Impact of dietary zinc on the growth performance, histopathological analysis, antioxidant capability, and inflammatory response of largemouth bass Micropterus salmoides.

Zinc plays a crucial role in the antioxidant capacity, and inflammatory response of aquatic species, but its impact on largemouth bass Micropterus salmoides is rarely reported. Therefore, this paper aimed to investigate the effects of different levels of zinc on the growth performance, histopathology, antioxidant capacity, and inflammatory cytokines of largemouth bass Micropterus salmoides. Fish with an initial weight of 7.84 ± 0.06 g were cultured for 10 weeks. Five experimental diets were prepared with supplemented proteinate Zn (Bioplex Zn, Alltech) (0, 30, 60, 90, and 120 mg/kg), which were named the Zn-42, Zn-73, Zn-103, Zn-133, and Zn-164 groups. No evident difference was found between the dietary zinc level and the survival rate, the crude lipid content of the whole fish, or the visceral somatic index. Weight gain, condition factor, whole-body crude protein content, interleukin-10, and transforming growth factor beta gene expression were gradually enhanced with up to 102.68 mg/kg zinc and decreased at higher levels. The hepatosomatic index, feed conversion ratio, malondialdehyde level in the liver, aspartate aminotransferase, and alanine transaminase activity in the serum, gradually decreased up to 102.68 mg/kg zinc, and gradually increased beyond this. Activation of the nuclear factor erythroid-derived 2-like 2/Kelch-like ECH-associated protein 1 signaling pathway gradually up-regulated the mRNA levels and activities of glutathione peroxidase, total antioxidant capacity, catalase, and superoxide dismutase in the liver, this antioxidant ability was lower when the zinc was greater than 102.68 mg/kg. The gene expressions of nuclear factor-k-gene binding and pro-inflammation cytokines (interleukin-1ß, interleukin-15, tumor necrosis factor alpha, and interleukin-8) were up-regulated up to 102.68 mg/kg zinc and then gradually repressed. In conclusion, using broken line analysis to estimate weight gain and Zn proteinate as the zinc source, the recommended dietary zinc for largemouth bass is 66.57 mg/kg zinc.

Dietary protein improves flesh quality by enhancing antioxidant ability via the NF-E2-related factor 2/Kelch-like ECH-associated protein 1 signaling pathway in softshell turtle (Pelodiscus sinensis).

An 8-week feeding trial was performed to assess the influence of a gradient of protein levels (14.38-45.23%) on flesh quality, skin color, amino acid profile, collagen, antioxidant capability, and antioxidant-related signaling molecule expression of the softshell turtle (Pelodiscus sinensis). Hardness, gumminess, chewiness, and yellowness values in the plastron and carapace, along with collagen, superoxide dismutase, catalase, total antioxidant capacity, and glutathione peroxidase, all improved with elevating dietary protein up to 26.19%, after which they leveled off. Additionally, total amino acids, flavor amino acids, essential amino acids, and non-essential amino acids in the muscle, as well as the expression of copper/zinc superoxide dismutase, glutathione peroxidase, catalase, manganese superoxide dismutase, NF-E2-related factor 2 were all enhanced by increasing the dietary protein level but not changed by higher protein levels. When dietary protein levels were less than 26.19%, the mRNA expression of Kelch-like ECH-associated protein 1, malondialdehyde, and redness values in the carapace and plastron were reduced, as was the lightness values of the carapace, all of which plateaued at higher protein levels. Using catalase activity and malondialdehyde as the indicators and applying a broken-line analysis, the optimal dietary protein level for P. sinensis was inferred to be 26.07 and 26.06% protein, respectively. In summary, an optimal protein input improved turtle flesh quality by strengthening antioxidant capacity in muscle tissue and by regulating the expression of antioxidant-related enzymes via the Nrf2/keap1 signaling pathway.

Effects of dietary protein on water quality, growth performance, RNA/DNA ratio and haemato-immunological indices of soft-shelled turtle (Pelodiscus sinensis).

In aquatic animals, dietary protein plays a crucial role in their growth and immunity. A feeding trial was conducted on soft-shelled turtles (Pelodiscus sinensis) to assess the effects of various levels of protein on the specific growth rate (SGR), ambient water quality (total ammonia nitrogen (TAN), total nitrogen (TN) and total phosphorus (TP)), hematological parameters (respiratory burst (RB), red blood cell count (RBC), albumin content (Alb), hemoglobin level (Hb) and osmolality), plasma immunoglobulin M (IgM) levels and lysozyme activity. Soft-shelled turtles weighing about 4.02 g were fed fish meal-based diets with 14.38%, 20.41%, 26.19%, 32.23%, 37.63% and 45.23% protein for 8 weeks. SGR, RBC, Hb, Alb, RB, IgM and lysozyme activity were enhanced as the dietary protein was increased from 14.38% to 26.19%, then reached a plateau. For identical feeding times, TAN and TN were increased with elevating dietary protein levels. While, no statistically significant differences were observed among the 26.19%, 32.23% and 37.63% groups. When the turtles were cultivated for 56 days and fed with 45.23% protein, the TP in the culturing water was higher than that in the other groups. An increase in dietary protein level up to 26.19% increased the RNA/DNA ratio, which subsequently plateaued at a steady level. The levels of dietary protein had no impact on osmolality or alkaline phosphatase (AKP) activity. On the basis of broken-line analyses derived from SGR, the optimum dietary protein level for soft-shelled turtles was found to be 27.11% protein.

Genome-wide identification and characterization of toll-like receptor 5 (TLR5) in fishes.

Toll-like receptors 5 (TLR5), a member of the toll-like receptors (TLRs) family, is a class of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). It responds to vertebrate recognition of bacterial flagellin and participates in innate immune responses. However, genome-wide identification and characterization of TLR5 in fishes have not been investigated. Here, three TLR5M isotypes (TLR5Ma, TLR5Mb1, and TLR5Mb2) and a TLR5S are all extracted from fish genomes on the basis of phylogenetic and synteny analyses. We confirmed that the non-teleost fishes have one TLR5M gene, as well as additional TLR5 genes (TLR5M and TLR5S) in teleost fishes. In addition, some special teleost fishes possess two to three TLR5 genes, which have undergone the fourth whole-genome duplication (WGD). According to our results, we inferred that the diversity of TLR5 genes in fishes seems to be the result of combinations of WGD and gene loss. Furthermore, TLR5 isoforms displayed differences at the flagellin interaction sites and viral binding sites, and showed lineage-specific, which indicated that TLR5 duplicates may generate functional divergence. Bacterial experiments also supported the idea that CiTLR5Ma and CiTLR5Mb are subfunctionalized to sense bacterial flagellin. In summary, our present comparative genomic survey will benefit for further functional investigations of TLR5 genes in fish.

Evaluation of dietary zinc on antioxidant-related gene expression, antioxidant capability and immunity of soft-shelled turtles Pelodiscussinensis.

Zinc (Zn) plays a role in the antioxidant capacity and immunity of aquatic animals. A twelve-week feeding experiment was performed to estimate the impact of dietary zinc on antioxidant enzyme-related gene expression, antioxidant enzyme activity and non-specific immune functions of soft-shelled turtles, Pelodiscus sinensis. Six fishmeal-based experimental diets with 32.45% protein were formulated, which contained 35.43, 46.23, 55.38, 66.74, 75.06 and 85.24 mg/kg Zn, respectively. Catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels improved with an elevation in dietary Zn from 35.43 to 55.38 mg/kg and then reduced when dietary Zn was further elevated. The expression levels of Nrf2 and antioxidant-related genes CuZnSOD, MnSOD, CAT, GPX1, GPX2, GPX3 and GPX4 escalated with elevating Zn concentration up to 55.38 mg/kg in diets and then reduced as dietary Zn elevated. The expression levels of Kelch-like ECH-associating protein 1 (keap1) showed a reverse trend with that of Nrf2. The contents of malondialdehyde (MDA) in the 55.38 and 66.74 mg/kg Zn diet-fed groups were the lowest. Alkaline phosphatase activity (AKP), superoxide anion (O2-), lysozyme activity and total antioxidant capacity (T-AOC) improved with an escalation in dietary Zn concentration up to 66.74 mg/kg. Optimal dietary Zn improved antioxidant capability, immunity, and antioxidant enzyme-related gene expression. The dietary Zn demand for soft-shelled turtles were 60.93 and 61.63 mg/kg, based on second regression analysis of SOD and T-AOC activity, respectively.

A Comparative Genomic and Transcriptional Survey Providing Novel Insights into Bone Morphogenetic Protein 2 (bmp2) in Fishes.

Intermuscular bones (IBs) are only found in the muscles of fish. Bone morphogenetic protein 2 (bmp2) is considered to be the most active single osteogenesis factor. It promotes cell proliferation and differentiation during bone repair, as well as inducing the formation of bones and cartilages in vivo. However, detailed investigations of this family in fish are incredibly limited. Here, we have used a variety of published and unpublished bmp2 sequences for teleosts and cartilage fish in order to explore and expand our understanding of bmp2 genes in fish. Our results confirmed that teleost genomes contain two or more bmp2 genes, and the diversity of bmp2 genes in vertebrates appears to be as a result of a combination of whole genome duplication (WGD) and gene loss. Differences were also observed in tissue distribution and relative transcription abundance of the bmp2s through a transcriptomic analysis. Our data also indicated that bmp2b may play an important role in the formation of IBs in teleosts. In addition, protein sequence alignments and 3D structural predictions of bmp2a and bmp2b supported their similar roles in fishes. To summarize, our existing work provided novel insights into the bmp2 family genes in fishes through a mixture of comparative genomic and transcriptomic analysis.

Inhibition of Cyclophilin A on the replication of red spotted grouper nervous necrosis virus associates with multiple pro-inflammatory factors.

Cyclophilin A (CypA) is a ubiquitously expressed cellular protein and involves in diverse pathological conditions, including infection and inflammation. CypA acts as a key factor in the replication of several viruses. However, little is known about the role of CypA in the replication of the red-spotted grouper nervous necrosis virus (RGNNV). In the present report, grouper CypA (GF-CypA) was cloned from the grouper fin cell line (GF-1) derived from orange-spotted grouper (Epinephelus coioides). Sequence analysis found that GF-CypA open reading frame (ORF) of 495 bp encodes a polypeptide of 164 amino acids residues with a molecular weight of 17.4 kDa. The deduced amino acid sequence shared highly conserved regions with CypA of other animal species, showing that GF-CypA is a new member of Cyclophilin A family. We observed that GF-CypA was up-regulated in the GF-1 cells infected with RGNNV. Additionally, overexpression of CypA could significantly inhibit the replication of RGNNV in GF-1 cells. By contrast, when the GF-CypA was knock-downed by siRNA in GF-1 cells, the replication of RGNNV was enhanced. Furthermore, the expressions of pro-inflammatory factors, such as TNF-2, TNF-α, IL-1b, and ISG-15, were increased in GF-CypA transfected GF-1 cells challenged with RGNNV, indicating that GF-CypA might be involved in the regulation of the host pro-inflammatory factors. Altogether, we conclude that GF-CypA plays a vital role in the inhibitory effect of RGNNV replication that might be modulating the cytokines secretion in GF-1 cells during RGNNV infection. These results will shed new light on the function of CypA in the replication of RGNNV and will pave a new way for the prevention of the infection of RGNNV in fish.

The immune function of prophenoloxidase from red swamp crayfish (Procambarus clarkii) in response to bacterial infection.

Prophenoloxidase (proPO) is the zymogen form of phenoloxidase (PO), a key enzyme in melanization cascade that has been co-opted in invertebrate immune reactions. There have been reported that proPO plays many essential roles in the crustacean immune system. However, little is known about the function of proPO from red swamp crayfish (Procambarus clarkii) which is an important cultured species worldwide. Here, we cloned and expressed proPO gene from red swamp crayfish (PcproPO). Subsequently, specific antibody against PcproPO was generated. The immune function of PcproPO was further characterized in vitro and in vivo. The results showed that the expression of PcproPO mRNA could be significantly up-regulated during the challenge of Gram-positive-negative (Vibrio parahaemolyticus) and Gram-positive-positive bacterial (Staphylococcus aureus). Furthermore, the purified recombinant PcproPO protein had a strong affinity binding to both bacteria and polysaccharides. In vivo knockdown of PcproPO could significantly reduce the crayfish bacterial clearance ability, resulting in the higher mortality of the crayfish during V. parahaemolyticus infection. In addition, in vitro knockdown of PcproPO in the hemocytes significantly reduced the phenoloxidase (PO) activity and the bacterial clearance ability, indicating that PcproPO might involve in hemocyte-mediated melanization. Our results will shed a new light on the immune function of PcproPO in the crayfish.

The RAG2 gene of yellow catfish (Tachysurus fulvidraco) and its immune response against Edwardsiella ictaluri infection.

Recombination-activating gene 2 (rag 2) allies with recombination-activating gene 1 (rag 1) and regulates the V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes. Being a key player in the adaptive immune response of vertebrates, functional characterization of rag 2 from yellow catfish is beneficial for understanding the biological response towards the pathogens. In this report, we have cloned and characterized the rag 2 gene of yellow catfish, and a particular pattern of expression was analysed in the major tissues of yellow catfish. The results showed that the open reading frame (ORF) of yellow catfish rag 2 was 1596 bp in length, which encodes a peptide of 531 amino acids. The multiple sequence alignment and phylogenetic analysis of rag 2 of yellow catfish with other species showed the conserved regions and the classical taxonomic evolution among the different vertebrate species. The qRT-PCR and Western blot results revealed that rag 2 transcripts and proteins were present in various tissues of yellow catfish with relatively high expression in the tissues of the thymus, head-kidney, and spleen. The systematic distribution analysis of the rag 2 expression by immunohistochemistry (IHC) using the rabbit polyclonal antibody, exposed relatively high expression in head kidney, spleen and thymus tissues after infected with Edwardsiella ictaluri. Moreover, the temporal expression of rag 2 and pro-inflammatory cytokines (IL-1ß and TNF-α) were significantly upregulated at different time points in the specific lymphoid tissues of yellow catfish following E. ictaluri infection. Our findings suggest that rag 2 potentially exhibited the immunological response in primary lymphoid tissues of yellow catfish against bacterial infection. This study will provide an essential source about rag 2 gene and its relationship with the inflammatory cytokines during infection.