Screening of temperature-responsive signalling molecules during sex differentiation in Asian yellow pond turtle (Mauremys mutica).

BACKGROUND: The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. This species is a highly sexually dimorphic species, with males growing at a faster rate than females and exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation in the Asian yellow pond turtle are remain a mystery. RESULTS: Temperature-specific gonadal transcriptomics of the Asian yellow pond turtle were performed during the thermosensitive period (stage 15) using RNA-seq technology to identify candidate genes that initiate gonadal differentiation. We uncovered candidates that were the first to respond to temperature. These candidates were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1). CONCLUSIONS: Our work will provide important genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.

Whole-Genome Identification and Characterization of the DKK Gene Family and Its Transcription Profiles: An Analysis of the Chinese Soft-Shell Turtle (Pelodiscus sinensis).

The DKK family is a canonical small family of WNT antagonists. Though recent studies have suggested that the DKK gene family may be involved in sex differentiation in Pelodiscus sinensis, there are still a lot of things about the DKK gene family that we do not know. In this study, we used bioinformatics methods to identify members of the DKK gene family in P. sinensis and analyzed their phylogeny, covariance, gene structure, structural domains, promoter conserved sites, signal peptides, gonadal transcription factors, transcriptional profiles, and tissue expression profiles. Additionally, qRT-PCR results were utilized for the validation and preliminary investigation of the function of the DKK gene family in P. sinensis. The results showed that the DKK gene family is divided into six subfamilies, distributed on six different chromosomal scaffolds containing different gene structures and conserved motifs with the same structural domains, and all of the members were secreted proteins. Our transcriptional profiling and embryonic expression analysis showed that DKKL1 and DKK4 were significantly expressed in the testes, whereas DKK1 and DKK3 were significantly upregulated in the ovaries. This suggests a potential function in sex differentiation in P. sinensis. Our results may provide a basic theoretical basis for the sex differentiation process in P. sinensis.

Identification of Sex-Specific Markers and Candidate Genes Using WGS Sequencing Reveals a ZW-Type Sex-Determination System in the Chinese Soft-Shell Turtle (Pelodiscus sinensis).

Male and female Chinese soft-shelled turtles (Pelodiscus sinensis) have sex-dimorphic growth patterns, and males have higher commercial value because of their larger size and thicker calipash. Thus, developing sex-specific markers is beneficial to studies on all-male breeding in P. sinensis. Here, we developed an accurate and efficient workflow for the screening of sex-specific sequences with ZW or XY sex determination systems. Based on this workflow, female and male P. sinensis reference genomes of 2.23 Gb and 2.26 Gb were obtained using de novo assembly. After aligning and filtering, 4.01 Mb female-specific sequences were finally identified. Subsequently, the seven developed sex-specific primer pairs were 100% accurate in preliminary, population, and embryonic validation. The presence and absence of bands for the primers of P44, P45, P66, P67, P68, and P69, as well as two and one bands for the PB1 primer, indicate that the embryos are genetically female and male, respectively. NR and functional annotations identified several sex-determining candidate genes and related pathways, including Ran, Eif4et, and Crkl genes, and the insulin signaling pathway and the cAMP signaling pathway, respectively. Collectively, our results reveal that a ZW-type sex-determination system is present in P. sinensis and provide novel insights for the screening of sex-specific markers, sex-control breeding, and the studies of the sex determination mechanism of P. sinensis.

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis).

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17ß-estradiol and 17α-methyltestosterone) and Wnt/ß-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/ß-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17ß-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/ß-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

A chromosome-level genome assembly of the Asian giant softshell turtle Pelochelys cantorii.

The Asian giant softshell turtle Pelochelys cantorii is one of the largest aquatic turtles in China and has been designated a First Grade Protected Animal in China. To advance conservation research, a combination of Illumina short-read, PacBio long-read, and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for P. cantorii. A total of 262.77 Gb of clean data were produced (121.6 × depth) and then the genome was assembled into 2.16 Gb with a contig N50 of 41.44 Mb and scaffold N50 length of 120.17 Mb, respectively. Moreover, about 99.98% assembly genome sequences were clustered and ordered onto 33 pseudochromosomes. Genome annotation revealed that 21,833 protein-coding genes were predicted, and 96.40% of them were annotated. This new chromosome-level assembly will be an enabling resource for genetic and genomic studies to support fundamental insight into P. cantorii biology.

Exploring the relationship between environmental DNA concentration and biomass in Asian giant softshell turtle (Pelochelys cantorii).

In recent years, environmental DNA (eDNA) technology has become an accepted approach for investigating rare and endangered species because of its economic efficiency, high sensitivity, and non-invasiveness. The Asian giant softshell turtle (Pelochelys cantorii) is a first-class protected aquatic animal in China, and traditional resource survey methods have not identified its natural populations for many years. In this study, primers and a TaqMan probe targeting ND5 were designed, reaction conditions were optimized, a standard curve was constructed using synthetic DNA, and an eDNA quantitative PCR (qPCR) detection method was established. The eDNA detection technology for P. cantorii revealed that the number of species in the experimental pools showed a significant linear relationship with the eDNA concentration (p < 0.05). The eDNA concentration was negatively correlated with the length of time after the removal of P. cantorii and retention in the water body for 9 days. The qPCR detection method for P. cantorii eDNA established in this study can be applied to the qualitative detection of P. cantorii in water bodies, as well as to preliminary evaluation of its relative biomass. This can serve as a baseline for the investigation of natural P. cantorii population and the evaluation of its wild release effects.

DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in Caenorhabditis elegans.

Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.

Chromosome-Level Analysis of the Pelochelys cantorii Genome Provides Insights to Its Immunity, Growth and Longevity.

The Asian giant soft-shelled turtle, Pelochelys cantorii (Trionychidae), is one of the largest aquatic turtles in China and was designated as a First-Grade Protected Animal in China in 1989. Previous investigation based on a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies acquired a high-quality chromosome-level genome of Pc. cantorii. In this study, comparative genomic analysis between Pc. cantorii and 16 other vertebrate genomes indicated that turtles separated from the ancestor of archosaurians approximately 256.6 (95% highest posterior density interval, 263.6-251.9) million years ago (Mya) (Upper Permian to Triassic) and that Pc. cantorii separated from the ancestor of Pd. sinensis and R. swinhoei approximately 59.3 (95% highest posterior density interval, 64.3-54.3) Mya. Moreover, several candidate genes, such as VWA5A, ABCG2, A2M and IGSF1, associated with tumor suppression, growth and age were expanded, implicating their potential roles in the exceptional longevity of turtles. This new chromosome-level assembly has important scientific value in the study of conservation of Pc. cantorii and also enriches the evolutionary investigation of turtle species.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2.

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Melanocortin-5 Receptor: Pharmacology and Its Regulation of Energy Metabolism.

As the most recent melanocortin receptor (MCR) identified, melanocortin-5 receptor (MC5R) has unique tissue expression patterns, pharmacological properties, and physiological functions. Different from the other four MCR subtypes, MC5R is widely distributed in both the central nervous system and peripheral tissues and is associated with multiple functions. MC5R in sebaceous and preputial glands regulates lipid production and sexual behavior, respectively. MC5R expressed in immune cells is involved in immunomodulation. Among the five MCRs, MC5R is the predominant subtype expressed in skeletal muscle and white adipose tissue, tissues critical for energy metabolism. Activated MC5R triggers lipid mobilization in adipocytes and glucose uptake in skeletal muscle. Therefore, MC5R is a potential target for treating patients with obesity and diabetes mellitus. Melanocortin-2 receptor accessory proteins can modulate the cell surface expression, dimerization, and pharmacology of MC5R. This minireview summarizes the molecular and pharmacological properties of MC5R and highlights the progress made on MC5R in energy metabolism. We poInt. out knowledge gaps that need to be explored in the future.

Regulation of melanocortin-1 receptor pharmacology by melanocortin receptor accessory protein 2 in orange-spotted grouper (Epinephelus coioides).

Melanocortin-1 receptor (MC1R) has important roles in regulating pigmentation and inflammation. Melanocortin receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of mammalian melanocortin receptors. However, the effect of MRAP2 on fish MC1R has not been extensively studied. Herein, we cloned the orange-spotted grouper (Epinephelus coioides) mc1r, which had a 972 bp open reading frame encoding a putative protein of 323 amino acids. Grouper mc1r was mainly expressed in the brain, skin, testis, spleen, head kidney, and kidney. EcoMC1R showed high constitutive activities in both Gs-cAMP and ERK1/2 pathways, which could be differentially modulated by grouper MRAP2 (EcoMRAP2). Three agonists, including α-melanocyte-stimulating hormone (MSH), ß-MSH, and ACTH, could bind to EcoMC1R and dose-dependently increase intracellular cAMP production. EcoMRAP2 had no effect on the IC50 in binding assay or EC50 in cAMP assay; however, it dose-dependently decreased the cell surface expression and maximal response to the three agonists. EcoMRAP2 increased basal ERK1/2 activation but did not alter α-MSH-stimulated ERK1/2 activation. This study extends the knowledge base of fish MC1R pharmacology and its regulation by MRAP2.

Comparative transcriptome analysis reveals the mechanism of ß-glucan in protecting rainbow trout (Oncorhynchus mykiss) from Aeromonas salmonicida infection.

To study the mechanism of ß-glucan in immune protection, rainbow trout were fed diets with or without 0.2% ß-glucan for 42 days and then infected with Aeromonas salmonicida. After that, spleen tissues were sampled on 4- and 6-days post infection (dpi). Transcriptome analysis was compared between control group (CG, without ß-glucan addition) and 0.2% ß-glucan group (BG). In CG vs BG, 378 and 406 DEGs were identified on 4 dpi and 6 dpi respectively; furthermore, 46 DEGs were shared on 4 dpi and 6 dpi, enriching in GO terms, such as complement activation, inflammatory response, and metabolic process. KEGG pathway analysis revealed that some DEGs in CG vs BG were involved in immune or metabolic signaling pathways such as complement and coagulation cascades, toll-like receptor signaling pathway, NF-κB signaling pathway, antigen processing and presentation, and platelet activation on 4 or 6 dpi. DEGs, such as fgg, fgb, f5, c9, c3, c5, tlr5, and myd88, were analyzed in CG vs BG on 4 dpi and 6 dpi, implying their potential roles in ß-glucan-modulated immunity. These results are beneficial to understand the mechanism of ß-glucan in resisting bacteria in fish.

Effect of flow velocity on the growth, stress and immune responses of turbot (Scophthalmus maximus) in recirculating aquaculture systems.

Land-based recirculating aquaculture systems (RAS) are widely utilized for turbot (Scophthalmus maximus) culture. Flow velocity in the tank is essential to maintain water quality, conservation of energy and fish welfare. However, little is known about how turbot respond to different velocities in the long term. In this study, water quality was kept constant, allowing the effect of flow velocity on the feeding intake, growth, plasma biochemical indexes, innate (non-specific) immunity and immune-related stress gene expressions in the skin to be examined in isolation in RAS. Turbot (average body length 20.10 cm) were reared for 60 days in RAS under three velocities, 0.06 m s-1, 0.18 m s-1, and 0.36 m s-1, corresponding to approximately 0.3 body length per second (bl s-1), 0.9 bl s-1 and 1.8 bl s-1, respectively. The results showed that at velocities of 0.36 m s-1 (1.8 bl s-1), juvenile turbot were subject to stress accompanied by a reduced growth rate. A velocity of 0.36 m s-1 was also found to significantly reduce SOD and GSH activity, and the concentration of total protein in plasma, while concentrations of urea nitrogen (BUN) and total bilirubin (TBIL) increased. There was an up-regulation of cathepsin D and lysozyme (LZM) in the skin at the highest velocity, implying the activation of stress and immune responses. At the medium velocity of 0.18 m s-1 (0.9 bl s-1), turbot increased their feed intake, obtained an elevated special growth rate (SGR), and exhibited significantly higher AKP and ACP activity in plasma. Overall, the results suggest that excessively high velocities are a stressor for turbot inducing an immune response in the skin, which is sensitive to environmental changes. A velocity of approximately 0.9 bl s-1 is suggested to promote growth and obtain better innate immunity of cultured turbot.

Effect of dietary ß-glucan on growth, survival and regulation of immune processes in rainbow trout (Oncorhynchus mykiss) infected by Aeromonas salmonicida.

The present study evaluated the effects of dietary ß-glucan (0, 0.05%, 0.1%, and 0.2%) on growth performance after 42 days of feeding. Thereafter, rainbow trout (Oncorhynchus mykiss) were infected with Aeromonas salmonicida, and survival rates as well as the regulating processes of stress- and immune-related factors were analyzed. In general, higher dietary ß-glucan levels obviously improved specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) (P ≤ 0.05). Survival rates in ß-glucan groups increased significantly compared with the control group after A. salmonicida infection (P ≤ 0.05). Serum total superoxide dimutase (T-SOD), peroxidase (POD) as well as catalase (CAT) activities, and their mRNA expressions in the head kidney of fish in the ß-glucan groups generally increased to higher levels after infection, and more quickly, compared with in the control group. Serum lysozyme (LSZ) and its expression in the head kidney in ß-glucan groups reached a higher peak earlier than in the control group. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in the ß-glucan groups were significantly lower than in the control group (P ≤ 0.05). The peak of heat shock protein 70 (HSP70) expression in the 0.2% ß-glucan group was higher and occurred earlier than in other groups (P ≤ 0.05). These results confirm that 0.1% and 0.2% dietary ß-glucan are beneficial for promoting growth in rainbow trout and enhancing resistance against A. salmonicida. Furthermore, ß-glucan could play an important role in regulating stress- and immune-related factors in rainbow trout to more quickly fight against bacterial infection.

MiR-136 contributes to pre-eclampsia through its effects on apoptosis and angiogenesis of mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) play an important role in the pathology of preeclampsia (PE). The survival of MSCs and angiogenesis in the maternal-fetal interface are important for a successful pregnancy. MicroRNA-136 (miR-136) is highly expressed in decidua-derived MSCs (MSCs) from PE compared with healthy donors (NC). The role of the MSCs aberrant expressed miR-136 in PE development is still unclear. In the present study, we examined the impact of miR-136 on the survival of MSCs and angiogenesis in the maternal-fetal interface. METHODS: MSCs were extracted and transfected with miR-136 mimic and interfering RNAs using lipofectamine-2000. Then cell apoptosis were tested using flow cytometry. HUVEC tube formation ability was tested on Matrigel co-cultured with conditioned MSCs supernatants. RESULTS: High level of miR-136 could suppress cell proliferation and promote apoptosis of MSCs through targeting BCL2. It could also impairs HUVEC capillary formation by suppressing VEGF. DISCUSSION: MiR-136 significantly increase the apoptosis and suppress the proliferation of MSCs. It could also inhibit the capillary formation and trophoblast cell invasion. These data suggest that decidua-derived miR-136 that is increased in PE is a potential causal factor of PE.

Genetic polymorphisms and DNA methylation in exon 1 CpG-rich regions of PACAP gene and its effect on mRNA expression and growth traits in half smooth tongue sole (Cynoglossus semilaevis).

The pituitary adenylate cyclase activating polypeptide (PACAP) is a new type of hypophysiotropic hormone and plays an important role in regulating the synthesis and secretion of growth hormone and gonadotropin. The research on the relationship between PACAP and different growth traits would contribute to explain its function during the process of growth. Moreover, epigenetic modifications, especially DNA methylation at the CpG sites of the SNPs, play important roles in regulating gene expression. The results suggest that a SNP mutation (c.C151G) in the PACAP gene of male half smooth tongue sole (Cynoglossus semilaevis) is significantly associated with growth traits and serum physiological and biochemical parameters such as inorganic phosphorus (P < 0.05). The SNP is located in a CpG-rich region of exon 1. Intriguingly, the transition (C→G) added a new methylation site of PACAP gene. This SNP was also significantly related to the expression and methylation level of PACAP (P < 0.05). Individuals with GG genotype had faster growth rates than those of CG and CC genotypes. Moreover, GG genotype had significantly higher PACAP expression level and lower methylation level than CG and CC genotypes. In the serum indexes, only inorganic phosphorus content within GG genotypes was significantly higher than CC genotypes. This implied that the mutation and methylation status of PACAP gene could influence growth traits and this locus could be considered as a candidate genetic or epigenetic marker for Cynoglossus semilaevis molecular breeding.

Development of carbon nanotubes/CoFe2O4 magnetic hybrid material for removal of tetrabromobisphenol A and Pb(II).

Multi-walled carbon nanotubes (MWCNTs) coated with magnetic amino-modified CoFe2O4 (CoFe2O4-NH2) nanoparticles (denoted as MNP) were prepared via a simple one-pot polyol method. The MNP composite was further modified with chitosan (CTS) to obtain a chitosan-functionalized MWCNT/CoFe2O4-NH2 hybrid material (MNP-CTS). The obtained hybrid materials were characterized by Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectrogram (FT-IR) Analysis and X-ray Photoelectron Spectroscopy (XPS) Analysis, Vibrating Sample Magnetometer (VSM) Analysis and the Brunauer-Emmett-Teller (BET) surface area method, respectively. The composites were tested as adsorbents for tetrabromobisphenol A (TBBPA) and Pb(II), and were investigated using a pseudo-second-order model. The adsorption of TBBPA was well represented by the Freundlich isotherm; the Langmuir model better described Pb(II) absorption. MNP-CTS adsorbed both TBBPA and Pb(II) (maximum adsorption capacities of 42.48 and 140.1mgg(-1), respectively) better than did MNP without CTS. Magnetic composite particles with adsorbed TBBPA and Pb(II) could be regenerated using 0.2M NaOH solution and were separable from liquid media using a magnetic field.