Integrative mRNA-miRNA interaction analysis associated with the immune response of Epinephelus coioddes to Vibrio alginolyticus infection.

MicroRNAs (miRNAs) are a kind of small non-coding RNAs that have been reported to play a vital role in mediating host-pathogen interactions. High-throughput sequencing technology was applied to identify and illuminate mRNAs and miRNAs from grouper infected with Vibrio alginolyticus. The KEGG pathway enrichment analysis showed that the most significate DEGs are associated with Toll-like receptor signaling pathway and NOD-like receptor signaling pathway. We obtained 374 known miRNAs and 116 novel miRNAs. During them, there are 31 up-regulated miRNAs and 93 down-regulated miRNAs. miRNA-mRNA GO and KEGG analysis show that there are 90 miRNAs associated with the immune system. The target genes of immune-related miRNAs (miR-142, miR-146, miR-150, miR-155, miR-203, miR-205, miR-24, miR-31) and genes (CD80, IL-2, AMPK, PI3K) in Epinephelus coioddes were predicted and validated. This study provides an opportunity to further understanding the molecular mechanisms especially the immune system of miRNA regulation in Epinephelus coioddes host-pathogen interactions.

Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides).

CD59, a multifunctional glycoprotein, not only plays a regulatory role in complement cascades, but also participates in modulation of teleostean immunity. In this study, full length sequence of EcCD59 was obtained, comprising a 5'UTR of 163 bp, an ORF of 354 bp and a 3'UTR of 559 bp. EcCD59 gene encoded a polypeptide of 117 amino acids. Tissue-specific analysis revealed that the highest expression of EcCD59 mRNA was observed in muscle. Vibrio alginolyticus challenge can significantly increase EcCD59 mRNA expression in liver, kidney and spleen. EcCD59 distribution was detected by a combined approach using GFP-overexpression, immunofluorescence and ELISA assay, indicating that EcCD59 may be predominantly aggregated in cellular membrane. Both EcCD59 and EcCD59delGPI can directly bind to V. alginolyticus and decrease the in vitro growth of V. alginolyticus. Additionally, vibrio injection experiment indicated that the binding of EcCD59 or EcCD59delGPI to V. alginolyticus can restrict its growth rate in vivo. In this study, we found that EcCD59 may be involved in immune defense against vibrio infection in a complement-independent manner.

N-terminal domain of EcC1INH in Epinephelus coioides can antagonize the LPS-stimulated inflammatory response.

Complement 1 inhibitor (C1INH) serving as a multifunctional factor can participate in the regulation of complement cascades and attenuate the activation of various proteases. In this study, we obtained EcC1INH cDNA and the tissue-specific analysis indicate that the highest expression level of EcC1INH mRNA was detected in liver. Moreover, Vibrio alginolyticus challenge can significantly increase EcC1INH mRNA expression in liver and kidney. N-terminal domain of EcC1INH could decrease LPS binding activity to cell surface, while loss of positively charged residues (PCRs) Arg21, His22, Lys50, Arg61 in N-terminal domain of EcC1INH can significantly reduce its interaction with LPS. Furthermore, LPS injection experiment indicated that the binding of EcC1INH N-terminal domain to LPS can antagonize LPS-induced inflammatory signaling pathway and attenuate the production of proinflammatory cytokines in vivo, indicating that EcC1INH was involved in negative regulation of inflammatory response.

Identifying the function of LvPI3K during the pathogenic infection of Litopenaeus vannamei by Vibrio alginolyticus.

It is well known that PI3K regulates various processes in mammalian cells by generating a secondary messenger that later activates AKT. However, its innate immune function in crustaceans remains unclear. We report the characterization of Litopenaeus vannamei PI3K (LvPI3K) for investigating how PI3K participates in the innate immunity of crustaceans. Full-length LvPI3K cDNA was 3357 bp long, with a 3222 bp open reading frame (ORF) that encodes a putative protein of 1292 amino acids. The PI3K catalytic domain (PI3Kc) of LvPI3K was found to be rather conserved when the PI3Ks from other species were analyzed. The LvPI3K protein was shown to be localized to the cytoplasm of Drosophila S2 cells, while LvPI3K mRNA was ubiquitously expressed in healthy L. vannamei, with the highest expression found in hemolymph. A dual luciferase reporter gene assay demonstrated that LvPI3K overexpression activated the promoter of antibacterial peptide LvPEN4 in a dose-dependent manner. However, the addition of PDTC, a specific inhibitor of NF-κB, suppressed the LvPI3K-induced LvPEN4 promoter activation. Moreover, Vibrio alginolyticus challenge induced a rapid up-regulation of LvPI3K expression. Further experiments showed that LvPI3K silencing in shrimp challenged with V. alginolyticus significantly increased Vibrio number, ROS production and DNA damage in the hemolymph, as well as significantly decreased total hemocyte count. The mRNA levels of certain molecules related to LvPI3K signaling, such as LvAKT and LvPEN4, also decreased following LvPI3K silencing. Taken together, these results suggest that LvPI3K regulates the downstream signal component LvPEN4 and functions in V. alginolyticus resistance.

Molecular characterization and function analysis of a nucleotide excision repair gene Rad23 from Litopenaeus vannamei after Vibrio alginolyticus challenge.

Nucleotide excision repair (NER) removes many different types of DNA lesions, and NER related host factors are reported to aid recovery steps during viral integration. Here, we report the identification and characterization of a DNA repair gene Rad23 from Litopenaeus vannamei and explore its role in innate immunity of crustaceans. LvRad23 contains a1149 bp open reading frame (ORF) which encodes a 382 amino acids protein with predicted theoretical isoelectric point of 4.21. LvRad23 was ubiquitously expressed in the muscle, eyestalk, gill, stomach, heart, legs, intestine, and hepatopancreas in order from high to low and LvRad23 protein was showed to be located in the cytoplasm of Drosophila S2 cells. The homology analysis showed that it has a high sequence homology with Rad23 protein from Marsupenaeus japonicus. Vibrio alginolyticus challenge induced a remarkable up-regulation of LvRad23 mRNA in hepatopancreas. Knocking down LvRad23can interfere the NER pathway by down regulating the expression of replication protein A (RPA) and proliferating cell nuclear antigen (PCNA). However it didn't cause any significant difference on total hemocyte count (THC) between LvRad23-silenced and non-silenced group.LvRad23-silenced then challenge with V. alginolyticus inducing high level of reactive oxygen species (ROS) and DNA damage in hemolymph. As well as decreased THC, which seriously diminished the innate immune system of L. vannamei. Meanwhile, the NER pathway was reactived by enhancing the expression of LvRad23 and promoting the production of LvPCNA to resist apoptosis and maintain proliferation of hemolymph cells in the later stage. Our results suggest that LvRad23 plays a vital role in shrimp specific immune response to V. alginolytcus through its participation in NER pathway.

A Novel Kelch-Like-1 Is Involved in Antioxidant Response by Regulating Antioxidant Enzyme System in Penaeus vannamei.

Heavy metals are typical cumulative pollutants that can enter and poison the human body through the food chain. However, the molecular mechanism of heavy metal-induced oxidative stress is unclear. In this study, we characterize PvKelch-like-1 from P. vannamei and explore its antioxidant roles in immune regulation of crustaceans. PvKelch-like-1 full length contains 2107 nucleotides, consists of a 5' untranslated region (UTR) of 79 bp, a 3' UTR of 180 bp, and a ORF of 1848 encoded 615 amino acids, which contain a BTB, BACK and Kelch motif, putative molecular mass and isoelectric point were 69 KDa and 6.54. PvKelch-like-1 mRNA was ubiquitously expressed in all detected tissue of P. vannamei, and mRNA expression levels were significantly up-regulated from 6 to 24 h after cadmium stress and reached the highest level (3.2-fold) at 12 h in the hepatopancreas. Subcellular localization analysis revealed that PvKelch-like-1 was localized in the nucleus. Silencing PvKelch-like-1 significantly increased reactive oxygen species (ROS) (1.61-fold) production and DNA damage (1.32-fold) in the shrimp hemolymph, and significantly decreased total hemocyte counts (THC) (0.64-fold) at 6 h in hemolymph. Additionally, the antioxidant genes PvCAT (0.43-fold), PvMnSOD (0.72-fold), PvGST (0.31-fold) and PvGPx (0.59-fold) at 6 h were decreased significantly in PvKelch-like-1 silenced shrimp after cadmium stress. Overexpression of PvKelch-like-1 has the opposite results in enzyme activity. The SOD (2.44-fold) and CAT (2.19-fold) activities were significantly increased after overexpressing PvKelch-like-1. These results suggest that PvKelch-like-1 plays a vital role in shrimp innate immune defense by positively regulating the expression of antioxidant enzyme genes to respond to cadmium stress.

Effect of food restriction on the energy metabolism of the Chinese bulbul (Pycnonotus sinensis).

Food resources play an important role in the regulation of animals' physiology and behavior. We investigated the effect of short-term food restriction on metabolic thermogenesis of Chinese bulbuls (Pycnonotus sinensis) by measuring changes in body mass, body fat, basic metabolic rate (BMR), and organ mass of wild-caught Chinese bulbuls from Wenzhou, China. Short-term food restriction induced a significant decrease in body mass and body fat but body mass returned to normal levels soon after food was no longer restricted. Food restriction caused a significant reduction in BMR after 7 days (P<0.05), which returned to normal levels after food restriction ceased. Log total BMR was positively correlated with log body mass (r(2)=0.126, P<0.05). The dry masses of livers and the digestive tract were higher in birds that had been subject to temporary food restriction than in control birds and those subject to continual food restriction (P<0.001 and P<0.05, respectively). There was also significant differences in the dry mass of the lungs (P<0.05), heart (P<0.01), and spleen (P<0.05) in birds subject to short-term food restriction compared to control birds and those subject to continual food restriction. BMR was positively correlated with body and organ (heart, kidney and stomach) mass. These results suggest that the Chinese bulbul adjusts to restricted food availability by utilizing its energy reserves, lowering its BMR and changing the weight of various internal organs so as to balance total energy requirements. These may all be survival strategies that allow birds to cope with unpredictable variation in food abundance.

Metabolic rate and evaporative water loss in the silky starling (Sturnus sericeus).

To better understand the physiological characteristics of the silky starling (Sturnus sericeus), its body temperature (Tb), basal metabolic rate (BMR), evaporative water loss (EWL) and thermal conductance (C) elicited by different ambient temperatures (Ta) (5-30 ℃) were determined in the present study. Our results showed that they have a high Tb (41.6 ± 0.1 ℃), a wide thermal neutral zone (TNZ) (20-27.5℃) and a relatively low BMR within the TNZ (3.37 ± 0.17 mL O2/g·h). The EWL was nearly stable below the TNZ (0.91 ± 0.07 mg H2O/g·h) but increased remarkably within and above the TNZ. The C was constant below the TNZ, with a minimum value of 0.14 ± 0.01 mL O2/g·h·â„ƒ. These findings indicate that the BMR, Tb and EWL of the silky starling were all affected by Ta, especially when Ta was below 20 ℃ and the EWL plays an important role in thermal regulation.