Dynamic transcriptome analysis reveals the gene network of gonadal development from the early history life stages in dwarf surfclam Mulinia lateralis.

BACKGROUND: Gonadal development is driven by a complex genetic cascade in vertebrates. However, related information remains limited in molluscs owing to the long generation time and the difficulty in maintaining whole life cycle in the lab. The dwarf surfclam Mulinia lateralis is considered an ideal bivalve model due to the short generation time and ease to breed in the lab. RESULTS: To gain a comprehensive understanding of gonadal development in M. lateralis, we conducted a combined morphological and molecular analysis on the gonads of 30 to 60 dpf. Morphological analysis showed that gonad formation and sex differentiation occur at 35 and 40-45 dpf, respectively; then the gonads go through gametogenic cycle. Gene co-expression network analysis on 40 transcriptomes of 35-60 dpf gonads identifies seven gonadal development-related modules, including two gonad-forming modules (M6, M7), three sex-specific modules (M14, M12, M11), and two sexually shared modules (M15, M13). The modules participate in different biological processes, such as cell communication, glycan biosynthesis, cell cycle, and ribosome biogenesis. Several hub transcription factors including SOX2, FOXZ, HSFY, FOXL2 and HES1 are identified. The expression of top hub genes from sex-specific modules suggests molecular sex differentiation (35 dpf) occurs earlier than morphological sex differentiation (40-45 dpf). CONCLUSION: This study provides a deep insight into the molecular basis of gonad formation, sex differentiation and gametogenesis in M. lateralis, which will contribute to a comprehensive understanding of the reproductive regulation network in molluscs.

Ancient homomorphy of molluscan sex chromosomes sustained by reversible sex-biased genes and sex determiner translocation.

Contrary to classic theory prediction, sex-chromosome homomorphy is prevalent in the animal kingdom but it is unclear how ancient homomorphic sex chromosomes avoid chromosome-scale degeneration. Molluscs constitute the second largest, Precambrian-originated animal phylum and have ancient, uncharacterized homomorphic sex chromosomes. Here, we profile eight genomes of the bivalve mollusc family of Pectinidae in a phylogenetic context and show 350 million years sex-chromosome homomorphy, which is the oldest known sex-chromosome homomorphy in the animal kingdom, far exceeding the ages of well-known heteromorphic sex chromosomes such as 130-200 million years in mammals, birds and flies. The long-term undifferentiation of molluscan sex chromosomes is potentially sustained by the unexpected intertwined regulation of reversible sex-biased genes, together with the lack of sexual dimorphism and occasional sex chromosome turnover. The pleiotropic constraint of regulation of reversible sex-biased genes is widely present in ancient homomorphic sex chromosomes and might be resolved in heteromorphic sex chromosomes through gene duplication followed by subfunctionalization. The evolutionary dynamics of sex chromosomes suggest a mechanism for 'inheritance' turnover of sex-determining genes that is mediated by translocation of a sex-determining enhancer. On the basis of these findings, we propose an evolutionary model for the long-term preservation of homomorphic sex chromosomes.

Discovery of Nanos1 and Nanos2/3 as Germ Cell Markers During Scallop Gonadal Development.

Nanos are conserved genes involved in germline cell specification and differentiation. However, little is known about the role of different members of Nanos family in germ cell development in mollusks. In the present study, we conducted genome-wide identification of Nanos family in an economically important scallop Patinopecten yessoensis, and detected their expression in adult tissues and during early development. Two Nanos genes (PyNanos1, PyNanos2/3) were identified, both of which have the N-terminal NOT1-interacting motif and C-terminal (CCHC)2 zinc finger domain. Expression profiles showed that PyNanos1 and PyNanos2/3 were primarily expressed in the gonads, with PyNanos1 being localized in the oogonia, oocytes, and spermatogonia, while PyNanos2/3 being specifically in spermatogonia. The results suggest that PyNanos are germ cell specific and may play crucial roles in gametogenesis in the scallop. PyNanos1 is a maternal gene, which is distributed uniformly at early cleavage, and restricted to 2-3 cell clusters from blastulae to trochophore larvae, suggesting its potential role in the formation of PGCs. Zygotically expressed PyNanos2/3 displayed a similar signal with PyNanos1 in the trochophore larvae, suggesting it may also participate in the formation and/or maintenance of PGCs. This study will benefit germplasm exploitation and conservation in bivalves, and facilitate a better understanding of the evolution of Nanos family and the role of different Nanos in germ cell development in mollusks.

Sexual Development of the Hermaphroditic Scallop Argopecten irradians Revealed by Morphological, Endocrine and Molecular Analysis.

Simultaneous or functional hermaphrodites possessing both ovary and testis at the same time are good materials for studying sexual development. However, previous research on sex determination and differentiation was mainly conducted in gonochoristic species and studies on simultaneous hermaphrodites are still limited. In this study, we conducted a combined morphological, endocrine and molecular study on the gonadal development of a hermaphroditic scallop Argopecten irradians aged 2-10 month old. Morphological analysis showed that sex differentiation occurred at 6 months of age. By examining the dynamic changes of progesterone, testosterone and estradiol, we found testosterone and estradiol were significantly different between the ovaries and testes almost throughout the whole process, suggesting the two hormones may be involved in scallop sex differentiation. In addition, we identified two critical sex-related genes FoxL2 and Dmrt1L, and investigated their spatiotemporal expression patterns. Results showed that FoxL2 and Dmrt1L were female- and male-biased, respectively, and mainly localized in the germ cells and follicular cells, indicating their feasibility as molecular markers for early identification of sex. Further analysis on the changes of FoxL2 and Dmrt1L expression in juveniles showed that significant sexual dimorphic expression of FoxL2 occurred at 2 months of age, earlier than that of Dmrt1L. Moreover, FoxL2 expression was significantly correlated with estradiol/testosterone ratio (E2/T). All these results indicated that molecular sex differentiation occurs earlier than morphological sex differentiation, and FoxL2 may be a key driver that functions through regulating sex steroid hormones in the scallop. This study will deepen our understanding of the molecular mechanism underlying sex differentiation and development in spiralians.

Expression of the Testis-Specific Serine/Threonine Kinases Suggests Their Role in Spermiogenesis of Bay Scallop Argopecten irradians.

Members of the testis-specific serine/threonine kinases (Tssk) family play critical roles in spermatogenesis in vertebrates. But in mollusks, research on Tssk family is still lagging. In this study, we systematically identified Tssk family based on the genomic and transcriptomic data from a commercially important scallop Argopecten irradians and detected the spatiotemporal expression in adult gonads. Five members were identified, with the gene length varying from 1,068 to 10,729 bp and the protein length ranging from 294 to 731 aa. All the Tssks possess a serine/threonine protein kinase catalytic (S_TKc) domain. Phylogenetic analysis revealed existence of four homologs of vertebrate Tssk1/2, Tssk3, Tssk4, Tssk5, and absence of Tssk6 in the scallop. The remaining gene (Tssk7) formed an independent clade with Tssks of other mollusks and arthropods, indicating that it may be a new member of Tssk family unique to protostomes. By investigating the expression of Tssks in four developmental stages of testes and ovaries, we found all five Tssks were primarily expressed in mature testis. In situ hybridization experiment revealed the five Tssks were localized in the spermatids and spermatozoa. The testis-predominant expression of Tssk family suggests Tssks may play pivotal roles in spermiogenesis in the scallop. Our study provides basic information on the characteristics and expression profiles of Tssk family of A. irradians. To our knowledge, it represents the first comprehensive analysis of Tssk family in mollusks.

The Effect of Temperature on Gonadal Sex Differentiation of Yesso Scallop Patinopecten yessoensis.

Many marine organisms are generally poikilotherms, making seawater temperature one of the most important environmental factors affecting gonadal sex differentiation. Mollusca is the second-largest animal phylum with diverse reproductive systems, but studies on the impact of temperature on sex differentiation are limited to a few sequential hermaphrodites. By combining morphological and molecular analyses, we investigated the effect of temperature on gonadal sex differentiation of a commercially important gonochoristic scallop Patinopecten yessoensis in the field and under laboratory conditions. Based on the relative expression of FoxL2 and Dmrt1L in the gonads of 6- to 12 month-old scallops, we found the scallops start to differentiate at 7 months old in September when the seawater temperature was 21°C. To eliminate the effect of factors other than temperature on sex differentiation, we compared the gonadal development of juvenile scallops at different temperatures (21, 16 and 11°C) under laboratory conditions. After 50 days of treatment, the 11°C group contain more germ cell types, and have higher sex differentiation rates than the 21°C group. But no obvious sex bias was observed. These results suggest that high temperature (21°C) inhibits sex differentiation, whereas low temperature (11°C) accelerates sex differentiation by 2 months for this cold-water species. It also supports juvenile P. yessoensis is gonochoristic rather than protandrous hermaphroditic. Our study addresses for the first time an environmental influence associated with genetic controls on scallop sex differentiation. It will facilitate a better understanding of how environmental factors affect gonadal development in poikilotherms, especially in the less studied molluscs.

Potential GnRH and steroidogenesis pathways in the scallop Patinopecten yessoensis.

Gonadotropin-releasing hormone (GnRH) controls synthesis of sex steroid hormones through hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. But in mollusks, research on GnRH and steroidogenesis pathways is still limited. In this study, we first identified two gonadotropin receptor like genes (LGR and LGR5L) and four steroidogenesis-related genes (CYP17A, HSD17B12, HSD3B1 and HSD3B2) in the scallop Patinopecten yessoensis. By examining the expression of 11 genes in the ganglia and/or gonad as well as the concentration of progesterone, testosterone and estradiol in the gonad, we postulate that a potential GnRH signaling pathway (GnRH-GnRHR-GPB5-LGR/LGR5L) in the cerebral and pedal ganglia (CPG) and steroidogenesis pathway (CYP17A, HSD17B12 and HSD3B1) in the gonad are involved in regulating sex steroid hormones. E2/T index that indicates aromatase activity is higher in the ovary than testis and is positively correlated with the expression of FOXL2 in the gonad, implying the presence of aromatase in the scallop. In addition, we confirmed that expression of most of the downstream genes in the two pathways was significantly elevated after injection of mature py-GnRH peptide. This study would contribute to a new understanding of the molecular basis underlying reproduction regulation by GnRH in mollusks.

Identification and expression profiles of Fox transcription factors in the Yesso scallop (Patinopecten yessoensis).

The forkhead box (Fox) gene family is a family of transcription factors that play important roles in a variety of biological processes in vertebrates, including early development and cell proliferation and differentiation. However, at present, studies on the mollusk Fox family are relatively lacking. In the present study, the Fox gene family of the Yesso scallop (Patinopecten yessoensis) was systematically identified. In addition, the expression profiles of the Fox gene family in early development and adult tissues were analyzed. The results showed that there were 26 Fox genes in P. yessoensis. Of the 26 genes, 24 belonged to 20 subfamilies. The Fox genes belonging to the I, Q1, R and S subfamilies were absent in P. yessoensis. The other 2 genes formed 2 independent clades with the Fox genes of other mollusks and protostomes. They might be new members of the Fox family and were named FoxY and FoxZ. P. yessoensis contained a FoxC-FoxL1 gene cluster similar in structure to that of Branchiostoma floridae, suggesting that the cluster might already exist in the ancestors of bilaterally symmetrical animals. The gene expression analysis of Fox showed that most of the genes were continuously expressed in multiple stages of early development, suggesting that Fox genes might be widely involved in the regulation of embryo and larval development of P. yessoensis. Nine Fox genes were specifically expressed in certain tissues, such as the nerve ganglia, foot, ovary, testis, and gills. For the 9 genes that were differentially expressed between the testis and ovary, their expression levels were analyzed during the 4 developmental stages of gonads. The results showed that FoxL2, FoxE and FoxY were highly expressed in the ovary during all developmental stages, while FoxZ was highly expressed in the testis during all developmental stages. The results suggested that these genes might play an important role in sex maintenance or gametogenesis. The present study could provide a reference for evolutionary and functional studies of the Fox family in metazoans.

Density functional study of Li/Na adsorption properties of single-layer and double-layer antimonenes.

ß-Antimonene, a stable two-dimensional material, has been successfully prepared recently. Experiments have demonstrated ß-antimonene's excellent Li storage properties. Based on first-principles density functional theory (DFT), the adsorption properties of Li/Na atoms on single-layer antimonene (SL-Sb) and double-layer antimonene (DL-Sb) have been studied. The optimal adsorption position of Li/Na atoms on SL-Sb is the V site with an adsorption energy of 1.91/1.46 eV. With the increase of Li adsorption density, the crystal structure of the antimonene changes significantly. The optimal adsorption position of Li on DL-Sb is the V2 site in the interlayer with an adsorption energy of 2.71 eV, and that of Na is the V1 site outside the surface with an adsorption energy of 1.72 eV. With the increase of the adsorption density of Li, the antimonene presented a trend of forming an alloy. Whereas with the increase of Na adsorption density, the antimonene retains its original structure. The diffusion barrier of Li/Na atoms on the SL-Sb surface is 0.22/0.13 eV, and outside the DL-Sb surface is 0.25/0.15 eV. In short, DL-Sb can maintain a stable structure with a large Li/Na storage density; the diffusion barriers of Li/Na atoms on antimonene are relatively low, which is beneficial to the rapid insertion/extraction.