acclimation of key physiological processes can provide short-term protection against extreme conditions. Importantly, these phenotypic modifications can be passed on to subsequent generations thereby priming offspring for increased tolerance. However, for most fish species the scope for phenotypic plasticity and the precise mechanisms of action are poorly understood. This severely limits our ability to anticipate responses in the majority of ecologically and economically important marine species. The overarching objective of this proposal is to investigate the potential for within-generational and multigenerational plasticity in response to co-occurring hypoxia and acidification in the forage fish Atlantic silverside (Menidia menidia). The Atlantic silverside is a foundational species and an essential trophic component of coastal food webs along the North American Atlantic coast and serves as key prey item for many seabirds and commercially important fish. Understanding the long-term bioenergetic impacts and the potential for rapid adaptation in this species will therefore fundamentally advance our understanding of the ecological consequences of rapid environmental change in coastal marine ecosystems.

The project will be centered around a series of laboratory exposure experiments and state-of-the-art metabolic assays utilizing wild-caught Atlantic silversides collected during their spring spawning season. The PI will investigate how parental environments influence offspring phenotype by conditioning mature wild Atlantic silversides (F0) to contrasting fluctuating CO2/O2 treatments. The F1 generation will be then reared in a reciprocal transplant experiment to quantify how parental and offspring treatment levels affect key life-history traits including survival, growth, and aerobic performance. Surviving F1 offspring will be reared until maturity to evaluate how environmental stress experienced during early development affects adult reproductive capacity. Furthermore, the PI will investigate the role of biological memory in multigenerational plasticity by exposing F2 offspring to factorial combinations of grandparental and parental environments. In parallel, the PI will investigate how molecular mechanisms may mediate rapid adaptation to changing environments using transcriptomics (RNAseq) and genome-wide DNA methylation profiling (Methylseq). Linking molecular changes with whole organism phenotypic responses will broaden our understanding of the effects of multiple stressors on an ecologically important species which will provide clues for developing mitigation plans to protect coastal food webs from various climatic factors.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.