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Fish exhibit diverse mechanisms of sex differentiation and determination, shaped by both external and internal influences, often regulated by distinct DNA methylation patterns responding to environmental changes. In S. senegalensis aquaculture, reproductive issues in captivity pose significant challenges, particularly the lack of fertilization capabilities in captive-bred males, hindering genetic improvement measures. This study analyzed the methylation patterns and transcriptomic profiles in gonadal tissue DNA from groups differing in rearing conditions and sexual maturity stages. RRBS (Reduced Representation Bisulfite Sequencing) was employed to detect notable methylation variations across groups, while RNA was extracted and sequenced for differential expression analysis. Our findings suggest that DNA methylation significantly regulates gene expression, acting as a mechanism that can both repress and enhance gene expression depending on the genomic context. The complexity of this epigenetic mechanism is evident from the varying levels of methylation and correlation rates observed in different CpGs neighboring specific genes linked to reproduction. Differential methylation comparisons revealed the highest number of differently methylated CpGs between maturation stages, followed by rearing conditions, and lastly between sexes. These findings underscore the crucial role of methylation in regulating gene expression and its potential role in sex differentiation, highlighting the complex interplay between epigenetic modifications and gene expression. [ABSTRACT FROM AUTHOR]

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Abstract Background The Nile tilapia (Oreochromis niloticus) is the second most farmed fish species worldwide. It is also an important model for studies of fish physiology, particularly because of its broad tolerance to an array of environments. It is a good model to study evolutionary mechanisms in vertebrates, because of its close relationship to haplochromine cichlids, which have undergone rapid speciation in East Africa. The existing genomic resources for Nile tilapia include a genetic map, BAC end sequences and ESTs, but comparative genome analysis and maps of quantitative trait loci (QTL) are still limited. Results We have constructed a high-resolution radiation hybrid (RH) panel for the Nile tilapia and genotyped 1358 markers consisting of 850 genes, 82 markers corresponding to BAC end sequences, 154 microsatellites and 272 single nucleotide polymorphisms (SNPs). From these, 1296 markers could be associated in 81 RH groups, while 62 were not linked. The total size of the RH map is 34,084 cR3500 and 937,310 kb. It covers 88% of the entire genome with an estimated inter-marker distance of 742 Kb. Mapping of microsatellites enabled integration to the genetic map. We have merged LG8 and LG24 into a single linkage group, and confirmed that LG16-LG21 are also merged. The orientation and association of RH groups to each chromosome and LG was confirmed by chromosomal in situ hybridizations (FISH) of 55 BACs. Fifty RH groups were localized on the 22 chromosomes while 31 remained small orphan groups. Synteny relationships were determined between Nile tilapia, stickleback, medaka and pufferfish. Conclusion The RH map and associated FISH map provide a valuable gene-ordered resource for gene mapping and QTL studies. All genetic linkage groups with their corresponding RH groups now have a corresponding chromosome which can be identified in the karyotype. Placement of conserved segments indicated that multiple inter-chromosomal rearrangements have occurred between Nile tilapia and the other model fishes. These maps represent a valuable resource for organizing the forthcoming genome sequence of Nile tilapia, and provide a foundation for evolutionary studies of East African cichlid fishes.

Abstract Background Members of the makorin (mkrn) gene family encode RING/C3H zinc finger proteins with U3 ubiquitin ligase activity. Although these proteins have been described in a variety of eukaryotes such as plants, fungi, invertebrates and vertebrates including human, almost nothing is known about their structural and functional evolution. Results Via partial sequencing of a testis cDNA library from the poeciliid fish Xiphophorus maculatus, we have identified a new member of the makorin gene family, that we called mkrn4. In addition to the already described mkrn1 and mkrn2, mkrn4 is the third example of a makorin gene present in both tetrapods and ray-finned fish. However, this gene was not detected in mouse and rat, suggesting its loss in the lineage leading to rodent murids. Mkrn2 and mkrn4 are located in large ancient duplicated regions in tetrapod and fish genomes, suggesting the possible involvement of ancestral vertebrate-specific genome duplication in the formation of these genes. Intriguingly, many mkrn1 and mkrn2 intronless retrocopies have been detected in mammals but not in other vertebrates, most of them corresponding to pseudogenes. The nature and number of zinc fingers were found to be conserved in Mkrn1 and Mkrn2 but much more variable in Mkrn4, with lineage-specific differences. RT-qPCR analysis demonstrated a highly gonad-biased expression pattern for makorin genes in medaka and zebrafish (ray-finned fishes) and amphibians, but a strong relaxation of this specificity in birds and mammals. All three mkrn genes were maternally expressed before zygotic genome activation in both medaka and zebrafish early embryos. Conclusion Our analysis demonstrates that the makorin gene family has evolved through large-scale duplication and subsequent lineage-specific retroposition-mediated duplications in vertebrates. From the three major vertebrate mkrn genes, mkrn4 shows the highest evolutionary dynamics, with lineage-specific loss of zinc fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos.

Abstract Background Large collections of expressed sequence tags (ESTs) are a fundamental resource for analysis of gene expression and annotation of genome sequences. We generated 116,899 ESTs from 17 normalized and two non-normalized cDNA libraries representing 16 tissues from tilapia, a cichlid fish widely used in aquaculture and biological research. Results The ESTs were assembled into 20,190 contigs and 36,028 singletons for a total of 56,218 unique sequences and a total assembled length of 35,168,415 bp. Over the whole project, a unique sequence was discovered for every 2.079 sequence reads. 17,722 (31.5%) of these unique sequences had significant BLAST hits (e-value < 10-10) to the UniProt database. Conclusion Normalization of the cDNA pools with double-stranded nuclease allowed us to efficiently sequence a large collection of ESTs. These sequences are an important resource for studies of gene expression, comparative mapping and annotation of the forthcoming tilapia genome sequence.

D'Cotta, Helena, Claudiane Valotaire, Florence Le Gac, and Patrick Prunet. Synthesis of gill [Na.sup.+]-[K.sup.+]-ATPase in Atlantic salmon smolts: differences in [Alpha]-mRNA and [Alpha]-protein levels. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 278: R101-R110, 2000.--Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill [Na.sup.+]-[K.sup.+]-ATPase activity of Atlantic salmon smolts. A major [Alpha]-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The [Alpha]-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in [Alpha]-protein levels, revealed by Western blots with specific antibodies and by an increase in gill [Na.sup.+]-[K.sup.+]-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in [Alpha]-mRNA abundance and had basal levels of [Alpha]-protein and enzyme activity. Measurement of the binding of [[sup.3]H]ouabain to [Na.sup.+]-[K.sup.+]-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20-23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in [Alpha]-mRNA levels after 24 h with a rise in [Na.sup.+]-[K.sup.+]-ATPase activity occurring only after 11 days. No qualitative change in [Alpha]-expression was revealed at either the mRNA or protein level. Immunological identification of the [Alpha]-protein was performed with polyclonal antibodies directed against the rat [Alpha]-specific isoforms, revealing that parr, freshwater, and seawater smolts have an [[Alpha].sub.3]-like isoform. This study shows that the increase in [Na.sup.+]-[K.sup.+]-ATPase activity in smolt gills depends first on an increase in the [Alpha]-mRNA expression and is followed by a slower rise in [Alpha]-protein abundance that eventually leads to a higher synthesis of [Na.sup.+]-[K.sup.+] pumps. sodium pump; salmonids; smoltification; [[sup.3]H]ouabain binding; transcript