The Acute Effects Of A
Handling Stressor On Select
Oreochromis aureus
Plasma 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
200
Change in glucose level mg/dL
control
150
stressed
100
50
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).
0
-50
-100
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
Control: 2.95 g/L
Stressed: 2.89 g/L
0.8
change in total protein g/L
0.6
Ave. decrease:
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
Average final
levels
control
stressed
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
200
change in iron ug/dL
150
100
50
0
-50
control
stressed
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
Change in Cholesterol mmol/L
60
40
20
0
-20
-40
-60
-80
control
stressed
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 punctatus nickel 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
change in lysozyme activity ug/mL
6
5
4
3
2
1
0
-1
-2
-3
control
stresssed
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
change in sodium level mmol/L
25
20
15
10
5
0
-5
-10
-15
-20
control
stressed
-25
-30
• Control: average loss of 2 mmol/L
• Stressed: average increase of 3 mmol/L
Chloride data
30
Change in chloride level mmol/L
20
10
0
-10
control
-20
stressed
-30
• 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.