2.9 MS Data
Processing
1. Proteome Discoverer 2.4 package implemented with
SEQUEST-HT license.
2. UniProt/TrEMBL database downloaded from Uniprot.org.
3 Methods
3.1 Parasite Isolation
and In Vitro Culture
1. The worms could be isolated from the Baltic herring (Clupea
harengus membras) or another paratenic host of Anisakis simplex s.s. L3 developmental stage, e.g., European hake (Merluccius merluccius) or horse mackerel (Trachurus trachurus).
2. Follow the fish longitudinal abdominal incision using surgical
scissors. Find free worms in the host body cavity (Fig. 3). For
the experiment, use 20 mm or more in length and collect with
the help of a tweezers, place on a Petri dish with 0.9% NaCl
solution, and wash in it several times.
3. Observe the worms individually under an inverted microscope
and discard those which show any kind of internal or external
damage.
4. Use ten larvae for taxonomical identification using conventional PCR to amplify the ITS-rDNA region (see Note 10
and ref. 8). Isolate gDNA with the use of a commercially
available kit and perform PCR reaction according to the manufacturer’s instruction. Set the PCR temperature conditions as
follows: 95
C for 10 min (initial denaturation), followed by
30 cycles at 95
C for 30 s (denaturation), 55
C for 30 s
(annealing), 72
C for 75 s (extension), and a final elongation
step at 72
C for 7 min. Then digest the PCR products using
the Hinf I restriction endonuclease. Perform the digestion
using 1 μL of restriction enzyme (Hinf I), 1 μL of buffer B
10Â, and 8 μL of PCR products up to a final volume of 10 μL.
Perform the digestion for 3 h at 37
C. Visualize restriction
patterns on 2% agarose gel under UV light (Fig. 4).
5. Divide the rest of the larvae into two groups. The first group is
stored separately for protein extraction and precipitation (see
Subheading 3.2), and the second group is used for in vitro
culture to L4 developmental stage.
6. Wash larvae intended for L4 stage culture for 30 min in a
bactericidal and fungicidal solution. Then put them into 0.9%
NaCl solution and wash in it several times.
7. Culture larvae on a sterile polystyrene 6-well culture plate.
Place the culture medium (3 mL) into each well with three
parasites. Then place the culture plate in an incubator at 37
C
and 5% CO 2 in humid air. Renew the culture medium every
2 days for 1 week. Observe the worms every 2 days for mobility,
molting, and survival.
64
Robert Stryin ´ ski et al.
Processing
1. Proteome Discoverer 2.4 package implemented with
SEQUEST-HT license.
2. UniProt/TrEMBL database downloaded from Uniprot.org.
3 Methods
3.1 Parasite Isolation
and In Vitro Culture
1. The worms could be isolated from the Baltic herring (Clupea
harengus membras) or another paratenic host of Anisakis simplex s.s. L3 developmental stage, e.g., European hake (Merluccius merluccius) or horse mackerel (Trachurus trachurus).
2. Follow the fish longitudinal abdominal incision using surgical
scissors. Find free worms in the host body cavity (Fig. 3). For
the experiment, use 20 mm or more in length and collect with
the help of a tweezers, place on a Petri dish with 0.9% NaCl
solution, and wash in it several times.
3. Observe the worms individually under an inverted microscope
and discard those which show any kind of internal or external
damage.
4. Use ten larvae for taxonomical identification using conventional PCR to amplify the ITS-rDNA region (see Note 10
and ref. 8). Isolate gDNA with the use of a commercially
available kit and perform PCR reaction according to the manufacturer’s instruction. Set the PCR temperature conditions as
follows: 95
C for 10 min (initial denaturation), followed by
30 cycles at 95
C for 30 s (denaturation), 55
C for 30 s
(annealing), 72
C for 75 s (extension), and a final elongation
step at 72
C for 7 min. Then digest the PCR products using
the Hinf I restriction endonuclease. Perform the digestion
using 1 μL of restriction enzyme (Hinf I), 1 μL of buffer B
10Â, and 8 μL of PCR products up to a final volume of 10 μL.
Perform the digestion for 3 h at 37
C. Visualize restriction
patterns on 2% agarose gel under UV light (Fig. 4).
5. Divide the rest of the larvae into two groups. The first group is
stored separately for protein extraction and precipitation (see
Subheading 3.2), and the second group is used for in vitro
culture to L4 developmental stage.
6. Wash larvae intended for L4 stage culture for 30 min in a
bactericidal and fungicidal solution. Then put them into 0.9%
NaCl solution and wash in it several times.
7. Culture larvae on a sterile polystyrene 6-well culture plate.
Place the culture medium (3 mL) into each well with three
parasites. Then place the culture plate in an incubator at 37
C
and 5% CO 2 in humid air. Renew the culture medium every
2 days for 1 week. Observe the worms every 2 days for mobility,
molting, and survival.
64
Robert Stryin ´ ski et al.