The role of antifreeze genes in the tolerance of cold stress in the Nile tilapia (Oreochromis niloticus)

Tilapia is one of the most essential farmed fishes in the world. It is a tropical and subtropical freshwater fish well adapted to warm water but sensitive to cold weather. Extreme cold weather could cause severe stress and mass mortalities in tilapia. The present study was carried out to investigate the effects of cold stress on the up-regulation of antifreeze protein (AFP) genes in Nile tilapia (Oreochromis niloticus). Two treatment groups of fish were investigated (5 replicates of 15 fish for each group in fibreglass tanks/70 L each): 1) a control group; the fish were acclimated to lab conditions for two weeks and the water temperature was maintained at 25 °C during the whole experimental period with feeding on a commercial diet (30% crude protein). 2) Cold stress group; the same conditions as the control group except for the temperature. Initially, the temperature was decreased by one degree every 12 h. The fish started showing death symptoms when the water temperature reached 6-8 °C. In this stage the tissue (muscle) samples were taken from both groups. The immune response of fish exposed to cold stress was detected and characterized using Differential Display-PCR (DD-PCR).The results indicated that nine different up-regulation genes were detected in the cold-stressed fish compared to the control group. These genes are Integrin-alpha-2 (ITGA-2), Gap junction gamma-1 protein-like (GJC1), WD repeat-containing protein 59 isoform X2 (WDRP59), NUAK family SNF1-like kinase, G-protein coupled receptor-176 (GPR-176), Actin cytoskeleton-regulatory complex protein pan1-like (PAN-1), Whirlin protein (WHRN), Suppressor of tumorigenicity 7 protein isoform X2 (ST7P) and ATP-binding cassette sub-family A member 1-like isoform X2 (ABCA1). The antifreeze gene type-II amplification using a specific PCR product of 600 bp, followed by cloning and sequencing analysis revealed that the identified gene is antifreeze type-II, with similarity ranging from 70 to 95%. The in-vitro transcribed gene induced an antifreeze protein with a molecular size of 22 kDa. The antifreeze gene, ITGA-2 and the WD repeat protein belong to the lectin family (sugar-protein).In conclusion, under cold stress, Nile tilapia express many defence genes, an antifreeze gene consisting of one open reading frame of approximately 0.6 kbp.