Integrative Metabolomics, Enzymatic Activity, and Gene Expression Analysis Provide Insights into the Metabolic Profile Differences between the Slow-Twitch Muscle and Fast-Twitch Muscle of Pseudocaranx dentex.

The skeletal muscles of teleost fish encompass heterogeneous muscle types, termed slow-twitch muscle (SM) and fast-twitch muscle (FM), characterized by distinct morphological, anatomical, histological, biochemical, and physiological attributes, driving different swimming behaviors. Despite the central role of metabolism in regulating skeletal muscle types and functions, comprehensive metabolomics investigations focusing on the metabolic differences between these muscle types are lacking. To reveal the differences in metabolic characteristics between the SM and FM of teleost, we conducted an untargeted metabolomics analysis using Pseudocaranx dentex as a representative model and identified 411 differential metabolites (DFMs), of which 345 exhibited higher contents in SM and 66 in FM. KEGG enrichment analysis showed that these DFMs were enriched in the metabolic processes of lipids, amino acids, carbohydrates, purines, and vitamins, suggesting that there were significant differences between the SM and FM in multiple metabolic pathways, especially in the metabolism of energy substances. Furthermore, an integrative analysis of metabolite contents, enzymatic activity assays, and gene expression levels involved in ATP-PCr phosphate, anaerobic glycolysis, and aerobic oxidative energy systems was performed to explore the potential regulatory mechanisms of energy metabolism differences. The results unveiled a set of differential metabolites, enzymes, and genes between the SM and FM, providing compelling molecular evidence of the FM achieving a higher anaerobic energy supply capacity through the ATP-PCr phosphate and glycolysis energy systems, while the SM obtains greater energy supply capacity via aerobic oxidation. These findings significantly advance our understanding of the metabolic profiles and related regulatory mechanisms of skeletal muscles, thereby expanding the knowledge of metabolic physiology and ecological adaptation in teleost fish.

DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

Mitochondrial Homeostasis Regulating Mitochondrial Number and Morphology Is a Distinguishing Feature of Skeletal Muscle Fiber Types in Marine Teleosts.

Fishes' skeletal muscles are crucial for swimming and are differentiated into slow-twitch muscles (SM) and fast-twitch muscles (FM) based on physiological and metabolic properties. Consequently, mitochondrial characteristics (number and morphology) adapt to each fiber type's specific functional needs. However, the mechanisms governing mitochondrial adaptation to the specific bioenergetic requirements of each fiber type in teleosts remain unclear. To address this knowledge gap, we investigated the mitochondrial differences and mitochondrial homeostasis status (including biogenesis, autophagy, fission, and fusion) between SM and FM in teleosts using Takifugu rubripes as a representative model. Our findings reveal that SM mitochondria are more numerous and larger compared to FM. To adapt to the increased mitochondrial number and size, SM exhibit elevated mitochondrial biogenesis and dynamics (fission/fusion), yet show no differences in mitochondrial autophagy. Our study provides insights into the adaptive mechanisms shaping mitochondrial characteristics in teleost muscles. The abundance and elongation of mitochondria in SM are maintained through elevated mitochondrial biogenesis, fusion, and fission, suggesting an adaptive response to fulfill the bioenergetic demands of SM that rely extensively on OXPHOS in teleosts. Our findings enhance our understanding of mitochondrial adaptations in diverse muscle types among teleosts and shed light on the evolutionary strategies of bioenergetics in fishes.

Linked selection, differential introgression and recombination rate variation promote heterogeneous divergence in a pair of yellow croakers.

Understanding the mechanisms underlying heterogeneous genomic divergence is of particular interest in evolutionary biology. Highly differentiated genomic regions, known as genomic islands, often evolve between diverging lineages. These genomic islands may be related to selection promoting adaptation or reproductive isolation. Based on whole genome assembly and genome-wide RAD sequencing in a pair of yellow croakers (genus: Larimichthys), we investigated the evolutionary processes shaping genomic landscapes of divergence. Demographic modelling indicated that the two species diverged following a secondary contact scenario, where differential introgression and linked selection were suggested to be involved in heterogeneous genomic divergence. We identified reduced recombination rate in genomic islands and a relatively good conservation of both genetic diversity and recombination landscapes between species, which highlight the roles of linked selection and recombination rate variation in promoting heterogeneous divergence in the common ancestral lineage of the two species. In addition, we found a positive correlation between differentiation (FST ) and absolute sequence divergence (Dxy ), and elevated Dxy in genomic islands, indicating that the genomic landscape of divergence was not shaped by linked selection alone. Restricted gene flow in highly differentiated regions has probably remodelled the landscape of heterogeneous genomic divergence. This study highlights that highly differentiated genomic regions can also arise from a combination of linked selection and differential gene flow in interaction with varying recombination rates.

Expression profiles and transcript properties of fast-twitch and slow-twitch muscles in a deep-sea highly migratory fish, Pseudocaranx dentex.

Fast-twitch and slow-twitch muscles are the two principal skeletal muscle types in teleost with obvious differences in metabolic and contractile phenotypes. The molecular mechanisms that control and maintain the different muscle types remain unclear yet. Pseudocaranx dentex is a highly mobile active pelagic fish with distinctly differentiated fast-twitch and slow-twitch muscles. Meanwhile, P. dentex has become a potential target species for deep-sea aquaculture because of its considerable economic value. To elucidate the molecular characteristics in the two muscle types of P. dentex, we generated 122 million and 130 million clean reads from fast-twitch and slow-witch muscles using RNA-Seq, respectively. Comparative transcriptome analysis revealed that 2,862 genes were differentially expressed. According to GO and KEGG analysis, the differentially expressed genes (DEGs) were mainly enriched in energy metabolism and skeletal muscle structure related pathways. Difference in the expression levels of specific genes for glycolytic and lipolysis provided molecular evidence for the differences in energy metabolic pathway between fast-twitch and slow-twitch muscles of P. dentex. Numerous genes encoding key enzymes of mitochondrial oxidative phosphorylation pathway were significantly upregulated at the mRNA expression level suggested slow-twitch muscle had a higher oxidative phosphorylation to ensure more energy supply. Meanwhile, expression patterns of the main skeletal muscle developmental genes were characterized, and the expression signatures of Sox8, Myod1, Calpain-3, Myogenin, and five insulin-like growth factors indicated that more myogenic cells of fast-twitch muscle in the differentiating state. The analysis of important skeletal muscle structural genes showed that muscle type-specific expression of myosin, troponin and tropomyosin may lead to the phenotypic structure differentiation. RT-qPCR analysis of twelve DEGs showed a good correlation with the transcriptome data and confirmed the reliability of the results presented in the study. The large-scale transcriptomic data generated in this study provided an overall insight into the thorough gene expression profiles of skeletal muscle in a highly mobile active pelagic fish, which could be valuable for further studies on molecular mechanisms responsible for the diversity and function of skeletal muscle.

Complete Mitochondrial Genome of Pseudocaranx dentex (Carangidae, Perciformes) Provides Insight into Phylogenetic and Evolutionary Relationship among Carangidae Family.

Pseudocaranx dentex (white trevally) which belongs to the Carangidae family, is an important commercial fishery and aquaculture resource in Asia. However, its evolution and population genetics have received little attention which was limited by the mitogenome information absence. Here, we sequenced and annotated the complete mitochondrial genome of P. dentex which was 16,569 bp in length, containing twenty-two tRNAs (transfer RNAs), thirteen PCGs (protein-coding genes), two rRNAs (ribosomal RNAs), and one non-coding region with conservative gene arrangement. The Ka/Ks ratio analysis among Carangidae fishes indicated the PCGs were suffering purify selection and the values were related to the taxonomic status and further influenced by their living habits. Phylogenetic analysis based on the PCGs sequences of mitogenomes among 36 species presented three major clades in Carangidae. According to the phylogenetic tree, we further analyzed the taxonomic confusion of Carangoides equula which was on the same branch with P. dentex but a different branch with Carangoides spp. We inferred Kaiwarinus equula should be the accepted name and belong to the independent Kaiwarinus genus which was the sister genus of Pseudocaranx. This work provides mitochondrial genetic information and verifies the taxonomic status of P. dentex, and further helps to recognize the phylogenetic relationship and evolutionary history of Carangidae.

Effects of osmotic stress on Na+/K+-ATPase, caspase 3/7 activity, and the expression profiling of sirt1, hsf1, and hsp70 in the roughskin sculpin (Trachidermus fasciatus).

Osmoregulation mechanism underlying acclimation of migratory fish to different salinities has been a classical research topic for decades. In this study, the roughskin sculpin (Trachidermus fasciatus) were subjected to two different acute osmotic treatments (one extreme acute and one acute treatment, i.e., E-acute and acute group). Comparisons of branchial enzyme activity, as well as the time-course expression profiling of sirt1, hsf1, and hsp70 were performed to reveal changes at the physiological and molecular levels. As a result, the branchial Na+/K+-ATPase activity was significantly inhibited and the caspase 3/7 relating to apoptosis was significantly induced in the E-acute group; no significant difference of branchial enzyme activity was detected in the acute group. These results suggested that T. fasciatus could keep stable physiological levels when experiencing the acute salinity change but not under extreme osmotic stress. Significant variations of sirt1, hsf1, and hsp70 expression were determined in the four target tissues (gill, intestine, kidney, and liver). Similar profiling was detected between the time-course expression of sirt1 and hsf1, suggesting their association in the osmoregulation process. Tissue-specific gene expression patterns in all the three target genes showed that each tissue possesses its own gene expression pattern in response to salinity changes. The overall different expression profiling of sirt1, hsf1, and hsp70 under the extreme acute and acute osmotic treatments might respectively represent the molecular regulation of stress response and acclimation. The findings make it possible to provide more reliable data to decipher the mechanism of osmoregulation in migratory fish.

Transcriptome analysis for identifying possible causes of post-reproductive death of Sepia esculenta based on brain tissue.

BACKGROUND: The subpeduncle lobe/olfactory lobe-optic gland axis is called the endocrine regulation center of cephalopods. However, little is known about the mechanism of the subpeduncle lobe/olfactory lobe-optic gland axis regulate the sexual maturation and post-reproductive death of Sepia esculenta Hoyle. OBJECTIVES: The primary objective of this study was to provide basic information for revealing the mechanism of the subpeduncle lobe/olfactory lobe-optic axis regulating the rapid post-reproductive death of S. esculenta. METHODS: In this paper, Illumina sequencing based transcriptome analysis was performed on the brain tissue of female S. esculenta in the three key developmental stages: growth stage (BG), spawning stage (BS), and post-reproductive death stage (BA). RESULTS: A total of 66.19 Gb Illumina sequencing data were obtained. A comparative analysis of the three stages showed 2609, 3333, and 170 differentially expressed genes (DEGs) in BG-vs-BA, BG-vs-BA, and BS-vs-BA, respectively. The Gene Ontology (GO) enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity, oxidative phosphorylation, and respiratory chain were significantly enriched. The significant enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway identified pathways associated with the regulation of death, such as the mammalian target of rapamycin (mTOR) signaling pathway, AMPK signaling pathway, oxidative phosphorylation, and cell cycle. CONCLUSION: The post-reproductive death of S. esculenta was found to be a complex energy steady-state regulation network system. The mTOR acted as an energy receptor and had a key role in regulating energy homeostasis.

Analyses of the molecular mechanisms associated with salinity adaption of Trachidermus fasciatus through combined iTRAQ-based proteomics and RNA sequencing-based transcriptomics.

Osmoregulation mechanism underlying acclimation and adaptation of migratory fish to different salinities has been studied for decades. Recently developed transcriptomic and proteomic techniques would make it possible to provide more reliable data to decipher the mechanism study at a molecular level. Here in this study, an integrative analysis of the kidney-specific transcriptome and proteome was performed to identify important regulators and pathways involved in salinity adaption of roughskin sculpin (Trachidermus fasciatus). Fish were subjected to seawater-to-freshwater transfer that was achieved in 24 h, samples were collected at 12, 24 and 48 h after the start of the experiment. Time course profiling of gene and protein expressions were examined using RNA-seq and iTRAQ methods. A total of 1504 differentially expressed genes (DEGs) and 378 differentially expressed proteins (DEPs) were identified at the three time points. 66 proteins were filtered by correlation of transcriptome and proteome results. When comparing the data obtained from the three time points, 12 h exhibited the largest number of DEGs and DEPs, suggesting the time course experiment is critical to clarify the framework of regulatory genes/proteins in response to salinity change. This study provides the first time-course, kidney-specific, combined transcriptomic and proteomic profiling associated with salinity adaption of Trachidermus fasciatus. The results revealed the possibility of T. fasciatus as an experimental animal for osmoregulation studies, showed the powerfulness of combining transcriptomic and proteomic approaches to provide molecular insights of osmoregulation mechanisms in migratory fish.

Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis).

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1-4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5'-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of -272 to +28 and -740 to -396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Sequencing of complete mitochondrial genome of brown algal Saccharina sp. ye- C12.

The complete sequence (37 654 bp) of the mitochondrial DNA (mtDNA) of the Saccharina sp. ye-C12 was determined using Illumina sequencing data. The genome contains 38 protein-coding genes (PCG), three ribosomal RNA (rRNA), 25 transfer RNA (tRNA) genes and the phylogenetic analysis based on the mitochondrial genomes of brown algae indicated that Saccharina sp. ye-C12 and Saccharina japonica are the most closely related species, which strongly supports their close phylogenetic affinity.

Saccharina genomes provide novel insight into kelp biology.

Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies.

Effects of CO2 and seawater acidification on the early stages of Saccharina japonica development.

In this paper, we demonstrated that ocean acidification (OA) had significant negative effects on the microscopic development of Saccharina japonica in a short-term exposure experiment under a range of light conditions. Under elevated CO2, the alga showed a significant reduction in meiospore germination, fecundity, and reproductive success. Larger female and male gametophytes were noted to occur under high CO2 conditions and high light magnified these positive effects. Under conditions of low light combined with high PCO2, the differentiation of gametophytes was delayed until the end of the experiment. In contrast, gametophytes were able to survive after having been subjected to a long-term acclimation period, of 105 days. Although the elevated PCO2 resulted in a significant increase in sporophyte length, the biomass abundance (expressed as individual density attached to the seed fiber) was reduced significantly. Further stress resistance experiments showed that, although the acidified samples had lower resistance to high light and high temperature conditions, they displayed higher acclimation to CO2-saturated seawater conditions compared with the control groups. These combined results indicate that OA has a severe negative effect on S. japonica, which may result in future shifts in species dominance and community structure.

Population genetic structure and larval dispersal strategy of portunid crab Charybdis bimaculata in Yellow sea and East China sea.

Larval dispersal may have an important effect on genetic structure of benthic species. However, different species may choose different larval dispersal strategy. To examine the population genetic structure and larval dispersal strategy of portunid crab Charybdis bimaculata, a 658 base pair (bp) fragment of mtDNA COI gene was sequenced in this species. In total, 67 individuals were collected from 5 locations in Yellow Sea and East China, and 24 haplotypes were obtained. Mean haplotype diversity and nucleotide diversity for the five populations ranged from 0.2000 ± 0.1541 (Zhoushan) to 0.8333 ± 0.1265 (Nanji island), and from 0.0003 ± 0.0005 (Zhoushan) to 0.0026 ± 0.0019 (Nanji island). Analysis of molecular variance and pairwise FST revealed no significant differentiation between the Yellow Sea and the East China Sea in C. bimaculata, supporting high larval dispersal ability in this species, rejecting larval retention. Mismatch distribution revealed that C. bimaculata had undergone population expansion. Larval drift in the ocean currents, and recent range expansion could be the reasons for little genetic structure in the studied area.

Long-term experiment on physiological responses to synergetic effects of ocean acidification and photoperiod in the Antarctic sea ice algae Chlamydomonas sp. ICE-L.

Studies on ocean acidification have mostly been based on short-term experiments of low latitude with few investigations of the long-term influence on sea ice communities. Here, the combined effects of ocean acidification and photoperiod on the physiological response of the Antarctic sea ice microalgae Chlamydomonas sp. ICE-L were examined. There was a general increase in growth, PSII photosynthetic parameters, and N and P uptake in continuous light, compared to those exposed to regular dark and light cycles. Elevated pCO2 showed no consistent effect on growth rate (p=0.8) and N uptake (p=0.38) during exponential phrase, depending on the photoperiod but had a positive effect on PSII photosynthetic capacity and P uptake. Continuous dark reduced growth, photosynthesis, and nutrient uptake. Moreover, intracellular lipid, mainly in the form of PUFA, was consumed at 80% and 63% in low and high pCO2 in darkness. However, long-term culture under high pCO2 gave a more significant inhibition of growth and Fv/Fm to high light stress. In summary, ocean acidification may have significant effects on Chlamydomonas sp. ICE-L survival in polar winter. The current study contributes to an understanding of how a sea ice algae-based community may respond to global climate change at high latitudes.

Population genetic studies revealed local adaptation in a high gene-flow marine fish, the small yellow croaker (Larimichthys polyactis).

The genetic differentiation of many marine fish species is low. Yet local adaptation may be common in marine fish species as the vast and changing marine environment provides more chances for natural selection. Here, we used anonymous as well as known protein gene linked microsatellites and mitochondrial DNA to detect the population structure of the small yellow croaker (Larimichthys polyactis) in the Northwest Pacific marginal seas. Among these loci, we detected at least two microsatellites, anonymous H16 and HSP27 to be clearly under diversifying selection in outlier tests. Sequence cloning and analysis revealed that H16 was located in the intron of BAHCC1 gene. Landscape genetic analysis showed that H16 mutations were significantly associated with temperature, which further supported the diversifying selection at this locus. These marker types presented different patterns of population structure: (i) mitochondrial DNA phylogeny showed no evidence of genetic divergence and demonstrated only one glacial linage; (ii) population differentiation using putatively neutral microsatellites presented a pattern of high gene flow in the L. polyactis. In addition, several genetic barriers were identified; (iii) the population differentiation pattern revealed by loci under diversifying selection was rather different from that revealed by putatively neutral loci. The results above suggest local adaptation in the small yellow croaker. In summary, population genetic studies based on different marker types disentangle the effects of demographic history, migration, genetic drift and local adaptation on population structure and also provide valuable new insights for the design of management strategies in L. polyactis.

Molecular cloning and expression analysis of major histocompatibility complex class IIB gene of the Whitespotted bambooshark (Chiloscyllium plagiosum).

Major histocompatibility complex (MHC) plays an important role in the immune response to antigenic peptides in vertebrates. In this study, the full length of MHC IIB cDNA was isolated from the Whitespotted bambooshark (Chiloscyllium plagiosum) by homology cloning, and the rapid amplification of cDNA ends polymerase chain reaction. As a result, the MHC IIB cDNA is 1,407 bp, which contains an open reading frame (ORF) of 831 bp encoding a protein of 276 amino acids. Furthermore, seven alleles of the complete MHC IIB ORF were detected and the variable sites were mainly located in the immunoglobulin-like (ß2) region. Tissue distribution analysis showed that MHC IIB can be detected in all the ten tissues examined, with the highest expression in the spleen and gill. Challenge of C. plagiosum with the pathogenic bacteria, Vibrio harveyi, resulted in significant changes in the expression of MHC IIB mRNA in the three immune-related tissues (gill, liver and spleen). These results show that the MHC IIB plays an important role in response to bacterial infection in elasmobranches.

De novo sequencing and analysis of the Ulva linza transcriptome to discover putative mechanisms associated with its successful colonization of coastal ecosystems.

BACKGROUND: The green algal genus Ulva Linnaeus (Ulvaceae, Ulvales, Chlorophyta) is well known for its wide distribution in marine, freshwater, and brackish environments throughout the world. The Ulva species are also highly tolerant of variations in salinity, temperature, and irradiance and are the main cause of green tides, which can have deleterious ecological effects. However, limited genomic information is currently available in this non-model and ecologically important species. Ulva linza is a species that inhabits bedrock in the mid to low intertidal zone, and it is a major contributor to biofouling. Here, we presented the global characterization of the U. linza transcriptome using the Roche GS FLX Titanium platform, with the aim of uncovering the genomic mechanisms underlying rapid and successful colonization of the coastal ecosystems. RESULTS: De novo assembly of 382,884 reads generated 13,426 contigs with an average length of 1,000 bases. Contiguous sequences were further assembled into 10,784 isotigs with an average length of 1,515 bases. A total of 304,101 reads were nominally identified by BLAST; 4,368 isotigs were functionally annotated with 13,550 GO terms, and 2,404 isotigs having enzyme commission (EC) numbers were assigned to 262 KEGG pathways. When compared with four other full sequenced green algae, 3,457 unique isotigs were found in U. linza and 18 conserved in land plants. In addition, a specific photoprotective mechanism based on both LhcSR and PsbS proteins and a C4-like carbon-concentrating mechanism were found, which may help U. linza survive stress conditions. At least 19 transporters for essential inorganic nutrients (i.e., nitrogen, phosphorus, and sulphur) were responsible for its ability to take up inorganic nutrients, and at least 25 eukaryotic cytochrome P450s, which is a higher number than that found in other algae, may be related to their strong allelopathy. Multi-origination of the stress related proteins, such as glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase and heat-shock proteins, may also contribute to colonization of U. linza under stress conditions. CONCLUSIONS: The transcriptome of U. linza uncovers some potential genomic mechanisms that might explain its ability to rapidly and successfully colonize coastal ecosystems, including the land-specific genes; special photoprotective mechanism based on both LhcSR and PsbS; development of C4-like carbon-concentrating mechanisms; muti-origin transporters for essential inorganic nutrients; multiple and complex P450s; and glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase, and heat-shock proteins that are related to stress resistance.

The co-existence of two growth hormone receptors and their differential expression profiles between female and male tongue sole (Cynoglossus semilaevis).

Growth hormone receptor (Ghr) is a single-transmembrane pass protein which is important in initiating the ability of growth hormone (Gh) to regulate development and somatic growth in vertebrates. In this study, molecular cloning, expression analysis of two different ghr genes (ghr1 and ghr2) in the tongue sole (Cynoglossus semilaevis) was conducted. As a result, the ghr1 and ghr2 cDNA sequences are 2364 bp and 3125 bp, each of which encodes a transmembrane protein of 633 and 561 amino acids (aa), respectively. Besides, the ghr1 gene includes nine exons and eight introns. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. Both the ghr1 and ghr2 were predominantly expressed in the liver, and the ghr1 expression level in normal males was 1.6 and 1.4 times as much as those in females and extra-large males, while the ghr2 mRNA expression level in normal males was 1.1 and 1.2 times as much as those in females and extra-large males, respectively. Ontogenetic expression analysis at early life stages indicated that the ghr1 and ghr2 mRNAs were detected at all of the 35 sampling points (from oosphere to 410days-old). Furthermore, the sex differences in ghr mRNA expressions were also examined by using a full-sib family of C. semilaevis. Significantly higher levels of ghr1 mRNA were observed in males than in females at most stages of the sampling period (P<0.01). The ghr2 mRNA expression at most stages exhibited a significant sexual difference at each sampling point (P<0.01) without any variation trend related with the sexes during the whole sampling period.

Development and characterization of EST-SSR markers from Scapharca broughtonii and their transferability in Scapharca subcrenata and Tegillarca granosa.

Twenty-five novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the ark shell Scapharca broughtonii. Polymorphisms of these EST-SSR markers were evaluated in 48 wild individuals collected from Shidao, Shandong Province, China. A total of 202 alleles were detected at 25 loci. The numbers of alleles per locus ranged from 4 to 14, with an average of 8.08. The observed and expected heterozygosities varied from 0.2917 to 1.000 and from 0.3570 to 0.9002, respectively. After sequential Bonferroni correction for multiple tests, only one locus was found to deviate from Hardy-Weinberg equilibrium. Twenty-five EST-SSR markers showed a high rate of across-species transferability (100%) in Scapharca subcrenata and a low rate of across-genus transferability (20%) in Tegillarca granosa. These EST-SSRs will be helpful for QTL mapping, molecular breeding and investigation of population genetic diversity in ark shell S. broughtonii and other Scapharca species.