The Acute Effects Of A Handling Stressor On Select Oreochromis aureusPlasma Components Nora E. Demers Florida Gulf Coast University Ft. Myers, FL
Does the fight or flight response enhance innate immunity? Or perhaps more realistically: Is it possible to obtain a resting fish?
Innate immune parameters • Lysozyme- published information: • Immuno-suppressive with exposure to toxins • Immuno-enhancing with exposure to toxins • Immuno-enhancing within minutes of stress Any conservation of response among species?
Possible role of other plasma components? • Transferrin? • Control iron available to pathogens • Cholesterol? • Gross indicator of organelle trafficking
Fish Acquisition and Acclimation Purchase Oreochromis aureus from local aquaculture facility. Relocate to Lee County Hyacinth Control Ponds. Seine net capture and transfer 10 fish to 400L circular tank with flow-through well water. Acclimate for 1-2 weeks.
Experimental Design Anesthetize all fish in tank with 2-PE. Bleed all fish from caudal vessel with heparinized needle. Revive 5 fish while confined in nets in freshwater tank.
Stress fish Stress fish by 30 seconds in air and 5 minutes in shallow bucket. Bleed again (same fish). Bleed “control” fish again – had been in tank containing anesthesia entire time. Spin blood and collect plasma.
Data acquired from plasma sample lysozyme activity --using fresh plasma • (kinetic assay- microplate) HEWL digestion of Micrococcus lysodeckticus measured by change in optical density
Automated Assays on Frozen Plasma- (Vitros product) (compliments of DSI laboratories) • Glucose-oxidase-based colorimetric test • Total Protein-biuret reaction cupric ion in alkaline medium • Iron-ferric iron removed from transferrin by acidic pH, reduced and bound by dye • Cholesterol-esterhydrolase, oxidase and peroxidase, leuco dye detected
Automated Ion Assays Potentiometric analytical element (compliments of DSI laboratories) • Sodium- methyl monensin • Chloride- layer to prevent interference from bromide and uric acid All proficiency limits are very low. Data shown are “real” changes in values, not machine noise.
Glucose Data Average final levels. Control: 121 mg/dL. Stressed 86 mg/dL. Ave. increase: Control: 43 mg/dL (28/30). Stressed 7 mg/dL (20/30). Initial to final. p <.0001 (control). Stressed n.s. Control to stressed n.s.
Glucose data interpretation? “Control” fish were stressed. Initial anesthesia took >10 min. and keeping fish anesthetized was a stressor. Stressed fish either had response inhibited OR they used up the plasma glucose that was released. There was a significant difference between control and stressed groups.
Total protein data Average final levels Control: 2.95 g/L Stressed: 2.89 g/L Ave. decrease: Control: -.075 g/L ( 15/30 ) Stressed -.174 g/L (17/30) Initial to final p =.05 (stressed) p =.02 (control) Control to stressed p=
Total protein interpretation? Expected to see increase in TP level. (if Ho was supported). Why did both groups show decreases? due to changes in plasma volumes? (hypervolume of blood is used to explain increases in some parameters but these are decreases…). Why was stressed decrease greater?
Iron data Average final levels. Control: 127.7 ug/dL. Stressed: 120.7 ug/dL. Ave. increase: Control: 21ug/dL (15/27). Stressed 4ug/dL (13/26). Initial to final p=.02 (control). Control to stressed n.s.
Iron interpretation? • There was an increase in total iron in all samples. • This could indicate an increase in plasma transferrin level. • Does the “lesser increase” in stressed fish indicate a suppressed response or a return from more elevated level? (How would you determine?
Cholesterol data Average final levels Control: 148.3 mmol/L Stressed 142 mmol/L Ave. decrease: Control: 11.5 mmol/L (21/30) Stressed 17.3 mmol/L (22/30) Initial to final p=.005 (control) =.0002 (stressed)
Cholesterol literature Toxicology. Plasma cholesterol-. Clarias -carbaryl 96 hours and 15 d sub- lethal dose (1 2 or 4 mg/L) no change (Sharma 1999). • Decreased of 15% (40 mg/dL) at 24 hour sub-lethal dose (15 ppm) of carbaryl or phorate (Jyothi 2001) attributed to liver dysfunction. Liver cholesterol-. Channa punctatusnickel exposure for 30d – increasing levels of cholesterol with higher concentrations attributed to hepatic dysfunction or blocking of cholesterol synthesis.
Cholesterol literature Exercise. Trout decreased total protein and cholesterol by 4 mmol/L within 1 hr (strenuous) w or w/out endrin (Grant & Mehrle 1973) (but they measured only 14 mmol/L as total level). Piaractus decreased plasma chol. after 6 hr. confinement (Krieger-Azzolini et al, 1989). Experimental: Perrier (1972) showed adrenaline I.M. injections (15 hr later) increases plasma cholesterol.
Cholesterol interpretation? Why do decreases in cholesterol occur within minutes? (hypervolume is used to explain increases) My Ho is that this demonstrates exocytosis of granules How can this be tested? Mark granule contents? Mark cholesterol itself (how)? Which question would yield more valuable information? Do these results add useful information to interpretation of toxicology data?
Lysozyme data Average final levels. Control: 11.57 ug/mL. Stressed 11.72 ug/mL. Ave. change: Control: -.126 ug/mL (18/30). Stressed +.327 ug/mL (17/30). * Fish with increase of 4.73 was blind in one eye. Initial to final n.s. Control to stressed n.s.
Lysozyme interpretation? Stressor of all samples may have exasperated interpretation of results- Saw decreases in 2/3 of fish in both groups. Perhaps window of enhancement had passed (for C3 increase at 10 min. return to resting at 20 minutes). Changes in lysozyme activity with stress do not appear to be very reproducible in these Oreochromis aureus -only 66% responded similarly. With variation in published data, my confidence in lysozyme as an indicator of stress is lessened every day.
Sodium data • Control: average loss of 2 mmol/L • Stressed: average increase of 3 mmol/L
Chloride data • Control: average decrease of 2mmol/L • Stressed: average increase of 5 mmol/L
Conclusions • Fish respond more quickly to handling stressors than much literature documents. • A closer look at the immediate response may help elucidate confounding variables that confuse other research results. • Cholesterol and total protein data look to be closely related. • The role of iron and cholesterol would be especially interesting to elucidate.