Effects of sodium butyrate stimulation on immune‐related mRNA‐miRNA network in intestine of grass carp

Sodium butyrate is one of the most popular feed additives in animal husbandry. In recent years, sodium butyrate has been increasingly used as supplement in aquaculture. The present study is to investigate the intestinal mRNA and microRNA response to diet with sodium butyrate in grass carp (Ctenopharyngodon idella), an important aquaculture species in China. mRNA and microRNA profiles of intestine of grass carp fed with diet contained 0, 1.0, 2.5, 5.0, 7.5 and 10.0 g/kg sodium butyrate were obtained by RNA‐seq using Illumina Hiseq 2,500 platform. The feeding trial was performed using 18 individuals of 1‐year‐old grass carp (n = 3 for each group) and lasted for 40 days in tanks in laboratory. A total of 349,860,852 sequence reads were generated from six intestinal libraries. Functional analysis of differentially expressed genes showed that genes participated in immune pathways tend to be activated by sodium butyrate supplementation. A total of 700 microRNAs were obtained, including 275 conserved microRNAs and 425 novel microRNAs which are potentially involved in regulating 14,300 genes. Spearman's correlation analysis identified 18 pairs of microRNA‐mRNA associated with immune pathways (p < .01 and R<?0.5). The potential genes targeted by microRNAs include CXCL12, AKT1S1, Cab39 and MHCII which are important genes associated with intestinal immune pathways. To our knowledge, this is the first integrated profiling of both mRNA and microRNA in intestine with supplementation of sodium butyrate in grass carp. The present results suggest that sodium butyrate affects intestinal immune system by regulating microRNA‐mRNA interaction in fish.     

Optimal dietary alpha‐linolenic acid/linoleic acid ratio improved digestive and absorptive capacities and target of rapamycin gene expression of juvenile grass carp (Ctenopharyngodon idellus)

Growth performance, digestive and absorptive capacities and target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and eIF4E‐binding protein (4E‐BP) gene expression in the hepatopancreas and intestine of juvenile grass carp (Ctenopharyngodon idellus) fed graded ratios of dietary alpha‐linolenic acid/linoleic acid (ALA/LNA) (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days were investigated. The results showed that ALA/LNA ratio of 1.03 significantly improved (i) per cent weight gain (PWG) and feed efficiency, (ii) hepatopancreatic trypsin, chymotrypsin, lipase, amylase and intestinal creatine kinase (CK) activities, (iii) hepatopancreatic trypsinogen‐2 and chymotrypsinogen mRNA levels. Meanwhile, fish fed with ALA/LNA ratio of 0.68 significantly enhanced, (iv) Na⁺/K⁺‐ATPase and γ‐glutamyl transpeptidase activities in whole intestine, and alkaline phosphatase activities in the proximal intestine (PI) and distal intestine, (v) amylase, intestinal Na⁺/K⁺‐ATPase alpha‐subunit isoform 1, Na⁺/K⁺‐ATPase alpha‐subunit isoform 8 and CK mRNA abundances, (vi) TOR and S6K1 gene expression in the hepatopancreas and intestine of juvenile grass carp. Based on the quadratic regression analysis of PWG, cholecystokinin and leptin contents in the PI, optimal dietary ALA/LNA ratio of juvenile grass carp (8.78–72.00 g) was estimated to be 1.08, 1.19 and 1.05, respectively.     

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp (Ctenopharyngodon idella) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean β‐conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g β‐conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary β‐conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS‐generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid‐intestine, dietary β‐conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase‐3, caspase‐8 and caspase‐9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease‐activating factor‐1 [Apaf1] and Bcl‐2‐associated X protein [BAX]) and mitogen‐activated protein kinase (MAPK)‐related signal molecules (Jun N‐terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho‐p38 MAPK. Moreover, in the distal intestine, dietary β‐conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase‐3, caspase‐8 and caspase‐9 activities, death ligand (TNF‐α) mRNA expression level, and p38 MAPK and phospho‐p38 MAPK protein levels. In summary, dietary soybean β‐conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS‐mediated MAPK signalling.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

A novel 79‐bp insertion/deletion polymorphism in 3′‐flanking region of IGF‐I gene is associated with growth‐related traits in common carp (Cyprinus carpio L.)

Insulin‐like growth factor‐I (IGF‐I) participates in the modulation of the cellular development, differentiation and proliferation in various vertebrate tissues. Therefore, variations in IGF‐I gene are good candidates as growth regulators. In this study, a novel 79‐bp insertion/deletion (I/D) polymorphism was identified in 3′‐flanking region of IGF‐I gene by polymerase chain reaction (PCR) and sequencing approaches in common carp Cyprinus carpio. To gain insight into the association of the 79‐bp I/D polymorphism with growth‐related traits, a total of 747 individuals from Heilongjiang carp Cyprinus cario haematopterus (YL) (n = 263), selective line of German mirror carp Cyprinus carpio L. mirror (JL) (n = 229), and cold‐resistance line of Hebao red carp Cyprinus carpio var. wuyuanensis (HL) (n = 255) were genotyped. The DD genotype of the polymorphism was significantly associated with higher body length (135), body weight (325, 385, and 445 days), net weight (445 days) and lower overwintering body loss rate in JL. In addition, in pooled population (YL+JL+HL), fish with DD genotype also showed significantly higher body weight (445 days) and net weight (445 days) than those with II genotype. Taken together, these results suggest that the novel polymorphism in IGF‐I gene could be considered as a potential genetic marker for selection of growth performance in common carp.     

Effects of dietary 60 g kg−1 dried distiller's grains in least‐cost practical diets on production and gut allochthonous bacterial composition of cage‐cultured fish: comparison among fish species with different natural food habits

Dried distiller's grain (DDG) is considered as an alternative ingredient of dietary feed due to its high contents of protein, fibre and fat. In this study, 60 g kg^‐1 of DDG was used to feed grass carp (Ctenopharyngodon idella), bluntnose black bream (Megalobrama amblycephala), gibel carp (Carassius gibelio) and black carp (Mylopharyngodon piceus) for 8 weeks, and its effect on fish production and gut allochthonous microbiota was investigated for the development of a suitable fish feed high in nutrients and low in cost for polyculture freshwater fish. DDG supplementation resulted in the less weight gain and higher feed conversion ratio of black carp (P < 0.05), but had no significant effects on other fish or parameters. PCR–denaturing gradient gel electrophoresis (DGGE) analysis indicated that all four fish species had some common and unique bacteria in their digestive tracts, and the gut microbiota of bluntnose black and gibel carp fed the control diet and DDG diets were very similar (Cs > 91%); of them, the total counts of intestinal bacteria studied by qPCR increased in grass carp (P < 0.05) and depressed in black carp (P < 0.05) when fed dietary DDG. Thus, we assumed that dietary DDG modulated production and gut microbiota of fish in a host‐specific way.     

Molecular characterization and induced expression analysis of the terminal complement component C9 in rohu,Labeo rohita

The complement component 9 (C9) plays a significant role in the formation of membrane attack complex (MAC) on the targeted cell surface. The current study is dealt with molecular characterization of C9 gene from rohu, Labeo rohita, an important cultured carp species in India. An open reading frame (ORF) of 1998 bp was amplified by polymerase chain reaction (PCR) that encodes a polypeptide of 666 amino acids having a signal peptide of 19 amino acids and a mature peptide of 647 amino acids. The SMART domain architecture analysis revealed two thrombospondin type‐1 domains (TSP1), a low‐density lipoprotein receptor domain class A (LDLa), a membrane attack complex and perforin (MACPF) domain, and an epidermal growth factor (EGF)‐like domain. Multiple sequence alignment and evolutionary analysis revealed a primitive C9 sequence of rohu with maximum similarity and clustering with common carp, grass carp and zebrafish. C9 was highly expressed in liver and constitutively expressed in wide array of tissues except in eye of rohu juveniles. It was expressed during early developmental days of rohu including in milt. A variable level of up‐regulation in C9 expression was noticed upon poly I:C induction, Aeromonas hydrophila and Argulus siamensis infections in liver, spleen and gill tissues of rohu at different time points. A constitutive expression of C9 in different stages of rohu during the ontogeny and in response to pathogen exposures along with high degree of sequence homology with other fish species proved it as an important primitive immune molecule of the complement system lytic pathway.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

Xylanase supplementation in plant protein‐enriched diets improves growth performance by optimizing the intestinal microflora and enhancing the intestinal immune function in grass carp grow‐out (Ctenopharyngodon idella)

This study investigated the effect of xylanase supplementation of a plant protein‐enriched diet on the growth performance and intestinal immune function of grass carp grow‐out (Ctenopharyngodon idella). Five hundred forty healthy grass carp grow‐out (232.74 ± 0.12 g) were fed diets containing graded levels of xylanase for 60 days. The fish were then challenged with Aeromonas hydrophila for 14 days. The results indicated that xylanase supplementation (a) improved fish growth performance and optimized the intestinal microflora; (b) increased lysozyme and acid phosphatase activities, complement and immunoglobulin M levels and antibacterial peptide hepcidin mRNA levels, thereby enhancing fish intestinal immune function; and (c) attenuated intestinal inflammation associated with the inhibition of the NF‐κB signalling pathway and activation of the TOR signalling pathway, resulting in the down‐regulation of pro‐inflammatory cytokines [except IL‐1β and IL‐12p40 in the distal intestine (DI) and IL‐6 in the proximal intestine (PI)] and up‐regulation of anti‐inflammatory cytokines [except IL‐4/13B and TGF‐β1 in the mid intestine (MI) and DI]. Finally, on the premise of a basal diet containing 45.8 g/kg araboxylans (AXs), the appropriate supplementation levels based on the ability to improve per cent weight gain (PWG) and prevent enteritis were estimated to be 1,527 and 1,608 U/kg, respectively.     

Effects of temperature and dietary protein on the growth performance and IGF‐I mRNA expression of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed with five different diets containing 303, 322, 341, 361 and 379 g kg^?1 protein and reared at three different water temperatures (18, 23 and 28 °C) for 60 days. We investigated the insulin‐like growth factor I (IGF‐I) mRNA expression, growth performance and the relationship between IGF‐I mRNA expression and the growth performance. The results indicated that the IGF‐I mRNA expression, final body weight, specific growth rate (SGR) and feed efficiency (FE) were enhanced significantly with increasing dietary protein levels (P < 0.05), whereas the protein efficiency ratio, hepatosomatic index (HSI) and viscerosomatic index (VSI) were decreased. Moreover, the IGF‐I mRNA expression, final body weight and SGR were increased significantly with temperature, whereas the HSI and VSI indices were decreased significantly with temperature. Correlation analysis showed that the IGF‐I mRNA expression levels in the brain and liver were positively related to the SGR and FE growth indices (P < 0.01). Finally, the optimal protein requirements for fish growth in different seasons were determined based on the values of SGR and FE, that is 343–348 g kg^?1 protein at 18 °C, 354–352 g kg^?1 at 23 °C and 371–362 g kg^?1 at 28 °C. In this way, we can adjust the dietary protein levels according to culture temperature to reduce any negative impacts on dietary costs and environmental pollution.     

Molecular cloning and characterization of p38 gene in the Chinese Mitten Crab,Eriocheir sinensis

p38 is an important component of the MAPK signaling pathways as a Ser/Thr protein kinase. We cloned the full length p38 (Es‐p38) cDNA sequence in Eriocheir Sinensis which contained a 1098‐nucleotide ORF that encoded a protein of 365 amino acids. Es‐p38 protein has a p38 MAPK functional domain (STKc_p38), it is a typical serine/threonine kinase and processes all p38 protein's conservative T‐G‐Y motif. Tissue expression of the Es‐p38 gene and expression during testis development stages were determined by quantitative real‐time PCR analysis. The results show that Es‐p38 gene is expressed in various tissues, but it expresses higher in the muscle, hepatopancreas, intestine, ovary and testis. The expression level of Es‐p38 gene at different stages of testis development shows no significant difference between the spermatocyte phase (July–August) and spermatid stage (August–October), but it rises significantly in the mid‐late period of sperm stage (December–January). This changing trend has a positive relationship with the E. Sinensis gonad development and reproduction stage, which preliminarily shows that Es‐p38 may function in the E. Sinensis testis development and the process of spermatogenesis.