Novel Insights for PI3KC3 in Mediating Lipid Accumulation in Yellow Catfish Pelteobagrus Fulvidraco

In this study, the transcriptional regulation of PI3KC3 by three transcript factors (PPARγ, PPARα and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5’-deletion assay, overexpression assay, site-mutation assay and electrophoretic mobility shift assay suggested that PPARα, PPARγ and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG), NEFA content, the mRNA level of pparα, pparγ, stat3 and dnmt3b, the protein level of PPARα, PPARγ and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.

Dietary Glutamine Inclusion Regulates Immune and Antioxidant System, as well as Programmed Cell Death in Fish to Protect against Flavobacterium columnare Infection

The susceptibility of animals to pathogenic infection is significantly affected by nutritional status. The present study took yellow catfish (Pelteobagrus fulvidraco) as a model to test the hypothesis that the protective roles of glutamine during bacterial infection are largely related to its regulation on the immune and antioxidant system, apoptosis and autophagy. Dietary glutamine supplementation significantly improved fish growth performance and feed utilization. After a challenge with Flavobacterium columnare, glutamine supplementation promoted il-8 and il-1β expression via NF-κB signaling in the head kidney and spleen, but inhibited the over-inflammation in the gut and gills. Additionally, dietary glutamine inclusion also enhanced the systematic antioxidant capacity. Histological analysis showed the protective role of glutamine in gill structures. Further study indicated that glutamine alleviated apoptosis during bacterial infection, along with the reduced protein levels of caspase-3 and the reduced expression of apoptosis-related genes. Moreover, glutamine also showed an inhibitory role in autophagy which was due to the increased activation of the mTOR signaling pathway. Thus, our study for the first time illustrated the regulatory roles of glutamine in the fish immune and antioxidant system, and reported its inhibitory effects on fish apoptosis and autophagy during bacterial infection.

Comparative Transcriptomic Analysis of Regenerated Skins Provides Insights into Cutaneous Air-Breathing Formation in Fish

Cutaneous air-breathing is one of the air-breathing patterns in bimodal respiration fishes, while little is known about its underlying formation mechanisms. Here, we first investigated the skin regeneration of loach (Misgurnus anguillicaudatus, a cutaneous air-breathing fish) and yellow catfish (Pelteobagrus fulvidraco, a water-breathing fish) through morphological and histological observations. Then, the original skins (OS: MOS, POS) and regenerated skins (RS: MRS, PRS) when their capillaries were the most abundant (the structural foundation of air-breathing in fish) during healing, of the two fish species were collected for high-throughput RNA-seq. A total of 56,054 unigenes and 53,731 unigenes were assembled in loach and yellow catfish, respectively. A total of 640 (460 up- and 180 down-regulated) and 4446 (2340 up- and 2106 down-regulated) differentially expressed genes (DEGs) were respectively observed in RS/OS of loach and yellow catfish. Subsequently, the two DEG datasets were clustered in GO, KOG and KEGG databases, and further analyzed by comparison and screening. Consequently, tens of genes and thirteen key pathways were targeted, indicating that these genes and pathways had strong ties to cutaneous skin air-breathing in loach. This study provides new insights into the formation mechanism of cutaneous air-breathing and also offers a substantial contribution to the gene expression profiles of skin regeneration in fish.

Dietary Nano-ZnO Is Absorbed via Endocytosis and ZIP Pathways, Upregulates Lipogenesis, and Induces Lipotoxicity in the Intestine of Yellow Catfish

Nano-sized zinc oxide (nano-ZnO) affects lipid deposition, but its absorption patterns and mechanisms affecting lipid metabolism are still unclear. This study was undertaken to investigate the molecular mechanism of nano-ZnO absorption and its effects on lipid metabolism in the intestinal tissues of a widely distributed freshwater teleost yellow catfish Pelteobagrus fulvidraco. We found that 100 mg/kg dietary nano-ZnO (H-Zn group) significantly increased intestinal Zn contents. The zip6 and zip10 mRNA expression levels were higher in the H-Zn group than those in the control (0 mg/kg nano-ZnO), and zip4 mRNA abundances were higher in the control than those in the L-Zn (50 mg/kg nano-ZnO) and H-Zn groups. Eps15, dynamin1, dynamin2, caveolin1, and caveolin2 mRNA expression levels tended to reduce with dietary nano-ZnO addition. Dietary nano-ZnO increased triglyceride (TG) content and the activities of the lipogenic enzymes glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and isocitrate dehydrogenase (ICDH), upregulated the mRNA abundances of lipogenic genes 6pgd, fatty acid synthase (fas), and sterol regulatory element binding protein 1 (srebp1), and reduced the mRNA expression of farnesoid X receptor (fxr) and small heterodimer partner (shp). The SHP protein level in the H-Zn group was lower than that in the control and the L-Zn group markedly. Our in vitro study indicated that the intestinal epithelial cells (IECs) absorbed nano-ZnO via endocytosis, and nano-Zn-induced TG deposition and lipogenesis were partially attributable to the endocytosis of nano-ZnO in IECs. Mechanistically, nano-ZnO-induced TG deposition was closely related to the metal responsive transcription factor 1 (MTF-1)-SHP pathway. Thus, for the first time, we found that the lipogenesis effects of nano-ZnO probably depended on the key gene shp, which is potentially regulated by MTF1 and/or FXR. This novel signaling pathway of MTF-1 through SHP may be relevant to explain the toxic effects and lipotoxicity ascribed to dietary nano-ZnO addition.

Withdrawal Interval Estimation of Doxycycline in Yellow Catfish (Pelteobagrus fulvidraco) Using an LC-MS/MS Method Based upon QuEChERS Sampling Preparation

Limited information on residue depletion of doxycycline (DC) is available in yellow catfish (Pelteobagrus fulvidraco) using the determination of LC-MS/MS based upon a rapid and simple method of sample preparation. This study collected plasma and tissue samples of yellow catfish at pre-determined time points following 3-day consecutive oral administration of DC at 20 mg/kg. The samples were prepared using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach. The limit of detection and the limit of quantitation were 2.5 and 5 µg/kg or µg/L, respectively, for DC in plasma and tissues (e.g., muscle + skin, liver, kidney, and gill). The recoveries of DC ranged from 67.2% to 86.2%. The decision limit (CCα) and the detection capability (CCβ) were from 106.2 to 127.8 μg/kg or μg/L. The withdrawal times of DC in muscle + skin were estimated to be 22 d based on the guidelines in China and Europe and 27 d based on Japan’s standard. Overall, this study not only provides an efficient method to rapidly determine the DC concentrations in fish-derived tissues but also provides important information on the safety assessment of DC in aquatic animal-derived food products.