284 Bulgarian Journal of Agricultural Science, 12 (2006), 284-289 National Centre for Agrarian Sciences Genetic – Biochemical Characteristic of the Hemoglobin and the Transferins in the Black Sea Turbot (Psetta maxima) P. IVANOVA, A. TSEKOV, I. DOBROVOLOV and S. ATANASOVA Institute of Fisheries and Aquaculture, BG - 9000 Varna, Bulgaria Abstract IVANOVA, P., A. TSEKOV, I. DOBROVOLOV and S. ATANASOVA, 2006. Ge- netic – biochemical characteristic of the hemoglobin and the transferins in the Black Sea turbot (Psetta maxima). Bulg. J. Agric. Sci., 12: 284-289 In the present work the genetic-biochemical analyses of transferins and hemoglobins of the Black Sea turbot from different localities (the “Drill” area, the Cape Emine and the mouth of Kamtchiya River) were carried out. Two different electrophoretical methods – starch gel electrophoresis and isoelectric focusing (IEF) are applied. Up to now the Black Sea turbot population structure is not studied, which is the mark for the significance of the problem we investigated. The main goal of the study is to found genetic-biochemical markers for the Black Sea turbot and genetical characteristic of this very valuable fish species. Key words: turbot, hemoglobin, transferins, electrophoresis, isoelectric focusing, population structure Introduction and more over the application of the popu- lation genetics of fish in the fishery man- The Black sea turbot is distributed along agement, as well as for the protection and the Mediterranean coast of Europe, Black developing of the aquacultures. When the and Azov Seas and specific meaning of genetic population structure of a species its artificial breeding has increased in the is known, the distribution of sub-popula- last decade (Șahin and Űstűndag, 2003). tions in mixed fisheries can be estimated It is one of the most valuable fish species (Utter, 1991). in the Black Sea. Electrophoresis is a good The population genetic analyses of spe- detector of variability and isozyme poly- cies in nature is of primary importance in morphisms have been widely used in ge- developing an optimal strategy for their netic studies (Rebordinos and Gross, 1999). effective management (Ciftci and Okumus, The genetic study of natural populations 2002). Bouza et al. (1997) on the base of is depending on the availability of polymor- allozyme analyses observed a lower dif- phic neutral markers (Neigel, 1997). Dur- ferentiation between the turbot popula- ing the last years the stress is falling more tions. Nielsen et al. (2004) examined popu- [email protected] Genetic – Biochemical Characteristic of the Hemoglobin... 285 lation – genetical structure of the turbot specimens inhabiting different regions are (Scophtalmus maximus L.) in the north- examined 12 specimens from the region east part of the Atlantic Ocean, using eight of “The Drill”; 15 specimens from the re- loci with high variability. The existence of gion of cape Emine and 14 specimens from two divergeted population connected by the region against the mouth of Kamtchia hybrid zone was found. Coughlan et al. River.Determination of enzyme polymor- (1998) investigated genetic variability on phism were found using two methods: hori- three polymorphic microsatelite loci in two zontal starch gel electrophoresis by Smith- wilds and two artificial turbot populations ies (1955) in the micromodyfication of in Ireland and Norway. Dobrovolov (1973) and isoelectric focus- The genetic heterogeny between the ing (IEF) on thin polyacrilamide Ampholine samples was determined. Imsland et al. gel plates with pH gradient 3.5 - 9.5 and (2003) examine the turbot (Scoptalmus LKB Stockholm, Sweden, equipment. The maximus) from five areas of North Eu- buffer systems according to Dobrovolov rope using an agaroze electrophoresis and (1976) and Clayton and Gee (1969) were isoelectric focusing of the hemoglobin (Hb) used. The visualization of nonenzyme pro- and proved that the turbot from the wa- tein fractions on thin polyacrilamide ters of North Europe belongs to more than ampholine gel was done by using one panmixed population.Suzuki et al. Coomassie brilliant blue R – 250, while on (2004) on the base of mtDNA analyses starch gel with Amido Black 10B. Enzyme proved existence of two genetic different systems (esterase – EST and malate de- turbot (Psetta maxima) lineages from west hydrogenase – MDH) were visualized and east part of the Mediterranean basin. according to Shaw and Prasad (1970). The population-genetic structure of the turbot in the Black Sea is not examined Results and Discussion until the present. Only the morphometric investigations and turbot stocks determi- After analyses of the proteins from nation of along the Bulgarian Black Sea blood on starch gel two polymorphic zones coast were done (Karapetkova, 1961, are determined. The closest to the start 1975; Ivanov and Karapetkova, 1981). fractions belongs to the transferines (Tf) More than 20 years the observation on the (Figures 1 and 2), followed by globulins, distribution, population structure, biology albumins and prealbulins. One-locus double and ecology of this valuable species were allele codominant inheritage is observed not carried out.The main purpose of the (Figure 1). The genetic frequency of Tf- presented work is to specify the genetic- 1*a is 0,773. The distribution of the phe- biochemical markers, which will be used notypes is presented on the Table 1. The for genetic characteristic of the turbot fractions of the globulins (Glob.) in the populations from the Bulgarian Black Sea turbot from the compared regions are with coast. different electroforetic movability (Figu- re 2). Material and Methods The fractions in the specimens from the region of the Cape Emine are faster than Blood samples for transferines and those from the region of “The Drill” (Fig- haemoglobin taken from Black Sea turbot ure 2). Parallel with starch gel electro- 286 P. Ivanova, A. Tsekov, I. Dobrovolov and S. Atanasova 0 Tf Glob Alb Prealb ab ab aa aa aa aa aa bb Tf ab ab bb ab aa aa bb bb Prealb Fig. 1. Starch gel electrophoresis on blood by the Black Sea turbot from the “Drill” area. On the electrophoregram two polymorphic zones of transferins (Tf) and prealbumins (Prealb.) are pre- sented, 0 – origin 0 Tf Glob Alb Prealb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Fig. 2. Starch gel electrophoresis on blood by the Black Sea from the “Drill” area (1-9) and from the Cape Emine (10-16), 0-origin Genetic – Biochemical Characteristic of the Hemoglobin... 287 0 Hb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Fig. 3. Isoelectric focusing (IEF) on hemoglobins (Hb) and general muscle proteins (PROT) on thin polyacrilamide Ampholine gel with pH gradients 3.5 – 10 by Black Sea turbot from the “Drill” area: (1 - 14) – Hb, (15 – 17) – PROT, 0 – origin 0 ЕST-1* EST-2* EST-3* EST-4* Fig. 4. Starch gel electrophoresis on esterases (EST) on blood by the Black Sea turbot from the “Drill” area. On the electrophoregram four zones with EST activity (EST – 1* and EST – 2* polymorphic) are presented, 0 – origin 288 P. Ivanova, A. Tsekov, I. Dobrovolov and S. Atanasova Table 1 Phenotypes arrangement of polymorphic loci by the Black Sea turbot Index The “Drill” area Phenotypes Tf* Prealb* aa 14 10 13.15 8.90 ab 6 8 7.72 10.19 bb 2 4 1.13 2.92 Numbers of the samples investigated 22 22 Criteria ?2 1.11 1.01 Probability (df=1) P> 0.25 P>0.25 Gene frequency a 0.773 0.636 b 0.227 0.364 phoresis, isoelectric focusing (IEF) of he- River) are good genetical markers for de- moglobins on Black Sea turbot was ap- termination of its population structure. The plied (Figure 3). Polymorphism was ob- polymorphism in the esterases, transferins, served, but for its decoding further ana- prealbumine and hemoglobin could also be lyzes should be carried out. used as markers for the Black Sea turbot The electroforetic spectra of the gen- populations. eral muscle protein (PROT) in the turbot consists of thirty-four fractions (Figure 3). References Differences in the electroforetic movabil- ity in the compared specimens from the Bouza, C., L. Sanchez and P. Martinez, 1997. region of “The Drill” are not obser- Gene diversity analysis in natural popula- ved.Regarding the esterase four zones are tions and cultured stocks of turbot (Scoph- determined, but only in two of them a poly- thalmus maximus L.). Animal Genetics, 28 morphism is observed (Figure 4). The poly- (1): 28. morphism found could be used for the Ciftici, Y. and I. Okumus, 2002. Fish popula- population genetic analysis of the turbot. tion genetics and application of molecular markers to fisheries and aqua-culture: I - Conclusions Basic principles of Fish popu-lation genet- ics. Turkish Journal of Fisheries and The fractions of the globulins (Glob.) Aquatic Sciences, 2: 145-155. observed in the Black Sea turbot from the Clayton, I. W. and G. Gee, 1969. Lactate dehy- compared regions (the “Drill” area, the drogenase isozymesin longnose and bla- Cape Emine and the mouth of Kamtchiya cknose dase (Rhinichtys cataracte and R. Genetic – Biochemical Characteristic of the Hemoglobin... 289 atratulus) and their hybrid. J. Fish. Res. Community Society, pp. 25-26. Bd. Canada, 26: 3049-3053. Neigel, J. E., 1997. A comparison of alterna- Coughlan, J. P., A. K. Imsland, P. T. Galvin, R. tive strategies for estimating gene flow from D. Fitzgerald, G. Naevdal and T. F. Cross, genetic markers. Annual Review of Ecol- 1998. Micrasatellite DNA variation in wild ogy and Systematics, 28: 105-128. populations and farmed strains of turbot Nielsen, Einar E., Nielsen, Peter H., Meldrup, from Ireland and Norway: a preliminary Dorte, Hansen and M. Michael, 2004. Ge- study. Journal of Fish Biology, 52 (5): netic population structure of turbot (Scop- 916-922.