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.

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.

Bacillus velezensis LG37: transcriptome profiling and functional verification of GlnK and MnrA in ammonia assimilation.

BACKGROUND: In recent years, interest in Bacillus velezensis has increased significantly due to its role in many industrial water bioremediation processes. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (from an aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, it is worth investigating the molecular mechanism of LG37 through ammonia nitrogen assimilation, where nitrogen in the form of molecular ammonia is considered toxic to aquatic organisms. RESULTS: Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown in minimal medium supplemented with ammonia (treatment) or glutamine (control) were obtained, from which 56 had Fold Change ≥2. BLAST-NCBI and UniProt databases revealed 27 out of the 56 DEGs were potentially involved in NH4+ assimilation. Among them, 8 DEGs together with the two-component regulatory system GlnK/GlnL were randomly selected for validation by quantitative real-time RT-PCR, and the results showed that expression of all the 8 DEGs are consistent with the RNA-seq data. Moreover, the transcriptome and relative expression analysis were consistent with the transporter gene amtB and it is not involved in ammonia transport, even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout and overexpression glnK mutants further evidenced the exclusion of amtB regulation, suggesting the involvement of alternative transporter. Additionally, in the transcriptomic data, a novel ammonium transporter mnrA was expressed significantly in increased ammonia concentrations. Subsequently, OEmnrA and ΔmnrA LG37 strains showed unique expression pattern of specific genes compared to that of wild-LG37 strain. CONCLUSION: Based on the transcriptome data, regulation of nitrogen related genes was determined in the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. Using genomics tools, the novel MnrA transporter of LG37 became apparent in ammonia transport instead of AmtB, which transports ammonium nitrogen in other Bacillus strains. Collectively, this study defines heterogeneity of B. velezensis LG37 through comprehensive transcriptome analysis and subsequently, by genome editing techniques, sheds light on the enigmatic mechanisms controlling the functional genes under different nitrogen sources also reveals the need for further research.

Antibacterial activity of erythrocyte from grass carp (Ctenopharyngodon idella) is associated with phagocytosis and reactive oxygen species generation.

Red blood cells (RBCs) are widely accepted as their primary function in respiration. Recent studies in mammals have revealed a vital role in immune responses of RBCs; however, little is known about immune function of teleost erythrocytes. Here we demonstrated that RBCs from grass carp (Ctenopharyngodon idella) were capable of binding and aggregating the bacteria with apparent morphological alterations. The phagocytosis by teleost RBCs (erythrophagocytosis) was visualized by confocal, scanning and transmission electron microscopy. Hb-FeII of hemoglobin (Hb) could quickly be auto-oxidated to Hb-FeIII (methemoglobin/metHb) in the presence of oxygen (O2), and release superoxide radical (O2-.) which could be spontaneously dismutated into H2O2 that could further oxidize Hb-FeIII to transient HbFeIV-OH (ferryl-Hb). Furthermore, bacterial extracellular proteases and pathogen-associated molecular patterns (PAMPs) binding to Hb could synergistically activate pseudoperoxidase, subsequently facilitated the generation of reactive oxygen species (ROS) which were toxic to the bacteria. Our results indicated that erythrocyte pertains anti-bacterial activity using unique ROS generation pathway via oxidation of hemoglobin and associated with its phagocytosis.