Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Chaperone-mediated autophagy protects against hyperglycemic stress.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.

Development of Ovaries and Sex Change in Fish: Bringing Potential into Action.

BACKGROUND: Encompassing about half of the 60,000 species of vertebrates, fish display the greatest diversity of sex determination mechanisms among metazoans. As such that phylum offers a unique playground to study the impressive variety of gonadal morphogenetic strategies, ranging from gonochorism, with either genetic or environmental sex determination, to unisexuality, with either simultaneous or consecutive hermaphroditism. SUMMARY: From the two main types of gonads, the ovaries embrace the important role to produce the larger and non-motile gametes, which is the basis for the development of a future organism. The production of the egg cells is complex and involves the formation of follicular cells, which are necessary for the maturation of the oocytes and the production of feminine hormones. In this vein, our review focuses on the development of ovaries in fish with special emphasis on the germ cells, including those that transition from one sex to the other as part of their life cycle and those that are capable of transitioning to the opposite sex depending on environmental cues. KEY MESSAGES: Clearly, establishing an individual as either a female or a male is not accomplished by the sole development of two types of gonads. In most cases, that dichotomy, be it final or transient, is accompanied by coordinated transformations across the entire organism, leading to changes in the physiological sex as a whole. These coordinated transformations require both molecular and neuroendocrine networks, but also anatomical and behavioural adjustments. Remarkably, fish managed to tame the ins and outs of sex reversal mechanisms to take the most advantages of changing sex as adaptive strategies in some situations.

Diving into the Evolutionary History of HSC70-Linked Selective Autophagy Pathways: Endosomal Microautophagy and Chaperone-Mediated Autophagy.

Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

Inactivation of the Anti-Müllerian Hormone Receptor Type 2 (amhrII) Gene in Northern Pike (Esox lucius) Results in Male-To-Female Sex Reversal.

BACKGROUND: The anti-müllerian hormone (Amh) pathway is crucial for sexual development in teleosts. A male-specific duplicate of anti-müllerian hormone (amhby) was previously identified as the northern pike (Esox lucius) master sex determination gene. However, the role of its putative cognate receptor, i.e., the anti-müllerian hormone receptor type 2 (amhrII) was unclear in this species. OBJECTIVE: Here, we investigated the role of amhrII during sexual development of northern pike. METHOD: We generated stable mutants with deletions in exon 9 of amhrII, inactivating the AmhrII protein using a CRISPR-Cas9-mediated gene knockout strategy. RESULT: The inactivation of amhrII in northern pike results in a high level of male-to-female sex reversal. CONCLUSION: This result demonstrates that amhrII is necessary for male sexual development in northern pike and supports the idea that AmhrII is a conserved regulator of the teleosts sex differentiation network.

A nonfunctional copy of the salmonid sex-determining gene (sdY) is responsible for the "apparent" XY females in Chinook salmon, Oncorhynchus tshawytscha.

Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sex-determining gene (sdY) that interacts at the protein level with Foxl2 leading to the blockage of the synergistic induction of Foxl2 and Nr5a1 of the cyp19a1a promoter. However, this hypothesis of a conserved master sex-determining role of sdY in salmonids is challenged by a few exceptions, one of them being the presence of naturally occurring "apparent" XY Chinook salmon, Oncorhynchus tshawytscha, females. Here, we show that some XY Chinook salmon females have a sdY gene (sdY-N183), with 1 missense mutation leading to a substitution of a conserved isoleucine to an asparagine (I183N). In contrast, Chinook salmon males have both a nonmutated sdY-I183 gene and the missense mutation sdY-N183 gene. The 3-dimensional model of SdY-I183N predicts that the I183N hydrophobic to hydrophilic amino acid change leads to a modification in the SdY ß-sandwich structure. Using in vitro cell transfection assays, we found that SdY-I183N, like the wild-type SdY, is preferentially localized in the cytoplasm. However, compared to wild-type SdY, SdY-I183N is more prone to degradation, its nuclear translocation by Foxl2 is reduced, and SdY-I183N is unable to significantly repress the synergistic Foxl2/Nr5a1 induction of the cyp19a1a promoter. Altogether, our results suggest that the sdY-N183 gene of XY Chinook females is nonfunctional and that SdY-I183N is no longer able to promote testicular differentiation by impairing the synthesis of estrogens in the early differentiating gonads of wild Chinook salmon XY females.

A supernumerary "B-sex" chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus.

Sex chromosomes are generally derived from a pair of classical type-A chromosomes, and relatively few alternative models have been proposed up to now.1,2 B chromosomes (Bs) are supernumerary and dispensable chromosomes with non-Mendelian inheritance found in many plant and animal species3,4 that have often been considered as selfish genetic elements that behave as genome parasites.5,6 The observation that in some species Bs can be either restricted or predominant in one sex7-14 raised the interesting hypothesis that Bs could play a role in sex determination.15 The characterization of putative B master sex-determining (MSD) genes, however, has not yet been provided to support this hypothesis. Here, in Astyanax mexicanus cavefish originating from Pachón cave, we show that Bs are strongly male predominant. Based on a high-quality genome assembly of a B-carrying male, we characterized the Pachón cavefish B sequence and found that it contains two duplicated loci of the putative MSD gene growth differentiation factor 6b (gdf6b). Supporting its role as an MSD gene, we found that the Pachón cavefish gdf6b gene is expressed specifically in differentiating male gonads, and that its knockout induces male-to-female sex reversal in B-carrying males. This demonstrates that gdf6b is necessary for triggering male sex determination in Pachón cavefish. Altogether these results bring multiple and independent lines of evidence supporting the conclusion that the Pachón cavefish B is a "B-sex" chromosome that contains duplicated copies of the gdf6b gene, which can promote male sex determination in this species.

The replaceable master of sex determination: bottom-up hypothesis revisited.

Different group of vertebrates and invertebrates demonstrate an amazing diversity of gene regulations not only at the top but also at the bottom of the sex determination genetic network. As early as 1995, based on emerging findings in Drosophila melanogaster and Caenorhabditis elegans, Wilkins suggested that the evolution of the sex determination pathway evolved from the bottom to the top of the hierarchy. Based on our current knowledge, this review revisits the 'bottom-up' hypothesis and applies its logic to vertebrates. The basic operation of the determination network is through the dynamics of the opposing male and female pathways together with a persistent need to maintain the sexual identity of the cells of the gonad up to the reproductive stage in adults. The sex-determining trigger circumstantially acts from outside the genetic network, but the regulatory network is not built around it as a main node, thus maintaining the genetic structure of the network. New sex-promoting genes arise either through allelic diversification or gene duplication and act specially at the sex-determination period, without integration into the complete network. Due to this peripheral position the new regulator is not an indispensable component of the sex-determining network and can be easily replaced. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Lessons from an unusual vertebrate sex-determining gene.

So far, very few sex-determining genes have been identified in vertebrates and most of them, the so-called 'usual suspects', evolved from genes which fulfil essential functions during sexual development and are thus already tightly linked to the process that they now govern. The single exception to this 'usual suspects' rule in vertebrates so far is the conserved salmonid sex-determining gene, sdY (sexually dimorphic on the Y chromosome), that evolved from a gene known to be involved in regulation of the immune response. It is contained in a jumping sex locus that has been transposed or translocated into different ancestral autosomes during the evolution of salmonids. This special feature of sdY, i.e. being inserted in a 'jumping sex locus', could explain how salmonid sex chromosomes remain young and undifferentiated to escape degeneration. Recent knowledge on the mechanism of action of sdY demonstrates that it triggers its sex-determining action by deregulating oestrogen synthesis that is a conserved and crucial pathway for ovarian differentiation in vertebrates. This result suggests that sdY has evolved to cope with a pre-existing sex differentiation regulatory network. Therefore, 'limited options' for the emergence of new master sex-determining genes could be more constrained by their need to tightly interact with a conserved sex differentiation regulatory network rather than by being themselves 'usual suspects', already inside this sex regulatory network. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Evolution of master sex determiners: TGF-ß signalling pathways at regulatory crossroads.

To date, more than 20 different vertebrate master sex-determining genes have been identified on different sex chromosomes of mammals, birds, frogs and fish. Interestingly, six of these genes are transcription factors (Dmrt1- or Sox3- related) and 13 others belong to the TGF-ß signalling pathway (Amh, Amhr2, Bmpr1b, Gsdf and Gdf6). This pattern suggests that only a limited group of factors/signalling pathways are prone to become top regulators again and again. Although being clearly a subordinate member of the sex-regulatory network in mammals, the TGF-ß signalling pathway made it to the top recurrently and independently. Facing this rolling wave of TGF-ß signalling pathways, this review will decipher how the TGF-ß signalling pathways cope with the canonical sex gene regulatory network and challenge the current evolutionary concepts accounting for the diversity of sex-determining mechanisms. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Expanding the classical paradigm: what we have learnt from vertebrates about sex chromosome evolution.

Until recently, the field of sex chromosome evolution has been dominated by the canonical unidirectional scenario, first developed by Muller in 1918. This model postulates that sex chromosomes emerge from autosomes by acquiring a sex-determining locus. Recombination reduction then expands outwards from this locus, to maintain its linkage with sexually antagonistic/advantageous alleles, resulting in Y or W degeneration and potentially culminating in their disappearance. Based mostly on empirical vertebrate research, we challenge and expand each conceptual step of this canonical model and present observations by numerous experts in two parts of a theme issue of Phil. Trans. R. Soc. B. We suggest that greater theoretical and empirical insights into the events at the origins of sex-determining genes (rewiring of the gonadal differentiation networks), and a better understanding of the evolutionary forces responsible for recombination suppression are required. Among others, crucial questions are: Why do sex chromosome differentiation rates and the evolution of gene dose regulatory mechanisms between male versus female heterogametic systems not follow earlier theory? Why do several lineages not have sex chromosomes? And: What are the consequences of the presence of (differentiated) sex chromosomes for individual fitness, evolvability, hybridization and diversification? We conclude that the classical scenario appears too reductionistic. Instead of being unidirectional, we show that sex chromosome evolution is more complex than previously anticipated and principally forms networks, interconnected to potentially endless outcomes with restarts, deletions and additions of new genomic material. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

The rise and fall of the ancient northern pike master sex-determining gene.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

Reconstruction of the birth of a male sex chromosome present in Atlantic herring.

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an ∼500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination.

Sex chromosome and sex locus characterization in goldfish, Carassius auratus (Linnaeus, 1758).

BACKGROUND: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of goldfish. RESULTS: Our results confirmed that sex determination in goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the goldfish amh gene. CONCLUSIONS: These results show that our goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of goldfish.

Lighting chaperone-mediated autophagy (CMA) evolution with an ancient LAMP: the existence of a functional CMA activity in fish.

Chaperone-mediated autophagy (CMA), as one of the main pathways of lysosomal catabolism, plays essential roles for the maintenance of cellular homeostasis. To date, the absence of any identifiable LAMP2A - the necessary and limiting protein required for CMA - in non-tetrapod lineages, led to the paradigm that this cellular process was restricted to mammals and birds. The recent findings of Lescat et al., demonstrating the existence of a CMA activity in fish, now reshuffle the cards regarding how the entire evolution of CMA function should be considered and appreciated across metazoans. Hence, beyond challenging the current tetrapod-centered accepted view, the work of Lescat et al. tackles the possibility - or the compelling need - of using complementary and powerful genetic models, such as zebrafish or medaka, for studying this fundamental function from an evolutionary perspective.

Chaperone-Mediated Autophagy in the Light of Evolution: Insight from Fish.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.