1SI Hydrobiological InstituteOhrid, Republic of Macedonia

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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,

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• 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.

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• 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.
  peloponnesius and 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).

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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.

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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 electrophoresis and
  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 barbels samples
  is very difficult to decode (Fig.7). A
  polymorphism in species Barbus meridionalis
  petenyi Heck. from Lake Ohrid is not established.

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• 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,(posit
  ions 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 subspecies of Barbus
  meridionalis in this lake.

• 1 2 3 4 5

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• 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

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• 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.

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