Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Experiments done at:

• Friday Harbor Laboratories Ocean Acidification Environmental Laboratory
• NOAA Northwest Fisheries Science Center Mukilteo Research Station

Krill for both experiments were collected from surface waters (upper 50 m) at approximately 48.01, -122.29 using a 60-cm diameter Bongo frame equipped with black 335-μm mesh nets and non-filtering cod ends towed for <10 min. The contents of each cod end were immediately diluted in coolers of seawater and sorted to remove healthy euphausiids. For the pH experiment krill were collected on 23 July 2017 between 23:00-00:30, stored in 1-4 L containers at a concentration of 5 ind L-1, sealed with all air removed to eliminate splashing, and transported in coolers to the University of Washington Friday Harbor Laboratories (FHL) where they were sorted for healthy female E. pacifica (obvious ovary) and put in in experimental chambers between 06:00-08:00 on 24 July 2017. For the DO experiment krill were collected on 20 Sept 2017 between 20:00 and 21:15, krill were transported in coolers to the NOAA Mukilteo Research Station within an hour, sorted for healthy female E. pacifica (obvious ovary), and immediately put in experimental chambers. Krill were kept chilled (10-14 °C) at all times and were gently transferred using broad spoons to prevent damage. During experiments krill were fed Shellfish Diet 1800 (Reed Mariculture) twice per day and newly hatched Artemia salina (San Francisco Bay Brand) nauplii once per day. All physiological measurements were taken after 10 days of acclimation; individuals were flash frozen in liquid nitrogen after oxygen consumption was measured.

Experimental acclimation to pH was conducted at the FHL Ocean Acidification Environmental Laboratory, using a flow-through system with pH controlled by CO2 bubbling. Target pH levels were 8.0, 7.5, and 7.2: three flow-through systems were used, one per pH treatment. Within each system, conditioned seawater was delivered to eight flow-through boxes that each contained two 500-mL mesh-sided experimental chambers with one individual krill per chamber, for a total of 16 individuals per system. Experimental systems were kept covered in black plastic to reduce light. Temperature was set to 12 °C and oxygen was maintained at ambient levels through bubbling, but not monitored.

Acclimation of krill to different oxygen levels in the laboratory was done at the NOAA Mukilteo Research Station in flow-through experimental systems with independent temperature, pH, and oxygen control. Target dissolved oxygen levels were 3, 5, and 9 mg DO L-1: nine systems were used, three per DO treatment, with krill held individually in a mix of both 250- and 500-mL flow-through containers within the experimental systems. Temperature was maintained at 12 °C and pH at ~7.82; temperature, pH, and dissolved oxygen were monitored continuously.

System indicates the system that generated controlled seawater conditions and represents the experimental replicates. Many individual krill were held within one experimental system. For the pH experiment 3 systems were used, only one per treatment condition (104A, 104B, 105A); for the DO experiment nine systems were used with three per treatment condition (M1, M2, M3, M4, M5, M9, M10, M11, M12). System names match the continuously recorded observations of seawater conditions: temperature and pH were monitored during the pH experiment (temperature was not logged in system 104A); DO, pH, and temperature were monitored during the oxygen experiment. 

Krill were measured for total length (from behind the eye to the end of the telson) at 6X using a calibrated eyepiece reticle then flash frozen individually in liquid nitrogen. Total length was converted to dry weight (DW) using a published length-weight regression (Feinberg et al. 2007).

Protein content was determined according to the bicinchoninic acid (BCA) method (Smith et al. 1985) using a Pierce BCA Protein Assay Kit (Thermo Scientific).

ETS activity was assayed using the method of Owens and King (1975), as modified by Gómez et al. (1996), and adapted for a 96-well plate. Assays and blanks were measured in triplicate at 25 °C, calculated according to Packard and Christensen (2004), and corrected to in situ temperatures (depth integrated) using the Arrhenius equation with an activation energy of 15 kcal mol-1 (Packard et al. 1975) and standardized to protein specific activity, spETS.

AARS was measured following the method of Yebra and Hernadez-León (2004), modified by Yebra et al. (2011), and adapted for a 96-well plate (Yebra et al. 2017). All activities were corrected to in situ temperatures with the Arrhenius equation using an activation energy of 8.57 kcal mol-1 (Yebra et al. 2005) and standardized to protein specific activity, spAARS. spAARS_1 was measured with the NADH Blank and calculated as described by Mclaskey et al. (2020). For each sample, assays and NADH Blanks were measured in triplicate.

Oxygen consumption of individual krill was measured at 12 °C by closed-cell respirometry in 22-mL vials containing optical oxygen sensors (PSt7 PreSens) and a 12-mm magnetic stir bar separated from the animal by 200-μm mesh. Measurements were taken every 15 minutes for 2 hours. Krill were kept in filtered seawater to clear their guts for ~1 hr prior to respirometry.

Description from NSF award abstract:
Low dissolved oxygen (hypoxia) is one of the most pronounced, pervasive, and significant disturbances in marine ecosystems. Yet, our understanding of the ecological impacts of hypoxia on pelagic food webs is incomplete because of our limited knowledge of how organism responses to hypoxia affect critical ecosystem processes. In pelagic food webs, distribution shifts of mesozooplankton and their predators may affect predator-prey overlap and dictate energy flow up food webs. Similarly, hypoxia may induce shifts in zooplankton community composition towards species that impede energy flow to planktivorous fish. However, compensatory responses by species and communities might negate these effects, maintaining trophic coupling and sustaining productivity of upper trophic level species. The PIs propose to answer the question "Does hypoxia affect energy flow from mesozooplankton to pelagic fish?" They approach this question with a nested framework of hypotheses that considers two sets of processes alternatively responsible for either changes or maintenance of pelagic ecosystem energy flows. They will conduct their study in the Hood Canal, WA. Unlike most hypoxia-impacted estuaries, hypoxic regions of Hood Canal are in close proximity to sites that are not affected. This makes it logistically easier to conduct a comparative study and reduces the number of potential confounding factors when comparing areas that are far apart.

Improved understanding of how hypoxia impacts marine ecosystems will benefit the practical application of ecosystem-based management (EBM) in coastal and estuarine ecosystems. Effective application of EBM requires that the impacts of human activities are well understood and that ecological effects can be tracked using indicators. This project will contribute to both of these needs. The PIs will share their findings on local and national levels with Federal, State, Tribal, and County biologists. To increase exposure of science to underrepresented groups, the PIs also will provide Native American youth with opportunities to participate in field collections and laboratory processing through summer internships. The PIs will collaborate with the NSF-funded Pacific Northwest Louis Stokes Alliance for Minority Participation and tribes from the Hood Canal region to recruit and mentor students for potential careers in marine science. This project will support several undergraduate researchers, two Ph.D. students, a post-doc, and two early-career scientists.