Available Online: http://www.ejpau.media.pl/volume16/issue3/art-01.html
INTRA AND INTER-STOCK VARIABILITY IN STERLET (ACIPENSER RUTHENUS) AS ASSESSED WITH BIOMETRIC AND GENETIC ANALYSES
Jolanta Kempter1, Paulina Hofsoe-Oppermann2, Antonina Neumann1, Remigiusz Panicz3, Sławomir Keszka4
1 Department of Aquaculture, Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
2 Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
3 Department of Meat Technology, Faculty of Food Technology and Fisheries,
West Pomeranian University of Technology, Szczecin, Poland
4 Division of Aquaculture, Faculty of Food Science and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
This study was carried out to determine biometric, morphometric,
and genetic features of two sterlet populations. The morphometric and biometric
analyses were performed to determine the degree of plasticity of individual characters
and the range of variability which in the sterlet require further analysis. The
purpose of the genetic analysis was to characterise the gene coding sequence
for the first subunit of cytochrome oxidase I (COI) in terms of its nucleotide
variability within and between populations. This study provided a comprehensive
characterisation of the two sterlet populations and defined the extent of their
variability (eg.: distance between tip of barbell and cartilaginous arch of mouth,
maximum width of head or number of dorsal plates) and homogeneity (eg.: horizontal
eye diameter, length of barbell or preanal distance).
This study was carried out to determine biometric, morphometric, and genetic features of two sterlet populations. The morphometric and biometric analyses were performed to determine the degree of plasticity of individual characters and the range of variability which in the sterlet require further analysis. The purpose of the genetic analysis was to characterise the gene coding sequence for the first subunit of cytochrome oxidase I (COI) in terms of its nucleotide variability within and between populations. This study provided a comprehensive characterisation of the two sterlet populations and defined the extent of their variability (eg.: distance between tip of barbell and cartilaginous arch of mouth, maximum width of head or number of dorsal plates) and homogeneity (eg.: horizontal eye diameter, length of barbell or preanal distance).
Key words: Acipenser ruthenus, sturgeon, biometric and genetic analyses, cytochrome oxidase I.
Acipenserids (Acipenseridae) are one of the oldest groups of fish. At present, there are a total of 27 acipenserid species worldwide [2, 4] which occur throughout the northern hemisphere, north of the Tropic of Cancer . Nine of those species occur in North America, the remaining ones inhabiting Eurasia [3, 4]. The only acipenserids in the Polish fish fauna were Acipenser sturio, A. oxyrinchus and A. ruthenus, the latter having been occasionally reported as a part of natural population from the River Bug . However, acipenserids have not reproduced naturally in Polish waters for years  but have become objectives of fish farming.
The sterlet is characterised by an elongated, spindle-shaped, body covered by bony plates (scutes) arranged in five rows, an elongated rostrum with four barbells, and a heterocercal fin. Among acipenserids, the sterlet has the highest number of lateral scutes (usually more than 56–71) . The mouth is protrudable, jawless and not equipped with teeth. It is located on the ventral side of the head. The sterlet is the smallest acipenserid within genus Acipenser. Mature individuals measure from 35 to 65 cm, their weight ranging from 0.25 to 1.5 kg . The species reaches up to 1m in length and 6 kg in body weight. Traits such as early maturity (genus Acipenser) and adaptation to freshwater habitats make sterlet a valuable object of fish farming practice .
Depleting natural populations require reliable fish culture supported with scientific analysis which aim at protecting genetic diversity. This analysis will help to increase number of individuals in populations or to reestablish those that were lost in the past. Recent years have witnessed great advances in the development of diagnostic methods to study DNA variability at the molecular level [37, 38]. Properties of mitochondrial DNA, allow to assess and evaluate possible differences between examined individuals, therefore are commonly used in research on intra-specific variability of fish .
The aim of this study was to carry out biometric, morphometric, and genetic comparative analyses of two sterlet stocks. Moreover, to determine the degree of plasticity of morphometric characters within two groups which in the sterlet should be described in more details. This study is an attempt to provide a comprehensive characterisation of the two sterlet populations and to define the extent of their variability/homogeneity with respect to related species from the standpoint of the stocks examined to provide broodstocks.
MATERIALS AND METHODS
The study involved two stocks of A. ruthenus. One (A) consisted of individuals kept in a cage culture at the Fisheries Research Station (FRS), Nowe Czarnowo (Poland) supplied with the Dolna Odra power station cooling water. The other (B) comprised individuals from a fish farm in Störnstein municipality in Bavaria (Germany). A total of 30 individuals aged 1+ were randomly picked from stock A in June 2009. The mean water temperature at which stock A individuals were kept was 17,9°C (SD = 7.9°C). The total length of the individuals ranged within 375–475 mm, the mean total length amounting to 422.06 mm. Weight ranged within 185–430 g, the mean individual weight being 297.66 g. Stock B yielded a total of 40 randomly picked individuals aged 3+ during April and May of 2009. The farm is located in Bavaria, the culture ponds are fed ground water. The total length of the these individuals was found to range within 352–461 mm, the mean total length amounting to 416.14 mm. The weight ranged between 163.1–309.1 g, with a mean weight of 227.04 g. The fish examined had not been feeding for 5 months. The water temperature was 12°C (SD = 3.6°C). Fish collected at both farms were measured using callipers and weighted with 0.01 mm and 1 g accuracy, respectively. Tissue samples to be used in genetic analyses consisted of fragments of the caudal fin, preserved in 96% alcohol and kept at 4°C until analysed.
Analysis of metric and meristic characters were carried out using live, etomidate-narcotised (Propiscin, manufactured by Department of Fish Immunology and Pathology in Żabieniec, Inland Fisheries Institute in Olsztyn, Poland) (5 mg/dm3) fish (30 from stock A and 40 from stock B). The fish morphometry was analysed following Krylova and Sokolov . A total of 35 measurements (to 0.01 mm) were taken with both an electronic and conventional callipers (Fig. 1). In addition, 2 meristic characters, i.e., the number of lateral plates (SL) and the number of dorsal plates (SD) were included . The metric characters were expressed as percentages of the total length (Lt.) and of the head length (C, defined at Fig. 1) . For each character, statistical metrics: arithmetic mean, standard deviation (SD), and coefficient of variation (CV) were calculated. When the coefficient of variation was equal or higher than 10% (the general rule), those characters considered as highly plastic [5, 32]. For comparisons of mean values for morphological characters Mann–Whitney U test was used .
L.t., Total length; L1, Fork length (Smitt’s length); L2, Standard length; L2C, Length of trunk; aD, Preanal distance; aA, Predeorsal distance; aV, Preventral distance; R, Preorbital distance (length of snout); C, Length of head; O, Horizontal eye diameter; iO, Interorbital distance (skull width); OP, Postorbital distance; Bc, Maximum width of head; lD, Length of dorsal fin; hD, Depth of dorsal fin; h, Maximum body depth (last depth of caudal penducle); H, maximum body depth; HC, Head depth (at nape); HCO, Head depth (at center of eye); Pl1, Length of caudal penducle; Pl2, Distance between anal fin tip and tip of middle rays in caudal fin; lA, Length of pectoral fin; hA, Depth of anal fin; VA, distance between ventral fin base and anal fin base; lV, Length of ventral (pelvic) fin; lVbs, Length of base of ventral (pelvic) fin; PV, Distance between pectoral fin base and ventral fin base; IP, Length of pectoral fin; SRr, Width of snout at the base of mouth; SO, Width of mouth; l1, Length of barbel;, rl, Distance between tip of snout and middle barbel; rr, Distance between tip of barbel and cartilaginous arch of mouth; rc, Distance between tip of snout and cartilaginous arch of mouth; SRC, Width of snout at base of barbels.
|Fig. 1. A schematic of acipenserid measurements after Krylova and Sokolov (1981), modified (Keszka 2000)|
The DNA from fish samples (30 mg of caudal fin) was extracted using peqGOLD Tissue DNA Mini Kit (PEQLAB Biotechnologie), according to manufacturer’s instructions. The PCR mixture, sequences of FishF2_t1 and FishR2_t1 primers  and cycling conditions were prepared and performed according to Ivanova et al. . Results of PCR were checked by running 1% gel electrophoresis, and analysed by means of the Gel-Doc XR software. The sequencing was carried out by Genomed S.A. company (Warsaw, Poland). Sequencing results were analysed with the BLAST, MEGA5, and BioEdit software [1, 10, 35].
Analysis of metric characters
Results of metric analyses performed on both the A and B sterlet stocks are summarised in Table 1. The plastic characters (CV equal to or higher than 10%) are marked in grey. Most of the metric characters were relatively stable, except of one character rr (distance between tip of barbel and cartilaginous arch of mouth), their coefficient of variation being usually lower than 10%. In both stocks, rr character presented as % of C varied and the ratios ranging between 15.42–58.0 (mean 52.06) and 51.1–60.82 (mean 56.44) in the A and B stocks, respectively.
Table 1. Metric characters of sterlets collected within two geographically distinct populations.
Absolute values (mm). % total length (L.t.). % head length (C). standard deviation (SD). coefficient of variation (CV). Grey-highlighted cells denote plastic characters (V>10%) (* significant differences p=0.05)
Analysis of meristic characters
Examination of live sterlet individuals allowed to identify two meristic characters regarded as deserving further analysis: the number of lateral scutes (SL) and the number of dorsal scutes (SD). In the B stock, individuals with 12 dorsal (mean = 12.55, CV = 8.45) and 54 (mean = 54.18, CV = 4.94) lateral scutes were most frequent. Whereas, in the A stock individuals with 14 dorsal (mean = 13.83, CV = 9.12) and 55 (mean = 55.20, CV = 3.85) lateral scutes were most frequent. Mann–Whitney U test revealed only significant differences between dorsal scutes in both stocks (Table 2).
Table 2. Comparison of meristic characters of the samples examined (* significant differences p=0.05)
SD – standard deviation; CV – coefficient of variation
Genetic analysis of the sterlet populations
The PCR product for each sample was analogous in size to that reported by Ward et al. . Therefore, further genetic analyses and sequencing were run on 9 and 6 isolates, randomly selected from the B and A samples, respectively. Once the readings had been checked and the complete 652 bp sequences processed, they were aligned. No differences whatsoever were found between the 6 sequences obtained for A stock. In the B group, 7 of the 9 sequences were in 100% identical with the samples discussed above (A stock). The remaining two sequences showed a (G→A) transition type mutation at position 463. The mutation was non-synonymous, whereby the glutamate acid-determining codon at Seq A determines lysine in SeqG.
The preliminary analyses performed with the aid of the BLAST software showed 13 sequences carrying the polymorphic site G (Seq G) to have a number of analogues in the NCBI base (e.g., HQ960593 – HQ960595). However, those sequences have not been provided with information as to what species they were obtained from, other than that indicating acipenserid fish as a source material. On the other hand, the two sequences with differing site A (Seq A) had no 100% concordant record in the base. Further comparisons of the COI gene sequences in the NCBI data base revealed the Seq G studied to show 99.6% similarity to those obtained from A. brevirostrum (EU523870, EU523871) and A. baerii (GQ328814, GQ328815). On the other hand, the Seq A’s were completely similar to those from A. brevirostrum, but the similarity with A. baerii was lower (95%; Table 3). Relationships between the sequences analysed are presented also on a dendrogram produced by the MEGA5 software (Neighbour-Joining/Jukes-Cantor) (Fig. 2). Polymorphic sites presented in table 4 were obtained after sequence alignment produced by the BioEdit software. The results of this study allowed to supply two sequences to the NCBI data base. The first sequence (access No. JF807060) derived from A. ruthenus has adenine base (Seq A) at site 463, the other (access No. JF807061) showing guanine (Seq G) as a result of substitution.
Table 3. Genetic distance between the sequences analysed as determined by the Jukes-Cantor model applied by MEGA5 software
|Fig. 2. Evolutionary relationships between sequences, constructed based on Neighbour-Joining method (MEGA5)|
Table 4. Summary of polymorphisms observed at individual nucleotide positions of the analysed sturgeon COI sequences
A. brevirostrum (EU523870) was taken as a reference (in bold). (*) indicates identity with the reference sequence.
In the sterlet, like in other acipenserids, variability of morphological characters among others is expressed, in the relative length of the rostrum. According to Sokolov and Vasilèv , the rostrum of sterlet from different areas accounts for 27.8–63.5% of the lateral head length. In our study, the rostrum made up 38.34–50.69% (mean 44.96%) and 44.46–55.34% (mean 49.73%) in the A and B samples, respectively. Hence, with the regard to the relative head length the A stock resembles, the characteristics of the Danube catchment (mean R=44.08% lc) , while the B stock resemble those from the north-western part of the Black Sea with estuaries of the Danube and Dnieper (mean R=48.63% lc) . Presented results indicate that both stocks possess characters similar with Danube populations. This is might be the reason of different morphological forms of sterlet that exist in Danube, and this is strictly strictly connected with adaptive changes of sturgeon and species conservation by stocking .
Earlier studies from the first half of the 20th century pointed to the existence of two forms differing in rostrum length and possessing a number of different biological traits . Those sterlets with a blunt, shorter rostrum were supposed to mature earlier, to grow faster, and to be more fertile than the sterlets with a longer, pointed rostrum. Later studies, however, did not confirm those findings; they revealed only that the rostrum length is a highly variable character [15, 16, 34]. Generally, however, sterlet populations are dominated by individuals whose rostrum is intermediate between sharp-tipped and blunt. Among populations from European rivers, those from the northern Daugava showed the shortest rostrum, the longest rostrum being typical of populations from the Danube and Dnieper. Ruban and Sokolov  suggested that warmer temperatures during early development of Siberian sturgeon increased growth rate and formed a blunt snout, whereas colder temperatures decreased growth rate and formed a pointed snout. Of the meristic characters discussed in the literature, it was only the pyloric caeca count that proved significantly different between juvenile and older sterlets . A similar trend was observed by Keszka  in the Siberian sturgeon. Brannon et al.  who studied White sturgeon (Acipenser transmontanus), considered that the number of dorsal scutes may vary due to water temperature during early rearing and may indicate more about early rearing conditions than genetic variability.
In the present study, as the sterlets were examined live, only the numbers of dorsal and lateral scutes were analysed (Table 2). In terms of the dorsal scutes number, the sterlets examined in this study were closest to those studied by Lukin et al.  in the Volga catchment (the Kuybyshev reservoir), but the mean numbers of lateral scutes found in this study (54.18–55.2) differed markedly from the literature data (62.41–64.27). This is perhaps a result of selection induced by aquaculture, as indicated by Ruban  who studied the farmed and natural populations of Siberian sturgeon, an effect of genotype changes resulting in a fixation of the initial modification reaction by stabilizing selection – the Baldwin effect, which results in the appearance of a new creode based on changes in a reaction norm. This suggestion seem to be confirmed by results of genetic studies on juvenile sevruga (A. stellatus) kept at high temperatures. Serov et al.  found high water temperature to induce genetic variability in the juvenile A. stellatus with respect to the position of the LDH locus. Perhaps other loci in the genotype undergo changes as well.
The population-level genetic variability of sterlet was investigated by Reinartz et al. . Using mtDNA, they determined the level of genetic variability of sterlet populations from the Danube, Volga, and Kuban. The study showed the Danube sterlets, as opposed to the Volga population, to be genetically homogenous. The Volga population was found to consist of 3 sub-populations. Due to genetic homogeneity (77%) of the Danube population, it is recommended by Reinartz et al.  as a source of broodstocks to avoid inbreeding depression. Guti and Gaebele  are of the opinion that, to protect the Danube sterlet population, methods more effective than, e.g., protective size or no-fishing seasons, should be applied. It is only a complete recreation of a natural habitat of the sterlet population in question that would make it possible to save it. In the case of conservation studies in sturgeons, the genetic and morphological studies have to be conducted to asses population structure by genetic profiling to serve as basics for selection programs which are aimed at sustaining wild populations.
Sterlet is listed as threatened in the IUCN Red Book of Endangered Species . This means that its effective size of populations (Ne) is low and it is necessary to conduct similar research as presented above. It seems then necessary to make an attempt at characterising the extant sterlet populations and at assessing their intra- and interpopulation variability. Moreover, the state of knowledge needs genetic and biometric data obtained from correctly classified sturgeon species. Additionally, correct identification of several morphologically similar sturgeons require further improvement or development of morphological tools to apply them jointly with genetic markers. This will make it possible to identify populations which, on account of their exceptional commercial value, should be used to derive broodstocks from. Breeding of A. ruthenus without maintaining the genetic pool diversity of the broodstock, with a particular emphasis on negative selection of hybrids, impedes production of fry with improved utility traits, higher survival rate, better growth rate, and lower susceptibility to pathogens.
In our study, variability of a series of meristic and morphological characteristics were compared. The rostrum made up 38.34–50.69% (mean 44.96%) and 44.46–55.34% (mean 49.73%) in the A and B samples, respectively. Hence, with the regard to the relative head length the A stock resembles, the characteristics of the Danube catchment (mean R=44.08% lc) , while the B stock resemble those from the north-western part of the Black Sea with estuaries of the Danube and Dnieper (mean R=48.63% lc) . In both populations, the distance between the tip of the snout and the cartilaginous arch of the mouth, the per cent ratios ranging within 5.91–9.42 (mean 7.78) and 5.26–8.87 (mean 6.87) were plastic characters in the B and A samples, respectively. In addition, the A samples showed another plastic character: the distance from the barbell-base to the cartilaginous arch of the mouth (rr). The results of the genetic study on the cytochrome oxidase I (COI) variability in allowed to supply two properly described sequences to the NCBI data base; (access No. JF807060) derived from A. ruthenus with adenine base (Seq A) at site 463, and the other (access No. JF807061) showing guanine (Seq G) as a result of substitution. In the case of conservation studies in sturgeons, the genetic and morphological studies have to be conducted to asses population structure by genetic profiling to serve as basics for selection programs which are aimed at sustaining wild populations.
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Department of Aquaculture, Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
phone: +48 91 449 66 63
Kazimierza Królewicza 4
Division of Aquaculture, Faculty of Food Technology and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
Department of Aquaculture, Faculty of Food Sciences and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland
phone: +48 91 449 66 63
Department of Meat Technology, Faculty of Food Technology and Fisheries,
West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4
Division of Aquaculture, Faculty of Food Science and Fisheries, West Pomeranian University of Technology, Szczecin, Poland
Kazimierza Królewicza 4, 71-550 Szczecin, Poland, phone: +48 91 449 66 36
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