Genetic Markers for Species Identification of Fishes from Family Siluridae

297

Bulgarian Journal of Agricultural Science, 12 (2006), 297-300 National Centre for Agrarian Sciences

А. TSEKOV, P. IVANOVA and I. DOBROVOLOV Institute of Fisheries and Aquaculture, BG - 9000 Varna, Bulgaria

Abstract

TSEKOV, А., P. IVANOVA and I. DOBROVOLOV, 2006. Genetic markers for species identification of fishes from Family Siluridae. Bulg.J. Agric. Sci.,12: 297-300

The genetic-biochemical analyses of two silurid species S. glanis and S. aristotelis are carried out. Eight enzyme and fourteen non-enzyme loci were investigated. The differences on the species level between S. glanis and S. aristotelis on some enzyme system (esterase - EST, malic enzyme – MEP, superoxide dismutase – SOD, 6-phosphogluconate dehydrogenase – 6- PGDH and malate dehydrogenase - MDH) and isoelectric focusing (IEF) of general muscle proteins (PROT) could be used for control of its posterities. The low genetic distance between the species examined, show that these species, belonging to one and the same Genus. Key words: silurus, electrophoresis, isoelectric focusing, genetic markers

Introduction and the Balkans. For the Balkans the fol- lowing two species are described – The catfish from Family Siluridae are Silurus glanis and Silurus aristotelis freshwater fish species, distributed in Eu- (Economidis, 1991; Economidis and rope and Asia with valuable market mean- Banarescu, 1991; Naumovsky, 1995; ing. According to Bleekeer (1862), Karapetkova and Zhivkov, 1995; Georgiev, Nickolskiy (1971), Banarescu (1989) the 1998). Siluridae Family is presented by two The species from two Genera have dif- Generas: Silurus and Parasilurus ferent number of barbels. The species Nickolskiy (1971) put in the Silurus Ge- from Silurus Genus have three pairs, while nus the species Silurus glanis (European these from Genus Parasilurus – two pairs catfish) and Silurus soldatovi (typical for of barbels. According to the classification the basin of the Amur River). In the of Nickolskiy (1971), Silurus aristotelis Parasilurus Genus the same author put belongs to the Parasilurus Genus. the amurian catfish – Parasilurus asotus Bleekeer (1982) and Banarescu (1989) are and some close species from South Asia agreeing with Nikolsky opinion. On the un- [email protected] 298 A Tsekov, P. Ivanova and I. Dobrovolov der chin on S. aristotelis only one pair of (1955), modified by Dobrovolov (1973). barbels is presented and the length on the Isoelectric focusing (IEF) on thin barbels on a upper lip do not outstrip the polyacrilamide Ampholine gel with pH gra- length of the pectoral fins, while by S. dients between 3.5 - 9.5 with the equip- glanis the barbels overtake the pectoral ment of LKB (Stockholm, Sweden) was fin length. used as well. The muscle proteins were According to Bornbusch (1995) the ge- stained with Amido Black- 10B or netical differences obtain between Genus Commassie Brilliant Blue R-250. The Parasilurus and Silurus are insufficient buffer systems according to Dobrovolov and the author thinks that they are identi- (1976) and Clayton and Gee (1969) were cal. Triantafyllidis et al. (1999a, b), con- used. sidered that both species are well differ- The visualization of the enzymes (es- entiated but the values of the genetic dis- terase - EST, malate dehydrogenase - tance between them is not enough and MDH, superoxide dismutase - SOD, malic accepted that Silurus glanis and Silurus enzyme – MEP, lactate dehydrogenase – aristotelis are from the same Genus. LDH and 6-phosphogluconate dehydroge- The market interest towards the cat- nase – 6-PGDH) is done on Shaw and fish (industrial production and sports fish- Prasad (1970). ing) has imposed introduction and accli- matization of new species in the area, in Results and Discussion which they are not typical. The new species, after the introduction and acclimati- In the examined specimens from both zation, enter the natural water basins of species (with weight 85 to 210 g) there the areas and this requires control over are no essential differences on the first their interaction with the local species. A sight. The same could be accepted for possibility for such control is the discover- posterity from only one species. With more ing of genetic markers for identification carefully observation the lack of the sec- of the species and their posterities, which ond pair of barbels on the under chin, a is the main purpose of the present work. smaller length on the barbels on a upper lip and a bigger width of the first ray of Material and Methods pectoral fin of Silurus aristotelis is found (Figure 1). The thing that makes impres- The analyses are done over five speci- sion is that the female and male individu- mens of Silurus glanis from a 2004 pos- als from this species, with the shown size terities and 10 specimens of Silurus are in the maturity. The female have a well aristotelis taken from the lake in the re- developed eggs and having the possibility gion of Agrino town (Greece) during April for a participation in a reproduction cam- 2005. These specimens from both species paign in 2005 year. have the weight from 85 to 210 g. For Six enzyme systems (eight loci) and one analysis of the enzyme and nonenzyme non-enzyme system (14 loci) were protein systems, a homogenate of white analysed. dorsal muscle was used. The proteins When the general muscle proteins were separated by horizontal starch gel (PROT) are examined differences in the electrophoresis according to Smithies electrophoretic mobility of the proteins on Genetic Markers for Species Identification of Fishes from Family Siluridae 299 a species level are observed. In Silurus netical markers found could be used for a glanis species specific fraction is found control of the species affiliation of the pos- out in the IV zone, which lacks in Silurus terity from these two species. The pre- aristotelis (Figure 2). The differences liminary calculations for the genetic dis- between the species on some enzyme sys- tance (DNei = 0.289), give us the reason to tems are presented on Figure 3. The ge- accept the Bornbusch (1995) and

Fig. 1. Photo of S. aristotelis (1), female and S. glanis (2)

ІІ ІІІ

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V VІ VІІ 00 VІІІ

ІХ Х 1 2 3 4 5 6 7 8 9 10

Fig. 2. Isoelectric focusing (IEF) of general muscle proteins (PROT) on thin polyacri- Fig. 3. Schema of zymograms of SOD, EST lamide Ampholine gel: S. glanis (1 - 2) and S. and MEP on starch gel: S. glanis (1) and S. aristotelis (9 - 10), 0 – origin aristotelis (2) 300 A Tsekov, P. Ivanova and I. Dobrovolov

Triantafyllidis et al. (1999) thesis for the Dobrovolov, I. S., 1976. Multiple forms of lac- insufficient genetic distance between the tate dehydrogenase in anchovy (Engaulis two Balkan species of catfish and their encrasiholus L.) from the Black Sea, the belonging to one and the same Genus. Sea of Azov and the Atlantic ocean. Comptes rendus de l’Academie bulgare Conclusions des Sciences, 29 (6): 877-880. Economidis, P. S., 1991. Check-list of fresh wa- The differences on the species level ter fishes of Greece. Recent status of between S. glanis and S. aristotelis on threats and protection. Hellenic Society some enzyme system (EST, MDH, MEP, for the Protection of Nature, pp. 29-30. 6-PGDH and SOD) and isoelectric focus- Economidis, P. S. and P. M. Banarescu, 1991. ing of general muscle proteins (PROT) The distribution and origins of freshwater give possibility for control of its posteri- fishes in the Balcan penisula especially in ties. Greece. Int. ges. Hydrobiol., 76 (2): The low genetic distance between two 257-283. species are in accordance with Bornbusch Georgiev, S., 1998. A key for fish (1995) and Trintafyllidis et al. (1999a, b) (Osteichthyes) and ells (Cephalaspi thesis that these two species, belonging to domorpha) determination in Macedonia, one and the same Genus. 177 pp. (Mc). Karapetkova, М. and М. Zhivkov, 1995. Bul- References garian Fish Species, 247 pp. (Bg). Naumovsky, М., 1995. Fishes of Macedonia. Banarescu, P. M., 1989. Zoogeography and Zhaki. Scopie, (Mc). History of the Freshwater Fish Fauna of Nickolskiy, G. V., 1971. Fam. Siluridae. Ich- Europe. In: J. Holcik (Editor), Freshwater thyology, Moskva, pp. 268-270 (Ru). Fishes of Europe, AULA Verlag, Smithies, O., 1955. Zone electrophoresis in Wiesbaden, 1 (II): 88-107. starch gels: group variations in the serum Bleeker, P., 1862. Notices sur les generes proteins of normal human adults. Biochem. Parasilurus, Eutropiichthys, Eudeutro- J., 61: 629-641. pius et Pseudopangasius. Versl .Akad. Amsterdam, 14: 390-399. Shaw, C. R. and R. Prasad, 1970. Starch gel Bornbusch, A. H., 1995. Phylogenetic relation- electrophoresis of enzymes-a compilation ships within the Eurasian catfish family of recipes. Biochem. Genet., 4: 297-320. Siluridae (Pisces: Siluriformes), with com- Triantaphyllidis, A., C. Ozouf-Costaz, P. Rab, ments on genetic validities and biogeog- R. Suciu and Y. Karakousis, 1999a. raphy. Zool . J. Linn. Soc., 115: 1-46. Allozyme variation in European silurid spe- Clayton, I. W. and G. Gee, 1969. Lactate dehy- cies S. glanis and S. aristotilis. Bioch. Syst. drogenase isozimes in longnose and and Ecol., 27: 487-489. blacknose dase (Rhinichtys cataracte and Triantafyllidis, A., T. J. Abatzopoulos and P. R. atratulus) and their hybrid. J. Fish. Res. Economidis, 1999b. Genetic differentiation Bd. Canada, 26: 3049-3053. and phylogenetic relationships among Dobrovolov, I. S., 1973. Micro starch gel elec- Greek S. glanis and S. aristotelis popula- trophoresis. Proceeding of the Institute of tions assessed by PCR-RFLP analysis of Oceanography and Fisheries, Varna, 12: mitochondrial RNA segments. Heredity, 157-162 (Bg). 82: 503-509.