Differential expression and alternative splicing analyses of multiple tissues reveal albinism-associated genes in the Wels catfish (Silurus glanis).

Albinism is a widespread departure from a typical body colouration due to altered melanin production. The Wels catfish (Silurus glanis) is among the largest freshwater fish species in the world, and albino individuals occur both in the wild and in aquaculture. Here, we performed transcriptome-wide analysis of albino and normally pigmented S. glanis using four tissues (skin, dorsal fin, whole eye and liver) to identify genes associated with albinism by exploring patterns of differential expression (DE) and differential alternative splicing (DAS). Multi-tissue analyses revealed a large number of genes in skin (n = 1355) and fin (n = 614) tissue associated with the albino phenotype in S. glanis, while the number of DE genes in eye and liver tissues was lower (n = 188, n = 189, respectively). Several DE genes across multiple tissues were detected as the most promising candidates (e.g., hsp4, hsp90b1, raph1, uqcrfs1, adcy-family and wnt-family) potentially causally linked to the albino phenotype in Wels catfish. Moreover, our findings supported earlier observations of physiological differences between albino and normally pigmented individuals, particularly in energy metabolism and immune response. In contrast, there were only a few pigmentation-related genes observed among DAS genes (4 in skin, 2 in fin), the overlap between DAS and DE genes was low (n = 25) and did not include known pigmentation-related genes. This suggests that DAS and DE in Wels catfish are, to a large extent, independent processes, and the observed alternative splicing cases are probably not causally linked with albinism in S. glanis. This work provides the first transcriptome-wide multi-tissue insights into the albinism of Wels catfish and serves as a valuable resource for further understanding the genetic mechanisms of pigmentation in fish.

Flow cytometry for assessing the efficacy of interspecific gynogenesis induction in sturgeon.

The efficacy of ploidy analysis for separating progeny of Siberian sturgeon Acipenser baerii after induced gynogenesis was demonstrated using sperm of a paternal species differing in ploidy level from the maternal species. Gynogenesis was induced in tetraploid A. baerii with UV-C irradiated sperm from the diploid sterlet Acipenser ruthenus and vice-versa. The success of sperm UV irradiation and diploidy restoration by heat-shock was estimated based on the ploidy level of progeny, confirmed by microsatellite parentage assignment. Hatching rates of interspecific gynogenotes were comparable with rates reported for gynogenesis induction using sperm and eggs of the same species. Juvenile mortality was similar to that observed in the control hybrids. The efficiency and reliability of this method may foster its use for production of gynogenotes in aquaculture, potentially allowing interspecific gynogenesis to replace intraspecific.

Technical note: Production of tetraploid sturgeons.

Studies and practical application of androgenesis and gynogenesis in sturgeon are significantly hindered by strong influence of ploidy restoration treatment on survivability of progeny; therefore, developed method of production of tetraploid broodstock and, consequently, use of their diploid gametes might help to avoid ploidy restoration treatment. In the present study, for the first time was developed a protocol for tetraploidy induction in 2 model sturgeon species, sterlet () and Siberian sturgeon (). A high efficiency of treatment was achieved by optimization of heat shock using a temperature of 37°C for 2 min timed between the end of female pronuclei formation and the beginning of pronuclei migration, that is, 0.8 to 1.0 τ (duration of 1 mitotic cycle during the period of synchronous cleavage division). Fertilized eggs developed in tetraploid larvae, up to 31 (89.6% in control) and 34% (70.9% in control) in sterlet and Siberian sturgeon, respectively. Most of the tetraploid larvae exhibited body malformations; as a result, consequent large scale study revealed high larval mortality, which drastically decreased after 2 mo of age. Consequent comparison of BW, length, and malformation rate and mortality between diploid and tetraploid progeny of sterlet did not reveal significant differences in fitness of diploid and tetraploid juveniles at 9 and 11 mo of age. The present study can be considered the first step towards improving the androgenesis methods of conservation of endangered sturgeons as well as understanding the sturgeon sex determination system through induction of mitotic gynogenesis.

Molecular cytogenetics in artificial hybrid and highly polyploid sturgeons: An evolutionary story narrated by repetitive sequences.

We applied comparative genomic hybridization (CGH) and genomic in situ hybridization (GISH) to examine genomes of artificially produced sturgeon hybrids between sterlet, Acipenser ruthenus female (∼120 chromosomes) or Russian sturgeon, A. gueldenstaedtii female (∼240 chromosomes) and a spontaneous triploid Siberian sturgeon A. baerii male (∼360 chromosomes), respectively. The ploidy levels of progenies were analyzed by karyotyping and flow cytometry. We found that the species-specific regions were surprisingly identifiable only on some micro- and small(er) macrochromosomes in hybrid metaphases. We hypothesize that these distinguishable regions are represented by species-specific repetitive sequences driven by more dynamic molecular evolutionary mechanisms. On larger chromosomes, GISH faintly visualized only blocks of pericentromeric and telomeric repetitive sequences, remaining regions were equally shared by both parental species. We concluded that the interspecies hybridization producing viable and even fertile progeny is enabled by the fact that genomes of the species involved are likely divergent at the level of the repetitive sequences only and probably highly conserved in the coding sequences. These small differences of coding sequences are in concordance with previous estimations of relatedness of examined species producing artificial as well as natural hybrids. CGH and GISH represent a challenge in sturgeon cytogenetics as a valuable though technically not simple tool to discriminate chromosomes of parental species in hybrids. The potentials and drawbacks of CGH and GISH application in sturgeons are discussed and further experimental possibilities are proposed.

First evidence of autotriploidization in sterlet (Acipenser ruthenus).

Polyploidization has played an important role in vertebrate evolution. Acipenseridae bring clear examples of polyploidy ancestry and, also, polyploidization seems to be an ongoing process in these fishes. In the present study, the genetic origin of six triploid specimens morphologically determined as Acipenser ruthenus from commercial aquaculture was analyzed using a combination of mitochondrial and nuclear markers. A further five successive statistical analyses including median joining of mitochondrial DNA control region sequences, principal coordinate analysis (PCA), factorial correspondence analysis (FCA), STRUCTURE assignation, and NewHybrids status determination for microsatellite data were applied for the clarification of the origin of one extra chromosome set added in these triploids genomes. Although interspecific hybridization had been suggested as a source of these triploids, the statistical analyses showed that the investigated triploids originate from autotriploidization rather than from interspecific hybridization. Therefore, we conclude that a combination of molecular markers with suitable statistical analyses should be used to verify the origin of unusual ploidy level. Evidently, such an approach is critically essential in aquaculture, where interspecific hybridization is very common and usually detected by changes in ploidy levels only.

Sympatric occurrence of triploid, aneuploid and tetraploid weatherfish Misgurnus fossilis (Cypriniformes, Cobitidae).

Ploidy analyses of 116 weatherfish Misgurnus fossilis individuals revealed the sympatric occurrence of triploid, intermediate aneuploid and tetraploid specimens in a 1:1:4 ratio. No diploids were detected and the sex ratio of triploids and tetraploids was 1:1, while that of aneuploids was skewed at 3:1 for males. An origin of intermediate aneuploids from mating triploids with tetraploids is hypothesized.

Frequency analyses of active NORs in nuclei of artificially induced triploid fishes.

The correspondence between increased numbers of both chromosomal and nuclear NORs and artificially induced triploidy in three fish species (rainbow trout, Oncorhynchus mykiss; common carp, Cyprinus carpio; and tench, Tinea tinea) has been confirmed by CMA3 fluorescence and Ag-staining. The frequencies of cell nuclei with one, two and three active NORs, as revealed by Ag-staining, has been analyzed statistically to find the minimum cell number which verifies the increased ploidy level. A minimum sample size of about 80 cells exhibiting three active NORs is sufficient to confirm triploidy in all three species and may be of use for categorising other ploidy-manipulated fish species.