Gui-Rong Zhang

Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream (Megalobrama amblycephala)

Liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 are widespread in fish and extremely important components of the host innate immune system. In this study, full-length cDNAs of LEAP-1 and LEAP-2 were cloned and sequenced from blunt snout bream, Megalobrama amblycephala. The open reading frames (ORF) of LEAP-1 and LEAP-2 genes encode putative peptides of 94 and 92 amino acids, which possess eight and four conserved cysteine residues, respectively. The homologous identities of deduced amino acid sequences show that the LEAP-1 and LEAP-2 of blunt snout bream share considerable similarity with those of grass carp. The mRNA expressions of LEAP-1 and LEAP-2 were detectable at different early developmental stages of blunt snout bream and varied with embryonic and larval growth. LEAP-1 and LEAP-2 were expressed in a wide range of adult tissues, with the highest expression levels in the liver and midgut, respectively. Bacterial challenge experiments showed that the levels of LEAP-1 and LEAP-2 mRNA expression were up-regulated in the liver, spleen, gill and brain of juvenile blunt snout bream. These results indicate that the LEAP-1 and LEAP-2 may play important roles in early development of embryos and fry, and may contribute to the defense against the pathogenic bacterial invasion. This study will further our understanding of the function of LEAP-1 and LEAP-2 and the molecular mechanism of innate immunity in teleosts.

Molecular cloning, tissue distribution, and ontogenetic expression of ghrelin and regulation of expression by fasting and refeeding in the grass carp (Ctenopharyngodon idellus).

Ghrelin is a recently discovered brain-gut peptide in fish and mammals. It has two main physiological functions-stimulating growth hormone secretion and regulating appetite. To investigate the biological function of ghrelin in appetite regulation in grass carp Ctenopharyngodon idellus, the full-length cDNA sequence of the ghrelin gene was obtained by RT-PCR and rapid amplification of cDNA ends methods. Homology analysis indicated that ghrelin cDNA sequence is conserved in teleosts. The grass carp ghrelin gene consists of four exons and three introns, which is similar to that of common carp, zebrafish and humans. Real-time quantitative PCR was used to detect ghrelin mRNA expression in the study. In adult tissues, high levels of gene expression were found in the foregut; moderate levels in the muscle, liver, hypothalamus, white adipose tissue, midgut, heart, and pituitary; and lower levels in the three other examined tissues. During embryonic development, ghrelin mRNA expression could be detected as early as fertilized egg stage and displayed an increase in expression until heart appearance stage. After hatching, the level of ghrelin mRNA expression decreased sharply to the lowest level at 1 day post-hatching (dph), then increased to a higher level at 7 dph and subsequently decreased to a relative stabilized level from 15 to 35 dph. The ghrelin mRNA expression was significantly upregulated in the brain and intestine during fasting and was downregulated after refeeding. The results suggest that ghrelin mRNA expression has obvious tissue specificity and may have a role in appetite regulation in grass carp.

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) in blunt snout bream (Megalobrama amblycephala): cDNA cloning, tissue distribution and mRNA expression changes responding to fasting and refeeding

Blunt snout bream (Megalobrama amblycephala Yih, 1955) is an endemic freshwater fish in China for which the endocrine mechanism of regulation of feeding has never been examined. Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) play important roles in the regulation of fish feeding. In this study, full-length cDNAs of ghrelin, NPY and CCK were cloned and analyzed from blunt snout bream. Both the ghrelin and NPY genes of blunt snout bream had the same amino acid sequences as grass carp, and CCK also shared considerable similarity with that of grass carp. The three genes were expressed in a wide range of adult tissues, with the highest expression levels of ghrelin in the hindgut, NPY in the hypothalamus and CCK in the pituitary, respectively. Starvation challenge experiments showed that the expression levels of ghrelin and NPY mRNA increased in brain and intestine after starvation, and the expression levels of CCK decreased after starvation. Refeeding could bring the expression levels of the three genes back to the control levels. These results indicated that the feeding behavior of blunt snout bream was regulated by the potential correlative actions of ghrelin, NPY and CCK, which contributed to the defense against starvation. This study will further our understanding of the function of ghrelin, NPY and CCK and the molecular mechanism of feeding regulation in teleosts.

Ontogeny expression of ghrelin, neuropeptide Y and cholecystokinin in blunt snout bream, Megalobrama amblycephala

Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding in fish and mammals. To better understand the role of the three peptides in appetite regulation in the early developmental stages of blunt snout bream (Megalobrama amblycephala), partial cDNA sequences of ghrelin, NPY and CCK genes were cloned. And then, real-time quantitative PCR and RT-PCR were used to detect and quantify the mRNA expressions of these genes from zygotes to larvae of 50 days after hatching (DAH). Ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. Ghrelin and NPY mRNA expressions were upregulated at 1, 3, 5 DAH, while CCK mRNA expression was reduced significantly at 3 DAH. The mRNA expression levels of three genes in larvae varied significantly until 30 DAH. In adult fish, all three peptides were detected to be expressed in brain and several peripheral tissues. Ghrelin mRNA was mainly expressed in the intestine, whereas NPY and CCK mRNAs were mainly expressed in the brain. Taken together, these results indicate that ghrelin, NPY and CCK may have roles in early development and participate in the regulation of feeding of larvae in blunt snout bream and will be helpful for further investigation into feed intake regulation in adults of this species.

Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge

Abstract Toll‐like receptors (TLRs) are important components of pattern recognition receptors (PRRs), which play significant roles in innate immunity to defense against pathogen invasion. Many TLRs have been found in teleosts, but there are no reports about cloning and expression of TLR genes in yellow catfish (Pelteobagrus fulvidraco). In this study, we analyzed the sequence characters and the relative mRNA expression levels of nine TLRs (TLR1, TLR2, TLR3, TLR4‐1, TLR5, TLR7, TLR8‐2, TLR9 and TLR22) in different tissues of yellow catfish. The results showed that all nine TLR genes are highly expressed in head kidney, trunk kidney, spleen and liver, all of which are related to host immunity. Subsequently we used Aeromonas hydrophila as a stimulating agent to detect the expression profiles of these nine TLRs in the liver, spleen, trunk kidney and head kidney of yellow catfish at different time points after injection with killed Aeromonas hydrophila. All nine TLRs responded to A. hydrophila challenge with tissue‐specific patterns in different immune tissues. The kinetics of up‐ or down‐regulation of these nine TLRs exhibited a similar trend, rising to an elevated level at first and then falling to the basal level, but the peak value differed at different time points in different tissues. The expression levels of the TLR3, TLR4‐1, TLR9 and TLR22 genes were significantly up‐regulated after bacterial challenge in the liver, spleen, head kidney and trunk kidney. The relatively high expression of TLR genes in the immune tissues in response to the A. hydrophila challenge indicated that TLRs may play important roles in the innate immune response against gram‐negative bacteria in yellow catfish. HighlightsNine TLR genes were identified in yellow catfish.Deduced amino acid sequences of nine TLRs were analyzed.All the Nine TLRs were mainly expressed in the liver, spleen, head kidney and trunk kidney.All the Nine TLRs responded to A. hydrophila challenge with different expression patterns in immune‐related tissues.

A Comparative Transcriptome Analysis between Wild and Albino Yellow Catfish (Pelteobagrus fulvidraco)

Body colours are important and striking features for individual survival and reproductive success, in particular in vertebrates where mating behaviour and mate preference may be strongly influenced by non-normal phenotypes. Pigmentation disorders may be generated by disruption of one or many independent genes as well as by environmental factors. The first discovery of albino yellow catfish (Pelteobagrus fulvidraco Richardson) with golden skin colour from fish farms in China provides us valuable material to study the molecular mechanism underlying the abnormalities of pigmentation. In this study, transcriptome sequencing of fin tissues corresponding to the distinct body colours, wild type and mutant albino yellow catfish, were performed using Illumina sequencing technology. Based on next-generation sequencing technology and de novo assembly, we generated a transcriptome of P. fulvidraco. A number of genes differentially expressed between the wild types and albinos were identified, suggesting their contribution to the different phenotypes and fitness. However, non-synonymous mutations result from single nucleotide substitutions residing in coding regions may not contribute to such differences. Based on the high-throughput expression data generated for the two different types of P. fulvidraco, we found that alterations of expression pattern may be more common than non-synonymous mutations. The transcriptome of P. fulvidraco will be an invaluable resource for subsequent comparative genomics and evolutionary analyses of this economically important fish.

Molecular characterization and expression analysis of three TLR genes in yellow catfish (Pelteobagrus fulvidraco): Responses to stimulation of Aeromonas hydrophila and TLR ligands

Abstract Toll‐like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf_TLR18 and Pf_TLR19 consist of leucine‐rich repeats (LRRs), a transmembrane domain and a Toll/interleukin‐I receptor domain, and the Pf_TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes could be detected from fertilized eggs to 30 day post‐hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up‐regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up‐regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic‐polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish. HighlightscDNAs of TLR18, TLR19 and TLR21 genes were cloned in yellow catfish.Expressions of three TLR genes were different in early embryonic stages and fry.Three TLR genes were highly expressed in immune‐related tissues.Three TLR transcripts were up‐regulated in five tissues after bacterial challenge.Three TLR transcripts were up‐regulated in lymphocytes after ligands stimulation.

Molecular cloning and expression analysis of two β-defensin genes in the blunt snout bream (Megalobrama amblycephala)

β-Defensins are a group of cysteine-rich, cationic antimicrobial peptides that play important roles in innate immune system against pathogenic microbes invading. In this study, the part-length cDNA sequences of two β-defensin genes (maΒD-1, maΒD-2) in blunt snout bream (Megalobrama amblycephala) were identified. Homology analysis showed that the cDNA sequences of maΒD-1 and maΒD-2 had high similarities to those in common carp and zebrafish. Real-time quantitative PCR results exhibited that expression level of maΒD-1 in juvenile tissues was the highest in skin, followed by blood and liver, whereas maΒD-2 was lowly expressed in liver, kidney, brain and foregut. In the early development period, fertilized eggs to 31-day post-hatching (dph) larvae, the expression levels of maΒD-1 were higher at the stage from heart beat stage to 3 dph with the highest value at 1 dph, whereas maΒD-2 was expressed higher at fertilized eggs and late cleavage stages. Following bacterial stimulation in vivo by Aeromonas sobria, maΒD-2 expressions were significantly up-regulated in liver, skin, gill, and foregut of juveniles, and maΒD-1 expressions were significantly up-regulated in liver and skin. The results suggest that maΒD-1 and maΒD-2 may play important roles in protecting blunt snout bream embryos, fry and juveniles from pathogenic microbe invading.

Phylogeography and population genetics of Schizothorax o’connori : strong subdivision in the Yarlung Tsangpo River inferred from mtDNA and microsatellite markers

The Qinghai-Tibet Plateau (QTP) is a biodiversity hotspot, resulting from its geological history, contemporary environment and isolation. Uplift of the QTP and Quaternary climatic oscillations are hypothesised to have influenced the genetic diversity, population structure and dynamics of all QTP endemic species. In this study, we tested this hypothesis by assaying variation at two mitochondrial DNA regions (cytochrome b and control region) and at 12 microsatellite loci of seven populations of the endemic fish, Schizothorax o’connori from the Yarlung Tsangpo River (YLTR) on the QTP. Analyses revealed one group of six populations to the west, above the Yarlung Tsangpo Grand Canyon (YTGC), and a second group to the east below the YTGC. Estimates of the timing of this east-west split indicate that these groups represent evolutionarily significant units that have evolved separately and rapidly in the middle Pleistocene, at the time of the Kunlun-Huanghe Movement A Phase and the Naynayxungla glaciation. Population dynamic analyses indicate that S. o’connori experienced a pronounced late Pleistocene expansion during the last interglacial period. The results of this study support the hypotheses that the QTP uplift and Quaternary climatic oscillations have played important roles in shaping the population genetics and dynamics of this endemic fish.

Molecular Characterization and Expression Analyses of the Complement Component C8α, C8β and C9 Genes in Yellow Catfish (Pelteobagrus fulvidraco) after the Aeromonas hydrophila Challenge

The complement components C8α, C8β and C9 have important roles in the innate immune system against invading microorganisms. Partial cDNA sequences of the Pf_C8α, Pf_C8β and Pf_C9 genes (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned from yellow catfish. The Pf_C8α, Pf_C8β and Pf_C9 genes showed the greatest amino acid similarity to C8α (54%) and C8β (62%) of zebrafish and to C9 (52%) of grass carp, respectively. Ontogenetic expression analyses using real-time quantitative PCR suggested that the three genes may play crucial roles during embryonic and early larval development. The mRNA expressions of the three genes were all at the highest levels in liver tissue, and at lower or much lower levels in 16 other tissues, demonstrating that the liver is the primary site for the protein synthesis of Pf_C8α, Pf_C8β and Pf_C9. Injection of Aeromonas hydrophila led to up-regulation of the three genes in the spleen, head kidney, kidney, liver and blood tissues, indicating that the three genes may contribute to the host’s defense against invading pathogenic microbes. An increased understanding of the functions of the Pf_C8α, Pf_C8β and Pf_C9 genes in the innate immunity of yellow catfish will help enhance production of this valuable freshwater species.