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.

Sex-specific metabolic dysregulation in digestive glands of green mussels following exposure to triazophos.

As a ubiquitous environmental pollutant in China, triazophos (TP) is known to have neurotoxicity, oxidative stress, and reproductive toxicity to mussels. To investigate the molecular mechanisms of TP toxicity, metabolic changes in the digestive glands of Perna viridis in different sexes were examined after treated with 35 µg/L TP. Notably, 158 significant different metabolites (SDMs) were detected in TP-treated mussels and more than half of the SDMs were lipids and lipid-like molecules, which suggested that TP disturbed the lipid metabolism of P. viridis. In addition, metabolites associated with neurotoxicity and reproductive disturbance were also detected in female and male mussels. Moreover, a larger number of SDMs were found in male mussels (120 SDMs) than females (99 SDMs), and 60 common metabolites exhibited consistent variation tendency and similar magnitude in both sexes. The metabolic alternations in female and male mussels displayed similar protective mechanisms and also sex-specific responses, male mussels were more sensitive to TP exposure. This research provided new data about the molecular mechanisms of TP toxicity and the gender specific changes in mussels after treated by chemicals.

Transcriptional response of four C1q domain containing protein (C1qDC) genes from Venerupis philippinarum exposed to the water soluble fraction of No.0 diesel oil.

As pattern recognitionreceptors, the C1q-domain-containing (C1qDC) proteins play an important role in the pathogen recognition and complement pathway activation. In the present study, four novel C1q domain containing proteins (designated as VpC1qDC1, VpC1qDC2, VpC1qDC3 and VpC1qDC4) were cloned and characterized from clam Venerupis philippinarum. The four VpC1qDCs all possessed the conserved features critical for the fundamental structure and function of the C1q family. The four VpC1qDCs genes showed differential response profiles after exposure to the water soluble fraction of No.0 diesel oil (WSFD). More notably, VpC1qDC1 and VpC1qDC3 were more sensitive to low concentration of WSFD, as their mRNA level changed by higher magnitudes. In addition, VpC1qDC2 and VpC1qDC4 displayed notable increases with larger amplitude to high concentration of WSFD. All these results suggested that the transcriptional response of VpC1qDCs genes were probably a protective mechanism of the cell to oils pollution. The diverse expression patterns of VpC1qDCs demonstrated that VpC1qDC1 and VpC1qDC3 were sensitive responders to environmental stress in V. philippinarum.

Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and Metoprolol.

ß-adrenergic receptor blockers (ß-blockers) are widely detected in the aquatic environment; however, the effects of these pharmaceuticals on aquatic organisms remain uncertain. In this study, adult zebrafish were exposed to two different ß-blockers, propranolol and metoprolol, for 96 h. After exposure, the transcriptional responses of genes encoding the ß-adrenergic receptor (i.e., adrb1, adrb2a, adrb2b, adrb3a and adrb3b), genes involved in detoxification and the stress response (i.e., hsp70, tap, mt1 and mt2), and genes related to the antioxidant system (i.e., cu/zn-sod, mn-sod, cat and gpx) were examined in the brain, liver and gonad. Our results show that both propranolol and metoprolol exposure changes the mRNA level of ß-adrenergic receptors, indicating clear pharmacological target engagement of the ß-blockers. The transcription of genes related to antioxidant responses and detoxification process were induced, suggesting that ß-blocker exposure can activate the detoxification process and result in oxidative stress in fish. Moreover, the transcriptional responses displayed substantial tissue- and gender-specific effects. Considering the environmental concentrations of propranolol and metoprolol, these results suggest that these pharmaceuticals are unlikely to pose a risk to fish. However, the impacts in prolonged exposure, along with other possible side effects due to ß-adrenergic receptor blockade, should be further assessed.

Novel ortho ester-based, pH-sensitive cationic lipid for gene delivery in vitro and in vivo.

CONTEXT: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower. OBJECTIVE: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo. MATERIALS AND METHODS: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control. RESULTS: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes. DISCUSSION AND CONCLUSION: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.

Distribution and typologies of anthropogenic seafloor litter in the Pearl River Estuary and adjacent coastal waters, China.

Marine litter pollution poses a significant threat to offshore ecosystems, eliciting widespread concern. We investigated seafloor litter patterns in the Pearl River Estuary and adjacent coastal waters of China in 2023 via bottom trawl survey. Average number and weight densities were found to be 154.34 ± 30.95 n/km2 and 2384.63 ± 923.98 g/km2, respectively. Plastic was the most abundant material by number density (79.07 %), and rubber the highest by weight density (22.93 %). Overall number density varied from 40.50 ± 22.50 to 221.13 ± 52.44 n/km2, with the highest in Daya Bay and the lowest in Guanghai Bay. Weight density varied from 189.93 ± 71.94 to 5386.70 ± 3050.30 g/km2, with the highest in Qiao Island and the lowest in Honghai Bay. The main source was plastic bags and wrappers. The Pearl River Delta and Daya Bay were identified as seafloor litter distribution hotspots. Controlling plastic waste input is crucial for reducing seafloor litter in the Pearl River Estuary.

Dynamic impacts of short-term bath administration of enrofloxacin on juvenile black seabream Acanthopagrus schlegelii.

Dynamic impacts of short-term enrofloxacin (ENR) exposure on juvenile marine fish are not well understood, and the underlying mechanisms remain unclear. We therefore investigated the accumulation and elimination of ENR in the liver of juvenile black seabream Acanthopagrus schlegelii. Meanwhile, the dynamic alterations of biochemical parameters and liver transcriptomes after short-term bath immersion and withdrawal treatment were explored. The results indicated that the contents of ENR in the liver were significantly increased after bath administration for 24 h, and then quickly declined to very low concentrations along with the decontamination time increasing. Judging from the changes in biochemical indicators and liver transcriptomic alterations, 0.5 and 1 mg/L ENR exposure for 24 h triggered oxidative stress, impairment of immune system, as well as aberrant lipid metabolism via differential molecular pathways. Interestingly, biochemical and transcriptome analysis as well as integrated biomarker response (IBR) values showed that more significant changes appeared in 1 mg/L ENR group at decontamination periods, which indicated that the impact of high dose ENR on juvenile A. schlegelii may persist even after depuration for 7 days. These results revealed that the risk of short-term bath of 1 mg/L ENR should not be overlooked even after depuration period. Therefore, attention should be paid to the dosage control when administering the drug to juvenile A. schlegelii, and the restoration of physiological disturbance may be an important factor in formulating a reasonable treatment plan.

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.

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.

Differential response of two ferritin subunit genes (VpFer1 and VpFer2) from Venerupis philippinarum following pathogen and heavy metals challenge.

As a principal extracellular iron storage molecule, ferritin plays an important role in the iron-withholding strategy of innate immunity and detoxification system. In this study, we cloned and characterized another ferritin from Venerupis philippinarum (designated as VpFer2), in addition to one previously reported (VpFer1). VpFer2 possessed all the conserved features critical for the fundamental structure and function of ferritin H subunit. VpFer1 and VpFer2 mRNA were both found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpFers, and VpFer2 showed more sensitive to Vibrio anguillarum infection. For heavy metals exposure, the expression level of VpFer1 was significantly induced by Cd at 48 h, but kept relatively constant after exposure to Cu. With regards to VpFer2, the expression level dropped significantly at 24 h, then began to increase to the peak value at 48 h under Cd exposure, while Cu exposure constantly depressed the expression level of VpFer2 throughout the time course. Similarly, VpFer2 seemed to be more sensitive to heavy metals exposure than VpFer1 as its mRNA level changed by higher magnitudes. All these results suggested that VpFers may be important proteins involved in host immune defense and heavy metals detoxification. The diverse expression patterns of VpFers demonstrated that VpFer2 was an early and sensitive responder to environmental stress in V. philippinarum.

Source apportionment of nitrate in the Pearl River Estuary using δ15N and δ18O values and isotope mixing model.

The mitigation of eutrophication in the Pearl River Estuary (PRE) has encountered numerous challenges in regards to source control. Herein, the isotope mixing model (SIAR) was used to quantify the primary nitrate sources in the PRE. The results showed that the nitrate levels were significantly higher in the high-flow season than in the low-flow season. Meanwhile, we found the most important nitrate sources were manure and sewage during the high-flow season, with a contribution ratio of 47 % in the low salt area (LSA) and 29 % in the high salt area (HSA). During the low-flow season, the primary nitrate sources were identified as reduced nitrogen fertilizer in the LSA and manure and sewage in the HSA, which accounted for 52 % and 44 %, respectively. Furthermore, we also suggest that a feasible measure might be to control the pollution caused in the PRE by manure and sewage as well as reduced nitrogen fertilizer.

Genome-wide identification, evolution and expression analysis of bone morphogenetic protein (BMP) gene family in chinese soft-shell turtle (Pelodiscus sinensis).

Introduction: Bone morphogenetic proteins (BMPs) play a crucial role in bone formation and differentiation. Recent RNA-Seq results suggest that BMPs may be involved in the sex differentiation of P. sinensis, yet more relevant studies about BMPs in P. sinensis are lacking. Methods: Herein, we identified BMP gene family members, analyzed the phylogeny, collinear relationship, scaffold localization, gene structures, protein structures, transcription factors and dimorphic expression by using bioinformatic methods based on genomic and transcriptomic data of P. sinensis. Meanwhile, qRT-PCR was used to verify the RNA-Seq results and initially explore the function of the BMPs in the sex differentiation of P. sinensis. Results: A total of 11 BMP genes were identified, 10 of which were localized to their respective genomic scaffolds. Phylogenetic analysis revealed that BMP genes were divided into eight subfamilies and shared similar motifs ("WII", "FPL", "TNHA", "CCVP", and "CGC") and domain (TGF-ß superfamily). The results of the sexually dimorphic expression profile and qRT-PCR showed that Bmp2, Bmp3, Bmp15l, Bmp5, Bmp6 and Bmp8a were significantly upregulated in ovaries, while Bmp2lb, Bmp7, Bmp2bl and Bmp10 were remarkable upregulated in testes, suggesting that these genes may play a role in sex differentiation of P. sinensis. Discussion: Collectively, our comprehensive results enrich the basic date for studying the evolution and functions of BMP genes in P. sinensis.

Transcriptome analysis reveals sex-specific alterations in gonads of green mussel exposed to organophosphorus insecticide triazophos.

Triazophos (TP) is a widespread pollutant in aquatic environments. A sex-specific metabolic response in green-lipped mussel Perna viridis to TP exposure was observed in our previous study, and this led us to investigate the mechanisms associated with its toxicity. P. viridis were subjected to chronic exposure (15 days) to TP at 35 µg/L to compare the sex-biased transcriptomic profiles in the gonads of male and female mussels. We identified 632 differentially expressed genes (DEGs) (348 up-regulated and 284 down-regulated) in TP-exposed males, and only 61 DEGs (9 up-regulated and 52 down-regulated) in TP-exposed females. Many DEGs were found to be involved in the nervous, reproductive endocrine, oxidative stress, and immune systems of P. viridis. Additionally, enzymatic activity analysis indicated TP induced neurotoxic effects and oxidative damage to the mussels. Our results demonstrate that the stress response and molecular mechanisms of TP toxicology are different between female and male mussels.

Oyster arsenic, cadmium, copper, mercury, lead and zinc levels in the northern South China Sea: long-term spatiotemporal distributions, combined effects, and risk assessment to human health.

Estuarine and coastal ecosystems are often considered vulnerable due to the complex biogeochemical processes and the human disturbances through a variety of pollution. Among environmental contaminants, heavy metals in estuarine and coastal ecosystems have been of increasing concern in environmental conservation. Long-term exposure to heavy metal contamination, mainly through food and water, could be harmful to human health. It is therefore critical to understand the quantitative comparisons and combined effects of different heavy metals in common seafood species, such as oysters. This work studied the long-term spatiotemporal trends and health risk assessment of oyster arsenic (As), cadmium (Cd), copper (Cu), mercury (Hg), lead (Pb), and zinc (Zn) levels in the coastal waters of northern South China Sea. Cultured oysters (Crassostrea rivularis) from 23 estuaries and harbors in the coastal areas of northern South China Sea in 1989-2015 were analyzed for the spatiotemporal trends of the six heavy metal levels. Metal pollution index (MPI), target hazard quotient (THQ), and hazard index (HI) were used for quantifying the exposure of the six heavy metals to human health through oyster consumption. Principal component analysis (PCA) was used for assessing the relative importance of the six metals in oyster heavy metal distribution patterns in the northern South China Sea. Overall, the As, Cd, Cu, Hg, Pb, and Zn levels in oysters from the northern South China Sea generally declined from 1989 to 2015, stayed relatively high (MPI = 2.42-3.68) during 1989-2000, gradually decreased since 2000, and slightly increased after 2010. Oyster heavy metal levels were highest in the Pearl River Estuary (MPI = 1.20-5.52), followed by west Guangdong and east Guangdong, Guangxi, and Hainan coastal waters. This pattern is probably because economics and industry around the Pearl River Estuary have been growing faster than the other areas of this work in the recent two decades, and it should be taken as a hotspot for the monitoring of seafood safety in southern China. Principal component analysis indicated that Cu, Zn, and Cd were the most important metals in the long-term distributions of oyster heavy metal levels in the northern South China Sea. Health risk assessment suggested that the risk of the six heavy metals exposure through oyster consumption were relatively high during 1989-2005 (THQ = 1.01-5.82), significantly decreased since 2005 (THQ < 1), and slightly increased after 2010.

Identification and functional analysis of miRNAs in skeletal muscle of juvenile and adult largemouth bass, Micropterus salmoides.

MicroRNAs (miRNAs) are considered key regulators to post-transcriptionally regulate gene expression affecting multiple biological activities. However, the developmental process of fish skeletal muscles is regulated by complicated molecular mechanism that has not been completely well-described. In this study, two small RNAs libraries from skeletal muscle of juvenile as well as adult largemouth bass (LMB) were obtained and sequenced using deep sequencing to investigate the development-related miRNAs. We identified an overall number of 486 already recognized miRNAs in addition to 43 novel miRNAs. Comparison of two different skeletal muscle development stages led to the identification of 220 differently expressed miRNAs between juvenile and adult LMB containing 116 up-regulated as well as 104 down-regulated miRNAs. Of them, confirmation of some differently expressed miRNAs was performed via a stem-loop qRT-PCR, which exhibited differently expressed level in juvenile and adult LMB. Furthermore, GO and KEGG enrichment analyses of targets of differently-expressed miRNAs were carried out. Additionally, the analysis of miRNAs-targets interaction network showed that miR-181b-5p_R-1, miR-725 and miR-103 as the nodal miRNAs has over 20 target genes. Moreover, miR-103 could bind the 3'-UTR of actr8, which was validated via dual-luciferase reporter assay. It has been reasonably hypothesized that miR-103 may play a crucial role, which regulate skeletal muscle development of LMB. The present study provides the first identification of miRNA expression profiles at two different skeletal muscle development stages in LMB. Results may be valuable in interpreting the regulatory role miRNAs plays in the growth and developmental process of skeletal muscle and its possible use in LMB breeding.

Tetrabromobisphenol A and hexabromocyclododecanes in sediments from fishing ports along the coast of South China: Occurrence, distribution and ecological risk.

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecanes (HBCDDs) have attracted extensive attention due to their strong persistence and toxicity. However, little has been known about their pollution status in fishing ports, which are typical sinks of land-sourced pollutants. In this study, we investigated the occurrence, distribution and ecological risk of TBBPA and HBCDDs in sediments from fishing ports along the coast of South China. The concentrations of TBBPA and ΣHBCDD (sum of α-, ß-, and γ-HBCDD) in the fishing-port sediments were in the ranges of 0.02-21.5 ng/g dw and 1.06-14.1 ng/g dw, respectively. γ-HBCDD was the predominant diastereoisomer in most fishing-port sediments. The enantiomeric analysis indicated a preferential enrichment of (-)-enantiomers for α-, ß-, and γ-HBCDD. The geographical location of fishing ports is a significant determinant of distribution for TBBPA and HBBCDs. The concentrations of TBBPA and HBCDDs in fishing-port sediments were strongly associated with local population density, but weakly correlated with total organic carbon content of the sediment. The mass inventories of TBBPA and ΣHBCDD were estimated to be 77.0 ng/cm2 and 141 ng/cm2, respectively. The ecological risk assessment demonstrated that TBBPA and HBCDDs in fishing-port sediments exhibited low risks to marine organisms. This study contributes to the understanding pollution situation of fishing ports, and provides a reference for environmental safety assessment and environmental pollution control.

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To understand the population characteristics of Collichthys lucidus, an important economic fish in the Pearl River Estuary, the biological characteristics and resource density distribution characteristics of C. lucidus were preliminarily analyzed using bottom trawling by cruises conducted in each spring and autumn during 2017 and 2020. The results showed that the body length and weight of C. lucidus ranged between 22-168 mm and 0.23-103.11 g, respectively. Female individuals were larger than the male ones. The length of sexually mature individuals intensively ranged between 90 mm and 140 mm. Neither of them evidenced the earlier of sexually maturity nor the minimizer of dominant group. The population of C. lucidus in Pearl River estuary still developed in safe status in all, but its habitat downgraded than in 1988, as indicated by the fact that the allometric growth factor (b=2.9057) of the body length to body weight had no significant annual variations, but the conditional factor (a=3.029×10-5) was drama-tically decreased than in 1988. The population was at a state of overexploitation due to the estimated exploitation rate of 0.67. The resource density averaged 77.73 kg·km-2, showing a pattern of higher in the middle and west than in the east and relatively uniform of latitudinal distribution. The four high densities of sampling zones suggested that the zone around Nansha Port was probably the core of spawning ground of C. lucidus. Considering the annual average resource density in 2017-2020 sharply decreased by 93.5% than in 1980 to 1982, it was pressing to establish the protection zone in spawning ground in spring to protect the recruiting and spawning stocks of C. lucidus population.

Chromosome-level genome assembly of Asian yellow pond turtle (Mauremys mutica) with temperature-dependent sex determination system.

Knowledge of sex determination has important implications in physiology, ecology and genetics, but the evolutionary mechanisms of sex determination systems in turtles have not been fully elucidated, due to a lack of reference genomes. Here, we generate a high-quality genome assembly of Asian yellow pond turtle (Mauremys mutica) using continuous long-read (PacBio platform), Illumina, and high-throughput chromatin conformation capture (Hi-C) technologies. The M. mutica haplotype has a genome size of 2.23 Gb with a contig N50 of 8.53 Mb and scaffold N50 of 141.98 Mb. 99.98% sequences of the total assembly are anchored to 26 pseudochromosomes. Comparative genomics analysis indicated that the lizard-snake-tuatara clade diverged from the bird-crocodilian-turtle clade at approximately 267.0-312.3 Mya. Intriguingly, positive selected genes are mostly enriched in the calcium signaling pathway and neuroactive ligand-receptor interaction, which are involved in the process of temperature-dependent sex determination. These findings provide important evolutionary insights into temperature-dependent sex determination system.

Whole-Transcriptome Analysis Identifies Gender Dimorphic Expressions of Mrnas and Non-Coding Rnas in Chinese Soft-Shell Turtle (Pelodiscus sinensis).

In aquaculture, the Chinese soft-shelled turtle (Pelodiscus sinensis) is an economically important species with remarkable gender dimorphism in its growth patterns. However, the underlying molecular mechanisms of this phenomenon have not been elucidated well. Here, we conducted a whole-transcriptome analysis of the female and male gonads of P. sinensis. Overall, 7833 DE mRNAs, 619 DE lncRNAs, 231 DE circRNAs, and 520 DE miRNAs were identified. Some "star genes" associated with sex differentiation containing dmrt1, sox9, and foxl2 were identified. Additionally, some potential genes linked to sex differentiation, such as bmp2, ran, and sox3, were also isolated in P. sinensis. Functional analysis showed that the DE miRNAs and DE ncRNAs were enriched in the pathways related to sex differentiation, including ovarian steroidogenesis, the hippo signaling pathway, and the calcium signaling pathway. Remarkably, a lncRNA/circRNA-miRNA-mRNA interaction network was constructed, containing the key genes associated with sex differentiation, including fgf9, foxl3, and dmrta2. Collectively, we constructed a gender dimorphism profile of the female and male gonads of P. sinensis, profoundly contributing to the exploration of the major genes and potential ncRNAs involved in the sex differentiation of P. sinensis. More importantly, we highlighted the potential functions of ncRNAs for gene regulation during sex differentiation in P. sinensis as well as in other turtles.

Expression and Characterization of the Spats1 Gene and Its Response to E2/MT Treatment in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

Spats1 (spermatogenesis-associated, serinerich 1) has been characterized as a male-biased gene which acts an important role in the germ cell differentiation of mammals. Nevertheless, the function of Spats1 in the Chinese soft-shelled turtle (P. sinensis) has not yet been reported. To initially explore the expression of Spats1 in P. sinensis and its response to sex steroid treatment, we cloned the CDS of Spats1 for the first time and analyzed its expression profile in different tissues, including the testes in different seasons. The Spats1 cDNA fragment is 1201 base pairs (bp) in length and contains an open reading frame (ORF) of 849 bp, which codes for 283 amino acids. Spats1 mRNA was highly expressed in the testes (p < 0.01) and barely detectable in other tissues. In P. sinensis, the relative expression of Spats1 also responsive to seasonal changes in testis development. In summer (July) and autumn (October), Spats1 gene expression was significantly higher in the testes than in other seasons (p < 0.05). Spats1 mRNA was found to be specifically expressed in germ cells by chemical in situ hybridization (CISH), and it was mainly located in primary spermatocytes (Sc1), secondary spermatocytes (Sc2) and spermatozoa (St). Spats1 expression in embryos was not significantly changed after 17α-methyltestosterone (MT)and 17ß-estradiol (E2) treatment. In adults, MT significantly induced Spats1 expression in male P. sinensis. However, the expression of Spats1 in testes was not responsive to E2 treatment. In addition, the expression of Spats1 in females was not affected by either MT or E2 treatment. These results imply that Spats1 is a male-specific expressed gene that is mainly regulated by MT and is closely linked to spermatogenesis and release in P. sinensis.