TL;DR - My students ran a variety of experiments using hairy shore crabs (Hemigrapsus oregonensis) this quarter. Some stand-out stressors were anti-biofouling paint, high temp coupled with pathogen exposure, hypoxia, NSAIDs, and caffeine. A scaled up resazurin protocol was successful as a proxy for respirometry. Use of hemocytometers, physiological assay kits, and handheld glucometers showed promise as quick and less invasive methods for measuring stress response.

Summaries of each of the projects ran in the class I TAed this spring.

Impacts of calcium buildup on Hemigrapsus oregonensis in recirculating aquaculture systems

  • Objective: Test how elevating dissolved CaCO₃ (to ~600 mg/L) affects molting, physiology, and survival in the shore crab under controlled RAS conditions.\
  • Setup: Hairy shore crabs held at 13 °C or 27 °C; one tub per temperature received extra CaCO₃; controls received none. Five crabs per tub; water and hemolymph sampled at weeks 1 and 2.\
  • Measurements: Molt counts, gill observations, hemolymph lactate (stress marker), righting response (behavioral burden), and mortality records.\
  • Key findings:
    • Total mortality in the hot-Ca²⁺ treatment by week 2; visible calcium deposits on gills in cold-Ca²⁺ group.\
    • No statistically significant changes in lactate or righting times (low sample size), but pronounced shell and gill mineral buildup preceded death.\
  • Conclusion: Excessive Ca²⁺ surpasses a threshold where crabs cannot molting-compensate, leading to gill fouling and mortality—critical for RAS mineral management.

Caffeine concentrations on the stress response of Hemigrapsus oregonensis

  • Objective: Determine how low (5 µg/L), medium (15 µg/L), and high (20 µg/L) caffeine exposures over 14 days alter stress physiology.\
  • Setup: Three treatment tanks plus control; six crabs each; caffeine solutions replaced at day 7; all held at constant temperature/salinity.\
  • Measurements:
    • Righting time (behavioral stress) at days 7 & 14\
    • Resazurin assay for oxygen consumption (metabolic rate)\
    • Hemolymph lactate concentration (anaerobic stress)\
    • Behavioral observations (escaping, cannibalism) and mortality\
  • Key findings:
    • Righting times varied non-monotonically across doses.\
    • Week 1: highest O₂ consumption in high dose; week 2: low dose peaked.\
    • Lactate highest in control, then high dose.\
    • Highest mortality in high dose; observed escape attempts and lethargy.\
  • Conclusion: Caffeine acts as a chronic stressor in shore crabs, affecting metabolism, behavior, and survival—sample size and replication should be increased for stronger inference.

The ecophysiological effects of water temperature and ibuprofen on Hemigrapsus oregonensis

  • Objective: Test individual and combined impacts of increased temperature (13 °C vs 27 °C) and ibuprofen dose (0, ~12.5 µg/L, ~62.5 µg/L) on crab physiology.\
  • Setup: Six tanks (2 × 3 design), three crabs each, two-week exposure; water changed weekly with re-dosing.\
  • Measurements:
    • Weekly resazurin assay for oxygen consumption rates\
    • Hemolymph glucose via colorimetric assay at study end\
  • Key findings:
    • Ambient: initial highest O₂ in controls, then high dose dominated by week 2.\
    • Elevated temp: control crabs always showed highest O₂; drug treatments showed metabolic depression.\
    • Glucose highest in ambient controls; ibuprofen and heat both tended to lower hemolymph glucose.\
  • Conclusion: Ibuprofen induces metabolic depression, modulating thermal-stress responses; suggests pharmaceutical pollutants can alter energy budgets in crustaceans.

The impacts of anti-fouling paint on the ecophysiology of Hemigrapsus oregonensis

  • Objective: Characterize stress (righting time, lactate) from exposure to cuprous-thiocyanate–based paint.\
  • Setup: Four tanks (control + three paint-exposure levels via coated foil of increasing area); one-week exposure at constant 15 °C/33 ppt.\
  • Measurements: Righting time flip assay; hemolymph lactate via Cayman L-Lactate kit; qualitative behavior; t-tests vs control.\
  • Key findings:
    • Righting time increased markedly with paint exposure (longest at highest surface area).\
    • Lactate levels decreased as exposure increased (opposite of expected anaerobic response).\
    • High exposure crabs were lethargic; one mortality and a non-extractable pregnant female occurred in mid-level treatment.\
  • Conclusion: Anti-fouling pigments induce behavioral stress and disrupt metabolic profiles; copper toxicity likely underlies observed effects.

Too close for comfort: effects of crowding on behavioral and physiological stress in Hemigrapsus oregonensis

  • Objective: Disentangle physical crowding vs waterborne cues on crab stress.\
  • Setup:
    1. Phase 1—crowded (9 crabs/200 mL) vs uncrowded (9/1000 mL) for 7 days\
    2. Phase 2—uncrowded + crowded water vs recrowded clean water for another week\
  • Measurements: Resazurin oxygen-consumption assay (30–90 min), righting time trials, mortality/escape/limb-loss events, salinity changes.\
  • Key findings:
    • Crowded crabs had the highest mass-specific respiration slopes; uncrowded + crowded-water matched controls.\
    • All non-control groups showed slower righting times; variation greatest in crowded.\
    • Mortality, escapes, and limb loss occurred only under physical crowding.\
    • Salinity rose to ~40–41 ppt in crowded treatments, adding osmotic stress.\
  • Conclusion: Physical crowding (and its byproducts) drives stress via aggression and salinity build-up; waterborne chemical cues alone insufficient to elevate metabolism.

How thermal stress and feeding state affect glucose levels in Hemigrapsus oregonensis

  • Objective: Examine interactions of temperature (13 °C vs 27 °C) and feeding (fed vs fasted) on hemolymph glucose; evaluate glucometer utility.\
  • Setup: Three treatment tanks plus large control; trial 1: glucose measured at 1.25 h post-feeding; trial 2: at ~4 h. Glucometer readings and parallel colorimetric assays.\
  • Measurements: Five time-points per trial; hemolymph sampled via leg-joint extraction; glucometer detection range 20–600 mg/dL.\
  • Key findings:
    • Most glucometer readings fell below device floor (<20 mg/dL); only fed 13 °C at trial 1 final point read ~22 mg/dL.\
    • Assay data showed fed groups had higher glucose peaks (~0.18–0.31 mg/dL) vs fasted (~0.006–0.1 mg/dL).\
    • Thermal stress altered glucose mobilization patterns; limited sample volumes reduced data completeness.\
  • Conclusion: Feeding state dominates hemolymph glucose, but high temperature may accelerate turnover; standard glucometers lack sensitivity for crustacean hemolymph—dedicated assays recommended.

Impacts of hard plastic exposure on Hemigrapsus oregonensis

  • Objective: Assess how exposure to ground HDPE bottle-cap particles (1–5 mm) via water versus food influences mortality and lactate levels.\
  • Setup: 20 crabs split into two groups:
    • MP-water: 2.5 mg plastic soaked in oyster juice added to water column\
    • MP-fed: 2.5 mg plastic mixed with 2.5 mg oyster muscle tissue
      Each crab in an isolated 250 mL jar (mesh top) at 25 °C; weekly water changes.\
  • Measurements:
    • Single terminal hemolymph extraction; lactate assays (two technical replicates per crab)\
    • Post-mortem dissection for plastic presence in gut and gills\
  • Key findings:
    • 88 % mortality in MP-fed group by end of week 1; dissection indicated hypoxia (grey/black gills) due to poor flow.\
    • MP-water individuals showed high variance in lactate (STDev = 108.1 µM); surviving MP-fed crab had low lactate (~36.9 µM).\
    • No plastics found in tissues—crabs largely ignored positively buoyant HDPE beads.\
  • Conclusion: Mortality driven by hypoxia overshadowed plastic effects; flotation behavior and lack of ingestion suggest need for varied particle types and improved flow in future studies.

The Physiological and Behavioral Effects of Copper Exposure on Hemigrapsus oregonensis

  • Objective: Evaluate how copper sulfate (CuSO₄·5H₂O) at 0, 25, and 167 mg/L affects metabolic activity and righting behavior.\
  • Setup:
    • Control: ≥ 20 crabs (0 mg/L Cu)\
    • Low Cu: 9 crabs at 25 mg/L\
    • High Cu: 9 crabs at 167 mg/L
      Standardized salinity, temperature, pH; 14 day exposure.\
  • Measurements:
    • Resazurin respirometry: 35 mL dye chambers, fluorescence sampled every 30 min over 90 min, normalized to body mass\
    • Righting time: time to self-right after inversion\
  • Key findings:
    • Mortality: Week 1 survival ~70 % (low) vs ~50 % (high); Week 2 ~10 % (low) vs 0 % (high).\
    • Metabolism: Week 1 control > low > high in fluorescence increase; Week 2 low ≈ control (~250 RFU).\
    • Righting: Week 1 control (1–4 s, SD 1.2), low similar (SD 1.6), high slower/more variable (SD 5.6, max ~16.5 s); Week 2 only low & control (<4 s), high singleton ~12.5 s.\
  • Conclusion: Copper exposure reduces aerobic metabolism, impairs motor function, and causes high mortality—underscoring need for tighter copper regulation in coastal environments.

Effects of Combined Thermal and Pathogen Stress on Immune Response in Hemigrapsus oregonensis

  • Objective: Investigate how temperature (13 °C vs 27 °C) and natural mud-borne pathogens affect hemocyte counts and metabolic rate.\
  • Setup: Four treatments (6 crabs each):
    • Mud + Heat (mud in 27 °C water)\
    • Mud + Cold (mud in 13 °C water)\
    • Heat-only (27 °C, no mud)\
    • Control (13 °C, no mud)
      Mud from Golden Gardens introduced ambient pathogens; 2 week exposure.\
  • Measurements:
    • Hemocyte counts via hemocytometer at weeks 1 & 2\
    • Resazurin assay: 35 mL working solution, sampling at 30, 60, 90 min, RFU normalized to mass\
  • Key findings:
    • Week 1 hemocytes: Mud + Heat ~1800 cells/µL; Mud + Cold ~400 cells/µL; Heat-only ~50–150 cells/µL; Control <200 cells/µL.\
    • Week 2 hemocytes: Declines across all—Mud + Heat ~600 cells/µL, Mud + Cold ~300 cells/µL, Heat-only ~350 cells/µL, Control ~<200 cells/µL.\
    • Metabolism: Highest RFU in Mud + Cold; lowest in Mud + Heat—suggesting metabolic suppression under combined stress.\
  • Conclusion: Pathogen exposure elicits strong immune response, amplified by heat; immune activity wanes by week 2 (possible immunosuppression); replication and controlled pathogen dosing recommended.

Physiological Stress Responses to Hypoxia and Emersion Breathing in Hemigrapsus oregonensis

  • Objective: Determine whether intertidal access (emersion breathing) mitigates hypoxia-induced stress compared to deepwater confinement.\
  • Setup: Three treatments (9 crabs each):
    • Control: airstone (DO₂ 6–7 ppm)\
    • Deepwater: sealed, no airstone\
    • Intertidal: mesh for surface access, no airstone
      2 week exposure.\
  • Measurements:
    • DO₂ monitoring; observation of surface-seeking behavior\
    • Righting time and resazurin assay at weeks 1 & 2\
    • Hemolymph lactate (Cayman kit) at week 2; Gill tissue grading (good/partial/bad)\
  • Key findings:
    • DO₂ dropped to ~3.2 ppm (intertidal) and ~3.4 ppm (deepwater) by week 2; deepwater had 100 % mortality, intertidal only one death.\
    • Righting times: no significant differences (ANOVA p = 0.329).\
    • Lactate: Intertidal 130 µM vs Control 300 µM (p = 0.016), indicating lower anaerobic stress.\
    • Metabolism: Week 1 both treatments > Control; week 2 intertidal still > Control but decreased.\
    • Gill wasting: 88 % deepwater fully wasted vs ~50 % intertidal partial wasting.\
  • Conclusion: Emersion access improves survival and reduces physiological damage under hypoxia; direct observation of emersion breathing and finer temporal monitoring advised.