Cloning and expression of cck and cart4 genes of 

Pampus argenteus and effects of jellyfish intake 

on their expression patterns

Abstract: Silver pomfret (Pampus argenteus) is one of the most valued and attractive food fish species in China. In spite of important technical developments and intense research in Pampus argenteus, some bottlenecks still limit the expansion of its industry. One of these limitations is the lack of a suitable formulated diet for broodstock, which leads to the poor quality of gonads in cultured broodstock. This fish species has a distinct preference for preying on jellyfish, and their daily intake of Aurelia aurita exceeds their body weight by more than 10 times. The feeding patterns of wild pomfret in the East China Sea were studied using carbon and nitrogen stable isotope approaches, and jellyfish contributed up to 54% of Pampus argenteus diet. This indicated the fact that Pampus argenteus preferentially hunted jellyfish. Jellyfish is a conspicuous, yet longoverlooked component of pelagic marine systems, and it is widely distributed in the marine ecosystem and is an important fishery resource in China. It has long been regarded as a “dead end” in marine food webs due to its high water content (> 95%) and low content of highvalue nutrients. In recent years, studies have reported that the role of jellyfish in the marine food chain is seriously underestimated, and this finding is of great importance to the entire marine food chain system. In the last few years, the notion of gelatinous species as merely carbon sinks or trophic dead ends has become largely obsolete, and there is renewed interest in their trophic ecology. Recent data show that they may represent a qualitatively important resource for growth, reproduction and developmental processes, comprising the diet of more than 120 fishes and other medusivorous vertebrates, such as sea turtles. Some predators, like the ocean sunfish, may feed selectively on specific tissues within large jellyfish. According to research on fish digestion and jellyfish absorption efficiency, the digestion rate of salmon jellyfish is more than 20 times that of shrimp flesh. This is because jellyfish tissues can be rapidly digested and assimilated, and therefore, their diluted nutritive value can be compensated when consumed in sufficient supply and processed at a higher rate. Addition of jellyfish to the diet stimulates the digestion and absorption efficiency of the digestive tract. Adding jellyfish to Pampus argenteus feed can successfully boost their growth rate and Pampus argenteus can better digest and absorb jellyfish by altering the intestinal microbial makeup, indicating that this fish species possesses a system for digesting and absorbing nutrients from jellyfish. There is now overwhelming evidence suggesting that jellyfish has considerable nutritional content and their contribution to the energy budget of Pampus argenteus has been categorical. However, the underlying mechanism of the feeding preference behavior of Pampus argenteus to jellyfish is still unclear.

Cholecystokinin and cocaine and amphetamineregulated transcript proteinlike play important roles in animal feeding regulation. The cck gene is a key regulator of gastrointestinal function and an important satiety signal in vertebrates, including fish, and is involved in appetite regulation. The cart4 gene is thought to be an important neurotransmitter regulating body weight and food intake in rodents and humans. In this study, the full cDNA sequences of cck and cart4 genes of Pampus argenteus were cloned. The cck gene was 1 125 bp in length, 5′untranslated region (5′UTR) was 42 bp in length, and 3′UTR was 679 bp in length. It contained a 405 bp open reading frame (ORF), which encoded and synthesized a 134 aa protein and contained a characteristic sequence of a typical Cck family. The highest homology of Cck between Pampus argenteus and Micropterus salmoides was 87.59%, and the homology between Pampus argenteus and other fish was also higher than 67.88%. The phylogenetic tree results also showed that Cck between Pampus argenteus and Micropterus salmoides clustered in the same branch and had the closest genetic relationship. The cart4 gene was 865 bp in length, 5′UTR was 133 bp in length, and 3′UTR was 400 bp in length. It contained a 333 bp open reading frame (ORF), which encoded and synthesized a 110 aa protein, a signal peptide composed of 24 amino acids and a domain named CART. The highest homology of Cart4 between Pampus argenteus and Xiphias gladius was 91.82%, and the homology between Pampus argenteus and other fish was also higher than 63.06%. The phylogenetic tree results also showed that Cart4 between Pampus argenteus and Xiphias gladiu clustered in the same branch and had the closest genetic relationship. Realtime PCR was used to detect the expression levels of cck and cart4 genes in different tissues and the effects of jellyfish ingestion on the expression levels of cck and cart4 genes in different tissues. The results showed that the expression of cck gene was the highest in midgut of noeating jellyfish group (P<0.05), and the highest in the brain of eating jellyfish group (P<0.05). Compared with noeating group, the expression levels of brain, liver and kidney in jellyfish feeding group significantly increased (P<0.01), and the expression levels of muscle also slightly increased (P>0.05), while the expression levels of midgut, gill and gonad in jellyfish feeding group significantly decreased (P<0.01). The expression of cart4 gene was the highest in brain (P<0.05). Compared with nofeeding group, the expression level of jellyfish in brain significantly increased (P<0.01), but significantly decreased in midgut, gill, liver and muscle (P<0.01). 

The results showed that jellyfish consumption resulted in a significant upregulation of cck and cart4 gene expression in the brain tissue of Pampus argenteus. It indicated that eating jellyfish would increase the satiety of Pampus argenteus. The expression levels of two genes in the intestinal tract were significantly downregulated, which might be due to the high intestinal congestion caused by the high water content in jellyfish. The results further confirmed that cck and cart4 genes played important roles in the regulation of feeding activities of Pampus argenteus.

Keywords: cck gene; cart4 gene; Pampus argenteus; feeding jellyfish; expression pattern