Effects of seawater and freshwater challenges on the Gh/Igf system in the saline-tolerant blackchin tilapia (Sarotherodon melanotheron).

Prolactin (Prl) and growth hormone (Gh) as well as insulin-like growth factor 1 (Igf1) are involved in the physiological adaptation of fish to varying salinities. The Igfs have been also ascribed other physiological roles during development, growth, reproduction and immune regulation. However, the main emphasis in the investigation of osmoregulatory responses has been the endocrine, liver-derived Igf1 route and local regulation within the liver and osmoregulatory organs. Few studies have focused on the impact of salinity alterations on the Gh/Igf-system within the neuroendocrine and immune systems and particularly in a salinity-tolerant species, such as the blackchin tilapia Sarotherodon melanotheron. This species is tolerant to hypersalinity and saline variations, but it is confronted by severe climate changes in the Saloum inverse estuary. Here we investigated bidirectional effects of increased salinity followed by its decrease on the gene regulation of prl, gh, igf1, igf2, Gh receptor and the tumor-necrosis factor a. A mixed population of sexually mature 14-month old blackchin tilapia adapted to freshwater were first exposed to seawater for one week and then to fresh water for another week. Brain, pituitary, head kidney and spleen were excised at 4 h, 1, 2, 3 and 7 days after both exposures and revealed differential expression patterns. This investigation should give us a better understanding of the role of the Gh/Igf system within the neuroendocrine and immune organs and the impact of bidirectional saline challenges on fish osmoregulation in non-osmoregulatory organs, notably the complex orchestration of growth factors and cytokines.

Polymorphism of Sex Determination Amongst Wild Populations Suggests its Rapid Turnover Within the Nile Tilapia Species.

Sex-determining regions have been identified in the Nile tilapia on linkage groups (LG) 1, 20 and 23, depending on the domesticated strains used. Sex determining studies on wild populations of this species are scarce. Previous work on two wild populations, from Lake Volta (Ghana) and from Lake Koka (Ethiopia), found the sex-determining region on LG23. These populations have a Y-specific tandem duplication containing two copies of the Anti-Müllerian Hormone amh gene (named amhY and amhΔY). Here, we performed a whole-genome short-reads analysis using male and female pools on a third wild population from Lake Hora (Ethiopia). We found no association of sex with LG23, and no duplication of the amh gene. Furthermore, we found no evidence of sex linkage on LG1 or on any other LGs. Long read whole genome sequencing of a male from each population confirmed the absence of a duplicated region on LG23 in the Lake Hora male. In contrast, long reads established the structure of the Y haplotype in Koka and Kpandu males and the order of the genes in the duplicated region. Phylogenies constructed on the nuclear and mitochondrial genomes, showed a closer relationship between the two Ethiopian populations compared to the Ghanaian population, implying an absence of the LG23Y sex-determination region in Lake Hora males. Our study supports the hypothesis that the amh region is not the sex-determining region in Hora males. The absence of the Y amh duplication in the Lake Hora population reflects a rapid change in sex determination within Nile tilapia populations. The genetic basis of sex determination in the Lake Hora population remains unknown.

Different transcriptomic architecture of the gill epithelia in Nile and Mozambique tilapia after salinity challenge.

Tilapiine fishes of the genus Oreochromis vary in their euryhaline capabilities, therefore inhabiting aquatic environments of different salinities across the African continent. We analyzed the differential gene expression in the gills before and after 6 weeks salinity challenge between the highly tolerant Mozambique tilapia (Oreochromis mossambicus) and the less tolerant Nile tilapia (O. niloticus). The pathways triggered by salinity in both tilapia species reveal immune and cell stress responses as well as turnover of ionocytes. Nevertheless, the actual differential expressed genes vary between these two species, pointing at differential transcriptomic architecture, which likely contribute to the species osmoregulation capabilities in elevated salinities.

Structure and Sequence of the Sex Determining Locus in Two Wild Populations of Nile Tilapia.

In domesticated strains of the Nile tilapia, phenotypic sex has been linked to genetic variants on linkage groups 1, 20 and 23. This diversity of sex-loci might reflect a naturally polymorphic sex determination system in Nile tilapia, or it might be an artefact arising from the process of domestication. Here, we searched for sex-determiners in wild populations from Kpandu, Lake Volta (Ghana-West Africa), and from Lake Koka (Ethiopia-East Africa) that have not been subjected to any genetic manipulation. We analysed lab-reared families using double-digest Restriction Associated DNA sequencing (ddRAD) and analysed wild-caught males and females with pooled whole-genome sequencing (WGS). Strong sex-linked signals were found on LG23 in both populations, and sex-linked signals with LG3 were observed in Kpandu samples. WGS uncovered blocks of high sequence coverage, suggesting the presence of B chromosomes. We confirmed the existence of a tandem amh duplication in LG23 in both populations and determined its breakpoints between the oaz1 and dot1l genes. We found two common deletions of ~5 kb in males and confirmed the presence of both amhY and amh∆Y genes. Males from Lake Koka lack both the previously reported 234 bp deletion and the 5 bp frameshift-insertion that creates a premature stop codon in amh∆Y.

Mismatches between the genetic and phenotypic sex in the wild Kou population of Nile tilapia Oreochromis niloticus.

Sex determination and sex chromosomes can be very diverse between teleost species. The group of tilapias shows a polymorphism in sex determination not only between closely related species but also between domestic strains within a species. In the Nile tilapia, the major effect genes and therefore the Y chromosome have been located on either linkage group 1 (LG1) or LG23 depending on the strains. In a Japanese strain, the sex determinant of LG23 (the amhY gene) has been identified as a duplicated amh (anti-Müllerian hormone) gene, with its gametolog found on the X chromosome (amhX). AmhY is located in tandem with the amhΔY gene (a truncated form) on the Y chromosome. X and Y chromosome markers based on the amh genes have been validated only on a few domestic strains but not in wild populations. Here, we used four of these markers in order to examine (1) the possible variation in sex determination of a wild population of Nile tilapia living in Lake Kou (Burkina Faso), (2) putative polymorphisms for these amh copies and (3) the existence of sex reversed individuals in the wild. Our genotyping of 91 wild Kou individuals with the amh sex-diagnostic markers of LG23 showed that while phenotypic females were all XX, phenotypic males were either XY or XX. Progeny testing of eight of these XX males revealed that one of these males consistently sired all-female progenies, suggesting that it is a wild sex reversed male (which could result from high temperature effects). The other XX males gave balanced sex ratios, suggesting that sex is controlled by another locus (possibly on another LG) which may be epistatically dominant over the LG23 locus. Finally, identification of unexpected amh genotypes was found for two individuals. They produced either balanced or female-biased sex ratios, depending on the breeder with whom they were crossed, suggesting possible recombination between the X and the Y chromosomes.

Species-Specific Marker Discovery in Tilapia.

Tilapias (family Cichlidae) are of importance in aquaculture and fisheries. Hybridisation and introgression are common within tilapia genera but are difficult to analyse due to limited numbers of species-specific genetic markers. We tested the potential of double digested restriction-site associated DNA (ddRAD) sequencing for discovering single nucleotide polymorphism (SNP) markers to distinguish between 10 tilapia species. Analysis of ddRAD data revealed 1,371 shared SNPs in the de novo-based analysis and 1,204 SNPs in the reference-based analysis. Phylogenetic trees based on these two analyses were very similar. A total of 57 species-specific SNP markers were found among the samples analysed of the 10 tilapia species. Another set of 62 species-specific SNP markers was identified from a subset of four species which have often been involved in hybridisation in aquaculture: 13 for Oreochromis niloticus, 23 for O. aureus, 12 for O. mossambicus and 14 for O. u. hornorum. A panel of 24 SNPs was selected to distinguish among these four species and validated using 91 individuals. Larger numbers of SNP markers were found that could distinguish between the pairs of species within this subset. This technique offers potential for the investigation of hybridisation and introgression among tilapia species in aquaculture and in wild populations.

Effects of the acclimation to high salinity on intestinal ion and peptide transporters in two tilapia species that differ in their salinity tolerance.

Tilapiine species, widely distributed across habitats with diverse water salinities, are important to aquaculture as well as a laboratory model. The effects of water salinity on two tilapia species, that differ in their salinity tolerance, was evaluated. Oreochromis niloticus reared in brackish-water, showed a significant decrease in growth and feed efficiency, whereas O. mossambicus reared in seawater did not show any significant changes. The expression and activity of Na+/K+-ATPase (NKA), V-type H+-ATPase (VHA) and carbonic anhydrase (CA), as well as expression levels of genes encoding two HCO3- and three peptide transporters (nbc1, slc26a6, slc15a1a, slc15a1b and slc15a2) were measured in three intestinal sections of these two species, grown in freshwater and brackish/sea-water. Overall, the spatial distribution along the intestine of the genes examined in this study was similar between the two species, with the exception of tcaIV. The salinity response, on the other hand, varied greatly between these species. In O. mossambicus, there was a salinity-dependent increased expression of most of the examined genes (except slc26a6 and slc15a2), while in O. niloticus the expression of most genes did not change, or even decreased (tcaIV, nbc1 and slc15a1b). This study highlighted differences in the intestinal response to salinity acclimation between closely- related species that differ in their salinity tolerance. O. mossambicus, which has a high salinity tolerance, showed expression patterns and responses similar to marine species, and differed from the low-salinity-tolerance O. niloticus, which showed a response that differed from the accepted models, that are based on marine and diadromous fishes.

The Reversible Sex of Gonochoristic Fish: Insights and Consequences.

Fish sex reversal is a means to understand sex determination and differentiation, but it is also used to control sex in aquaculture. This review discusses sex reversal in gonochoristic fish, with the coexistence of genetic and environmental influences. The different periods of fish sensitivity to sex reversal treatments are presented with the mechanisms implicated. The old players of sex differentiation are revisited with transcriptome data and loss of function studies following hormone- or temperature-induced sex reversal. We also discuss whether cortisol is the universal mediator of sex reversal in fish due to its implication in ovarian meiosis and 11KT increase. The large plasticity in fish for sex reversal is also evident in the brain, with a reversibility existing even in adulthood. Studies on epigenetics are presented, since it links the environment, gene expression, and sex reversal, notably the association of DNA methylation in sex reversal. Manipulations with exogenous factors reverse the primary sex in many fish species under controlled conditions, but several questions arise on whether this can occur under wild conditions and what is the ecological significance. Cases of sex reversal in wild fish populations are shown and their fitness and future perspectives are discussed.

Comparative analysis of a sex chromosome from the blackchin tilapia, Sarotherodon melanotheron.

BACKGROUND: Inversions and other structural polymorphisms often reduce the rate of recombination between sex chromosomes, making it impossible to fine map sex-determination loci using traditional genetic mapping techniques. Here we compare distantly related species of tilapia that each segregate an XY system of sex-determination on linkage group 1. We use whole genome sequencing to identify shared sex-patterned polymorphisms, which are candidates for the ancestral sex-determination mutation. RESULTS: We found that Sarotherodon melanotheron segregates an XY system on LG1 in the same region identified in Oreochromis niloticus. Both species have higher densities of sex-patterned SNPs, as well as elevated number of ancestral copy number variants in this region when compared to the rest of the genome, but the pattern of differentiation along LG1 differs between species. The number of sex-patterned SNPs shared by the two species is small, but larger than expected by chance, suggesting that a novel Y-chromosome arose just before the divergence of the two species. We identified a shared sex-patterned SNP that alters a Gata4 binding site near Wilms tumor protein that might be responsible for sex-determination. CONCLUSIONS: Shared sex-patterned SNPs, insertions and deletions suggest an ancestral sex-determination system that is common to both S. melanotheron and O. niloticus. Functional analyses are needed to evaluate shared SNPs near candidate genes that might play a role in sex-determination of these species. Interspecific variation in the sex chromosomes of tilapia species provides an excellent model system for understanding the evolution of vertebrate sex chromosomes.

Endocrine and Local IGF-I in the Bony Fish Immune System.

A role for GH and IGF-I in the modulation of the immune system has been under discussion for decades. Generally, GH is considered a stimulator of innate immune parameters in mammals and teleost fish. The stimulatory effects in humans as well as in bony fish often appear to be correlated with elevated endocrine IGF-I (liver-derived), which has also been shown to be suppressed during infection in some studies. Nevertheless, data are still fragmentary. Some studies point to an important role of GH and IGF-I particularly during immune organ development and constitution. Even less is known about the potential relevance of local (autocrine/paracrine) IGF-I within adult and developing immune organs, and the distinct localization of IGF-I in immune cells and tissues of mammals and fish has not been systematically defined. Thus far, IGF-I has been localized in different mammalian immune cell types, particularly macrophages and granulocytes, and in supporting cells, but not in T-lymphocytes. In the present study, we detected IGF-I in phagocytic cells isolated from rainbow trout head kidney and, in contrast to some findings in mammals, in T-cells of a channel catfish cell line. Thus, although numerous analogies among mammals and teleosts exist not only for the GH/IGF-system, but also for the immune system, there are differences that should be further investigated. For instance, it is unclear whether the primarily reported role of GH/IGF-I in the innate immune response is due to the lack of studies focusing on the adaptive immune system, or whether it truly preferentially concerns innate immune parameters. Infectious challenges in combination with GH/IGF-I manipulations are another important topic that has not been sufficiently addressed to date, particularly with respect to developmental and environmental influences on fish growth and health.

The sensitive period for male-to-female sex reversal begins at the embryonic stage in the Nile tilapia and is associated with the sexual genotype.

In this study, we sought to determine the mechanism of early sex reversal in a teleost by applying 4 hr feminization treatments to XY (17α-ethynylestradiol 2000 µg L(-1) ) and YY (6500 µg L(-1) ) Nile tilapia embryos on the first day post-fertilization (dpf). We then searched for changes in the expression profiles of some sex-differentiating genes in the brain (cyp19a1b, foxl2, and amh) and in sex steroids (testosterone, 17ß-estradiol, and 11-ketotestosterone) concentrations during embryogenesis and gonad differentiation. No sex reversal was observed in YY individuals, whereas sex-reversal rates in XY progeny ranged from 0-60%. These results, together with the clearance profile of 17α-ethynylestradiol, confirmed the existence of an early sensitive period for sex determination that encompasses embryonic and larval development and is active prior to any sign of gonad differentiation. Estrogen treatment induced elevated expression of cyp19a1b and higher testosterone and 17ß-estradiol concentrations at 4 dpf in both XY and YY individuals. foxl2 and amh were repressed at 4 dpf and their expression levels were not different between treated and control groups at 14 dpf, suggesting that foxl2 did not control cyp19a1b in the brains of tilapia embryos. Increased cyp19a1b expression in treated embryos could reflect early brain sexualization, although this difference alone cannot account for the observed sex reversal as the treatment was ineffective in YY individuals. The differential sensitivity of XY and YY genotypes to embryonic induced-feminization suggests that a sex determinant on the sex chromosomes, such as a Y repressor or an X activator, may influence sex reversal during the first steps of tilapia embryogenesis.

Genetic inactivation of European sea bass (Dicentrarchus labrax L.) eggs using UV-irradiation: observations and perspectives.

Androgenesis is a form of uniparental reproduction leading to progenies inheriting only the paternal set of chromosomes. It has been achieved with variable success in a number of freshwater species and can be attained by artificial fertilization of genetically inactivated eggs following exposure to gamma (γ), X-ray or UV irradiation (haploid androgenesis) and by restoration of diploidy by suppression of mitosis using a pressure or thermal shock. The conditions for the genetic inactivation of the maternal genome in the European sea bass (Dicentrarchus labrax L.) were explored using different combinations of UV irradiation levels and durations. UV treatments significantly affected embryo survival and generated a wide range of developmental abnormalities. Despite the wide range of UV doses tested (from 7.2 to 720 mJ x cm(-2)), only one dose (60 mJ x cm(-2) x min(-1) with 1 min irradiation) resulted in a small percentage (14%) of haploid larvae at hatching in the initial trials as verified by flow cytometry. Microsatellite marker analyses of three further batches of larvae produced by using this UV treatment showed a majority of larvae with variable levels of paternal and maternal contributions and only one larva displaying pure paternal inheritance. The results are discussed also in the context of an assessment of the UV-absorbance characteristics of egg extracts in this species that revealed the presence of gadusol, a compound structurally related to mycosporine-like amino acids (MAAs) with known UV-screening properties.

The genomic substrate for adaptive radiation in African cichlid fish.

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.

Oestrogen and insulin-like growth factors during the reproduction and growth of the tilapia Oreochromis niloticus and their interactions.

Oestrogens and insulin-like growth factors (Igfs) play both a central role in the regulation of reproduction and growth and can interact especially in species showing a clear-cut sex-linked growth dimorphism (SGD) like in tilapia. Aromatase is essential in ovarian differentiation and oogenesis since it controls oestrogen synthesis. During tilapia sex differentiation, aromatase cyp19a1a expression increases from 9 days post-fertilization (dpf), resulting in high oestradiol level. High temperature, exogenous androgens or aromatase inhibitors override genetic sex differentiation inducing testes development through the suppression of cyp19a1a gene expression and aromatase activity. Supplementation with 17ß-oestradiol (E2) of gonadectomized juveniles induced a sustained and higher E2 plasma level than in intact or gonadectomized controls and both sexes showed reduced growth. Juvenile and mature females treated with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione had 19% lower E2 plasma level compared to controls and they showed a 32% increased growth after 28 days of treatment. Altogether, these data suggest that E2 inhibits female growth leading to the SGD. Regarding Igf-1, mRNA and peptide appeared in liver at ∼ 4 dpf and then in organs involved in growth and metabolism, indicating a role in early growth, metabolism and organogenesis. Gonad igf-1 showed an early expression and the peptide could be detected at ∼ 7 dpf in somatic cells. It appeared in germ cells at the onset of ovarian (29 dpf) and testicular (52 dpf) meiosis. In testis, Igf-1 together with steroids may regulate spermatogenesis whereas in ovary it participates in steroidogenesis regulation. Igf-1 and Igf-2 promote proliferation of follicular cells and oocyte maturation. Igf-3 expression is gonad specific and localized in the ovarian granulosa or testicular interstitial cells. In developing gonads igf-3 is up-regulated in males but down-regulated in females. In contrast, bream Gh injections increased igf-1 mRNA in male and female liver and ovaries but gonadal igf-3 was not affected. Thus, local Igf-1 and Igf-2 may play crucial roles in the formation, development and function of gonads while Igf-3 depending on the species is involved in male and female reproduction. Furthermore, precocious ethynylestradiol (EE) exposure induced lasting effects on growth, through pituitary gh inhibition, local suppression of igf-1 expression and in testis only down-regulation of igf-3 mRNA. In conclusion, SGD in tilapia may be driven through an inhibitory effect due to E2 synthesis in female and involving Igfs regulation.

Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions.

Nile tilapia has primarily a XX/XY sex determining system but minor genetic factors as well as temperature can override the major factors. Female XX progenies can be sex-reversed into functional males by rearing at high temperatures (>34°C) from 10dpf onwards. Temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. The temporal expression profiles of cyp19a1a and foxl2, two ovarian-developmental genes and dmrt1 and amh, two testes-developmental genes were analysed during key stages of the sex differentiation of genetic all-females, all-males and temperature-masculinised XX females (TM) tilapia. Overall QPCR analysis was similar between gonads and trunks. Both amh and dmrt1 expressions were up-regulated simultaneously in TM already at 13-15dpf. Dmrt1 expression became markedly elevated ∼3-fold higher than XY male levels at 20-26dpf whereas amh had similar levels to XY males. Foxl2 and cyp19a1a expression profiles were similar. Both were up-regulated at early stages in TM but repressed after 17-19dpf, whilest levels continued to increase in XX-females. Our results show that temperature action on tilapia testis development induces the rapid increase of both dmrt1 and amh expressions followed by the down-regulation of foxl2 and cyp19a1a. This suggests that dmrt1 and/or amh may be the modulator(s) of the down-regulation of foxl2 and/or cyp19a1a.

A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs.

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 cR(3500) 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.

Genetic and physical mapping of sex-linked AFLP markers in Nile tilapia (Oreochromis niloticus).

Identification of the sex-determining genes of the Nile tilapia (Oreochromis niloticus) has important implications for commercial aquaculture. We previously identified an XX/XY sex-determining locus in this species within a 10-cM interval between markers GM201 and UNH995 on linkage group one (LG1). In order to refine this region, we developed new AFLP markers using bulked segregant analysis of the mapping families. We identified three AFLP markers that showed a sex-specific pattern of segregation. All three mapped near, but just outside, the previously identified sex-determining region on LG1. Hybridization of BAC clones containing these markers to chromosome spreads confirmed that the XX/XY sex-determining locus is on one of the small chromosomes in O. niloticus.

The vertebrate makorin ubiquitin ligase gene family has been shaped by large-scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene.

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.

Comparative physical maps derived from BAC end sequences of tilapia (Oreochromis niloticus).

BACKGROUND: The Nile tilapia is the second most important fish in aquaculture. It is an excellent laboratory model, and is closely related to the African lake cichlids famous for their rapid rates of speciation. A suite of genomic resources has been developed for this species, including genetic maps and ESTs. Here we analyze BAC end-sequences to develop comparative physical maps, and estimate the number of genome rearrangements, between tilapia and other model fish species. RESULTS: We obtained sequence from one or both ends of 106,259 tilapia BACs. BLAST analysis against the genome assemblies of stickleback, medaka and pufferfish allowed identification of homologies for approximately 25,000 BACs for each species. We calculate that rearrangement breakpoints between tilapia and these species occur about every 3 Mb across the genome. Analysis of 35,000 clones previously assembled into contigs by restriction fingerprints allowed identification of longer-range syntenies. CONCLUSIONS: Our data suggest that chromosomal evolution in recent teleosts is dominated by alternate loss of gene duplicates, and by intra-chromosomal rearrangements (~one per million years). These physical maps are a useful resource for comparative positional cloning of traits in cichlid fishes. The paired BAC end sequences from these clones will be an important resource for scaffolding forthcoming shotgun sequence assemblies of the tilapia genome.

Seawater and freshwater challenges affect the insulin-like growth factors IGF-I and IGF-II in liver and osmoregulatory organs of the tilapia.

Contradictory studies suggest IGF-I in fish liver and gills is involved in osmoregulation, but nothing is known about the kidney and intestine's role nor about IGF-II's role in any organ. Tilapia were transferred from freshwater (FW) to seawater (SW) for 1week (wk) and retransferred to FW for another week. At 4h, 1d, 2d, 3d and 1wk after SW-transfer and FW-retransfer IGF-I, IGF-II and growth hormone receptor (GHR1) mRNA were measured by real-time PCR. Hepatic IGF-I, IGF-II and GHR1 mRNA were downregulated in parallel after SW-transfer, recovered and were again downregulated after FW-retransfer. In gills, IGF-I, IGF-II and GHR1 were upregulated synchronously after SW-transfer and, partially also after FW-retransfer. The renal genes were downregulated after SW-transfer and partially upregulated after FW-retransfer. Persisting upregulation in intestinal IGF-I mRNA occurred after FW-retransfer. Thus, endocrine and auto/paracrine IGF-I and IGF-II seem to be involved in fish osmoregulation in an organ-specific manner.