Chuanjie Qin

Effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae.

The objective of this study was to investigate the effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae. After ammonia-N exposure at a constant concentration of 7.21 ± 0.10 mg L(-1) for 96 h, B. reevesae was challenged with Aeromonas hydrophila. Quantitative PCR analysis showed predominant and significant expression of HSP70 in liver, gill, skin, spleen and kidney (P < 0.05), with significantly upregulated expression of the mRNA transcript in these tissues after sublethal ammonia-N exposure and A. hydrophila challenge. Furthermore, following A. hydrophila challenge after ammonia-N exposure, HSP70 mRNA expression was significantly upregulated in kidney and gill tissues, although its expression levels were significantly lower than those detected following A. hydrophila challenge or ammonia-N exposure individually. These results indicate that B. reevesae HSP70 is involved in resistance to pathogenic bacteria. It is hypothesized that ammonia-N results in the downregulation of HSP70 mRNA in immune organs after an A. hydrophila challenge, thus lowering their resistance to pathogenic stress.

Effect of ammonia-N and pathogen challenge on complement component 8α and 8β expression in the darkbarbel catfish Pelteobagrus vachellii

ABSTRACT The complement components C8&agr; and C8&bgr; mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full‐length complement C8&agr; (Pv‐C8&agr;) and C8&bgr; (Pv‐C8&bgr;) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia‐N and pathogen treatment. The Pv‐C8&agr; gene contained 1983 bp, including a 1794‐bp open reading frame (ORF) encoding 598 amino acids. The Pv‐C8&bgr; gene contained 1952 bp, including a 1761‐bp ORF encoding 587 amino acids. Pv‐C8&agr; and Pv‐C8&bgr; had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8&agr; (62%) and Japanese flounder Paralichthys olivaceus C8&bgr; (83%), respectively. Sequence analysis indicated that both Pv‐C8&agr; and Pv‐C8&bgr; contained a thrombospondin type‐1 (TSP1) domain, a low‐density lipoprotein receptor class A (LDLR‐A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor‐like (EGF‐like) domain. In addition, Pv‐C8&agr; and Pv‐C8&bgr; were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia‐N stress, the Pv‐C8&agr; and Pv‐C8&bgr; mRNA levels significantly decreased (P < 0.05), with minimum levels, respectively, attained at 24 and 48 h in the liver, 48 and 24 h in the head kidney, and 24 and 24 h in the spleen. After Aeromonas hydrophila challenge, the Pv‐C8&agr; and Pv‐C8&bgr; mRNA levels significantly increased (P < 0.05), with maximum levels, respectively, attained at 48 and 24 h in the liver, 24 and 48 h in the head kidney, and 48 and 48 h in the spleen. The present study indicated that Pv‐C8&agr; and Pv‐C8&bgr; exhibited important immune responses to infection and that ammonia‐N in water decreased the immune responses of Pv‐C8&agr; and Pv‐C8&bgr;. HighlightsComplement component 8&agr; and 8&bgr; genes were cloned and identified.C8a and C8&bgr; were mainly distributed in the liver and also detected in eggs.Aeromonas hydrophila significantly increased the mRNA levels of C8a and C8&bgr; in the tissues.Ammonia‐N stress significantly decreased the mRNA levels of C8a and C8&bgr; in the tissues.

Transcriptome analysis of the spleen of the darkbarbel catfish Pelteobagrus vachellii in response to Aeromonas hydrophila infection

ABSTRACT Intensive aquaculture has increased the susceptibility of fish to Aeromonas hydrophila, and this has led to severe economic damage. There has been little study of the host defense mechanism against A. hydrophila infection in scaleless fish. Therefore, in the present study, the transcriptome profiles of the spleen of Pelteobagrus vachellii were examined after infection with A. hydrophila. In total, 37,730 unigenes from 322 KEGG pathways were identified. Following A. hydrophila infection, 27,803 differentially expressed genes were identified, including 13,934 upregulated and 13,869 downregulated genes. Significant enrichment analysis of these differentially expressed unigenes showed that the major immune pathways were involved, including toll‐like receptor pathways, B‐cell receptor signaling pathways, Fc&ggr; receptor‐mediated phagocytosis, complement and coagulation cascades, and natural killer cell‐mediated cytotoxicity pathways. From these pathways, 59 key immune‐related differentially expressed genes were selected: 53 genes that were upregulated, including those coding for complement components, interferons, and interleukins, and six DEGs that were downregulated, including inhibitor of nuclear factor kappa‐B kinase. Finally, nine DEGs, which were randomly selected, were confirmed by qRT‐PCR to be differentially expressed. The results indicated that complement components, interferons and Fc&ggr; receptor‐mediated phagocytosis played key role in the response to A. hydrophila infection in the spleen of P. vachellii, which may prove useful in the future for the development of therapeutic regimens. Highlights27,803 DEGs were identified from spleen transcriptome of Pelteobagrus vachellii.362 DEGs were annotated in 10 immune‐related KEGG pathways.Complement components, interleukin played key role to response to A. hydrophila infection.

Comparative analysis of the liver transcriptome of Pelteobagrus vachellii with an alternative feeding time

Pelteobagrus vachellii, an important freshwater fish in China, shows predominantly nocturnal behavior. To better understand the growth and molecular mechanisms underlying altered feeding times in this species, we studied the growth and liver transcriptome of P. vachellii with shifted feeding times. In this study, a 9-week growth trial was conducted on male P. vachellii (mean weight±S.E.=1.05±0.36g) with commercial feed. Two triplicate groups of fish were fed either at 0800 (day group, control) or at 2000 (night group) with the same amount of feed. After nine weeks, a significant increase in growth was observed in the night group, demonstrated by the specific growth rate (SGR). Using high-throughput RNA-seq, 70,793,844 and 67,930,610 paired-end clean reads were obtained from six cDNA libraries of P. vachellii liver, and 60,069 unigenes were assembled. Gene expression comparison revealed that 122 genes were significantly up-regulated and 59 genes were significantly down-regulated in the night group. Gene pathway and GO enrichment analyses revealed metabolic responses of genes and gene networks related to protein, lipid and carbohydrate metabolism and rhythms. This study indicates that an alternative feeding time can improve growth and create metabolic alterations in the liver of P. vachellii.

Uncoupling protein 1 in snakehead (Channa argus): Cloning, tissue distribution, and its expression in response to fasting and refeeding

In mammals, uncoupling protein 1 (UCP1) is well known for its thermogenic role in brown adipose tissue (BAT). However, the UCP1 physiological roles are still unclear in fish, although several teleost ucp1 genes have been identified. The aim of this study is to investigate the potential roles of fish UCP1 involved in food intake regulation and energy homeostasis. We herein report on the molecular cloning, tissue distribution and the effect of fasting and refeeding on the expression of ucp1 in Channa argus. UCP1 consisted of a 921 bp open reading frame predicted to encode 306 amino acids. Sequence analysis revealed that snakehead UCP1 was highly conserved (>80%) with teleost UCP1, but shared a lower identity (60-72%) with mammals. Phylogenetic analysis supported that snakehead UCP1 was closely related to piscine UCP1. In addition, ucp1 was found to extensively expressed in all detected tissues, with the highest level in liver. Futhermore, the hepatic ucp1 was found to significantly increased after short-term and long-term food deprivation, and dramatically increased following refeeding. These findings suggested that snakehead UCP1 might play important roles in food intake regulation and fatty acid metabolism in snakehead fish, and it could be as a potential target locus to improve commercial production of this kind of fish.

Molecular cloning, tissue distribution, and effect of fasting and refeeding on the expression of neuropeptide Y in Channa argus

Neuropeptide Y (NPY) is a 36 amino-acid amidated peptide of the pancreatic polypeptide (PP) family, which plays an important role in appetite regulation and energy expenditure in mammals. Although several teleost NPY have been identified, its roles remain unclear in fish. We herein reported on the molecular cloning, tissue distribution and the effect of fasting on the expression of NPY in Channa argus, and designated as CaNPY. It consisted of a 300 bp open reading frame predicted to encode a prepro-NPY of 99 amino acids. Sequence analysis revealed that CaNPY was highly conserved (>60%) with other vertebrate NPY. Phylogenetic analysis highly supported CaNPY was closely related to piscine NPY. In addition, except for muscle and spleen tissues, CaNPY was found to extensively expressed in all other detected tissues, with the highest level in brain. Futhermore, the CaNPY transcript was found to significantly increase after short-term and long-term food deprivation, and dramatically decrease following refeeding. These findings suggested that CaNPY might be involved in food intake regulation and it could be as a potential target locus to improve commercial production of this kind of fish.

Molecular characterization and expression of toll-like receptor 5 genes from Pelteobagrus vachellii

ABSTRACT Toll‐like receptor 5 (TLR5) is an important pathogen recognition receptor (PRR) that recognizes the flagellin protein of pathogenic bacteria and plays a fundamental role in activating the innate immune response. In this study, full‐length pvTLR5m (membrane) and pvTLR5s (soluble) genes were cloned from darkbarbel catfish Pelteobagrus vachellii, and their expression and that of downstream genes were analyzed following exposure to the Aeromonas hydrophila pathogen. The 3009 bp pvTLR5m cDNA includes a 2652 bp open reading frame (ORF) encoding 884 amino acids. The 2422 bp pvTLR5s cDNA includes a 1944 bp ORF encoding a predicted protein of 648 amino acids. The genes are most closely related to TLR5m (75%) and TLR5s (69%) from Ictalurus punctatus, respectively, and both have a typical TLR structure. Both genes were constitutively expressed in all examined tissues, and most abundantly in the head kidney and spleen. Following pathogen challenge, pvTLR5m and pvTLR5s expression was increased significantly (P < 0.05) and peaked at 24 and 12 h post‐exposure in the liver, 24 and 12 h in the head kidney, and 48 and 24 h in the spleen, respectively. The downstream genes interleukin‐1&bgr; (IL‐1&bgr;), IL‐12 and tumor necrosis factor‐alpha (TNF‐&agr;) were significantly up‐regulated following pathogen exposure in spleen, and the NF‐kB inhibitor (I&kgr;B) was down‐regulated. These findings indicated that pvTLR5 may play an important role in the immune responses to A. hydrophila. These results provide new insight to elucidate the immune signalling pathways of fish TLR. HighlightsIdentification and characterization of TLR5m and pvTLR5s in Pelteobagrus vachellii.The pvTLR5m and pvTLR5s were mainly distributed in the head kidney and spleen.A. hydrophila significantly increased the mRNA levels of pvTLR5 in the tissues.A. hydrophila induce the expression of TLR signaling pathway related genes.

Molecular characterization and expression of complement factor I in Pelteobagrus vachellii during Aeromonas hydrophila infection

ABSTRACT Complement factor I (CFI) is a novel regulatory serine protease that plays an important role in resistance to pathogen infection. In this study, the CFI gene of Pelteobagrus vachellii (Pv‐CFI) was sequenced and characterized. The full‐length cDNA of 2320 bp includes a 155 bp 5′‐untranslated region (UTR), a 164 bp 3′‐UTR, and a 2001 bp open reading frame (ORF) encoding a 667 amino acids. Multiple sequence alignment revealed five highly conserved domains with a typical modular architecture and identical active sites in vertebrates, indicating a conserved function. Pv‐CFI mRNA was constitutively expressed in all examined tissues and most abundant in liver. During infection with Aeromonas hydrophila, Pv‐CFI mRNA expression was significantly up‐regulated in liver at 3–24 h, spleen at 3–48 h and head kidney at 3–48 h. The results suggest Pv‐CFI plays an important role in resistance to pathogenic bacteria in P. vachellii. HighlightsComplement factor I was identified and characterized in Pelteobagrus vachellii.Pv‐CFI mRNA is constitutively expressed in all tissues and most abundant in liver.Pv‐CFI mRNA is up‐regulated in pathogen‐infected tissues.

RESTORATION OF FLESH FATTY ACID COMPOSITION IN DARKBARBEL CATFISH ( PELTEOBAGRUS VACHELLI ) USING A FINISHING FISH OIL DIET

This study aimed to evaluate the effects of 50%-100% soybean oil on growth performance and flesh fatty acid composition of darkbarbel catfish ( Pelteobagrus vachelli ), so as to assess the effects of refeeding fish oil (FO) on flesh fatty acid composition. Four isonitrogenous, isolipidic diets, i.e., FO, soybean oil (SO), 50% FO+50% SO (S1), and 25% FO+75% SO (S2), were fed to triplicate groups of 40 juvenile P. vachelli [(1.10±0.12) g] for 80d. At the end of the 80d period, all fish were fed with FO for 30d. The results showed that growth rates, hepatosomatic index ( HSI ), and proximate composition in darkbarbel catfish were not affected by SO. With increasing SO levels, the percentages of oleic acid, arachidonic acid, and monounsaturated fatty acids significantly increased ( P P P < 0.05), but not to the same extent as those in the FO-containing groups except S1. The results revealed that it was possible to substitute almost 100% of FO with SO in the diets of darkbarbel catfish without affecting growth performance. A refeeding period of 30d with 100% FO significantly increased flesh levels of Σn-3 HUFA, 20:5n-3, and 22:6n-3 in fish which were fed diets containing SO in the first stage.