Structural characteristics of orexin receptor type 2 in Pacific abalone and its diurnal expression pattern after fasting and re-feeding.

Pacific abalone (Haliotis discus hannai) is a typical nocturnal organism. To examine the circadian expression pattern of orexin receptor type 2 (OX2 R) and its potential effect on the feeding behavior of abalone, the coding region sequence of OX2 R that is 1215 bp in length and encodes 404 amino acids was first cloned using the rapid amplification of cDNA ends technique. A recombinant expression vector was constructed for H. discus hannai based on the OX2 R protein, obtaining a recombinant protein with a molecular weight of 46 kDa. Polyclonal antibody was prepared with the purified recombinant protein used as the antigen, and the antibody titer of ≥512 K was detected by enzyme-linked immunosorbent assay. The expression levels of OX2 R determined using western blotting were highest in the intestinal tract (P < 0.05), but they were not significantly different from the levels in the pedal. Immunofluorescence experiments affirmed that OX2 R was widely expressed in the columnar cells of the intestinal mucosal epithelium. To further account for the relationship between the onset of feeding behavior and the expression level of OX2 R in abalone, the circadian expression characteristics of OX2 R were analyzed by dissecting the intestinal tissues after three days of normal feeding and fasting and following the refeeding treatment. The expression levels of OX2 R in the refeeding group were significantly higher than those in the normal feeding and fasting groups at any time point (P < 0.05). The cosine curve analysis revealed that the expression levels of OX2 R lost rhythmicity after fasting. Based on the quantification of behavioral data for abalone after fasting and refeeding, the cumulative movement distance and movement duration in each group followed a significant cosine rhythm (P < 0.05), which is consistent with abalone's nocturnal ecological habits. However, the cumulative movement distance and movement duration in the fasting group were significantly lower than those in the normal feeding and refeeding groups (P < 0.05). The peak phases of the cumulative movement distance and movement duration in the refeeding group (ZT08:22 and ZT08:44) shifted backward compared to the normal feeding group (ZT07:33 and ZT07:39). The above results first identified the structural characteristics and circadian expression patterns of OX2 R in the marine mollusk abalone, which may reveal the molecular mechanism behind the generation of a feeding rhythm in marine nocturnal organisms and serve as a tool helping to maintain the diversity of marine benthic resources.