High resolution genomes of multiple Xiphophorus species provide new insights into microevolution, hybrid incompatibility, and epistasis.

Because of diverged adaptative phenotypes, fish species of the genus Xiphophorus have contributed to a wide range of research for a century. Existing Xiphophorus genome assemblies are not at the chromosomal level and are prone to sequence gaps, thus hindering advancement of the intra- and inter-species differences for evolutionary, comparative, and translational biomedical studies. Herein, we assembled high-quality chromosome-level genome assemblies for three distantly related Xiphophorus species, namely, X. maculatus, X. couchianus, and X. hellerii Our overall goal is to precisely assess microevolutionary processes in the clade to ascertain molecular events that led to the divergence of the Xiphophorus species and to progress understanding of genetic incompatibility to disease. In particular, we measured intra- and inter-species divergence and assessed gene expression dysregulation in reciprocal interspecies hybrids among the three species. We found expanded gene families and positively selected genes associated with live bearing, a special mode of reproduction. We also found positively selected gene families are significantly enriched in nonpolymorphic transposable elements, suggesting the dispersal of these nonpolymorphic transposable elements has accompanied the evolution of the genes, possibly by incorporating new regulatory elements in support of the Britten-Davidson hypothesis. We characterized inter-specific polymorphisms, structural variants, and polymorphic transposable element insertions and assessed their association to interspecies hybridization-induced gene expression dysregulation related to specific disease states in humans.

Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Vitellogenin uptake activity in the intestinal ducts of intraovarian embryos in a viviparous teleost Xenotoca eiseni.

In the viviparous teleost species belonging to the family Goodeidae, intraovarian embryos absorb maternal supplements while they grow during the gestation period. They take up the components via trophotaeniae, a hindgut-derived placental structure. Our previous study using a goodeid species Xenotoca eiseni revealed that intraovarian embryos absorb the yolk protein vitellogenin (Vtg) via the trophotaenia. However, another group indicated yolk components accumulate in the intestinal lumen of X. eiseni embryos. Here, we investigated whether the intestinal duct is capable of protein uptake, as is the trophotaenia. Immunohistochemical studies indicated that endogenous vitellogenin is detected in the intestinal epithelial cells of the intraovarian embryo. Tracer analysis using FITC-Vtg also indicated that intestinal tissues can take up protein. The endocytosis-related genes expressed in trophotaenia were also detected in the intestinal tissues of the embryo. Lipid transporter genes which are not expressed in the trophotaenia were detected in the embryonic intestine. This evidence suggests that the intraovarian embryo of X. eiseni possesses two distinct sites for uptake of the maternal proteins. However, the presumed functions of the embryonic intestine and trophotaenia might be not identical. The study provides a new perspective on how mother-to-embryo matrotrophic interactions have changed in the evolution of viviparous teleosts.

Genomic organization and transcription of superoxide dismutase genes (sod1, sod2, and sod3b) and response to diazinon toxicity in platyfish (Xiphophorus maculatus) by using SOD enzyme activity.

The aim of this study is to determine the effects of 50% of 96 h LC50 (5.25 ppm) diazinon on the expression of superoxide dismutase (SOD) enzyme genes (sod1, sod2, and sod3b) and SOD enzyme activity at the end of 24, 48, 72, and 96 h in platyfish liver and gill tissues. To this end, we determined the tissue-specific distribution of sod1, sod2, and sod3b genes and performed in silico analyses in platyfish (Xiphophorus maculatus). It was determined that malondialdehyde (MDA) level and SOD enzyme activity were increased in the liver [(43.90 EU mg protein-1 (control), 62.45 EU mg protein-1 (24 h), 73.17 EU mg protein-1 (48 h), 82.18 EU mg protein-1 (72 h), 92.93 EU mg protein-1 (96 h)] and gill [(16.44 EU mg protein-1 (control), 33.47 EU mg protein-1 (24 h), 50.38 EU mg protein-1 (48 h), 64.62 EU mg protein-1 (72 h), 74.04 EU mg protein-1 (96 h)] tissues of platyfish exposed to diazinon, while the expression of the sod genes was down-regulated. The tissue-specific distribution of the sod genes varied, with the tissues and the sod genes expression were being predominant in the liver (628.32 in sod1, 637.59 in sod2, 888.5 in sod3b). Thus, the liver was considered a suitable tissue for further gene expression studies. Based on the phylogenetic analyses, platyfish sod genes can be reported to be orthologs of sod/SOD genes from other vertebrates. Identity/similarity analyses supported this determination. Conserved gene synteny proved that there are conserved sod genes in platyfish, zebrafish, and humans.

Physiological and transcriptional effects in the male western mosquitofish (Gambusia affinis) following exposure to dexamethasone.

Dexamethasone (DEX) is a synthetic glucocorticoid widely found in a variety of aquatic environments and has potential adverse effects on aquatic organisms. This study was to assess the toxic effects of exposure to different concentrations (0, 5 and 50 µg/L) of DEX for 60 days on adult male mosquitofish (Gambusia affinis). Morphological analyses of skeleton and anal fin, histological effects of testes and livers, and transcriptional expression levels of genes related to reproductive and immune system were determined. The results showed that exposure to DEX significantly increased 14L and 14D values of hemal spines, which suggested DEX could affect skeleton development and result in more masculine characteristics in male fish. In addition, the damage to testis and liver tissue was observed after DEX treatment. It also enhanced mRNA expression of Erß gene in the brain and Hsd11b1 gene in the testis. The findings of this study reveal physiological and transcriptional effects of DEX on male mosquitofish.

Toxicological effects of polystyrene nanoplastics and perfluorooctanoic acid to Gambusia affinis.

Plastic pollution has attracted huge attention from public and scientific community in recent years. In the environment, nanoplastics (NPs, <100 nm) can interact with persistent organic pollutants (POPs) such as perfluorooctanoic acid (PFOA) and may exacerbate associated toxic impacts. The present study aims to explore the single and combined ecotoxicological effects of PFOA and polystyrene nanoplastics (PS-NPs, 80 nm) on the PI3K/AKT3 signaling pathway using a freshwater fish model Gambusia affinis. Fish were exposed individually to PS-NPs (200 µg/L) and PFOA (50, 500, 5000 µg/L) and their chemical mixtures for 96 h. Our results showed that the co-exposure significantly altered the mRNA relative expression of PI3K, AKT3, IKKß and IL-1ß, compared to corresponding single exposure and control groups, indicating that the PFOA-NP co-exposure can activate the PI3K/AKT3 signaling pathway. The bioinformatic analyses showed that AKT3 had more probes and exhibited a significantly sensitive correlation with DNA methylation, compared to other genes (PIK3CA, IKBKB, and IL1B). Further, the mRNA expressions of PIK3CA, AKT3, and IKBKB had a significant correlation with copy number variation (CNV) in human liver hepatocellular carcinoma (LIHC). And PIK3CA had the highest mutation rate among other genes of interest for LIHC. Moreover, AKT3 showed a relatively lower expression in TAM and CAF cells, compared to PIK3CA, IKBKB, and IL1B. Besides, hsa-mir-155-5p was closely correlated with AKT3, PIK3CA, IKBKB, and IL1B. In summary, these results provide evidence that NPs could enhance the carcinogenic effects of POPs on aquatic organisms and highlight possible targets of LIHC induced by PFOA-NP co-exposure.

The Effect of Meclofenoxate on the Transcriptome of Aging Brain of Nothobranchius guentheri Annual Killifish.

Annual fish of the genus Nothobranchius are promising models for aging research. Nothobranchius reproduces typical aspects of vertebrate aging, including hallmarks of brain aging. Meclofenoxate (MF) is a well-known compound that can enhance cognitive performance. The drug is prescribed for asthenic conditions, trauma, and vascular diseases of the brain. It is believed that MF is able to delay age-dependent changes in the human brain. However, until now, there has been no study of the MF effect on the brain transcriptome. In the present work, we performed an RNA-Seq study of brain tissues from aged Nothobranchius guentheri, which were almost lifetime administered with MF, as well as young and aged control fish. As expected, in response to MF, we revealed significant overexpression of neuron-specific genes including genes involved in synaptic activity and plasticity, neurotransmitter secretion, and neuron projection. The effect was more pronounced in female fish. In this aspect, MF alleviated age-dependent decreased expression of genes involved in neuronal activity. In both treated and untreated animals, we observed strong aging-associated overexpression of immune and inflammatory response genes. MF treatment did not prevent this effect, and moreover, some of these genes tended to be slightly upregulated under MF treatment. Additionally, we noticed upregulation of some genes associated with aging and cellular senescence, including isoforms of putative vascular cell adhesion molecule 1 (VCAM1), protein O-GlcNAcase (OGA), protein kinase C alpha type (KPCA), prolow-density lipoprotein receptor-related protein 1 (LRP1). Noteworthy, MF treatment was also associated with the elevated transcription of transposons, which are highly abundant in the N. guentheri genome. In conclusion, MF compensates for the age-dependent downregulation of neuronal activity genes, but its effect on aging brain transcriptome still cannot be considered unambiguously positive.

Neuronal Phenotype of col4a1 and col25a1: An Intriguing Hypothesis in Vertebrates Brain Aging.

Collagens are the most abundant proteins in vertebrates and constitute the major components of the extracellular matrix. Collagens play an important and multifaceted role in the development and functioning of the nervous system and undergo structural remodeling and quantitative modifications during aging. Here, we investigated the age-dependent regulation of col4a1 and col25a1 in the brain of the short-lived vertebrate Nothobranchius furzeri, a powerful model organism for aging research due to its natural fast-aging process and further characterized typical hallmarks of brain aging in this species. We showed that col4a1 and col25a1 are relatively well conserved during vertebrate evolution, and their expression significantly increases in the brain of N. furzeri upon aging. Noteworthy, we report that both col4a1 and col25a1 are expressed in cells with a neuronal phenotype, unlike what has already been documented in mammalian brain, in which only col25a1 is considered a neuronal marker, whereas col4a1 seems to be expressed only in endothelial cells. Overall, our findings encourage further investigation on the role of col4a1 and col25a1 in the biology of the vertebrate brain as well as the onset of aging and neurodegenerative diseases.

Molecular Signatures of Placentation and Secretion Uncovered in Poeciliopsis Maternal Follicles.

Placentation evolved many times independently in vertebrates. Although the core functions of all placentas are similar, we know less about how this similarity extends to the molecular level. Here, we study Poeciliopsis, a unique genus of live-bearing fish that have independently evolved complex placental structures at least three times. The maternal follicle is a key component of these structures. It envelops yolk-rich eggs and is morphologically simple in lecithotrophic species but has elaborate villous structures in matrotrophic species. Through sequencing, the follicle transcriptome of a matrotrophic, Poeciliopsis retropinna, and lecithotrophic, P. turrubarensis, species we found genes known to be critical for placenta function expressed in both species despite their difference in complexity. Additionally, when we compare the transcriptome of different river populations of P. retropinna, known to vary in maternal provisioning, we find differential expression of secretory genes expressed specifically in the top layer of villi cells in the maternal follicle. This provides some of the first evidence that the placental structures of Poeciliopsis function using a secretory mechanism rather than direct contact with maternal circulation. Finally, when we look at the expression of placenta proteins at the maternal-fetal interface of a larger sampling of Poeciliopsis species, we find expression of key maternal and fetal placenta proteins in their cognate tissue types of all species, but follicle expression of prolactin is restricted to only matrotrophic species. Taken together, we suggest that all Poeciliopsis follicles are poised for placenta function but require expression of key genes to form secretory villi.

First baseline for bioenergetic biomarkers in Cnesterodon decemmaculatus as test organism in ecotoxicological studies.

Cnesterodon decemmaculatus is a Neotropical teleost fish frequently used in ecotoxicological evaluations, whose biology has been thoroughly studied. Although there is considerable information on its response to different toxicants, no range of reference values has been so far established for the different biological parameters proposed as biomarkers of effect or exposure. Moreover, no study has yet examined the possible influence of the metabolic status of the exposed animals on their response to toxic stress. Therefore, the aim of this work was to provide a first baseline for a set of bioenergetic biomarkers in C. decemmaculatus adults exposed to a control medium under previously standardized conditions, and to assess their possible intrinsic seasonal variability. The responses of the biomarkers obtained from the controls were contrasted with those from the reference toxicant (Cadmio-Cd) and receiving waters (surface waters of the Reconquista River RR, Buenos Aires Province, Argentina). We conducted four 12-day assays (one in each season) of exposure to control media, (reconstituted moderate hard water, MHW) and two assays of exposure to Cd in MHW and surface river water (RR) in both summer and autumn. The variables recorded were: Food intake (In), fecal production (F), specific assimilation (A) and cumulative mortality, oxygen extraction efficiency (OEE), specific metabolic rate (SMR), ammonia excretion (N), ammonia quotient (AQ) and scope for growth (SFG). The seasonal variation shown by some physiological parameters, points to the need for establishing a baseline obtained from standardized media, preferably on a seasonal basis. Moreover, SFG and A appeared as the most sensitive biomarkers, emphasizing the importance to consider the metabolic status of the test organisms for the appropriate interpretation of results from ecotoxicological studies performed under controlled experimental conditions. The obtained results provide useful information on C. decemmaculatus as model species in ecotoxicological bioassays involving biomarkers of early effect.