Journal of Environmental Protection and Ecology 13, No 3A, 1856–1864 (2012) Marine biological research Landings, distribution, size structure and genetics of Pontic shad (Alosa immaculata b e n n e t t, 1835) in the buLgarian bLack sea area M. Panayotova*, v. Raykov, P. Ivanova, I. DobRovolov Institute of Oceanology, Bulgarian Academy of Sciences, P. O. Box 152, 40 Parvi May Street, 9000 Varna, Bulgaria E-mail: mpanayotova@io-bas.bg; vio_raykov@abv.bg; pavl_petya@yahoo.com abstract. Pontic shad is an economically important ish species targeted in the Bulgarian isheries since 1925. Statistical data of catches show variation of quantities between 2 t (1970) and 439.19 t (1954) during the period 1925–2010. Regime shift in landings was observed in 1951–1955, when the average catch reached maximum of 217.50 t. Seasonal distribution pattern of shad embraces both coastal and open sea areas where species is dispersed. Size structure of catches includes individuals with fork length between 9 and 35.5 cm. Due to dificulties in morphological identiication of the shads in the Black Sea, the genetical markers found during the study give possibility to distinguish 2 species – A. immaculata and A. caspia in the Black Sea. Lactate dehydrogenase (LDH) and esterases (EST) are species speciic markers for distinguishing A. immaculata and A. caspia in the black Sea. Polymorphic variation in MEP-2* locus by A. immaculata could be used for analyses of the Black Sea shad populations. Keywords: Pontic shad, Black Sea, distribution map, length–weight relationships, genetics markers. aIMS anD backgRounD Study is aimed to analyse the trends in Pontic shad landings during the period 1925–2010 and to compare the species distribution areas and size structure of landings in the period 1956–2011 as well as to clarify the genetic structure of the shads in the black Sea. Pontic shad (Alosa immaculata B e n n e t t, 1835) is an economically important ish species from genus Alosa, represented in the Black Sea by several species or subspecies, but the taxonomic status of them is still unclear. Genetic analyses are essential due to dificulties in species differentiation, owing to the variability of morphological characteristics. Molecular markers were applied to evaluate the genetic difference between Alosa species and understand their phylogenetic position1–3. Species A. immaculata, A. maeotica and A. caspia conspeciity were conirmed in the Azov–Black Sea basin based on allozyme data4. Recent studies5 * For correspondence. 1856 pointed Alosa immaculatа (B e n n e t t, 1835) as a valid name of previous Аlosa pontica (E c h w a l d, 1938). Pontic shad is anadromous pelagic species, inhabiting the Black Sea and the Sea of Azov and penetrating upriver for reproduction6–11. In front of the Bulgarian coast, shad is dispersed during the winter, feeding actively12. Spawning migrations of shoals towards the Danube river starts in spring (March–May) and reproduction takes place in the river. The shad ishery is carried out mainly during the spawning migration8, 12 using uncovered poundnets and gillnets, but shad is also caught as by-catch of trawl isheries. Bulgarian statistics of shad landings started in 1925 (Ref. 13) and covers catches in the Black Sea and the Danube river14,15. till now, the catches are dominated by A. immaculata. Biological peculiarities of the species make it vulnerable to different threats16, but the major ones are overishing and loss of spawning grounds. For that reason, species is the subject of special concern under Directive 92/43/EEC and specially managed NATURA 2000 sites were designated in Bulgaria. In the context of ishery management that takes ecological and ecosystem considerations, protected areas are highly important17. Recent data on shads stocks, distribution, population parameters and genetics in the Bulgarian Black Sea area urgently are needed for the species conservation and management issues. ExPERIMEntal Statistical data about the Bulgarian Pontic shad landings and discharges for the period 1925–2010 were collected from different sources13,15,18–20. the Rodionov method21 for regime shifts was applied for investigation of trend in Bulgarian shad landings in the Black Sea. Biological data were gathered during the research cruises and from market and commercial ishery sampling in the period 1956–2011. Historical data about distribution and size structure of shad population embraced about 30 000 individuals, for each fork length (cm) and weight were measured (g) (Ref. 14). Survey data covered the Bulgarian Black Sea area up to 100 m depth. Electrophoretical analysis of 150 shad samples collected during the period 2010–2011 from the Bulgarian Black Sea area (poundnets, bottom and pelagic trawl gears) was carried out. For the analyses of enzymes as esterase, lactate dehydrogenase, malic enzyme, fumarate hydratase, glycerol-3-phosphate dehydrogenase and non-enzyme protein systems, a homogenates of white dorsal muscle and eye (retina) were used. Proteins were separated by horizontal starch gel electrophoresis22,23. Isoelectric focusing (IEF) on thin polyacrylamide Ampholone gel with pH gradients between 3.5–10.0 was used. The proteins were stained with the Commassie brilliant blue R-250. Staining of different enzymes was performed24. The buffer 1857 systems25,26 were used for the electrophoresis. The nomenclature of loci and alleles used here followed the recommendation of Shaklee27. RESultS anD DIScuSSIon The Bulgarian Pontic shad landings are realised both in the Black Sea and the Danube river. Average values over the period 1950–2010 amounts at 55.47 t in the Black Sea and 34.70 t in the Danube river20. the black Sea catches dominated with share of 61.52% compared to those from the Danube river – 38.48%, respectively. Landings in the Black Sea varied between 2 t (1970) and 439.19 t (1954) over the period 1925–2010 (Fig. 1). For that period, average catch was estimated at 62.67 t. The tendency in the catches was analysed by the method of Rodionov21, using the 6-year cut off period. Regime shift was detected within the years 1951–1954 (Fig. 1), when the average catch reached maximum of 217.50 t. For the period before regime shift (1925–1950), the average landings amount at 77.15 t and after the period of regime shift (1955–2010), mean landings decreased to 39.93 t. Distribution of landings by regions show clear dominance of northern region (Krapets – cape Emine) compared to the southern region (cape Emine – Ahtopol) for the period 1956–1982. Before 1956, the situation was opposite8 and the landings in southern region prevailed in total catch. The period of 1956–1966 was characterised with relatively high winter temperatures, which possibly enabled shad population to be moved in northern direction8. According to recent data for the period 2006–2010, the trend was retained and the northern region has major importance for the Bulgarian shad isheries. 2010 2005 2000 1995 1990 1985 1980 1975 1970 1965 1960 1955 1950 1945 1940 1935 1930 500 450 400 350 300 250 200 150 100 50 0 1925 landings (t) shifts in the mean for pontic shad landings, 1925–2010 probability = 0.05, cutoff length = 6, the Huber parameter = 1 year fig. 1. Bulgarian landings of A. immaculata in the Black Sea and detected regime shifts during the period 1925–2010 Distribution pattern of shad population in front of Bulgarian coast was analysed in seasonal aspect – spring and autumn seasons, using combined data for 2 1858 different periods: historical (1956–2005) and recent (2006–2011). The records of the observed individuals of A. immaculata are presented in Fig. 2. fig. 2. Distribution of A. immaculata records during the period 1956–2011 Shad records in spring season had dispersed distribution both in coastal and open sea areas up to 100 m. Because research cruises cover only shelf area, the presence of species in deeper waters is still unknown. Shads present in trawl and poundnet catches. Most abundant records in spring were observed off Varna, cape Emine and Sozopol (Fig. 2). In autumn season, shad inhabits whole marine area, but ish were withdrawn predominantly after 20 m isobath. Most abundant records were made off Varna, Kaliakra and the Burgas bay. Because the shad population, inhabiting and migrating along the Bulgarian Black Sea coast is relatively low abundant, special measures and designation of marine protected areas are required for conservation of this valuable species. Size structure of A. immaculata during the periods 1984–1985 and 2010–2011 was analysed on the basis of 3000 individuals. Distribution and share of each length class (FL) is presented in Fig. 3. Similar size structures of landings were 1859 observed for the both periods with majority of ish with fork length between 24 and 30 cm. According to the data, for the last 25-year period, negative trend in length structure was not observed, probably due to relatively low quantities of landings and seems that the shad population in front of the Bulgarian coast was not strongly affected by the isheries. In the recent period, all length classes present in the landings, both young ish and larger size groups which will ensure further sustainability of shad stock. Alosa immaculata 10 share (%) 9 8 2010–2011 7 1984–1985 6 5 4 3 2 1 38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 0 fork length (cm) fig. 3. Size structure of A. immaculata during the periods 1984–1985 (Ref. 14) and 2010–2011 Length–weight relationships of shad population during 3 different periods (1959–1979, 1984–1985 and 2010–2011) were analysed and negative allometric relationship was observed for the 1955–1979 and 1984–1985 periods28 (Fig. 4). Lower weights than those corresponding to the shad lengths during the period 1959–1985 were probably effect of relatively higher density of shad population, the state of trophic base and food competition with other species with similar diet18. Recent data (2010–2011) show positive allometric L–W relationship (Fig. 4) due to good food availability. genetics markers for species differentiations of A. immaculata and A. caspia were investigated. Lactate dehydrogenase (LDH) – the two duplicated genes LDH-А*1 and LDH-A*2 were established (Fig. 5 A and B). Differences in the electrophoretic mobility of LDH-А*1 and LDH-A*2 between A. immaculata and A. caspia were registered (Fig. 5 A and B) and they relected also on their hybride interloci fractions. Because the hybrid fractions between the faster and slower electromorphism were not found we supposed that they belong to 2 different species. 1860 1959–1979 1984–1985 600 600 W = 0.0629 FL 2.574 R2 = 0.9938 500 400 weight (g) 400 weight (g) W = 0.0185 FL 2.882 R2 = 0.9196 500 300 300 200 200 100 100 0 0 6 10 14 18 22 26 30 6 34 10 14 18 22 26 30 34 FL (cm) FL (cm) 2010–2011 500 W = 0.0037 FL3.345 R2 = 0.9801 weight (g) 400 300 200 100 0 6 10 14 18 22 26 30 34 FL (cm) fig. 4. Length–weight relationship for A. immaculata during the periods 1959–1979, 1984–1985 (Refs 14 and 28) and 2010–2011 fig. 5. A – Zymograms on lactate dehydrogenase (LDH) on starch gel: 1–2 – A. immaculata, 3 – A. caspia (Black Sea), 4 – A.caspia (Caspian Sea); B – isoelectric focusing (IEF) of LDH on thin polyacrylamide Ampholine gel plate with pH range 3–10: 1–2 – Alosa immaculata, 3–4 – Alosa caspia; C – zymograms on malic enzyme (MEP) on starch gel by A. immaculata (kranevo). Polymorphism in MEP-2* locus – bb and ab – phenotypes, 0 – origin 1861 The faster LDH-A*1 and LDH-A*2 electromorphs were typical for A. caspia (the Caspian and Black Seas), while the slower – for the A. immaculata (Fig. 5A). The species differences were also well visible using isoelectric focusing (IEF), (Fig. 5B). After analyses of eye tissue (retina) of the Black Sea shads a new duplicated LDH*С locus (LDH*С1 and LDH*С2) was found but differences in the electrophoretical mobility of compared species were not observed29. the esterases enzyme system analysed also showed species speciic spectres for the 2 species compared. Genetics markers for distinguishing A. immaculata populations were studied. The mаlic enzyme (MEP) is useful for the analyses of genetical structure of A. immaculata population along the Bulgarian Black Sea coast. The enzyme are visualised with 2 fractions, determined from 2 loci (sMEP-1* and sMEP-2*). Polymorphism in sMEP-1* locus was found29 by the samples (the cape Kaliakra and the Danube river). Polymorphism in locus sMEP-2* by the samples from Balchik, Kranevo, Kaliakra, Sozopol, in front of the Kamchia river mouth was found (Fig. 5C, Table 1). table 1. Calculated allele frequencies of malic enzyme (sMEP-2*) polymorphic locus on the A. immaculata from different localities Locus Allele sMEP-2* a* b* Kranevo Kaliakra 0.024 0.976 0.182 0.818 Allele frequencies Balchik Sozopol Trawl, the kamchia river mouth 0.071 0.071 0.038 0.928 0.928 0.962 According to the results, allelic frequencies of MEP-2* could be used for distinguishing of A. immaculata populations. On the basis of the data received at least 3 populations are registered (Table 1). Two new enzyme systems as glycerol3-phosphate dehydrogenasae (G3PDH) and fumarate hydratase (FH) were tested as potential genetic markers. These 2 enzymes are encoded from 4 alleles each but differences between the species were not found. Conservation status of the shads in the Black Sea characterised species as vulnerable and listed them in the Council Directive 92/43/EEC, Convention on the Conservation of European Wildlife and Natural Habitats (the Bern Convention, 1982), IUCN Red List of Threatened species, etc. Genus Alosa species are extremely vulnerable to anthropogenic changes, especially related to access and quality of their spawning grounds2. The major threats include industrial and domestic pollution, acidiication, river barriers, drainage, ish farming and the introduction of new species. Recently, restoration and conservation programs as NATURA 2000 were initiated in Bulgaria and special sites for protection of marine habitats were designated. Till now, 14 sites with marine area of 610 km2 (9.4% from territorial waters and 6% from Bulgarian shelf zone) are protected30, which is not suficient for the effective conservation of 1862 marine biodiversity and ecosystems in the Bulgarian Black Sea area31. contemporary investigations on shad stocks, distribution, population parameters and genetic structure are necessary for the implementation of adequate ishery management and conservation measures concerning Alosa species. concluSIonS The Pontic shad is valuable species for the Bulgarian isheries and at the same time possesses important conservation status. Analyses of A. immaculata distribution during the period 1959–2011 showed that the species is dispersed in the whole Bulgarian shelf area. Recent investigations on shad size structure and length–weight relationships manifested good status of the population, probably due to low quantities of landings and favourable environmental conditions in the Black Sea. Genetic markers (LDH and EST) could be used for species identiication of A. immaculata and A. caspia in the Black Sea. Polymorphism observed in the MEP-2* locus by A. immaculata is useful for analyses of shad population structure in the Black Sea. High vulnerability of shad population requires special measures for protection of its stocks and the exploitation must be accomplished in sustainable manner. acknowledgements. Study was conducted with the inancial support of EMEPA, Ministry of Environment and Waters of Bulgaria (Contract No 7976/04.04.2011). The authors acknowledge Prof. Kolarov for making available historical data for shad distribution and size structure. Genetic studies were supported by the project CEBDER, National Science Fund at the Ministry of Education, Youth and Science of the Republic of Bulgaria. REFEREncES 1. R. FARIA, B. WALLNER, S. WEISS, P. 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