Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

FTR33, a member of fish-specific TRIM (finTRIM) subfamily, regulates negatively type I IFN antiviral immunity in zebrafish.

In mammals, the tripartite motif (TRIM) proteins have been identified as critical factors involved in various cellular processes, including antiviral immunity. In teleost fish, a subfamily of fish-specific TRIM (finTRIM, FTR) has emerged in genus- or species-specific duplication. In this study, a finTRIM gene, called ftr33, was identified in zebrafish (Danio rerio), and phylogenic analysis revealed that FTR33 is closely related with zebrafish FTR14. The FTR33 protein contains all conservative domains reported in other finTRIMs. The ftr33 has a constitutive expression in embryos and in tissues/organs of adult fish, and its expression can be induced following spring viremia of carp virus (SVCV) infection and interferon (IFN) stimulation. The overexpression of FTR33 significantly downregulated the expression of type I IFNs and IFN-stimulated genes (ISGs) both in vitro and in vivo, respectively, leading to the increased replication of SVCV. It was also found that FTR33 interacted with melanoma differentiation associated gene 5 (MDA5) or mitochondrial anti-viral signaling protein (MAVS) to weaken the promoter activity of type I IFN. It is thus concluded that the FTR33, as an ISG, in zebrafish can negatively regulate IFN-mediated antiviral response.

Molecular and functional characterization of zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1, ZDHHC1 in Chinese perch Siniperca chuatsi.

In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

Orf1B controls secretion of T3SS proteins and contributes to Edwardsiella piscicida adhesion to epithelial cells.

Edwardsiella piscicida is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish. The type III secretion system (T3SS) is one of its two most important virulence islands. T3SS protein EseJ inhibits E. piscicida adhesion to epithelioma papillosum cyprini (EPC) cells by negatively regulating type 1 fimbria. Type 1 fimbria helps E. piscicida to adhere to fish epithelial cells. In this study, we characterized a functional unknown protein (Orf1B) encoded within the T3SS gene cluster of E. piscicida. This protein consists of 122 amino acids, sharing structural similarity with YscO in Vibrio parahaemolyticus. Orf1B controls secretion of T3SS translocon and effectors in E. piscicida. By immunoprecipitation, Orf1B was shown to interact with T3SS ATPase EsaN. This interaction may contribute to the assembly of the ATPase complex, which energizes the secretion of T3SS proteins. Moreover, disruption of Orf1B dramatically decreased E. piscicida adhesion to EPC cells due to the increased steady-state protein level of EseJ within E. piscicida. Taken together, this study partially unraveled the mechanisms through which Orf1B promotes secretion of T3SS proteins and contributes to E. piscicida adhesion. This study helps to improve our understanding on molecular mechanism of E. piscicida pathogenesis.

Gene synteny, evolution and antiviral activity of type I IFNs in a reptile species, the Chinese soft-shelled turtle Pelodiscus sinensis.

Type I interferons (IFNs) are critical cytokines for the establishment of antiviral status in fish, amphibian, avian and mammal, but the knowledge on type I IFNs is rather limited in reptile. In this study, seven type I IFN genes, designed as IFN1 to IFN7, were identified from a reptile species, the Chinese soft-shelled turtle (Pelodiscus sinensis). These identified type I IFNs have relatively low protein identity, when compared with those in human and chicken; but they possess conserved cysteines, predicted multi-helix structure and N-terminal signal peptide. The Chinese soft-shelled turtle IFN1 to IFN5 have two exons and one intron, but IFN6 and IFN7 are the single-exon genes. Chinese soft-shelled turtle type I IFNs are located respectively on the two conserved reptile-bird loci, named as Locus a and Locus c, and are clustered into the four of the five reptile-bird groups (named as Groups I-V) based on phylogenetic evidence, due to the lack of IFNK in the turtle. Moreover, the Chinese soft-shelled turtle type I IFNs can be induced by soft-shelled turtle iridovirus (STIV) infection and show antiviral activity in soft-shelled turtle artery (STA) cells, except IFN6. In addition, due to the difference in genome organizations, such as the number of exons and introns of type I IFN genes from fish to mammal, the definition and evolution of 'intronless' type I IFN genes were discussed in lineages of vertebrates. Thus, the finding of type I IFNs on two different loci in P. sinensis sheds light on the evolution of type I IFN genes in vertebrates.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco).

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of -245 to -50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

Synthesizing the First Phase of Dynamic Sequences of Breast MRI for Enhanced Lesion Identification.

OBJECTIVE: To develop a deep learning model for synthesizing the first phases of dynamic (FP-Dyn) sequences to supplement the lack of information in unenhanced breast MRI examinations. METHODS: In total, 97 patients with breast MRI images were collected as the training set (n = 45), the validation set (n = 31), and the test set (n = 21), respectively. An enhance border lifelike synthesize (EDLS) model was developed in the training set and used to synthesize the FP-Dyn images from the T1WI images in the validation set. The peak signal-to-noise ratio (PSNR), structural similarity (SSIM), mean square error (MSE) and mean absolute error (MAE) of the synthesized images were measured. Moreover, three radiologists subjectively assessed image quality, respectively. The diagnostic value of the synthesized FP-Dyn sequences was further evaluated in the test set. RESULTS: The image synthesis performance in the EDLS model was superior to that in conventional models from the results of PSNR, SSIM, MSE, and MAE. Subjective results displayed a remarkable visual consistency between the synthesized and original FP-Dyn images. Moreover, by using a combination of synthesized FP-Dyn sequence and an unenhanced protocol, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MRI were 100%, 72.73%, 76.92%, and 100%, respectively, which had a similar diagnostic value to full MRI protocols. CONCLUSIONS: The EDLS model could synthesize the realistic FP-Dyn sequence to supplement the lack of enhanced images. Compared with full MRI examinations, it thus provides a new approach for reducing examination time and cost, and avoids the use of contrast agents without influencing diagnostic accuracy.

Retroposition of the Long Transcript from Multiexon IFN-ß Homologs in Ancestry Vertebrate Gave Rise to the Proximal Transcription Elements of Intronless IFN-ß Promoter in Humans.

IFN-ß is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-ß and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-ß in chicken and humans.

MORPHOLOGY OF ADULTS AND IMMATURES OF THE ACANTHOCEPHALAN, POMPHORHYNCHUS FUHAIENSIS YUE, 1998 (ACANTHOCEPHALA: POMPHORHYNCHIDAE) FROM CYPRINID FISH IN NORTHWEST CHINA.

Adult specimens of Pomphorhynchus fuhaiensis were identified from common carp (Cyprinus carpio L.) in Ulungur Lake of northwest China, and acanthors, acanthellae, cystacanths dissected from Gammarus lacustris in a small tributary of Ulungur River for the first time. The acanthocephalans were also found in crucian carp (Carassius carassius L.), tench (Tinca tinca L.), oriental bream (Abramis brama orientalis Berg), and ide (Leuciscus idus L.) in the lake. This species is distinguished from other species in Pomphorhynchus by its large, spherical bulb and very long neck as well as by a cylindrical proboscis armed with 15-17 longitudinal rows of 9-12 hooks each. The anterior proboscis hooks are almost uniform in size and shape, the sixth hook in a short row and the seventh hook in long row decrease abruptly in size posteriorly with the last end hook being a little larger than the prebasal hook, and in a ring; posterior proboscis hooks much more widely spaced. Furthermore, the lemnisci are claviform. The mean neck:trunk ratio is about 0.5, which is larger than most other species in Pomphorhynchus. Females are larger than males. In males, the testes are in one-third to one-half of the trunk, equal, ovoid-spheroid, usually contiguous, and small relative to the body size, and there are 6 ovoid cement glands. Pomphorhynchus fuhaiensis is similar to Pomphorhynchus laevis but can be distinguished by the number of longitudinal rows of hooks. Pomphorhynchus laevis is armed with 18-20 longitudinal rows of 11-13 hooks, P. fuhaiensis is armed with 15-17 longitudinal rows of 9-12 hooks.

Specific bioactivity of IL-22 in intestinal cells as revealed by the expression of IL-22RA1 in Mandarin fish, Siniperca chuatsi.

IL-22, a multifunctional cytokine, acts as an important regulator in host immunity in mammals. IL-22 homologues have been characterized in several species of fish, with its expression found in multiple tissues/cells in fish, but its target cells have not been fully analyzed. In the present research, different organ/tissue isolated cells were examined for the expression of IL-22 and the induced IL-22 responses in mandarin fish. The mandarin fish IL-22 was found to be expressed in all these tested cells with high basal expression in intestinal cells. The HKLs showed low basal expression but significant increase in expression of IL-22 after LPS treatment or bacterial infection. Only intestinal cells showed response to IL-22 by enhanced expression of hepcidin, LEAP2 and IL-22BP, with unresponsiveness observed in other tested cells, which indicated the cell-specificity of IL-22 bioactivity in mandarin fish. One of the heterodimeric receptor components for IL-22, the IL-22RA1, was cloned in mandarin fish, with four tandem fibronectin type III (FNIII) domains identified in its extracellular part. IL-22RA1 exhibited an intestinal cell-specific expression pattern, although another receptor component of IL-22, IL-10R2, displayed constitutive expressions in all these tested cells. The present study reveals that the mandarin fish IL-22 exhibits its bioactivity in a cell-specific manner in intestinal cells, which is reflected in the restrictive expression of its receptor unit, IL-22RA1.

Identification and Characterization of EvpQ, a Novel T6SS Effector Encoded on a Mobile Genetic Element in Edwardsiella piscicida.

In this study, a hypothetical protein (ORF02740) secreted by Edwardsiella piscicida was identified. We renamed the ORF02740 protein as EvpQ, which is encoded by a mobile genetic element (MGE) in E. piscicida genome. The evpQ gene is spaced by 513 genes from type VI secretion system (T6SS) gene cluster. Low GC content, three tRNA, and three transposase genes nearby evpQ define this MGE that evpQ localizes as a genomic island. Sequence analysis reveals that EvpQ shares a conserved domain of C70 family cysteine protease and shares 23.91% identity with T3SS effector AvrRpt2 of phytopathogenic Erwinia amylovora. Instead, EvpQ of E. piscicida is proved to be secreted at a T6SS-dependent manner, and it can be translocated into host cells. EvpQ is thereof a novel T6SS effector. Significantly decreased competitive index of ΔevpQ strain in blue gourami fish (0.53 ± 0.27 in head kidney and 0.44 ± 0.19 in spleen) indicates that EvpQ contributes to the pathogenesis of E. piscicida. At 8-, 18-, and 24-h post-subculture into DMEM, the transcription of evpQ was found to be negatively regulated by Fur and positively regulated by EsrC, and the steady-state protein levels of EvpQ are negatively controlled by RpoS. Our study lays a foundation for further understanding the pathogenic role of T6SS in edwardsiellosis.

Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China.

Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae, which stimulate the cleavage of caspase-8.

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild-type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining and cleaved caspase-3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase-8, caspase-9, and caspase-3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre-treatment of macrophages with caspase-8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase-8, caspase-9, and caspase-3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.

Transcriptomic responses of S100 family to bacterial and viral infection in zebrafish.

The S100 family proteins are a group of small acidic polypeptides and have diverse functions in regulating many aspects of physiological processes. They are structurally conserved and possess two EF-hands which are central for calcium-mediated functions. In this study, 14 S100 cDNA sequences were determined in zebrafish and their genomic organizations confirmed. Re-analyzing the gene synteny of the S100 loci identified two major S100 loci in Chr16 and Chr19 which share remarkable conservation with the S100 locus in human Chr1, suggesting they may have evolved from a single locus during the teleost specific whole genome duplication event. It appears that the homologues of human S100G and S100P have been lost in zebrafish. Expression analysis reveals that S100W, ICN1 and ICN2 are markedly expressed in embryos. Further, the transcripts of S100 genes are relatively abundant in mucosal tissues such as gills and gut. Intraperitoneal injection of poly(I:C) resulted in up-regulation of most S100 genes in the gut and spleen, with highest induction of S100V2 and S100Z detected. In fish challenged with spring viremia of carp virus (SVCV), expression of most S100 family genes was increased in the spleen between day 1 and 7 post infection, with consistent induction seen for the S100A1, S100A10b, S100B, S100ICN1, S100T, S100U, S100V1 and S100Z. Interestingly, intraperitoneal injection of Edwardsiella tarda down-regulated S100 expression in the gut but resulted in induction in the spleen. The results demonstrate that the S100 family genes are differentially modulated by bacterial and viral pathogens in zebrafish.

Functional characterization of IL-10 and its receptor subunits in a perciform fish, the mandarin fish, Siniperca chuatsi.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1ß, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

The Edwardsiella piscicida Type III Effector EseJ Suppresses Expression of Type 1 Fimbriae, Leading to Decreased Bacterial Adherence to Host Cells.

The type III secretion system (T3SS) of Edwardsiella piscicida plays a crucial role in its pathogenesis. Our previous study indicated that the T3SS effector protein EseJ inhibits the bacterium's adhesion to epithelioma papillosum cyprini (EPC) cells, while the mechanism of the inhibition remains elusive. In this study, we revealed that EseJ negatively regulates the fimA gene, as demonstrated by comparative transcription analysis of ΔeseJ and wild-type (WT) strains. As well, the dramatically increased production of FimA was detected in the absence of EseJ compared to that by the WT strain. The adherence of the ΔeseJ strain decreased far below that of the WT strain in the absence of FimA, demonstrating that FimA plays a pivotal role in the hyperadhesion of the ΔeseJ strain. Adherence analysis with a strain with truncated eseJ demonstrated that the C-terminal region of EseJ (Gly1191 to Ile1359) is necessary to inhibit the transcription of the type 1 fimbrial operon. Binding between the EseJ fragment from amino acid residues 1191 to 1359 and the DNA fragment upstream of fimA was not detected, indicating that EseJ might indirectly regulate the type 1 fimbrial operon. Our study reveals that EseJ controls E. piscicida adherence to EPC cells by negatively regulating the type 1 fimbrial operon.

Molecular and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.

Characterization of Sexual Trait Development in cyp17a1-Deficient Zebrafish.

Cytochrome P450 (Cyp)17A1 has both 17α-hydroxylase and 17,20-lyase activities, which are involved in the steroidogenic pathway that produces androgens and estrogens. Previously, a phenotype of all-male cyp17a1-deficient zebrafish generated by transcription activatorlike effector nuclease has been reported. In the current study, the mechanisms relating to Cyp17a1 that are involved in the development of sexual traits, especially gonadal differentiation and testicular development, were characterized. We found that the cyp17a1-deficient fish at 3 months postfertilization (mpf) were all fertile males with normal testis and spermatogenesis but compromised male-typical mating behaviors and secondary sex characters (SSCs), including breeding tubercles, body pigmentation, and anal fin coloration. These results demonstrate that spermatogenesis and testicular development are not as susceptible to androgen deficiency compared with the formation of male-typical SSCs and mating behaviors in zebrafish. The differentiation of the juvenile ovary into the mature ovary failed during the critical sexual differentiation stage. This all-male phenotype of the cyp17a1-deficient fish could be restored with testosterone or estradiol treatment. For testicular development in cyp17a1-deficient fish, a gradually increasing number of spermatozoa and testis hypertrophy from 3 to 6 mpf were observed, accompanied by constitutively upregulated pituitary gonadotropin FSH subunit ß (fshß). The hypertrophic testis and enhanced spermatogenesis in the cyp17a1-deficient fish at 6 mpf could be effectively rescued by fshß depletion. These results confirm that adequate estrogen is essential for maintaining ovarian differentiation, and they provide new insight into the role of FSHß in male testicular development and spermatogenesis.

Characterization of grass carp CD40 and CD154 genes and the association between their polymorphisms and resistance to grass carp reovirus.

In bony fish, CD40 and CD154 are two very important costimulatory molecules involved in T and B cell cooperation in thymus-dependent antibody production. In the current study, we identified the cDNAs of CD40 and CD154 and analyzed their genomic structures in grass carp. Quantitative real-time PCR indicated that the CD40 and CD154 were mainly expressed in immune organs. After challenge with grass carp reovirus (GCRV), these two genes were up-regulated at 72 h in head kidney and spleen. Moreover, seven and five single nucleotide polymorphisms (SNPs) were identified in the CD40 and CD154 respectively. Statistical analysis indicated that three SNPs in the coding region of the CD40 were significantly associated with the resistance of grass carp against GCRV. These results indicated that CD40 and CD154 play important roles in the responses to GCRV in grass carp. The SNP markers in the CD40 associated with the resistance to GCRV may facilitate the disease-resistant breeding of grass carp.

RIP2 Is a Critical Regulator for NLRs Signaling and MHC Antigen Presentation but Not for MAPK and PI3K/Akt Pathways.

RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as "Antigen processing and presentation" and "NOD-like receptor signaling pathway" and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.