Kai-Jian Wei

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.

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.

The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet

Abstract The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.

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.

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.

Molecular cloning and expression analysis of interleukin-1β and interleukin-1 receptor type I genes in yellow catfish (Pelteobagrus fulvidraco): Responses to challenge of Edwardsiella ictaluri

Interleukin-1β (IL-1β) is one of the pivotal early pro-inflammatory cytokines, which play important roles in regulating immune response and inducing a series of inflammatory reactions to infections. Interleukin-1 type I receptor (IL-1RI) is a receptor of the IL-1β that can mediate IL-1-dependent activation. In this study, partial cDNA sequences of the Pf_IL-1β and Pf_IL-1RI genes were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORF) of Pf_IL-1β and Pf_IL-1RI genes encode putative peptides of 280 and 543 amino acids, respectively. The deduced amino acid sequences of these two genes shared highly conserved structures with those from other teleosts. Quantitative real-time PCR results showed that the Pf_IL-1β mRNA had relatively high expression levels in trunk kidney and blood, and the Pf_IL-1RI mRNA was highly expressed in blood and had relatively high expression level in liver. Ontogenetic expression analyses indicate that the Pf_IL-1β and Pf_IL-1RI genes may play important roles during the embryonic developmental stages. The mRNA expression levels of Pf_IL-1β and Pf_IL-1RI genes were up-regulated in the trunk kidney, head kidney, blood, spleen, heart and liver after Edwardsiella ictaluri challenge. Western blot analyses showed that Pf_IL-1β protein was highly expressed in the spleen and head kidney, but not in the fin of adult individuals. These results suggest that the Pf_IL-1β and Pf_IL-1RI genes may play significant roles in the immune regulation and defense against E. ictaluri in the yellow catfish.

Molecular characterization and expression analysis of IL-22 and its two receptors genes in yellow catfish (Pelteobagrus filvidraco) in response to Edwardsiella ictaluri challenge

&NA; Interleukin (IL)‐22, as a member of the interleukin (IL)‐10 family, is an important mediator between the immune cells and epithelial tissues during infection and inflammation. This study reported the characterization and mRNA expression patterns of Pf_IL‐22 gene and its cell surface‐associated receptors Pf_IL‐22RA1 and soluble Pf_IL‐22RA2 genes in yellow catfish (Pelteobagrus filvidraco). The open reading frames (ORFs) of the Pf_IL‐22, Pf_IL‐22RA1 and Pf_IL‐22RA2 genes were 546 bp, 1740 bp and 690 bp in length, encoding 181, 579 and 229 amino acids, respectively. Alignments of the deduced amino acid sequences present that the Pf_IL‐22 has a conserved IL‐10 family signature motif, and the Pf_IL‐22RA1 and Pf_IL‐22RA2 have two conserved fibronectin type‐III domains. Quantitative real‐time PCR (qPCR) analyses showed that the Pf_IL‐22 and Pf_IL‐22RA1 mRNAs were highly expressed in mucosal tissues such as the fin, gill, intestine, skin mucus and stomach, and were weakly expressed in the kidney, liver and head kidney of adult yellow catfish, indicating that the Pf_IL‐22 transcripts may be mainly produced by mucosal immune cells/tissues in healthy yellow catfish. The mRNA expression levels of the Pf_IL‐22RA2 gene were high in the muscle and liver, and were relatively low in the spleen and kidney. The mRNA expression levels of the Pf_IL‐22 and its two receptor genes were significantly up‐regulated in both mucosal tissues (gill, hindgut, and skin mucus) and systemic immune tissues (spleen, head kidney and blood) after Edwardsiella ictaluri challenge. These results indicated that the Pf_IL‐22 and its two receptors genes might play an important role in the innate immune defense against bacterial invasion. HighlightsIL‐22, IL‐22RA1 and IL‐22RA2 genes were identified in yellow catfish.Deduced amino acid sequences of three genes were analyzed.IL‐22 and IL‐22RA1 genes were highly expressed in mucosal immune‐related tissues.Three genes expression were up‐regulated in six tissues after bacterial challenge.

Genetic diversity and population structure of the northern snakehead (Channa argus Channidae: Teleostei) in central China: implications for conservation and management

Major threats to freshwater fish diversity now include loss of native genetic diversity as a consequence of translocations of fishes between sites and from hatcheries to sites, and small effective population sizes resulting from overfishing and/or habitat loss. Ten polymorphic microsatellite markers were employed to evaluate genetic diversity, population genetic structure and gene flow amongst nine populations of the ecologically and economically important fish, the northern snakehead (Channa argus), in three river systems in central China. Multiple analyses revealed evidence of high genetic diversity and pronounced subdivision based on both regional separation and on river systems. A lack of evidence of genetic bottleneck over recent generations was consistent with the long-term stability of population size and contemporary distribution. The effective population sizes for most C. argus populations were small, suggesting the need for future conservation efforts focusing on these populations. Different lines of evidence point to the local enhancement of stocks by both aquaculture-reared fish and the transfer of wild fish. This study illustrates how human activities may affect genetic diversity and population genetic structure of C. argus populations, and highlights the need for new management regimes to protect native freshwater fish genetic diversity.

Development and characterization of 32 SNP markers for the northern snakehead (Channa argus) using high resolution melting (HRM)

Channa argus is an important commercial fish species in the aquaculture industry in China, however, it is declining in numbers and population size due to overfishing and environmental change. Based on the transcriptome database of C. argus from the Illumina Hiseq 2000 platform, 32 polymorphic single nucleotide polymorphism (SNP) markers were developed and characterized in 36 individuals using high resolution melting analysis. The observed and expected heterozygosities ranged from 0 to 0.694 and from 0.106 to 0.507, respectively, and the minor allele frequency ranged from 0.056 to 0.486. Only two loci showed significant deviation from Hardy–Weinberg equilibrium after Bonferroni correction. These SNP markers will serve as a valuable tool for population genetic analysis, natural resource conservation and selective breeding of this species.