DIFFERENCES BETWEEN THE BALTIC COD GADUS MORHUA CALLARIAS L. AND ATLANTIC COD GADUS MORHUA MORHUA L., BY SKELETAL MUSCLE PROTEIN POLYMORPHISM, ANALYZED WITH CLASSICAL SDS-PAGE ELECTROPHORESIS

Artur Antoszek¹, Jolanta Antoszek², Beata Więcaszek¹, Radosław Drozd², Sebastian Suszycki^2
1 Department of Fish Systematics, West Pomeranian University of Technology, Szczecin, Poland
² Department of Immunology, Microbiology and Physiological Chemistry, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

The taxonomic status of cod from the Baltic Sea has not yet been fully recognized. The aim of this study was to determine the suitability of regular SDS-PAGE electrophoresis to differentiate between two cod sub-species from the Baltic Sea. The examined fishing grounds were the Barents Sea (the region of Bear Isle), the Strait of Sund and Puck Bay (Baltic Sea). In muscle extracts from individuals from the Barents Sea and the Strait of Sund, SDS-PAGE analyses showed protein profile consisting of 25 proteins compare to 16 proteins from cod from the Baltic Sea. Fourteen of them were common for both groups. The results obtained in this study show that cod from the Strait of Sund show a high similarity (and are in fact identical) to cod from the Barents Sea. SDS-PAGE protein profiles of proteins from the Baltic Sea cod are different from cod from the Barents Sea and the Strait of Sund. Therefore it may be a strong premise for the statement that G. morhua callarias is a local Baltic subspecies.

Key words: Atlantic cod, Baltic cod, Gadus morhua, polymorphism, skeletal muscle protein.

INTRODUCTION

The Atlantic cod is a migratory marine predator, living in the northern part of the Atlantic Ocean and in the seas of northern Europe. It tolerates various degrees of salinity, lives down to 400 m, and preys mainly at the sea bottom. The Atlantic cod has two subspecies (geographical races): Gadus morhua morhua and G. m. callarias, which are different in their range, environmental preferences (salinity, temperature and feeding waters), colouration and body sizes [6, 4, 14]. Gadus m. morhua live in the northern part of the Atlantic Ocean, the North Sea, the western part of the Arctic Sea, near Iceland, Greenland, Newfoundland, and in the western part of the Baltic Sea, and show a high genetic variability of schools [7, 11]. Gadus m. callarias live in the Baltic Sea from 15°E up to the Gulf of Bothnia. It has smaller body size (the average body length of cod living in the North Sea is 30–80 cm, and in the Baltic Sea 30–70 cm) and is adapted to living in waters with lower salinity. In no other area has any subspecies of cod adapted to life in such a low salinity. Its spawning, from March to August, occurs in deep waters (Gotland, Bornholm and Gdańsk deeps) where a higher salinity ensures the pelagity of spawn [1, 2]. The International Council for the Exploration of the Sea (ICES) divides the population of the Baltic cod into two subpopulations, Western Baltic Cod (ICES SD 22–24) and the Eastern Baltic Cod (ICES SD 25–32), west and east of Bornholm (55°N, 15°E), different in their location and spawning times.

The taxonomic status of cod living in the Baltic Sea has not yet been fully recognized. It has been determined based on hemoglobin polymorphism [15, 16] that Baltic cod are more closely related to cod from the Barents Sea than those from the North Sea. It is possible as the history of the Baltic Sea dates back as early as the time when the Baltic Sea was connected with the Barents Sea (8000 B.C.) and when the marine fauna from this basin colonized the area of the present Baltic. It concerns mainly the stock in the eastern part of the Baltic Sea [17]. In opposition to the hypothesis by Sick [15] and Sobecka [17], the results of Nielsen et al. [10] came from microsatellite loci examination to prove a closer affinity of Baltic cods to those from the North Sea than from the Barents Sea. However, considering the fact that adaptation to an environment with lower salinity is a long-term process, associated with a reduction in body weight and a temporary decrease in immunity and tolerance to environmental pollution [17], the massive secondary migration of cod to the Baltic Sea does not seem very realistic.

In this study, the response was given to the growing need for monitoring the world cod population, decreasing and threatened by overfishing [10, 11], which also concerns local Baltic cod populations. As classic monitoring methods based mostly on morphometric examinations are imperfect [14, 9], molecular biology methods such as SDS-PAGE electrophoresis, using two dimensional electrophoresis, has been applied successfully to difference wild cod from farmed cod [8].

The aim of this study was to analyse the suitability of regular SDS-PAGE electrophoresis to differentiate between two sub-species of cod.

MATERIALS AND METHODS

The cod were collected from fishing grounds in the Barents Sea (the region of Bear Isle: 73°30 N, 19°00 E, depth: 300 m), the Strait of Sund (55°49 N, 12°42 E, depth: 30 m), and Puck Bay (Baltic Sea, 54°41 N, 18°40 E, depth: 3 m).

The morphology of the cod was examined, including the number of rays on the first dorsal fin (D1) and the second dorsal fin (D2), according to the criterion provided by Müller [9]. Muscle tissues samples were taken from 20 individuals in good condition (without visible wounds or injuries) from each fishery and stored at -20°C until analysis. The muscle extracts were prepared according to the following procedure: 20 mg of freshly defrosted tissue were homogenized on ice with 0.2 cm³ of 50 mmol/dm³ phosphate buffer, pH 7.8, and then centrifuged 10 000 rpm for 10 min. Supernatants of samples were mixed with equal amounts of denaturing loading buffer (62 mM TrisCl, pH 6.8, 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.001% bromphenol blue) and boiled in a water bath for 5 min at 100°C. Three µg of denatured protein extract was loaded on gels. The electrophoresis was carried out using an omniPAGE mini 10×10 cm in 10% polyacrylamide gel in the presence of 0.01% SDS. The separation time was 50 min (75V) in the stacking gel and 270 min (100V) in the resolving gel. Gels were stained by Coomassie Brilliant Blue R-250. After destaining gels were dried and scanned. The results were analyzed using UN-SCAN-IT gel 6.1 software (Silk Scientific Corporation, USA). The gels were calibrated with a set of molecular markers (Sigma, SDS6H2), myosin 205 kDa porcine, ß-galactosidase 116 kDa (E. coli), phosphorylase B 97.4 kDa rabbit muscle, bovine serum albumin 66 kDa, chicken ovalbumin 45 kDa, carbonic anhydrase, from bovine erythrocytes 29 kDa.

Protein concentration assay in supernatans were done using the Breadford method with bovine serum albumin as standard [3].

RESULTS

The selected individuals from three samples (Baltic Sea, Barents Sea and Strait of Sund) with regard to their morphometric characteristics were examined. The morphometric examination confirmed the affiliation of cod from the eastern part of the Baltic Sea (Puck Bay) to the subspecies G. m. callarias. The number of rays in the first (D1) and second (D2) dorsal fins were within the range provided by Müller [9] (D1>14, D2<18), D1 = 14.22 ± 0.11.21 D2 = 17.63 ± 0.16 (n = 40). The morphometric examination of cod from two remaining samples confirmed their affiliation to the subspecies G. m. morhua (D1<14, D2>18: Strait of Sund  D1 = 13.87 ± 0.12, D2 = 18.98 ± 0.17 (n = 54); D1>14, D2>18: Barents Sea: D1 = 14.32 24 ± 0.08, D2 = 19.58 ± 0.16 (n = 57). The condition of cod from the Barents Sea and Puck Bay was very good. However, cod from the Strait of Sund had numerous effusions (in 81.5% of the examined individuals) and ulcerations (10%); 53.7% of the cod from that area was parasitized with digenetic trematode metacercariae.

In muscle extracts from individuals from the Barents Sea and the Strait of Sund, SDS-PAGE analysis showed protein profile consisting of 26 proteins compare to 16 proteins from cod from the Baltic Sea. Fourteen of them were common for both groups (Table 1).

Table 1. Molar mass (kDa) of skeletal muscle proteins from G. morhua morhua and G. morhua callarias subject to SDS-PAGE under denaturating conditions

Fraction	Pertinence	G. morhua morhua (G.mm)	G. morhua callarias (G.mc)
		(kDa)	(kDa)
1	G.mm	196.9
2	G.mc		179.2
3	G.mm	145.9
4	G.mc		136.9
5	G.mm	133.9
6	G.mm	127.4
7	G.m	117.7	115.7
8	G.m	102.7	100.2
9	G.m	92.2	88.7
10	G.mm	84.4
11	G.mm	80.5
12	G.mm	76.3
13	G.mm	70.3
14	G.m	58.3	58.3
15	G.m	54.5	52.5
16	G.m	51.0
17	G.m	46.7	49.3
18	G.m	43.2	46.0
19	G.m	39.6	41.8
20	G.m	37.3	40.3
21	G.m	33.6	36.1
22	G.m	32.4	34.3
23	G.mm	30.9
24	G.m	29.4	30.2
25	G.mm	28.4
26	G.m	27.4	27.3
27	G.mm	26.7
28	G.m	26.1	25.9

Selected  electropherograms obtained in the experience are presented in Fig.1. Compared protein profiles were more diverse in range of molecular mass from 196.9 to 51.0 kDa, where differences were observed between proteins from cod from the Barents Sea and the Strait of Sund (G. morhua morhua) and the cod from the Baltic Sea (G. morhua callarias). Protein profiles differed also for protein bands 23–28 corresponding to the molecular mass of 30.9–25.9 kDa (Table 1). The comparison of protein profiles carried out for individual cod coming from various fishing grounds showed monomorphism with respect to 15 protein bands, 7, 8, 9, 14, 15, 17, 18, 19, 20, 21, 22, 24, 26, 27, and 28. In the pattern of proteins from G. m. morhua, there are eleven protein bands (1, 3, 5, 6, 10, 11, 12, 13, 16, 23, 25), with corresponding molecular masses: 196.9 kDa, 145.9 kDa, 133.9 kDa, 127.4 kDa, 84.4 kDa, 80.5 kDa,76.3 kDa, 70.3 kDa, 51.0 kDa, 30.9 kDa and 28.4 kDa. These protein bands were not detected in extracts from G. m. callarias. Two protein bands characteristic for cod from the Baltic Sea 179.2 kDa (2) and 136.9 kDa (4) differentiate G. m. callarias from cod in the remaining fishing grounds (G. m. morhua) in the Strait of Sund and the Barents Sea.

Fig. 1. Electrophoregrams of skeletal muscle proteins from cod: a) from Barents Sea, b) from  Strait of Sund, c, d) from Puck Bay

DISCUSSION

The Baltic Sea is a basin with a considerable degree of isolation. It receives only a small amount of salty ocean water through the Danish Straits (periodic oceanic inflows from the North Sea) which moves in the demersal zone in a north-easterly direction. As cod live mostly near the bottom, they can use this route to reach the Baltic Sea from the Atlantic Ocean. Migration in the opposite direction is practically impossible, due to the small size of the straits, higher salinity of the ocean waters, and the periodicity and the direction of oceanic inflows. The range of the hybrid zone, the area of possible permeation and mixing of subspecies, is limited to the Western Baltic (Meklenburg Bay, Arkona Basin, Pomeranian Bay). It was confirmed by the variability of hemoglobin in two different populations of cod from the Danish Belt Sea and the Baltic Sea, and demonstrated that these two populations do mix but not interbreed Sick [14, 15]. In later reports, Aro [1] and Müller [9] demonstrated that the mixing of the cod subspecies is restricted to the western part of the Baltic Sea. This observation is supported by Nielsen et al. [10] who, in their analysis of nine microsatellite loci in the North Sea and Baltic Sea, showed the presence of parental individuals from two different populations, and also hybrid offspring constituting hybrid-stocks. According to that report, the level of mixing or the location of the mixing area can have different ranges depending on the inflow volume from the Kattegat. Bleil and Oeberst [2] reported that mixing of G. morhua morhua and G. morhua callarias occurs in the Arkona Basin (near Cape Arkona 54°41'N, 13°26'E) during spring and summer spawning. It creates a strong premise that the western stock consists mainly of G. morhua morhua (Belt Sea cod), and the eastern stock G. morhua callarias (Baltic cod). In 1982 Grant and Stahl [cited in 13] reported that the polymorphism of skeletal muscle proteins in cod may be associated with environmental conditions affecting the examined individuals. It suggests the possibility of variability at a metabolic level as a result of secondary adaptation to the environment of the individual populations. Similar conclusions can be drawn from the report by Renaud et al. [13] who used IEF (isoelectrofocusing electrophoresis) to analyze the polymorphism of enzymes extracted from cod muscle tissues from distant fishing grounds.

The reliability of the obtained samples was the most important condition for the correct assessment of fractionation results (on proteins extracted from G. morhua skeletal muscles using SDS-PAGE) and the application of the gathered date for the identification of the G. morhua callarias subspecies. In the first approximation the assumption was that cod from the eastern part of the Baltic Sea basin significantly differ from Atlantic cod, and belong to the subspecies G. morhua callarias [1, 2]. The same samples were also subjected to microsatellitary examination (6 loci), and a similar result was obtained (Kijewska et al. unpublished). In this case, the material taken from cod from the south-eastern Baltic (Puck Bay) was compared with material from the south-western Baltic (the Strait of Sund). The study showed a high level of heterozygosity, and also a slight deficiency in heterozygotes, which means that the selected geographic distance was an effective barrier that inhibited the transfer of genes between the examined populations, what was also in agreement with Pogson et al. [12]. This effect was especially visible in cod from Puck Bay which were observed to have a deficiency of heterozygotes at locus Gmo8 (Kijewska et al. unpublished). Confirmation of these results can be found in an analysis of the population of higher taxa of parasites living in the examined cod. That study showed that cod from the Barents Sea and the Strait of Sund differed significantly from cods living in the Baltic Sea (eastern stock) with regard to the composition of higher-rank taxa [17]. The assessment of the general condition of the individuals from the examined populations also showed distinct differences. Cod from the Strait of Sund was in the worst condition. According to Sobecka [17], the reason is the lower immunity associated with the abrupt changes in the environment, which is proof of the fact that these individuals came from the North Sea to the Baltic Sea together with oceanic inflows. It confirms the hypothesis assumed in this paper that cod from the Barents Sea belongs to the subspecies G. morhua morhua, and cod from Puck Bay (the Baltic Sea, eastern stock) are from the subspecies G. morhua callarias. Cod from the Puck Bay may represent proteins associated with the very low salinity of this area and it suggests that individuals from the Baltic Sea are a subspecies that do not migrate beyond the Baltic Sea. These fractions did not occur in cod from the Strait of Sund and from the Barents Sea (G. morhua morhua), with  a distinctly higher salinity and richer nutrition. The results obtained in this paper show that cod from the Strait of Sund show a high similarity (and are in fact identical) to cod from the Barents Sea. SDS-PAGE protein profiles of proteins from the Baltic Sea cod are different from cod from the Barents Sea and the Strait of Sund. Therefore it may be a strong premise for the statement that G. morhua callarias is a local Baltic subspecies.

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17. Sobecka E., 2007. Pasożyty dorsza atlantyckiego z podgatunków Gadus morhua morhua L. i Gadus morhua callarias L. z wybranych rejonów Atlantyku i Bałtyku [Parasite fauna of two subspecies of the Atlantic cod Gadus morhua morhua L. and Gadus morhua callarias L. from selected areas of Atlantic and Baltic Sea]. Post-doctoral thesis. 245. Agriculture University of Szczecin. pp. 82. [in Polish, with English summary].

 

 

Accepted for print: 30.09.2011

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Artur Antoszek
Department of Fish Systematics,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland

Jolanta Antoszek
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin, Poland
2, Judyma st., 71-466 Szczecin, Poland

Beata Więcaszek
Department of Fish Systematics,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland
email: bwiecaszek@zut.edu.pl

Radosław Drozd
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin, Poland
2, Doktora Judyma st., 71-466 Szczecin

Sebastian Suszycki
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin, Poland
2, Doktora Judyma st., 71-466 Szczecin

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