1SI Hydrobiological Institute-Ohrid, Republic of Macedonia

1. BIOCHEMICAL - GENETICAL MARKERS AS USEFUL TOOLS FOR DETERMINATION OF POPULATION STRUCTURE OF BARBUS MERIDIONALIS PETENYI IN LAKE OHRID Lydia Velkova-Jordanoska1, Petya P. Ivanova2, Ivan S. Dobrovolov2 1SI Hydrobiological Institute-Ohrid, Republic of Macedonia 2 Institute for fisheries and aquaculture-Varna, Bulgaria,

2. Very often phenotype differences in different organisms can not be markers for differences in their genotype. In fact, in the final formation of some species phenotype, except genetical factors, is impacted by the environmental factors too. On the account of this, more variations of the phenotype in just one species are possible. In the literature there is data about using allozyme electrophoresis for identification of fish species and using the technique of multilocus enzyme electrophoresis addressing the issues related to systematic of fish. The conservation of animals is focused on a species or a strains of animals, aiming towards maintaing of a group as a genetic resources. Protein polymorphism may be very good indicator of genetic diversity of fish populations.

3. Dimovski and Grupce (1987) consider that Lake Ohrid is populated with only one, not two subspecies, or certain intermediate population between the two subspecies. Two subspecies of barbel present in Lake Ohrid are mentioned in the work of Naumovski (1995) as follows: Barbus meridionalis petenyi Heckel and Barbus meridionalis rebeli Koeller. Georgiev (1998) holds the view that in accordance with the principles of modern systematics, evolution and genetics, two subspecies of the same species cannot exist in the respective watershed. That implies the fact that Lake Ohrid can be populated with only one subspecies of barbel, not two. Karaman (1971), Economidis (1989), Kotellat (1997), and Georgiev (1998) use the name of black barbel Barbus peloponnesius Valenciennes, 1842 instead of Barbus meridionalis petenyi Heckel. According to Karakousis et al. (1995), B. peloponnesiusand B. m. petenyi are one and the same species. • Genetic-biochemical markers could be used for resolution of taxonomical problems and for clarifying of the population structure of fish species (Dobrovolov, 1992, 1996, 1999, Ivanova and Dobrovolov, 2002).

4. Material and methods • A total of 22 samples of species Barbus meridionalis petenyi Heck. from Lake Ohrid were collected during 2003 and 2004. Parts of white muscle of each freshly frozen specimen were homogenised, centrifuged, and a supernatant was used for starch-gel electrophoresis. Miogens (sarcoplasmic proteins) and five enzymes: esterase (EST), malat dehydrogenase (MDH), lactat dehydrogenase (LDH), malic enzyme (ME) and superoxide dismutase (SOD) were analysed, based on protein starch-gel electrophoresis and isoelectric focusing (IEF) on thin polyacrilamide ampholine gel with pH gradient between 3-10.

5. Results and discussion • Miogens: Each species of Barbus possessed a specific myogenic spectrum, which might be very useful tools for detection of interspecies polymorfism. Starch-gel electrophoresisand isoelecrtic spectrum can not display differences in electrophoretic mobility of miogen fractions among investigated samples. • EST ( E.C. 3.1.1.1.). The polymorphism in the esterase loci was not observed. • ME (E.C. 1.1.1.40). Electrophoretic mobility of ME fractions was tipical and equal for all of investigated samples. • SOD (E.C. 1.15.1.1.) According starch-gel electrophoregram of this enzyme system, differenses among investigated barbel’s samples is very difficult to decode (Fig.7). A polymorphism in species Barbus meridionalis petenyi Heck. from Lake Ohrid is not established.

6. LDH (E.C. 1.1.1.27). Enzymographic analyses of starch-gel electrophoresis detect species differensis and series of isoenzymes among investigated specimens. Several specimens are polimorfic for enzime lactat dechydrogenase,(positions 1,2, and 3) and other are monomorphic for them. Lactat dehydrogenase in fishes has been used as genetic marker, because its tetrameric structure is able to dissociate and hybridize without lossing in enzymatic activity, and it is occures in nature in many variant forms. We need much more investigation on barbel populations in Lake Ohrid, to resolve the question, about presence of one or two subspeciesof Barbus meridionalis in this lake. 1 2 3 4 5

7. MDH (E.C. 1.1.1.37). Starch-gel electrophoresis discovere the multiple isosymes and presence of several phenotypes of malate dehydrogenase in investigated samples. The locus MDH-1 is monomorphic and MDH-2 displey polimorfism. Positions 1, 3 and 5 displey MDH fractions from heterozigotes organisms. 1 2 3 4 5

8. The application of more modern methods in the resolution of taxonomical problems, for instance biochemical and molecular – genetic analyses will lead to obtaining a more accurate picture of the presence of certain species, their taxonomical affiliation and contribute to solve a greater number of questions which cannot be explained by morphological and osteological analyses. • The ultimate objective of all the investigations is to find a relevant, stable marker or markers that would enable a prompt and precise identification of the species of genus Barbus and alongside that provide valid proofs for the taxonomy of this fish genus.