Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

Characteristics of conserved microRNAome and their evolutionary adaptation to regulation of immune defense functions in the spleen of silver carp and bighead carp.

Immune defense functions of silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) have shown obvious evolutionary divergence. MiRNAs participate in the fine regulation of immune function. However, the evolutionary adaptation of miRNAs in the regulation of immune defense function is still poorly understood in silver carp and bighead carp. Here, small RNA libraries were constructed from the spleen tissue of one-year-old and three-year-old healthy silver carp and bighead carp, 424 and 422 known conserved miRNAs were respectively identified from the spleen of silver carp and bighead carp by bioinformatic analysis, which 398 were shared between the two species. These conserved miRNAs showed highly similar expression patterns between silver carp and bighead carp, but the abundance in spleen varied greatly in different species. Family analysis showed that miRNA families including mir-8, mir-7, mir-23, mir-338, mir-30, mir-27, mir-221, mir-19, mir-181, mir-17, mir-15, mir-148, mir-130, mir-10 and let-7 were the main miRNAs in the spleen of silver carp and bighead carp. 27 and 51 significant differentially expressed (SDE) miRNAs were identified from silver carp and bighead carp, respectively. Evolution analysis for the predicted target genes of SDE-miRNAs showed that ten biological processes such as blood coagulation, cell adhesion mediated by integrin and adaptive immune response were positively selected. In addition, immune genes including TLR3, NFATC3, MALT1, B2M, GILT and MHCII were positively selected only in silver carp, and they were specifically targeted by the SDE-miRNAs including miR-9-5p, miR-196a-5p, miR-375, miR-122, miR-722, miR-132-3p, miR-727-5p, miR-724, miR-19d-5p and miR-138-5p, respectively. PLA2G4 in Fc epsilon RI signaling pathway was positively selected only in bighead carp and was specifically targeted by the SDE-miRNAs including miR-222b, miR-22b-5p, miR-15c, miR-146a, miR-125c-3p, miR-221-5p, miR-2188-5p, miR-142a-3p, miR-212, miR-138-5p and miR-15b-5p. In particular, SDE-miRNAs such as miR-144-3p, miR-2188-3p, miR-731, miR-363-3p and miR-218b could simultaneously target multiple evolutionarily differentiated immune-related genes. These results indicated that in the spleen of silver carp and bighead carp, conserved miRNAs have obvious evolutionary adaptations in the regulation of immune defense function. The results of this study can provide valuable resources for further revealing themechanism of miRNA in the formation of resistance traits evolution between silver carp and bighead carp.

Screening and Stability Analysis of Reference Genes for Gene Expression Normalization in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) Fed Diets Containing Different Soybean Meal Levels.

In this study, we screened the expression stability of six reference genes (18S rRNA, ß-actin, GAPDH, EF1a, B2M, and HPRT1) in hybrid yellow catfish (n = 6), considering the SBM levels, sampling time points, and different tissues. Four different statistical programs, BestKeeper, NormFinder, Genorm, and Delta Ct, combined with a method that comprehensively considered all results, were used to evaluate the expression stability of these reference genes systematically. The results showed that SBM levels significantly impacted the expression stability of most of the reference genes studied and that this impact was time-, dose-, and tissue-dependent. The expression stability of these six reference genes varied depending on tissue, sampling time point, and SBM dosage. Additionally, more variations were found among different tissues than among different SBM levels or sampling time points. Due to its high expression, 18S rRNA was excluded from the list of candidate reference genes. ß-actin and GAPDH in the liver and ß-actin, HPRT1 and EF1a in the intestine were the most stable reference genes when SBM levels were considered. HPRT1, and EF1a in tissues sampled at 2 W and EF1a and ß-actin in tissues sampled at 4 and 6 W were proposed as two stable reference genes when different tissues were considered. When the sampling time points were considered, ß-actin, EF1a, and HPRT1 were the top three stable reference genes in the intestine. In contrast, ß-actin and B2M are the most stable reference genes in the liver. In summary, ß-actin, EF1a, and HPRT1 were the more stable reference genes in this study. The stability of reference genes depends on the tissues, sampling time points, and SBM diet levels in hybrid yellow catfish. Therefore, attention should be paid to these factors before selecting suitable reference genes for normalizing the target genes.