Molecular characterization of a teleost-specific toll-like receptor 22 (tlr22) gene from yellow catfish (Pelteobagrus fulvidraco) and its transcriptional change in response to poly I:C and Aeromonas hydrophila stimuli.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that can recognize pathogen-associated molecular patterns (PMPs) and play important roles in the innate immune system in vertebrates. In this study, we identified a teleost-specific tlr22 gene from yellow catfish (Pelteobagrus fulvidraco) and its immune roles in response to different pathogens were also determined. The open reading frame (ORF) of the tlr22 was 2892 bp in length, encoding a protein of 963 amino acids. Multiple protein sequences alignment, secondary and three-dimensional structure analyses revealed that TLR22 is highly conserved among different fish species. Phylogenetic analysis showed that the phylogenetic topology was divided into six families of TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11, and TLR22 subfamily was clustered into TLR11 family. Meanwhile, synteny and gene structure comparisons revealed functional and evolutionary conservation of the tlr22 gene in teleosts. Furthermore, tlr22 gene was shown to be widely expressed in detected tissues except barbel and eye, with highest expression level in liver. The transcription of tlr22 was significantly increased in spleen, kidney, liver and gill tissues at different timepoints after Poly I:C infection, suggesting TLR22 plays critical roles in defensing virus invasion. Similarly, the transcription of tlr22 was also dramatically up-regulated in spleen, kidney and gill tissues with different patterns after Aeromonas hydrophila infection, indicating that TLR22 is also involved in resisting bacteria invasion. Our findings will provide a solid basis for the investigation the immune functions of tlr22 gene in teleosts, as well as provide useful information for disease control and treatment for yellow catfish.

Transcriptome analysis provides insights into the molecular mechanism of liver inflammation and apoptosis in juvenile largemouth bass Micropterus salmoides fed low protein high starch diets.

The present study was conducted to investigate the regulatory mechanism of liver injury in largemouth bass Micropterus salmoides (LMB) fed low protein high starch diets. Two isolipidic and isoenergetic diets were formulated with different protein and starch ratios, being named as diets P49S9 (48.8 % protein and 9.06 % starch) and P42S18 (42.4 % protein and 18.2 % starch). Each diet was fed to triplicate replicates of LMB (initial body weight, 4.65 ± 0.01 g) juveniles. Fish were fed to visual satiation for 8 weeks. The results indicated that though the P42S18 fish up-regulated the feeding ratio to meet their protein requirements, feeding efficiency ratio and growth performance were impaired in treatment P42S18 as compared to treatment P49S9. Periodic acid-Schiff (PAS) staining showed glycogen accumulated in the liver of LMB fed low protein high starch diets, and the reason should be attributed to down-regulated expression of the glycogenolytic glycogen debranching enzyme. Lower liver lipid level was associated with feeding low protein high starch diets in LMB, which should be resulted from the changes in hepatic glycerolipid metabolism regulated by lipoprotein lipase (representative of triglyceride synthesis, up-regulated) and diacylglycerol acyltransferase (representative of triglyceride breakdown, down-regulated). Though fasting plasma glucose level was comparable, treatment P42S18 performed inferior glucose tolerance to treatment P49S9. Hematoxylin-eosin (HE) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining suggested that feeding low protein high starch diets induced disruption of structural integrity, inflammation and apoptosis in the hepatocytes of LMB. As expected, KEGG pathways analysis indicated that many of the up-regulated differentially expressed genes were enriched in AGE (advanced glycation end product)/RAGE (receptor for AGE), Toll-like receptor and apoptosis signaling pathways. Our transcriptome data revealed that feeding low protein high starch diets might promote the accumulation of AGEs in LMB, which bound to RAGE and subsequently induced PI3K/Akt signal pathway. The activation of Akt induced NF-κB translocation into the nucleus thus releasing proinflammatory factors including tumor necrosis factor-α (TNF-α) and interleukin-8. The release of these inflammatory factors concomitantly induced T cell stimulation and natural killer cells chemotactic effects through Toll-like receptor signaling pathway. Besides mediating inflammation and immune response, TNF-α signal transduction participated in mediating apoptosis through the receptor of TNF (TNF-R1) pathway by up-regulating the expression of caspase 8 and cytochrome c. In conclusion, our results demonstrated that feeding low protein and high starch diets induced hepatocytes inflammation and apoptosis in LMB through the PI3K/Akt/NF-κB signaling pathway.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

MRAP2 Interaction with Melanocortin-4 Receptor in SnakeHead (Channa argus).

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of MC4R. The Northern snakehead (Channa argus) is an economically important freshwater fish native to East Asia. To explore potential interaction between snakehead MC4R and MRAP2, herein we cloned snakehead mc4r and mrap2. The snakehead mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids, while snakehead mrap2 contained a 693 bp open reading frame encoding a protein of 230 amino acids. Synteny analysis indicated that mc4r was highly conserved with similar gene arrangement, while mrap2 contained two isoforms in teleost with different gene orders. Snakehead mc4r was primarily expressed in the brain, whereas mrap2 was expressed in the brain and intestine. Snakehead mc4r and mrap2 expression was modulated by fasting and refeeding. Further pharmacological experiments showed that the cloned snakehead MC4R was functional, capable of binding to peptide agonists and increasing intracellular cAMP production in a dose-dependent manner. Snakehead MC4R exhibited high constitutive activity. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. These findings suggest that snakehead MC4R might be involved in energy balance regulation by interacting with MRAP2. Further studies are needed to elucidate MC4R in regulating diverse physiological processes in snakehead.

Molecular characterization of two leptin genes and their transcriptional changes in response to fasting and refeeding in Northern snakehead (Channa argus).

Leptin has been proved to play critical roles in energy metabolism, body weight regulation, food intake, reproduction and immunity in mammals. However, its roles are still largely unclear in fish. Here, we report two leptin genes (lepA and lepB) from the Northern snakehead (Channa argus) and their transcriptions in response to different feeding status. The snakehead lepA is 781 bp in length and contains a 480 bp open reading frame (ORF) encoding a 159-aa protein, while the snakehead lepB is 553 bp in length and contains a 477 bp ORF encoding a 158-aa protein. Multi-sequences alignment, three-dimensional (3D) model prediction, syntenic and genomic comparison, and phylogenetic analysis confirm two leptin genes are widely existing in teleost. Tissue distribution revealed that the two leptin genes exhibit different patterns. In a post-prandial experiment, the hepatic lepA and brain lepB showed a similar transcription pattern. In a long-term (2-week) fasting and refeeding experiment, the hepatic lepA and brain lepB showed a similar transcription change pattern induced by food deprivation stimulation but differential changes after refeeding. These findings suggest snakehead lepA and lepB are differential both in tissue distribution and molecular functions, and they might play as an important regulator in energy metabolism and food intake in fish, respectively.

Complete mitochondrial genome of Palaemonetes sinensis and its phylogenetic consideration.

The aim of this study was to determine the complete mitochondrial genome (mitogenome) and the phylogenetic location of the Palaemonetes sinensis. The mitogenome was 15,736 bp in length, containing 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), 2 ribosome RNA genes (rRNAs), and a control region (CR). The overall nucleotide composition is as follows: A, 35.69%; C, 21.66%; G, 12.39%; T, 30.26%. Nine and four PCGs were encoded on the heavy and light strands, respectively. Phylogenetic analysis suggested that P. sinensis shares a close relationship with Palaemon serenus and Palaemon capensis. These findings are helpful for better understanding the phylogenetic relationship among Caridea, as well as investigating the population genetics of P. sinensis in the future.

Complete mitochondrial genome of a kind of snakehead fish Channa siamensis and its phylogenetic consideration.

The snakehead fish, Channa siamensis, belongs to the genus of Channa (perciformes: Channidae) and was first reported by Günther in 1861. Despite it has been described approximately for 15 decades, the genetic information is limited and the taxon status of this kind of fish is still unclear. The primary objective of this study is to get more genomic data and calculate the taxon location of this kind of fish. The next generation sequencing method was used to obtain the whole mitochondrial DNA information, and bioinformatic analysis was performed to investigate the evolutionary status and taxon location of C. siamensis. The circular mitochondrial DNA was 16,570 bp in length, and which showed typical piscine structure and arrangement. The overall nucleotide composition was 29.28% A, 24.72% T, 30.71% C, 15.29% G, with 54.1% AT, respectively. Phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Maximum likelihood and Bayesian analysis) both highly supported C. siamensis belongs to the genus Channa and shows a close relationship with C. micropeltes. These data will provide more useful information for a better understanding of the mitochondrial genomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.

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.