Diurnal rhythm and pathogens induced expression of toll-like receptor 9 (TLR9) in Pelteobagrus vachellii.

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces the production of inflammatory cytokines. In this study, the darkbarbel catfish Pelteobagrus vachellii TLR9 cDNA was cloned and sequenced. The daily expression pattern of TLR9 mRNA was investigated in various tissues. Furthermore, its expression was analyzed following exposure to the pathogen Aeromonas hydrophila. The 4249 bp cDNA includes a 3201 bp open reading frame (ORF) encoding 1067 amino acids. The predicted amino acid sequence comprises a leucine-rich domain (LRD), a toll/interleukin-1 receptor (TIR), and a transmembrane domain. P. vachellii TLR9 showed 42-87% amino acid sequence identity with TLR9 sequences of Ictalurus punctatus, Rhincodon typus, and Miichthys miiuy. The P. vachellii TLR9 mRNA was highly expressed in intestines, head kidney, and spleen in an apparently healthy fish. Following pathogen challenge, TLR9 expression increased significantly (P < 0.05) and peaked at 48 h post-exposure in the liver, at 24 in the head kidney, and at 12 h in the spleen. In addition, the pattern of TLR9 expression over a 24-h period showed a circadian rhythm in the head kidney, spleen, and intestine, with the acrophase at 20:34, 18:45, and 3:50, respectively. This result provided the basis for further study of the rhythm of innate immunity against bacteria in catfish.

Discovery of differentially expressed genes in the intestines of Pelteobagrus vachellii within a light/dark cycle.

In aquaculture, it is necessary to determine of the diurnal biological variations in the intestines to determine an appropriate feeding schedule. The present study aimed to examine the transcriptomes of the Pelteobagrus vachellii intestines at four time points (0 h, 6 h, 12 h, and 18 h) within a light/dark cycle. In comparison with the zeitgeber time 0 (ZT0) transcriptomes, we identified 37,842 unigenes with significant differential expression, including 6,638; 9,626; and 7,938 that genes upregulated, and 3,507; 4,703; and 5,412 genes that were down regulated at 4, 12, and 24 h respectively. The differentially expressed unigenes were subjected to enrichment analysis, which indicated the involvement of the major digestive pathways, including digestion of protein, lipid and carbohydrate, catabolic process (protein, carbohydrate and lipid), and circadian rhythm. We selected 73 key differentially expressed genes (DEGs) from among these pathways and identified DEGs that showed increased expression at night, including those encoding trypsin-3, chymotrypsinogen 2, amino acid transporter, maltase-glucoamylase, facilitated glucose transporter, lipase, phospholipase, fatty acid-binding protein, fatty acid synthase, long-chain fatty acid transport protein, and apolipoprotein. Moreover, DEGs involved of circadian rhythm were identified, including brain-muscle-Arnt-like 1 (BMAL1), cryptochrome-1, circadian locomoter output cycles protein kaput (CLOCK) and period circadian protein homolog 1-3. Finally, the expression levels of 12 unigenes were analyzed using quantitative real-time PCR, which were in accordance with RNA-sequencing analysis. In general, the expression of genes related to the digestion of proteins, lipids, and carbohydrates showed upregulated expression at night; however, the peak time of expression of transporters for different nutrition molecules showed more diversification within the light/dark cycle.