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Wang P.,Xiamen University | Wang J.,Xiamen University | Su Y.-Q.,Xiamen University | Mao Y.,Xiamen University | And 6 more authors.
Fish and Shellfish Immunology | Year: 2016

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics. © 2015 Elsevier Ltd.


Jiang B.,South China Agricultural University | Jiang B.,Sun Yat Sen University | Li Y.W.,South China Agricultural University | Abdullahi A.Y.,South China Agricultural University | And 5 more authors.
Aquaculture | Year: 2016

Cryptocaryoniasis is a severe parasitic disease caused by ciliate Cryptocaryon irritans in farmed marine fish. To control this disease, we established a method to interrupt the life cycle of C. irritans via the removal of its tomonts. Marine fish Plectorhinchus cinctus were divided randomly into four groups, including placemat removal group (Group I), rotational culturing group (Group II), infection control group (IF group) and blank control group (Control); the former three groups were infected with C. irritans at low dose. A daily food consumption (DFC), relative infection intensity (RII) and survival rate of each group were observed. At 4-week post infection, some fish were challenged with C. irritans theronts at a lethal dose. Results showed that the fish' DFC in IF Group, Group I and II decreased significantly on 2nd day post infection, and gradually restored food intake when the trophonts exited the fish on the 5th day. However, their DFC in IF Group decreased significantly on 7th day, and eventually stopped feeding. Trophonts on fish' body of the three groups began to reduce from the 4th day and those of Group I and II almost disappeared on the 7th day, conversely the trophonts of IF group increased ten times. At 2-week post infection, the survival rates of Group I, II, IF and Control were 97%, 98%, 0%, and 100%, respectively. The survival rates of Group I, II and Control were 88.3%, 93.3%, 66.7%, respectively after challenge. Results demonstrate that placemat and rotational culturing method to remove tomonts can effectively control C. irritans infection. Statement of relevance: Cryptocaryoniasis is a severe parasitic disease caused by ciliate Cryptocaryon irritans in farmed marine fish. To control this disease, we established a method to interrupt the life cycle of C. irritans via the removal of its tomonts. Marine fish P. cinctus were divided randomly into four groups, including placemat removal group (Group I), rotational culturing group (Group II), infection control group (IF group) and blank control group (Control); the former three groups were infected with C. irritans at low dose. A daily food consumption (DFC), relative infection intensity (RII) and survival rate of each group were observed. At 4-week post infection, some fish were challenged with C. irritans theronts at a lethal dose. Results showed that the fish' DFC in IF Group, Group I and II decreased significantly on 2nd day post infection, and gradually restored food intake when the trophonts exited the fish on the 5th day. However, their DFC in IF Group decreased significantly on 7th day, and eventually stopped feeding. Trophonts on fish' body of the three groups began to reduce from the 4th day and those of Group I and II almost disappeared on the 7th day, conversely the trophonts of IF group increased ten times. At 2-week post infection, the survival rates of Group I, II, IF and Control were 97%, 98%, 0%, and 100%, respectively. The survival rates of Group I, II and Control were 88.3%, 93.3%, 66.7%, respectively after challenge. Results demonstrate that placemat and rotational culturing method to remove tomonts can effectively control C. irritans infection. This method is an mechanical preventive one, not only provides an ideal means for controlling C. irritans in small scale culture systems, but also solves food safety problems. © 2016 Elsevier B.V.


Chen R.-N.,Xiamen University | Su Y.-Q.,Xiamen University | Wang J.,Xiamen University | Liu M.,Xiamen University | And 7 more authors.
Fish and Shellfish Immunology | Year: 2015

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans. © 2015 Elsevier Ltd.


PubMed | Ningde Fufa Fisheries Co., Zhejiang Ocean University and Xiamen University
Type: | Journal: Fish & shellfish immunology | Year: 2016

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C.irritans, we first performed a comparative gene transcription analysis using livers from C.irritans-immunized L.croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360contigs were obtained, with an average length of 1066.93bp. Further, a blast analysis indicates that 30747contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR<0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L.croceas liver transcriptome. This study characterized a gene expression pattern for normal and C.irritans-immunized L.croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C.irritans interaction but also facilitates future studies on L.croceas gene expression and functional genomics.


PubMed | Fujian Academy of Agricultural science, Ningde Fufa Fisheries Co., CAS East China Sea Fisheries Research Institute and Sun Yat Sen University
Type: | Journal: Veterinary parasitology | Year: 2016

The present study was conducted aiming to evaluate the in vitro and in vivo anti-parasitic efficacy of an isolated compound against the ciliate Cryptocaryon irritans. The compound was previously isolated from fermentation products of Streptomyces sp. CJK17 and designated as SFrD. Toxicity of the compound SFrD against the fish hosts (Larimichthys crocea) was also tested and its chemical structure was elucidated. The obtained results showed that the compound has potent anti-parasitic efficacy with the 10 min-, 1 h-, 2 h-, 3 h- and 4 h-LC50 (95% Confidence Intervals) of 6.8 (6.5-7.1), 3.9 (2.8-5.0), 3.3 (2.6-4.0), 2.7 (2.3-3.1) and 2.5 (2.2-2.8) mg L(-1) against theronts of C. irritans and the 6h-LC50 (95% CI) of 3.0 (2.8-3.2) mg L(-1) against the tomonts, respectively. Exposure of the compound SFrD remarkably reduced the mortality of fish infected with C. Irritans, from 100% in the control group to 61.7% and 38.3% in groups of 3.1 mg L(-1) and 6.3 mg L(-1), respectively. In the test of exposing fish to 40 mg L(-1) compound SFrD for 24h, no visible effects were observed affecting the normal behavior or any macroscopic changes. By spectrum analysis (EI-MS, (1)H NMR and (13)C NMR), the compound SFrD was identified as Leptomycin B. This study firstly demonstrated that Leptomycin B has potent anti-parasitic efficacy against ciliates in cultured marine fish.


Jiang Y.,Xiamen University | Jiang Y.,Jimei University | Han K.,Jimei University | Han K.,Ningde Fufa Fisheries Company Ltd | And 4 more authors.
Gene | Year: 2015

Sox genes play important roles in various developmental processes such as sex determination, embryogenesis, oogenesis, neurogenesis, and larval development. In order to clarify the roles of Sox genes in the developmental process of large yellow croaker, the full-length cDNA of the Sox11a gene (Lc-Sox11a) was cloned for the first time. Bioinformatics analysis indicated that Lc-Sox11a contains a protein of 366 amino acids with a Ser-rich region, a C-terminal conserved region, and a high mobility group box. The expression of Lc-Sox11a in different tissues of both sexes and in different developmental embryonic stages revealed that Lc-Sox11a were expressed with tissue and gender specificity, of which the expression level in female was ovary. > brain > eye > gill; in male was brain > testis > gill. The gender differences occurred in the brain and eye with the male brain > female brain, female eye > male eye. Moreover, the expression of Lc-Sox11a in the gonad and brain at different growth stages was detected. Significant up-regulated expression of Lc-Sox11a was found in the ovary and the male brain at 1000. dph (days post hatching) compared with 270. dph and 635. dph. However, significant down-regulated expression of Lc-Sox11a occurred in the testis with growth. Besides, the expression of Lc-Sox11a in the female brain showed a trend of first rising then falling, with the highest peak in 635. dph. The results of in situ hybridization displayed that Lc-Sox11a was widely distributed only in cytoplasm of oocytes at each stage in oogenesis. In early stage of oocytes, Lc-Sox11a was expressed weakly and evenly. As the appearance of vacuoles and synthesis of yolks, positive signals of Lc-Sox11a distributed intensively in the residual cytoplasm. In spermatogenesis, Lc-Sox11a was distributed in cytoplasm of all male germ cells except spermatozoon with spermatogonium > spermatocyte > spermatid. During embryogenesis, Lc-Sox11a was expressed in most embryonic stages, the highest expression occurred in the formation-of-eye-lens stage, closely followed by the closure-of-blastopore stage, then the beginning-of-heart-pulsation stage. The results of whole mount in situ hybridization showed that the expression of Lc-Sox11a began to increase beginning with the multiple-cell stage, with the major distribution of Lc-Sox11a in the brain and eye areas in the pre-hatching stage. © 2015 Elsevier B.V.


PubMed | Fujian Medical University, Ningde Fufa Fisheries Company Ltd and Xiamen University
Type: | Journal: Scientific reports | Year: 2016

The research on resistance genes (R-gene) plays a vital role in bioinformatics as it has the capability of coping with adverse changes in the external environment, which can form the corresponding resistance protein by transcription and translation. It is meaningful to identify and predict R-gene of Larimichthys crocea (L.Crocea). It is friendly for breeding and the marine environment as well. Large amounts of L.Croceas immune mechanisms have been explored by biological methods. However, much about them is still unclear. In order to break the limited understanding of the L.Croceas immune mechanisms and to detect new R-gene and R-gene-like genes, this paper came up with a more useful combination prediction method, which is to extract and classify the feature of available genomic data by machine learning. The effectiveness of feature extraction and classification methods to identify potential novel R-gene was evaluated, and different statistical analyzes were utilized to explore the reliability of prediction method, which can help us further understand the immune mechanisms of L.Crocea against pathogens. In this paper, a webserver called LCRG-Pred is available at http://server.malab.cn/rg_lc/.


Jiang Y.,Jimei University | Han K.,Jimei University | Han K.,Ningde Fufa Fisheries Company Ltd | Chen S.,Jimei University | And 3 more authors.
Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology | Year: 2016

The characterization and expression of Sox4 in large yellow croaker (Lc-Sox4) were studied in this paper. Lc-Sox4 contains a protein of 371 amino acids with a conserved high mobility group box. Quantitative real-time PCR displayed that the expression of Lc-Sox4 had tissue and gender specificity existing in brain, gonad, heart, intestine, and head kidney with male > female, in eye with female > male. During embryogenesis, Lc-Sox4 was expressed highest in one-day-post-hatching stage, next in formation-of-eye-lens stage. The expression pattern of Lc-Sox4 was different from that of Lc-Sox11a. The expression of Lc-Sox4 was significantly lower than that of Lc-Sox11a in the all tested tissues and embryonic stages except in heart, spleen, mutiple-cell, formation-of-eye-lens, and one-day-post-hatching stages (with Lc-Sox4 higher than Lc-Sox11a). There was overlapping expression between Lc-Sox4 and Lc-Sox11a in brain, gill, female eye, testis, formation-of-eye-lens stage and one-day post hatching stage. The whole mount in situ hybridization results indicated that Lc-Sox4 was expressed at all embryonic stages except 2-cell stage. The positive signals were mainly distributed in the central nervous system and notochord at one-day-post-hatching stage. In short, we first identified and analyzed the temporal and spatial expression patterns of Lc-Sox4 to elucidate its important influence on the development of nervous system, visual system and heart. We also detected the overlapping expression between Lc-Sox4 and Lc-Sox11a which may reveal the functional redundancy of them. These data would shed light on the molecular mechanism of development in large yellow croaker. © 2016 Published by Elsevier Inc.


Chen S.,Jimei University | Pu L.,Jimei University | Xie F.,Jimei University | Zou Z.,Jimei University | And 5 more authors.
General and Comparative Endocrinology | Year: 2015

The biological activity of estrogens in target organs is mainly mediated by estrogen receptors (ERs). Herein, we addressed the isolation and expression analysis of three nuclear estrogen receptors, namely LcERα, LcERβ1, and LcERβ2 from Larimichthys crocea by means of SMART-RACE, qRT-PCR, and in situ hybridization. Results in different tissues showed that both LcERα and LcERβ2 had the highest expression levels in female liver, followed by testis, but LcERβ1 expression level was significantly higher in testis and ovary than in other tissues. Expression of LcERα and LcERβ2 was significantly higher than LcERβ1 in female liver, and LcERβ2 was significantly higher than LcERα and LcERβ1 in male liver. Moreover, we analyzed the expression of LcERs in gonad and liver at three different growth stages during the same breeding season. Significant up-regulated expression of LcERα and LcERβ2 were found in female liver at 1000. dph compared with at 270. dph. The expression of LcERβ2 was prominently higher in male liver than LcERα, LcERβ1 and LcAR, while LcERβ1 was lower than other receptors in male and female liver at all the three stages. In ovary, LcERα at 270. dph was lower than at 635. dph and 1000. dph, but had no significant change in testis. The two LcERβ subtypes and LcAR highly expressed in the early testis, and gradually decrease with the development of testis. In embryogenesis, a significant increase in the expression of LcERα and LcERβ2 were observed after appearance of optic vesicles phase (11.8. hpf). LcERβ1 gradually decrease with the embryogenesis but increased dramatically at 1dph. Results of in situ hybridization showed that signals of LcERα and LcERβ1 mRNA were mainly detected in Stage I-Stage IV oocytes, as well as in follicle cells around the Stage II-Stage IV and degenerated oocytes. Signals of LcERβ2 were detected in the cytoplasm of Stage I and Stage II oocytes but not in the follicle cells of all oocytes stages. In parallel, LcERα and LcERβ1 were detected in all cell types of spermatogenesis, but in terms of LcERβ2, little or no signals were detected during spermatogenesis. Based on these results, we deduced that both LcERα and LcERβ2 play a major role in mediating the physiological effects of estrogen in female liver, and LcERβ2 maybe also play an important role in regulation of vitellogenesis in male liver. Differential expression of LcERs and LcAR imply their physiological functions during development and differentiation of gonad. The signals for LcERα and LcERβ1 in follicle cells suggested that the follicle cell maybe an important site of estrogen action, by which estrogens exert influences on the maturation oocytes and ovulation. Furthermore, the steroid hormones produced by follicle cells may be related to the differential distributions among ER subtypes. Besides, we deduced that LcERα and LcERβ1 rather than LcERβ2 may play a major role in spermatogenesis of croaker. However, the differential expression of LcERβ2 during gametogenesis also implicates its certain functions in mediating physiological process of estrogen action. © 2015 Elsevier Inc.


Yin F.,CAS East China Sea Fisheries Research Institute | Gao Q.,CAS East China Sea Fisheries Research Institute | Tang B.,CAS East China Sea Fisheries Research Institute | Sun P.,CAS East China Sea Fisheries Research Institute | And 4 more authors.
Fish and Shellfish Immunology | Year: 2016

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed. © 2016 Elsevier Ltd.

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