Analysis of Molecular Genetic, Morphometric and Sexual Characteristics of the Beluga (Huso huso L., 1758) Grown in the Aquaculture of the Republic of Belarus

Testing For Hybrid Presence

Microsatellite loci were used for individual genotyping. Allelic variants were determined for four STR loci (An20, AoxD161, AoxD165, AfuG41) and compared with the data published by Barmintseva AE, et al. [6]. The analysis did not reveal any allelic variants not corresponding to the Beluga (H. huso) species. A combination of occurrence with high frequency of Beluga-specific allelic variants (145 An20, 98 AoxD161, 178 AoxD165, 269 AfuG41) and the absence of allelic variants specific for other species indicates with a high probability the species purity of the studied beluga individuals. Sturgeon species-specific

Тable 1: Characteristics of 6 microsatellite loci investigated in 118 Huso huso L. specimens.

Allelic variants and the allelic variants identified in our research are listed in the Table 2. Crossing Huso huso x Acipenser ruthenus is very common in fish farming for obtaining highly productive hybrids. Therefore, the broodstock was examined for hybrids with A. ruthenus using the method specially developed for this purpose [9]. This method also allows distinguishing H. huso and A. ruthenus from some other sturgeon species. When using the positive primer mix, the DNA of non-hybrid H. huso individuals is amplified resulting in a fragment of 153 bp, and when using the negative primer mix, in a fragment of 247 bp. On the contrary, A. ruthenus samples give a 247 bp fragment using the positive primer mix and a 153 bp fragment using the negative primer mix. The samples of hybrid (H. huso x A. ruthenus) specimens produce two products using both primer mixes. Obtaining two PCR products using the negative primer mix and no products using the positive primer mix is a sign of other sturgeon species (e.g. H. dauricus, A. schrenckii, A gueldenstaedtii (Figure 1). It should be noted that this method does not allow distinguishing H. huso and A. nudiventris (NUD on Figure 1).

The third method for the species verification was developed specifically for the Beluga (H. huso L.) by Boscari

E, et al. [10] and Mugue NS [15]. It is based on the detection of Beluga-specific SNP in the 2nd intron of the nuclear ribosomal protein S6 (RP2S6). PCR was carried out using the RP2S6_huso_F and RP2S6_groupA_R primer [10].

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Abbreviations: HUS – Huso huso; RUT – Acipenser ruthenus; hybrid – Huso huso x Acipenser ruthenus; DAU–Huso dauricus; SCH– Acipenser schrenckii; GUE–Acipenser gueldenstaedtii; NUD–Acipenser nudiventris. Figure 1: Electrophoresis of PCR product samples of sturgeon species with positive and negative primer mixes.

mtDNA Analysis

In order to establish the population identity, we sequenced the 367 bp region of mitochondrial D-loop using LproF and DL651 primers [11]. In total, we studied 118 Beluga individuals. The sequences were equal and matched haplotype 3 according to the internal database of the Russian Federal Research Institute of Fisheries and Oceanography (Figure 2). This haplotype corresponds to the Caspian population of Beluga. The obtained sequences also correspond to the sequence (Huso huso caspicus voucher nC12 D-loop, partial sequence) published in the GenBank AY846650.1 [14].

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Figure 2: Comparison of two haplotypes #1 and #3 of the beluga sturgeon (Huso huso L., 1758) (haplotype #1 occurs in the Azov Sea beluga population; haplotype #3 is typical for the Caspian beluga population and found in the analyzed individuals at the OAO “Experimental fish farm “Selets”). *Differences in the nucleotide sequences of two haplotypes are marked as rectangles.

Population Structure Identification

Six STR loci (An20, AoxD161, AoxD165, AfuG41, Aox23,

Spl106) were analyzed from the chipped Beluga breeders (n=118) kept in warm and cold water (Table 3).

Population Structure Identification

Six STR loci (An20, AoxD161, AoxD165, AfuG41, Aox23, Spl106) were analyzed from the chipped Beluga breeders (n=118) kept in warm and cold water (Table 3).

Five out of six alleles in the studied (Huso huso L.) (n = 118) were polymorphic. It should be noted that alleles 229 (AfuG41) and 129 (Aox23) are registered only once in the sample analyzed in the same female specimen. Hardy- Weinberg expectations for genotype frequencies are shown in the Table 4.

Abbreviations: DF, the degrees of freedom = [Na(Na-1)]/2; Prob, probability of the observed numbers deviating as far from the expected numbers by chance. Table 4: Summary of Chi-Square Tests (χ2) for Hardy-Weinberg Equilibrium.

It was revealed a significant difference between the observed and expected according to Hardy-Weinberg model number of allele variance. Considering the fact that the main variety of allelic variants in the analyzed group is limited to 1-4, it is probable that most of individuals are the offspring’s of the same broodstock pair. The statistical parameters for population structure analysis shown in the Table 5.

Beluga from the fish farm “Selets” was compared with the domesticated Caspian Beluga population from a number of fish farms (the Russian Federation). The conducted analyses showed a lack of the gene pool diversity of Beluga sturgeon broodstock from the fish farm “Selets”. A number of identified allelic variants (Na) at the fish farm “Selets”/domesticated Beluga are as follows: An20 3/12, AoxD161 2/5, AoxD165 1/8, AfuG41 4/14. It should be noted that at the fish farm “Selets” Beluga showed a negative sign of the fixation index in contrast with a positive index for the domesticated Beluga population. It indicates the genetic balance displacement towards the heterozygote excess in the population of the fish farm “Selets” and shows the unequal allele distribution in the population, which may be a result of the specimens’ close relation. The intrapopulation breeding of these specimens would lead to a decrease in heterozygosity that increases the probability of recessive genetic abnormalities in their offspring.

Morphometric Analysis and Sexing

The morphometric analysis of Beluga (n=118) from the broodstock from the fish farm “Selets” revealed their unsatisfactory physiological state after winter and during the feeding period. Comparatively high variability (Cv) of the body mass (16,3%), Fulton’s condition factor (1,5%) W, kg L, cm l, cm H, cm O, cm K, % A Сv, % A Сv, % A Сv, % A Сv, % A Сv, % $$ \begin{array}{c c c c c c c c c c c} 6 0, 3 \pm 9, 8 & 1 6, 3 & 1 9 2, 2 \pm 1 8, 3 & 9, 5 & 1 6 1, 6 \pm 1 1, 2 & 6, 9 & 3 3, 2 \pm 2, 9 & 8, 7 & 9 1, 3 \pm 7, 6 & 8, 4 & 1, 5 \end{array} $$ and external traits may be a result of unfavorable feeding conditions (Table 6), since genetic evidence shows a low allelic variation in the Beluga broodstock. Using ultrasound scanning, we found that the broodstock of Beluga at the fish farm” Selets” was represented by 61.9% of females (73 pcs. ♀) and 38.1% of males (45 pcs. ♂). In our research, we observed the following stages of gonadal maturity: stages II, III and IV for males and II, II semi-fatty, II fatty, III and IV in females. Starting from the maturity stage II, testicular tissue was easily visible in frontal and transverse sections (Figure 3). The testicular part was hyperechoic and had distinct margins. The fat part was underdeveloped or poorly developed from the medial side and practically not visible. The margins of the gonad were smoothly curved, while a bright hyperechoic tunic of testis was clearly seen.

At maturity stage III, the echogenicity of testicular tissue increases significantly. The testes appeared on echograms as a homogeneous structure of a light grey (in some cases white) colour with distinct hyperechoic margins. In some cases, two clear hyperechoic lines, gonad margins and peritoneal lining, were well discernable. On the echogram, the testes at stage IV appeared as a bright, hyperechoic, fine-grained homogeneous structure with clear margins and well-defined tunics. Hyperechogenicity of the testis reached its maximum at stage IV. The ripe male maturity status and readiness to spawn were assessed by the brightness of the testis image. On the ultrasound image, the ovarian tissue (stage II) appeared as a grainy “cloud-like” structure of mixed echogenicity with uneven boundaries without a tunic. The fatty portion of the ovary was slight and was visualized in the shape of darker areas as distinct from the lighter ovarian tissue.

At maturity stage II semi-fatty, on the ultrasound image, single ovigerous lamellae appeared as areas of higher echogenicity (of a grey or light-grey colour) alternating with hypoechoic (dark) fat regions. Thus ovigerous lamellae “grew” from the lateral to the medial part of the gonad. At maturity stage II fatty, in contrast to previous stages, the proportion of the ovarian and fat tissue visible on echograms was different. The ovarian tissue was surrounded by fat both from the medial and lateral sides (dark, anechoic regions).

The dark anechoic fat layer was well discernable between the muscles and the gonads. At maturity stage II-III, an ovary seen in the ultrasound image showed moderate echogenicity (grey or light grey). Ovigerous lamellae “penetrated” the body of the gonad and appeared as a brachiate vertical structure (“coral-like” or “fringed” in shape) of higher echogenicity, spreading to the dark hypoechoic region (the fat tissue).

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