Tissue homogenization

Weight approximately 200-400 mg of live tissue (wet weight), fresh or frozen at -20°C immediately upon
sampling or at -80°C if prolonged storage is needed (>one month). Add 10x volume of homogenization
buffer (PBS in the case of downstream ELISA assays (as commonly suggested by ELISA kit
manufacturers) or 50mM Tris-HCl, pH 7 + 2 mM mannitol in the case of downstream enzyme assays).
Homogenize on ice using 40 strokes with pestle A and 10 strokes with pestle B (7 ml dounce grinder set,
D9063, Sigma). Leave the extract to settle down for a few minutes, then take the upper phase and leave at
-20°C for 1 h. Then thaw and place again at -20°C for 1 h. Then repeat the freeze-thawing cycle another
once and centrifuge the samples at +4°C, 15 500 g (maximal speed) for 15 min. Aliquot the supernatants
in at least two separate tubes (to avoid repeated thawing cycles). Store the samples at -20°C or at -80°C,
in the case of prolonged storage (>one week).

References: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

Protein assay, Analyticon Biotechnologies, 9106

Mix 10 μl of the Samples and Standard (R4) with 190 μl of R1 reagent, incubate 10 min at room
temperature (RT), then read the absorbance at 546 nm. Blank should contain a homogenization buffer
instead of Samples. For new samples (new fish species and different developmental phase) perform the
dose response analysis using concentrated homogenates, 10x and 100x dilution.

ELISA assay for thyroxine (T4), BioMatik EKC40454-96T

Dilute Wash Buffer in distilled water 20x. Add 50 μl of Standards and Samples, then add 50 μl of
conjugate (biotin-conjugated T4) (not in Blanks) and incubate for 60 min at 37°C. Blank should contain a
homogenization buffer instead of Sample. Aspirate and wash the wells using 200 μl of the Wash Buffer
3x. After decanting carefully all the fluid after the last wash, blot it against the paper towel and add 50 μl
of HRP-avidin to all wells except for the Blank well. Incubate 30 min at 37°C. Aspirate and repeat the
washing procedure 3x. Add 50 μl of Substrate A and 50 μl of Substrate B to each well, including Blank
and incubate 15 min at 37°C in the dark. Add 50 μl of Stop Solution and measure the absorbance at 450
nm within 10 min. For new samples (new fish species and different developmental phases) perform the
dose response analysis using concentrated homogenates, 10x and 100x dilution. Unused plates should be
sealed with an adhesive strip and stored at +4°C up to one month, in the presence of desiccant.

ELISA assay for tri-iodothyronine (T3), BioMatik EKC40453-96T

Dilute Wash Buffer in distilled water 20x. Add 50 μl of Standards and Samples, then add 50 μl of
conjugate (biotin-conjugated T3) (not in Blanks) and incubate for 60 min at 37°C. Blank should contain a
homogenization buffer instead of Sample. Aspirate and wash the wells using 200 μl of the Wash Buffer
3x. After decanting carefully all the fluid after the last wash, blot it against the paper towel and add 50 μl
of HRP-avidin to all wells except for the Blank well. Incubate 30 min at 37°C. Aspirate and repeat the
washing procedure 3x. Add 50 μl of Substrate A and 50 μl of Substrate B to each well, including Blank
and incubate 15 min at 37°C in the dark. Add 50 μl of Stop Solution and measure the absorbance at 450
nm within 10 min. For new samples (new fish species and different developmental phases) perform the
dose response analysis using concentrated homogenates, 10x and 100x dilution. Unused plates should be
sealed with an adhesive strip and stored at +4°C up to one month, in the presence of desiccant.

1
 ELISA assay for cholecystokinin (CCK), BioMatik EKC32146-96T

Dilute Wash Buffer in distilled water 20x. Add 50 μl of Standards and Samples, then add 50 μl of
HRP-conjugate (conjugated CCK) (not in Blanks) and incubate for 60 min at 37°C. Blank should contain
a homogenization buffer instead of Sample. Aspirate and wash the wells using 200 μl of the Wash Buffer
3x. After decanting carefully all the fluid after the last wash, blot it against the paper towel and add 50 μl
of Substrate A and 50 μl of Substrate B to each well, including Blank, and incubate 15 min at 37°C in the
dark. Add 50 μl of Stop Solution and measure the absorbance at 450 nm within 10 min. For new samples
(new fish species and different developmental phases) perform the dose response analysis using
concentrated homogenates, 10x and 100x dilution. Unused plates should be sealed with an adhesive strip
and stored at +4°C up to one month, in the presence of desiccant.

Phospholipase A2 (PLA2) assay, Abcam ab133089

Dilute the Assay Buffer 9x using distilled water. Reconstitute the contents of one vial of PLA2-DTNB
using 1 ml of distilled water and keep the reconstituted solution up to 8 h on ice in the dark. Evaporate the
ethanolic solution of diheptanoyl thio-PC (Substrate) at RT until complete dryness, then add 12 ml of
diluted Assay Buffer per vial. The Substrate can be stored at -20°C up to two weeks. Add 10 µl of
PLA2-DTNB, 10 μl of Sample, Bee Venom PLA2 (positive control) or Assay Buffer (blank) and 5 µl of
Assay Buffer. Then add 200 μl of Substrate and read the absorbance at 405 or 414 nm at 0 h and each
minute for the next 10 min. Determine the change in the absorbance between two arbitrary selected time
points on the linear portion of the curve using the equation:

∆A414/min = (A414 (Time 2) - A414(Time 1))/(Time 2 (min.) - Time 1 (min.))

Calculate ∆A414/min both for Samples and for the Blank and subtract the Blank values from the Sample
values (∆A414/min_Bl) Then calculate PLA2 activity using the equation:

PLA2 Activity (µmol/min/ml) = ((∆A414/min_Bl x 0.225 ml)/ 10.66 mM-1) x (Sample dilution/0.01 ml)

DTNB extinction coefficient at 414 nm is 10.66 mM-1 and at 405 nm is 10.0 mM-1.

For new samples (new fish species and different developmental phases) perform the dose response
analysis using concentrated homogenates, 10x and 100x dilution. Dilute the samples in the diluted Assay
Buffer.

Lipase assay

Mix 10 μl of sample with 190 μl of the reaction buffer consisting of: 1 mM ρ-nitrophenyl palmitate
(ρ-NPP) in 50 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 0.3 % Triton X - 100 (prepare 20 mM stock of ρ-NPP
in isopropanol). Read the absorbance at 410 nm immediately (0 h) and each minute during subsequent 10
min, keeping the 37°C incubation temperature. Prepare blanks using the homogenization buffer only. Find
the difference between two points at the linear part of the curve both for Samples and the Blank, then
subtract Blank values from Sample values. For new samples (new fish species and different
developmental phases) perform the dose response for one representative sample using concentrated
homogenates, 10x and 100x dilution. Then proceed with the above reaction using the dose with optimal
readings. Calculation formula:

2
 Enzyme activity (mU/mg of protein) = (((ΔAhomogenate – ΔAblank)/time (min)) x 1000 x total vol of
the reaction mixture)/(ε x vol of the homogenate x prot conc (mg/ml))

- ΔA represents a difference in the absorbance between two time points

- ε is the extinction coefficient of the product released in the reaction, with ε - 15 000 M-1 cm-1

References: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

Trypsin assay

Mix 10 μl of sample with 190 μl of the reaction buffer consisting of: 1 mM

Nα-benzoyl-DL-arginine-ρ-nitroanilide hydrochloride (BAPNA) dissolved in 50 mM Tris-HCl, pH 8.2 +
20 mM CaCl2 + 1 % dimethyl sulfoxide (DMSO). Read the absorbance at 410 nm immediately (0 h) and
each minute during subsequent 10 min, keeping the 37°C incubation temperature. Prepare blanks using
the homogenization buffer diluted in the reaction buffer at the same proportion as the samples. Find the
difference between two points at the linear part of the curve both for samples and the blank, then subtract
blank values from sample values. For new samples (new fish species and different developmental phases)
perform the dose response for one representative sample using concentrated homogenates, 10x and 100x
dilution. Then proceed with the above reaction using the dose with optimal readings. Calculation formula:

Enzyme activity (mU/mg of protein) = (((ΔAhomogenate – ΔAblank)/time (min)) x 1000 x total vol of
the reaction mixture)/(ε x vol of the homogenate x prot conc (mg/ml))

- ΔA represents a difference in the absorbance between two time points

- ε is the extinction coefficient of the product released in the reaction, with ε - 8 800 M-1 cm-1

Reference: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

Chymotrypsin assay

Mix 10 μl of sample with the reaction mixture consisting of: 190 μl of 0.5 mM
succinyl-(ala)2-pro-phe-ρ-nitroanilide (SAPNA) dissolved in in 50 mM Tris-HCl, pH 8.2 + 20 mM CaCl2
+ 1% dimethyl sulfoxide (DMSO). Read the absorbance at 410 nm immediately (0 h) and each minute
during subsequent 10 min, keeping the 37°C incubation temperature. Prepare blanks using the
homogenization buffer only (diluted at the same proportion as the samples in the reaction buffer). Find the
difference between the two points at the linear part of the curve both for samples and the blank, then
subtract the blank values from the sample values. For new samples (new fish species and different
developmental phases) perform the dose response analysis for one representative sample using
concentrated homogenates, 10x and 100x dilution (in the reaction buffer). Then proceed with the above
reaction using the dose with optimal readings. Calculation formula:

Enzyme activity (mU/mg of protein) = (((ΔAhomogenate – ΔAblank)/time (min)) x 1000 x total vol of
the reaction mixture)/(ε x vol of the homogenate x prot conc (mg/ml))

- ΔA represents a difference in the absorbance between two time points

- ε is the extinction coefficient of the product released in the reaction, with ε - 8 800 M-1 cm-1

References: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

3
 Alkaline phosphatase assay

Mix 10 μl of sample with 190 μl of 5 mM p-nitrophenyl phosphate (p-NPP) in 30 mM carbonate buffer,

pH 9.8, 1 mM MgCl2. Read the absorbance at 407 nm immediately (0 h) and each minute during
subsequent 10 min, keeping the 37°C incubation temperature. Prepare blanks using the homogenization
buffer only (diluted at the same proportion as the samples in the reaction buffer). Find the difference
between two points at the linear part of the curve both for Samples and the Blank, then subtract Blank
values from Sample values. For new samples (new fish species and different developmental phases)
perform the dose response for one representative sample using concentrated homogenates, 10x and 100x
dilution (in the reaction buffer). Then proceed with the above reaction using the dose with optimal
readings. For new samples (new fish species and different developmental phases) perform the dose
response using concentrated homogenates, 10x and 100x dilution. Calculation formula:

Enzyme activity (mU/mg of protein) = (((ΔAhomogenate – ΔAblank)/time (min)) x 1000 x total vol of
the reaction mixture)/(ε x vol of the homogenate x prot conc (mg/ml))

- ΔA represents a difference in the absorbance between two time points

- ε is the extinction coefficient of the product released in the reaction, with ε - 18 300 M-1 cm-1

References: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

Amylase assay

Mix 50 μl of samples with 50 μl of 1 % starch solution. Starch was dissolved in distilled water by boiling
for 15 min. Incubate the samples with 1 % starch for 15 min at RT. Afterwards, add 50 μl of stop solution
and boil the mixture for 15 min. Finally, mix 50 μl of the reaction mixture with 150 μl of water and
measure the absorbance at 540 nm. In blank samples, add homogenates after the addition of stop reagent,
before the boiling step. Stop reagent is made by mixing distilled water, Component A and Component B
in a ratio of 1:1.7:1.7 (vol:vol:vol). Component A consists of 96 mM 3,5- dinitrosalicylic acid (DNSA)
dissolved in 20 mM sodium phosphate buffer, pH 7, by heating at 50-70°C. Component B consists of
sodium-potassium tartarate dissolved in 2 M NaOH in a ratio of 1.5:1 (w:vol) by heating at 50-70°C. For
the standard curve pippete 50 μl of maltose standard (1, 10, 100 and 1 000 mg/ml), 50 μl of water and 50
μl of stop reagent, boil for 15 min, mix 50 μl of the mixture with 150 μl of water and measure the
absorbance at 540 nm. Express amylase activity as nmol of maltose released per min per mg of protein.
For new samples (new fish species and different developmental phases) perform the dose response
analysis using concentrated homogenates, 10x and 100x dilutions in the homogenization buffer.

References: http://dx.doi.org/10.1016/j.rvsc.2017.07.018

RNA isolation

Before the RNA isolation, prepare the Denaturing buffer and acidic phenol solution.

Denaturing buffer: 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.1 M β-mercaptoethanol (stock is

14.3 M), 0.5% [wt/vol] N-lauroylsarcosinate sodium salt.

Acidic phenol solution (prepare in laminar flow-hood): Thaw the crystal phenol at 55°C, the immediately
add 1 volume of 50mM sodium acetate pH 4, mix vigorously in the dark bottle and leave overnight at
+4°C for the phases to separate. Then aspirate the upper (water) phase and add again 1 volume of 50 mM

4
sodium acetate, shake vigorously and leave again overnight for the phases to separate. Repeat the
procedure another once and the phenol is ready for use.

Place 100-200 mg of larvae in the mortar, homogenize using liquid nitrogen, add 10 x Denaturing buffer
(the Denaturing buffer will freeze), then remove the frozen mixture using a scalpel blade and place it in a
50 ml Falcon tube on ice to thaw slowly. After complete thawing, aliquot into two 1.5 ml tubes and add 1
volume of acidic phenol solution, 1/10 volume of 2 M sodium acetate, pH 4 and 1/5 volume of
chloroform. Mix vigorously using hand shaking, leave 10 minutes on ice and place in the centrifuge
(maximal speed, +4°C, 15 min). Take carefully the upper phase using two tips (pool the samples from the
two tubes into one tube) and then repeat the phenol extraction. After second phenol extraction add 1
volume of isopropanol and leave the samples at -20°C for 30 min. Then cfg 15 min, +4°C, max speed and
wash the pellet one using 70 % ethanol (cfg at maximal speed, +4°C, 15 min). Aspirate all the remaining
fluid and dry the pellets at RT for 15 min. Dissolve the pellets in 20 μl of distilled water, measure RNA
concentration and store the samples at -80°C until use.

Reverse transcription, Xpert cDNA Synthesis Kit, GK80.0100

Mix 1 μg of RNA template with 4 μl of 5 x Reaction Buffer, 1 μl of dNTP mix, 1 μl of Random Hexamer,
1 μl of Xpert RTase and fill up the distilled water to 20 μl. Prepare the control by using heat inactivated
the Xpert RTase (at 85°C for 10 min). Incubate the mixtures at 25°C for 10 min, then at 55°C for 15 min
and then at 85°C for 5 min. Then chill the samples on ice and store at -20°C until use.

Real-time PCR (qPCR)

Prepare 100 mM working stocks of primers. Mix 10 μl of Xpert Fast SYBR 2X Mastermix (uni) with
ROX* with 2 μl of each primer (Fw and Rev), add 2 μl of template DNA and add PCR –grade water up to
20 µl. Use the following cycling conditions:

1x 95°C, 2-3min

40x 95°C, 5 sec

60-65°C, 20-30sec

Calculate ΔCt for each sample by subtracting Ct values of target gene and housekeeping gene. Then
subtract ΔCt of treatments to the ΔCt of the control group (or reference group) to obtain ΔΔCt for each
sample (relative quantification). Prior to setting up the relative gene expression test, perform primer
efficiency analysis with 1 x, 10 x, 100 x and 1000 x dilution of cDNA to check the slope of the curve (log
DNA copy number versus Ct). The slope should be somewhere between -2.5 and -3.5. If that is the case,
proceed to relative quantification using the selected cDNA concentrations. Perform at least two technical
replicates for each sample.

5
6
7
8