A novel C-type lectin with a YPD motif from Portunus trituberculatus (PtCLec1) mediating pathogen recognition and opsonization.

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that function as pattern recognition receptors (PRRs) in innate immune system. In this study, a new C-type lectin was identified from the swimming crab Portunus trituberculatus (PtCLec1). The full-length cDNA of PtCLec1 was 873 bp encoding 176 amino acids. The predicted PtCLec1 protein contained a signal peptide and a single carbohydrate-recognition domain with a special YPD motif. The PtCLec1 transcripts were mainly detected in hepatopancreas and its relative expression levels were significantly up-regulated after the challenges of Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtCLec1 (rPtCLec1) could bind all the tested pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and microorganisms, including V. alginolyticus, V. parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, M. luteus and P. pastoris. It also exhibited strong activity to agglutinate bacteria and yeast in a Ca2+-dependent manner, and such agglutinating activity could be inhibited by d-galactose and LPS. Moreover, rPtCLec1 revealed antimicrobial activity against the tested Gram-negative (V. alginolyticus, V. parahaemolyticus and P. aeruginosa) and Gram-positive bacteria (S. aureus and M. luteus), and promoted the clearance of V. alginolyticus in vivo and hemocyte phagocytosis in vitro. Knockdown of PtCLec1 could down-regulate the expression of phagocytosis-related genes, but enhance the expression levels of prophenoloxidase (proPO) system-related genes, mannose-binding lectin (MBL), antimicrobial peptides (AMPs), MyD88 and Relish. All these results indicate that PtCLec1 might act as a PRR in immune recognition and an opsonin in pathogen elimination.

Two alpha-2 macroglobulin from Portunus trituberculatus involved in the prophenoloxidase system, phagocytosis and regulation of antimicrobial peptides.

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.

Characterization and functional analysis of a novel gC1qR in the swimming crab Portunus trituberculatus.

The receptor for the globular head of complement component C1q, gC1qR, is a multifunctional and multiligand binding protein with a crucial role in host defense. In the present study, a full-length cDNA sequence of a gC1qR homolog (PtgC1qR) in Portunus trituberculatus was identified. PtgC1qR was a 268-amino-acid polypeptide with a conserved MAM33 domain and a mitochondrial targeting sequence in the first 56 amino acids. The transcripts of PtgC1qR were detected in all examined tissues with the highest level detected in the hepatopancreas. Compared with other early embryonic stages, PtgC1qR was highly expressed in the fertilized eggs and embryos at the cleavage stage, which suggest PtgC1qR may be a maternal gene. The transcripts of PtgC1qR in hemocytes exhibited time-dependent response expression pattern after challenged with bacteria (Vibrio alginolyticus, Micrococcus luteus) and fungi (Pichia pastoris). Moreover, the recombinant PtgC1qR (rPtgC1qR) exhibited strong antibacterial activity and microbial-binding activity, suggesting its crucial role in immune defense and recognition. Further phenoloxidase (PO) assay showed that rPtgC1qR could suppress the crab PO activity in vitro in a dose-dependent manner, and it could result in nearly 100% inhibition of PO activity under the concentration of 11.65 µM. Knockdown of PtgC1qR could significantly enhance the expression of serine protease related genes (PtSP1-3 and PtSPH), proPO-associated genes (PtproPO and PtPPAF) and C3-like genes (Ptα2M1 and PtTEP). However, the phagocytosis related genes (PtMyosin, PtRab5 and PtArp) and Ptα2M2 were significantly down-regulated in the PtgC1qR silenced crabs. These findings together demonstrate that PtgC1qR might function in crab immune response via its antibacterial activity, immune recognition or regulating the proPO system, complement pathway and phagocytosis.

Characterization and expression pattern of r-spondin1 in Cynoglossus semilaevis.

r-spondin1 (rspo1) encodes a secreted protein that is involved in the determination and differentiation of the mammalian ovary. However, little information is yet available for teleosts. Here, we identified a homologue of rspo1 in Cynoglossus semilaevis. The full-length cDNA of rspo1 had a length of 2,703 bp with an open reading frame of 834 bp, encoding a protein with a length of 277 amino-acids. rspo1 expression was detected via qRT-PCR in various tissues, and significant sexually dimorphic expression was observed in the gonads. Furthermore, ISH located rspo1 in germ cells such as spermatogonia, spermatocytes, spermatids, spermatozoa, and oocytes, as well as in somatic cells of the gonads. Following knockdown of rspo1 in an ovarian cell line, the expressions of wnt4a, ß-catenin, foxl2, and StAR were highly affected; wnt4a and ß-catenin were significantly downregulated, whereas foxl2 and StAR were significantly upregulated. In summary, these data suggest that rspo1 may be involved in the regulation of ovarian development and differentiation through a conserved pathway, while the function of the gene in the testis remains elusive.

Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole (Cynoglossus semilaevis).

Aquaporin 1 (AQP1) is a member of the transmembrane water channel family of proteins with special structural features, and two AQP1 paralogous genes (aqp1aa and aqp1ab) are reported in teleosts. In the present study, the aqp1aa gene of half-smooth tongue sole (Cynoglossus semilaevis) was cloned and characterized. The full-length cDNA of aqp1aa is 1411 bp with a 786 bp open reading frame encoding a 261-amino acid putative protein with a characteristic structure consisting of 6 membrane-spanning α-helical domains and two highly conserved asparagine-proline-alanine motifs. Real-time quantitative PCR revealed that aqp1aa mRNA is expressed predominantly in the testis of males and pseudo-males, while its expression is low in the ovary and lowest in doublesex and mab-3-related transcription factor 1(DMRT1) knock out fish and triploid males. In situ hybridization indicated that aqp1aa mRNA is expressed mainly in the germ cells of males and pseudo-males, especially in spermatozoa and spermatids. These results suggest that the aqp1aa may play a role in spermatogenesis of C. semilaevis.

Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis).

Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.

Establishment and characterization of a gonad cell line from half-smooth tongue sole Cynoglossus semilaevis pseudomale.

A new cell line was established from half-smooth tongue sole Cynoglossus semilaevis pseudomale gonad (CSPMG). Primary culture was initiated from gonad tissues pieces, and the CSPMG cells were cultured at 24 °C in Dulbecco's modified Eagle medium/F12 medium (1:1) (pH7.0), supplemented with 20 % fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, 2-mercaptoethanol, penicillin and streptomycin. The cultured CSPMG cells, in fibroblast shape, proliferated to 100 % confluency 10 days later and had been subcultured to passage 109. Chromosome analyses indicated that the CSPMG cells exhibited chromosomal aneuploidy with a modal chromosome number of 42, which displayed the normal diploid karyotype of half-smooth tongue sole (2n = 42t, NF = 42). Reverse transcription polymerase chain reaction revealed CSPMG cells could express gonad somatic cell functional genes Sox9a, Wt1a and weakly germ cell marker gene Vasa, but not male specific gene Dmrt1. Transfection experiment demonstrated that CSPMG cells transfected with pEGFP-N3 plasmid and small RNA could express green and red fluorescence signals with high transfection efficiency. In conclusion, a continuous CSPMG cell line has been established successfully. The cell line might serve as a valuable tool for studies on the mechanism of sex determination, sex reversal and gonad development in flatfish.

A first generation BAC-based physical map of the half-smooth tongue sole (Cynoglossus semilaevis) genome.

BACKGROUND: Half-smooth tongue sole (Cynoglossus semilaevis Günther) has been exploited as a commercially important cultured marine flatfish, and female grows 2-3 times faster than male. Genetic studies, especially on the chromosomal sex-determining system of this species, have been carried out in the last decade. Although the genome of half-smooth tongue sole was relatively small (626.9 Mb), there are still some difficulties in the high-quality assembly of the next generation genome sequencing reads without the assistance of a physical map, especially for the W chromosome of this fish due to abundance of repetitive sequences. The objective of this study is to construct a bacterial artificial chromosome (BAC)-based physical map for half-smooth tongue sole with the method of high information content fingerprinting (HICF). RESULTS: A physical map of half-smooth tongue sole was constructed with 30, 294 valid fingerprints (7.5 × genome coverage) with a tolerance of 4 and an initial cutoff of 1e-60. A total of 29,709 clones were assembled into 1,485 contigs with an average length of 539 kb and a N50 length of 664 kb. There were 394 contigs longer than the N50 length, and these contigs will be a useful resource for future integration with linkage map and whole genome sequence assembly. The estimated physical length of the assembled contigs was 797 Mb, representing approximately 1.27 coverage of the half-smooth tongue sole genome. The largest contig contained 410 BAC clones with a physical length of 3.48 Mb. Almost all of the 676 BAC clones (99.9%) in the 21 randomly selected contigs were positively validated by PCR assays, thereby confirming the reliability of the assembly. CONCLUSIONS: A first generation BAC-based physical map of half-smooth tongue sole was constructed with high reliability. The map will promote genetic improvement programs of this fish, especially integration of physical and genetic maps, fine-mappings of important gene and/or QTL, comparative and evolutionary genomics studies, as well as whole genome sequence assembly.

Construction of high-density genetic linkage maps and mapping of growth-related quantitative trail loci in the Japanese flounder (Paralichthys olivaceus).

High-density genetic linkage maps were constructed for the Japanese flounder (Paralichthys olivaceus). A total of 1624 microsatellite markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 resulted in the mapping of 1487 markers to 24 linkage groups, a result which was consistent with the 24 chromosomes seen in chromosome spreads. The female map was composed of 1257 markers, covering a total of 1663.8 cM with an average interval 1.35 cM between markers. The male map consisted of 1224 markers, spanning 1726.5 cM, with an average interval of 1.44 cM. The genome length in the Japanese flounder was estimated to be 1730.3 cM for the females and 1798.0 cM for the males, a coverage of 96.2% for the female and 96.0% for the male map. The mean recombination at common intervals throughout the genome revealed a slight difference between sexes, i.e. 1.07 times higher in the male than female. High-density genetic linkage maps are very useful for marker-assisted selection (MAS) programs for economically valuable traits in this species and for further evolutionary studies in flatfish and vertebrate species. Furthermore, four quantiative trait loci (QTL) associated with growth traits were mapped on the genetic map. One QTL was identified for body weight on LG 14 f, which explained 14.85% of the total variation of the body weight. Three QTL were identified for body width on LG14f and LG14m, accounting for 16.75%, 13.62% and 13.65% of the total variation in body width, respectively. The additive effects were evident as negative values. There were four QTL for growth traits clustered on LG14, which should prove to be very useful for improving growth traits using molecular MAS.

Construction of a high-density microsatellite genetic linkage map and mapping of sexual and growth-related traits in half-smooth tongue sole (Cynoglossus semilaevis).

High-density genetic linkage maps of half-smooth tongue sole were developed with 1007 microsatellite markers, two SCAR markers and an F1 family containing 94. The female map was composed of 828 markers in 21 linkage groups, covering a total of 1447.3 cM, with an average interval 1.83 cM between markers. The male map consisted of 794 markers in 21 linkage groups, spanning 1497.5 cM, with an average interval of 1.96 cM. The female and male maps had 812 and 785 unique positions, respectively. The genome length of half-smooth tongue sole was estimated to be 1527.7 cM for the females and 1582.1 cM for the males. Based on estimations of the map lengths, the female and male maps covered 94.74 and 94.65% of the genome, respectively. The consensus map was composed of 1007 microsatellite markers and two SCAR markers in 21 linkage groups, covering a total of 1624 cM with an average interval of 1.67 cM. Furthermore, 159 sex-linked SSR markers were identified. Five sex-linked microsatellite markers were confirmed in their association with sex in a large number of individuals selected from different families. These sex-linked markers were mapped on the female map LG1f with zero recombination. Two QTLs that were identified for body weight, designated as We-1 and We-2, accounted for 26.39% and 10.60% of the phenotypic variation. Two QTLs for body width, designated Wi-1 and Wi-2, were mapped in LG4f and accounted for 14.33% and 12.83% of the phenotypic variation, respectively. Seven sex-related loci were mapped in LG1f, LG14f and LG1m by CIM, accounting for 12.5-25.2% of the trait variation. The results should prove to be very useful for improving growth traits using molecular MAS.

Polymorphism and Balancing Selection of MHC Class II DAB Gene in 7 Selective Flounder (Paralichthys olivaceus) Families.

In order to determine the genetic variation of the MHC class IIB exon2 allele in the offspring, 700 fry from seven families of Japanese flounder challenged with V. anguillarum were studied, and different mortality rates were found in those families. Five to ten surviving and dead fry from each of the seven families were selected to study the MHC class II B exon2 gene with PCR and a direct sequencing method. One hundred and sixteen different exon2 sequences were found and 116 different alleles were identified, while a minimum of four loci were revealed in the MHC class II B exon2 gene. The ratio (d(N)/d(S)) of nonsynonymous substitution (d(N)) to synonymous substitutions (d(S)) in the peptide-binding region (PBR) of the MHC class IIB gene was 6.234, which indicated that balancing selection is acting on the MHC class IIB genes. The MHC IIB alleles were thus being passed on to their progeny. Some alleles were significantly more frequent in surviving than dead individuals. All together our data suggested that the alleles Paol-DAB*4301, Paol-DAB*4601, Paol-DAB*4302, Paol-DAB*3803, and Paol-DAB*4101 were associated with resistance to V. anguillarum in flounder.