1. Introduction
Acipenseriformes are one of the oldest groups of fish, having evolved for more than 200 mya, and are extensively distributed in the northern hemisphere [
1]. Sturgeons (family Acipenseridae) have been classified as critically endangered fishes on the IUCN (International Union for Conservation of Nature) Red List [
2], and the populations of these species have drastically decreased mostly due to overfishing, the destruction of their natural habitat, and water pollution. The overexploitation of wild populations has led to the listing of all sturgeon species in the Appendices to CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) over the past four decades for restocking and caviar production [
2]. Besides this, sturgeons are artificially propagated in aquaculture, mainly for the production of black caviar and boneless meat. Around 102,327 t of sturgeon meat and 364 t of black caviar is estimated to have been produced in 2017 [
3]. The most commercially important sturgeon species reared for caviar and meat production are
A. baerii, A. gueldenstaedtii, and the hybrid
H. dauricus × A. schrenckii [
3].
The sterlet
Acipenser ruthenus is one of several sturgeon species found in Eurasian waters, inhabiting rivers flowing into the Caspian, Black, Azov, Baltic, White, Barents, and Kara Seas [
4]. Compared to other sturgeon species, it is relatively small and has a shorter reproduction cycle. The onset of sexual maturity occurs from 3 to 7 years in males and 5 to 12 years in females [
5,
6]. Although the commercial importance of
A. ruthenus in aquaculture is low, the species is often used in hybridization programs—for example, in the production of the bester, a hybrid of the great sturgeon (beluga)
Huso huso female and a sterlet male [
7]. It is listed as threatened on the IUCN Red List [
2].
Overfishing, poaching, habitat destruction, and other anthropogenic causes have led to the profound decline of sterlet in the Upper and Middle Danube River from the years 1996 to 2010 [
8,
9]. The annual catches of sterlet in the Danube river varied between 36 and 117 metric tons from 1958 to 1981, and the construction of the Derdap dam in 1970 led to a more than 50% decrease in sterlet catches [
10]. Restocking is being undertaken in the Danube using remaining specimens as broodstock along with non-native stock from neighboring river systems in Romania and Hungary [
11,
12,
13]. Projects such as STURGENE conduct targeted restocking in the lower Danube [
14]. Recently, “LIFE Sterlet: Restoration of sterlet populations in the Austrian Danube” has begun restocking of sterlet in the Upper Danube [
15]. Reinartz et al. [
16] revealed that the wild population of Danube sterlet currently includes about 23% non-native sterlet carrying a partial or complete Volga genotype, pointing to a need for the genetic analysis of fish used for restocking.
Genetic rescue is nowadays considered as an effective conservation measure to address genetic destruction associated with habitat loss and fragmentation [
17]. The genetic variability in wild populations of fish at risk of extinction is considered a key element of their adaptability to extreme climate change and anthropogenic pressure and, hence, may play a crucial role in stocking success [
15,
18,
19]. However, we should note that the crossing of two genetically distinct individuals can both reduce the probability of extinction and dramatically increase fitness, which can lead to elevated population size. The major river catchments occupied by large historical populations of European sturgeon flow to either the northeast Atlantic Ocean or to one of the surrounding seas (Baltic, Black, Mediterranean, North). Many locally adapted sturgeon populations have become extinct or decreased to the point that genetic heterogeneity is extremely limited [
20]. The question of the optimal source of recovery stock is critical to the initial phase of recovery programs [
21], and the investigation of genetic patterns within and among populations is a prerequisite for a successful restocking program [
15,
16]. A potential option is to expand genetic heterogeneity through intraspecific hybridization. Recently, a genetic rescue study on
Poecilia reticulata concluded that the gene flow between small populations may be essential for providing the necessary variation for populations to persist and adapt to fast-paced environmental change [
22]. Unlike interspecific hybridization, within-species crosses usually produce viable and fertile individuals; however, the fitness of offspring can be higher (heterosis) or lower (outbreeding depression) than that of their parent populations.
The production of intraspecific hybrids of different populations is also an efficient strategy in aquaculture. The most widely recognized short-term benefit of intraspecific crossing is hybrid vigor, or heterosis, which is exhibited in the superiority of phenotypic traits of the hybrids over those of their parent stock [
23,
24,
25]. The phenomenon can exert impacts on fitness traits that are independent of adaption to a local habitat as well as to those that are environment-specific. A feasible option for conservation stocking may be the mixing of fish from isolated populations to increase genetic diversity, allowing future selection to play out under local natural pressures [
15].
Heterosis is not considered detrimental to conservation efforts, especially when it is undertaken using different populations within species with same chromosome numbers, which leads to the production of fertile offspring. The primary aim of selecting potential populations should be to avoid the outbreeding depression that may result from cross-breeding populations with fixed chromosome differences originating from different environments for over 20 generations or with no gene flow for over 500 years [
26,
27]. To avoid outbreeding depression when more than one population is used for restocking, it is best to choose parent populations that show low genetic divergence and are derived from similar local habitats [
27,
28,
29].
There are currently no reports of the evaluation of introgression asymmetry through hybridization in sturgeon associated with the extinction risk and fitness of future back-crossed generations. It is essential to expand the knowledge of genetic characteristics and fitness fate of hybrids produced in natural ecosystems. There have also been no studies documenting the implications of the interbreeding of divergent sterlet populations in the controlled environment of a hatchery.
We evaluated the effects of intraspecific hybridization at the species level with respect to the fitness-related traits such as body weight and specific growth rate, along with growth heterogeneity and cumulative survival at the population level within a closed aquaculture system provided with common environment, using sterlet as a model species. In addition, the genetic polymorphism and heterozygosity were assessed in purebred fish and intraspecific hybrids using six microsatellite markers.
2. Materials and Methods
This experiment was conducted at the Genetic Fisheries Center, Faculty of Fisheries and Protection of Waters (FFPW) in Vodňany, Czech Republic.
2.1. Ethical Statement
The experiment was carried out in compliance with the criteria of the Animal Research Committee of the FFPW. The fish were maintained in accordance with the principles of the animal welfare act of the Czech Republic and laboratory animal care in compliance with the law on the protection of animals against cruelty (Act no. 246/1992 Coll., ref. number 16OZ15759/2013-17214).
2.2. Parental Populations
We used adult sterlet broodstock from separate captive-bred, hatchery-reared populations originating from the Danube and Volga Rivers to produce hybrid and purebred offspring. The Volga broodstock was obtained from the Genetic Fisheries Center, FFPW, Vodňany, and the Danube broodstock from the Velký Dvůr fish hatchery operated by Rybnikářstvi, Pohořelice a.s., Czech Republic. The genetic origin of the Danube broodstock was the Danube River at the territory of the Slovak Republic, and the stock had been bred in captivity for a minimum of three generations. The Volga population was descended from specimens imported from Russia and bred in captivity for at least three generations. The Volga and Danube rivers are the two largest river systems in Europe with varying water flow regimes in their ranges. The Danube River is located in central and eastern Europe, flowing east from the north slopes of the Alps to the western Black Sea. The Volga River is located in central Russia and flows south from a broad interior region to the northern Caspian Sea. Reinartz et al. [
16] have already demonstrated the genetic differences among the sterlet stocks originating from the Danube and Volga rivers based on microsatellite markers.
2.3. Sampling for the Assessment of Population Divergence
We collected fin clips from 100 brooders of each population and stored them in 96% ethanol for use in microsatellite genotyping. Twelve males and 12 females from any population that expressed discriminatory alleles in at least one microsatellite loci were used as the broodstock to produce six purebred and six hybrid groups of Danube and Volga sterlet.
2.4. Broodstock Handling and Hormone Induction
The broodstock were held in the controlled conditions of a re-circulating water system of 5 m3 indoor tanks maintained at 15 °C for a seven-day acclimation period prior to hormone stimulation. The fish were immersed in 0.07 mL L−1 of clove oil for anesthesia before handling.
Spermiation was induced by an intramuscular injection of 4 mg kg
−1 body weight (BW) carp pituitary suspension in physiological saline 36 h before the intended sperm sampling [
30]. Ovulation was stimulated with an initial injection of 0.5 mg kg
−1 BW carp pituitary suspension in physiological saline 42 h before the intended ovulation, followed by a second injection after 12 h of 4.5 mg kg
−1 BW with the same suspension [
30]. The eggs were collected via the micro-incision of oviducts following the protocol described by Štěch et al. [
31] and maintained in aerobic conditions at temperatures below 16 °C during the evaluation of gamete parameters, such as spermatozoon motility and egg quality, prior to fertilization.
2.5. Fertilization and Hatching
To establish hybrid and purebred crosses, a coordinated simultaneous breeding program was organized at Vodňany and Pohořelice. Twelve males and 12 females from each population were used to produce two purebred and reciprocal hybrid crosses. Three sub crosses were established in a main cross. Each sub cross was established using four females and four males out of 12 females and 12 males of the respective population. The breeding program is shown in
Figure 1. Milt from the Volga males was held separately at 4 °C and transported from Vodňany to fertilize the Danube eggs in the fish hatchery at Pohořelice. Three Danube (♀) × Danube (♂) and three Danube (♀) × Volga (♂) crosses were produced and incubated at Pohořelice. Likewise, the milt from individual males of the Danube population was separately held at 4 °C and transported to fertilize Volga eggs at the Genetic Fisheries Center, Vodňany. Three Volga (♀) × Volga (♂) and three Volga (♀) × Danube (♂) crosses were produced and incubated at Vodňany. The fertilized eggs from the six established crosses at Pohořelice were transferred at the neurula stage to Vodňany in oxygenated water at 15 °C. The hatching and initial rearing was conducted in Vodňany.
For fertilization, 50 g of eggs were collected from each of four females. The eggs were pooled, divided into four plastic beakers in 50 g aliquots, and placed on an electronic shaker at 200 rpm and 10 mm deflection. The aliquots were separately fertilized by individual males to avoid sperm competition and to balance the genetic contribution of individual males. A clay suspension (20 g L
−1) was subsequently added to eliminate egg stickiness [
24], and, after shaking for an additional 45 min, the eggs were repeatedly washed in water, pooled, and incubated in glass jar incubators in triplicate. Incubators were supplied with UV-sterilized re-circulating tap water at 15.0 °C, 9 mg L
−1 O
2. For estimating the fertilization rate, ~100 eggs from each incubator were randomly sampled at 6 h post-fertilization in triplicate, and the live embryos were counted at the 2nd or 3rd cleavage division [
32]. The sub crosses were pooled, resulting in the establishment of two purebred groups (Danube and Volga) and two reciprocal hybrid groups (D × V and V × D). Forty-five swim-up larvae from each group were collected and preserved in 96% ethanol for molecular analysis. Larval rearing was conducted at the Genetic Fisheries Center in Vodňany.
2.6. Rearing of Progeny Groups
The fish were reared according to standard aquaculture procedures in the indoor recirculating aquaculture system and outdoor tanks. The fish groups were reared in three tank replicates throughout the experiment, be it indoor or outdoor tanks, or separate or communal rearing. After the complete absorption of the yolk sac, the larvae were fed with diced sludge worms
Tubifex tubifex for two weeks ad libitum and then shifted to co-feeding with dry feed. After four weeks of co-feeding, the larvae were completely transferred to dry feed (Alltech Coppens, Helmond, The Netherlands) and fed daily at a feeding rate of 8% of the fish biomass for the first three months (
Table 1). Approximately 30% mortality was recorded during the shift from live to dry feed. During the first three months, the stock in 0.5 m
3 recirculating indoor tanks was regularly reduced in the following manner to accommodate growth and limit the density during the critical growth period. After the initial three months of rearing in indoor troughs, the fish were transferred to 3.2 m
3 separate indoor circular tanks at an initial density of ~7 kg m
−3 and fed daily at a feeding rate of 4% of the fish biomass. At 175 days post-hatching (dph), the surviving fish were implanted with subcutaneous individual passive integrated transponder tags (134.2 kHz; AEG, Germany). At 229 dph, they were moved to 3.2 m
3 outdoor circular tanks for communal rearing in a density of ~10 kg m
−3 and fed daily at a feeding rate of 4% of the fish biomass. The water-dissolved oxygen and temperature were kept optimum throughout the rearing period. The feed pellet size was adjusted according to the fish developmental stage (
Table 1).
2.7. Measurement of Performance and Mean Heterosis
The fish were measured to calculate the mean weight and cumulative survival at 77, 175, 229, 325, 386, and 504 dph. The specific growth rate (SGR, % day−1) was calculated as SGR = (lnWf – lnWi × 100)/t, in which Wi and Wf are the initial and final mean BW and t is the time interval between the samplings in days. The growth heterogeneity (GH) was calculated from CVFBW/CVIBW, where CV is the coefficient of variation (100 × SD/mean) and IBW and FBW are the initial and final mean BW. The average heterosis for the mean BW and cumulative survival rate of the hybrid fish were calculated as average heterosis = [(F1 − MP)/MP] × 100, in which F1 = the value of hybrid, MP = the mean value of the purebred crosses.
2.8. Microsatellite Marker Analysis
Whole genomic DNA was extracted using the Nucleo Spin
® Tissue kit (MACHEREY-NAGEL GmbH and Co. KG, Düren, Germany) from fin clips for the population divergence study and from swim-up larvae for the assessment of heterozygosity in groups. Six microsatellite markers, AciG 35 [
33], AfuG 135 [
34], Aox 45 [
35], Spl 101, Spl 163, and Spl 173 [
36] were used for PCR amplification carried out according to Havelka et al. [
37]. A microsatellite fragment analysis was conducted on a 3500 ABI Genetic Analyzer (Applied Biosystems, Waltham, USA) using the GeneScan LIZ 600 size standard (Applied Biosystems), and genotypes were identified in the Genemapper 4.1 software (Applied Biosystems, Waltham, USA). The mean number of effective alleles (N
A), pairwise G
ST, D
A matrix, gene diversity at each locus, fixation index, and expected (H
e) and observed (H
o) heterozygosity of the Danube and Volga populations were calculated using GeneAlex [
38]. Likewise, the mean number of alleles (N
A) and expected (H
e) and observed (H
o) heterozygosity used to assess the level of polymorphism in the analyzed progeny fish groups were calculated using GeneAlex [
38]. The visualization of the genetic relationships among the progeny fish groups and two populations based on the multilocus genotypes were performed by a factorial correspondence analysis (FCA) in the GENETIX software (Version 4.05, 2004) for MS Windows. This enabled the visualization of the data in multidimensional space with no a priori assumptions on grouping, using each allele as an independent variable.
2.9. Statistical Analysis
A statistical analysis was conducted using Statistica 13 (STATISTICA advanced module STATISTICA Multivariate Exploratory Technique; Statsoft). The data were analyzed for normal distribution using the Kolmogorov–Smirnov test. Multiple comparisons were carried out by a one-way analysis of variance (ANOVA) at a significance of p = 0.05, and Tukey’s post-hoc (parametric data), Kruskal–Wallis, and Dunn’s post-hoc tests were used for the non-parametric data fertilization rate, the hatching rate, the mean BW, the growth heterogeneity, and the specific growth rate. The differences in survival between the three tank replicates were evaluated using a Pearson’s Chi-square test at a significance of p = 0.05. The significance of the differences between the individual groups was tested by an ANOVA with a significance at p = 0.05. The significance of the differences in NA, Ho, and He between the Danube and Volga populations and between the hybrids and purebreds was tested with a one-way ANOVA at a significance of p = 0.05.