Genetic variability in Oligosarcus paranensis ( Teleostei : Characiformes ) from the São Francisco river , Ivaí river basin – Paraná State , Brazil

The genetic variability of Oligosarcus paranensis was estimated from a population collected in São Francisco river, Prudentópolis county in Paraná State (Brazil) using the electrophoresis in starch gel technique. Eleven enzymatic systems were analyzed: Aspartate aminotransaminase (AAT; E. C. 2.6.1), Alcohol dehydrogenase (ADH; E. C. 1.1.1.1), Esterase (EST; E. C. 3.1.1.1), Glucose-6-phosphate isomerase (GPI; E. C. 5.3.1.9), Glycerol-3-Phosphate dehydrogenase (G3PDH; E. C. 1.1.1), Isocitrate dehydrogenase (IDH; E. C. 1.1.1.42), L-lactate dehydrogenase (LDH; E. C. 1.1.1.27), Malate dehydrogenase (MDH; E. C. 1.1.1.37 ), Malate dehydrogenase NADP (ME; E. C. 1.1.1.40), Phosphoglucomutase (PGM; E. C. 5.4.2.2) and Sorbitol dehydrogenase (SORB; E.C. 1.1.1.14). Twenty loci were identified through 15% corn starch gel electrophoresis of which nine (45%) were polymorphic. The average expected heterozygosity was estimated as 0.1229 ± 0.1728, and the observed was 0.0586 ± 0.1069, indicating high genetic variability. The average value of FIS = 0.5145 indicates homozygote excess.


Introduction
Fish are important for human consumption, however this importance are preventing many species to go through the extinction process.Fishes are also indicators of environmental pollution (NELSON, 2006).They play a key role as components of the ecosystem, participating in the nutrient cycling and energy flow, such as staple food for many birds and animals.Due to their impressive diversity of species, the fish fauna is a valuable gene bank, strategy for future applications.
The fish that have a great diversity of shapes, sizes and habitats.Of the 54,711 known species of vertebrates, fish are 27,977 of which 11,952 are freshwater ones (NELSON, 2006).South America is dominated by the order Characiformes, with comprises approximately 1,200 species and by the Siluriformes, with 1,300 species (AGOSTINHO et al., 2007).Despite the prevalence of these two orders, the composition and number of species among different basins varies considerably (AGOSTINHO et al., 2007).
The São Francisco river is a tributary of the Ivaí river, that is, in turn, a tributary of the Paraná river.The fish fauna of the Paraná river basin is estimated in 310 species (LANGEANI et al., 2007), but this number may be much higher (AGOSTINHO et al., 2007).Although many species are not yet described, the antropogenic changes in aquatic environments in recent decades have been threatening the perpetuation of natural populations.To accomplish its conservation, the true value of the fish fauna needs to be urgently better appreciated in the economic, scientific and ecological fields (AGOSTINHO et al., 2007).The Genus Oligosarcus represents 16 species, characterized as agile predators, preying on small fish and insects (RUBERT; MARGARIDO, 2007).They can be found in the river basins of the Paraná and Paraguay rivers and in the coast of south and southeastern Brazil (MENEZES, 1969).
The species Oligosarcus paranensis Menezes and Géry, 1983, reaches the standard length of 20.5 cm (OLIVEIRA et al., 2002), and has a diploid number of 2n = 50 (RUBERT; MARGARIDO, 2007).Despite of its small size and low commercial value it is of great ecological importance because it is a predator of smaller animals.
Abundance is a way to measure the species adaptedness -the state of being adapted to their environment (FISCH-MULLER, 2003), as well as adaptability (ability to survive and reproduce in different environments), which are closely linked to genetic variability of the species.The future of a species diversity depends upon the genetic diversity of that species (VIDA, 1994).The greater the genetic diversity maintained the greater adaptability, and thus, the probability of survival in an unstable environment.It is important to estimate the genetic diversity degree in this species to support conservation programs.
The objective of this study was to estimate the genetic variability of Oligosarcus paranensis in São Francisco river of the Paraná river basin and compare it with other species from the upper Paraná river basin to support preservation programs.

Material and methods
Thirty specimens of Oligosarcus paranensis (Figure 1) were collected just above the São Francisco falls (25° 8' 21.27'' S/51° 18'4.81''W) an approximately 190 meters high waterfall, in São Francisco river, located at Prudentópolis (Paraná State) as shown in Figure 2. The São Francisco river is a tributary of the Ivaí river, that is, in turn, a tributary of the Paraná river.The individuals were collected in February 2007 and were immediately frozen in liquid nitrogen, transported to the laboratory and stored in liquid nitrogen.
Samples were homogenized with plastic sticks in 1.5 mL Eppendorf tubes with 100 μL of Tris / HCl 0.02 M, pH 7.5 buffer, in order to be analyzed.Since the liver has a lot of fat, carbon tetrachloride was added to liver samples at a ratio of 1:2 (tissue: tetrachloride).The starch gel was prepared by making a solution of 15% corn starch (Penetrose 50) in buffer solution (VAL et al., 1981).The solution was heated to boiling and then poured into a glass plate with 0.6 cm edges.After cooling, the solution was consistent with 0.7 cm thick gel.The gel remained in the refrigerator for 24 hours.
Samples from each fish were loaded onto the gel through strips of paper filter 3mm and subjected to horizontal electrophoresis with a continuous current of 200V and 50V at source, measured at the ends of the gel.The starch gels were sliced horizontally and revealed to the desired enzyme.Tissues of gills, white muscle, heart, liver and kidney were subjected to electrophoresis in Tris 0.0135 M/0.043 M citrate, pH 7.0 (diluted 15 times in the gel) and 0.18 M Tris / 0.1 M borate / EDTA 0.004 M, pH 8.7 (diluted 4 times in the gel) to visualize the expression patterns of enzyme and choose the most suitable tissue for the analysis population.Since there were no major differences in the expression of enzymes in the two buffers, we used only Tris / citrate for the population analysis.The loci and alleles were named according to Murphy et al. (1996).The data were analyzed by PopGene 1.31 (YEH et al., 1997).

Results
Through the starch gel electrophoresis technique, 11 enzyme systems of Oligosarcus paranensis, collected in São Francisco ri ver, were analyzed.Twenty loci were detected, with a total of thirty alleles.
ADH -This enzyme showed a pattern of several bands with a higher expression in the liver.EST -The expression of this monomeric enzyme was detected in the liver with TBE 8.7 and in the gills by TC 7.0 buffer, with a greater expression of these tissues.
G3PDH -A dimeric enzyme, with expression of three monomorphic loci Gpd-1, Gpd-2 and Gpd-3 (allele a), expressed in the liver of all individuals, while in muscle tissue, only two loci Gpd-2 and Gpd-3 were expressed.
GPI -A dimeric enzyme encoded by two loci Gpi-A and Gpi-B expressed in all tissues, with the presence of heterodimers between them.
IDH -This dimeric enzyme showed differential expression among tissues.A polymorphic locus with three alleles was expressed in gill and liver, while another monomorphic locus was detected in heart and white muscle.Both (Idh-1 and Idh-2) were expressed weakly in the kidney.
LDH -This tetrameric enzyme was detected as the result of the expression of two homozygous loci.The two loci are expressed in all tissues, but Ldh-B is best expressed in muscle tissue.
MDH -This dimeric enzyme showed four bands of activity in the gills, heart and white muscle.This pattern was interpreted as the expression of two loci, Mdh-a and Mdh-b.
ME -This tetrameric enzyme showed a pattern of bands with two loci in the two buffers used.
PGM -A single-banded pattern was observed for all individuals inliver and muscle; typical of a monomeric enzyme encoded by a polymorphic locus with two alleles.
SORB -This enzyme is presented as encoded by two polymorphic loci, presenting greater intensity in the liver.This enzyme system was not tested in TBE buffer.
Figure 3 shows the electrophoretic patterns of 11 enzyme systems in Tris / citrate pH 7.0 and also the designation of the detected loci and alleles.Table 1 shows the allele frequencies for each detected locus and Table 2 measures the genetic variability.Nine (45%) loci (Aat-2, Adh, Gpd-2, Gpi-A, Gpi-B, Idh-2, Mdh-B, Pgm-1, Sorb-2) were polymorphic.Five loci (Adh, Idh-2, Me-2, Pgm-1, and Sorb-2), are not in Hardy-Weinberg equilibrium (Table 2).homozygotes.These results can be due to many causes for this excess, such as inbreeding, natural selection, fragmentation of the population, and assortative mating.The results of this work, through the enzyme electrophoresis in starch gel technique, showed that Oligosarcus paranensis is a species with high genetic variability and deserves proper environmental management attention in order to be preserved.

Figure 2 .
Figure 2. Geographic representation of the Ivaí river basin showing the location of collection (*) of Oligosarcus paranensis in the São Francisco river.

Figure 3 .
Figure 3. Gel photos of 11 enzymatic systems showing the loci and alleles detected in a population of Oligosarcus paranensis from São Francisco river, Prudentópolis county, Ivaí river basin (Paraná State, Brazil).