ABSTRACT

   Extrachromosomal circular DNAs (eccDNAs) play a significant role in
   regulating various biological processes, including abnormal muscle
   development. The molecular functions and impact of eccDNAs in the
   muscle development of fish are poorly understood. To investigate the
   potential roles of eccDNAs in the muscle development of fish, we
   analyzed and compared the expression profile of muscle eccDNAs of crisp
   grass carp, fed a faba bean meal‐based diet, and ordinary grass carp,
   fed a practical diet. Using the Circle‐seq strategy, we found the
   eccDNA abundance in crisp grass carp (211,920 eccDNAs) was
   significantly higher than that in ordinary grass carp (25,857 eccDNAs),
   suggesting that the faba bean diet likely independently influences
   eccDNA production. Compared to ordinary grass carp, crisp grass carp
   exhibited 10,565 upregulated and 129 downregulated eccDNAs, indicating
   eccDNAs were possibly associated with the muscle development of grass
   carp. GO and KEGG enrichment analyses indicated that the upregulated
   eccDNAs were related to muscle fiber development, cellular structure,
   and cell junctions. Based on our results, we speculated that the
   overexpression of genes involved in muscle fiber, calcium metabolism,
   and collagen driven by eccDNAs likely contributes to the observed
   increase in muscle fiber density, calcium levels, and collagen content
   in crisp grass carp, thereby enhancing muscle hardness. Notably,
   eccDNAs were identified as potential innate immunostimulants capable of
   eliciting immune responses in fish. In summary, our findings
   demonstrate that eccDNAs are aberrantly expressed in the muscles of
   fish fed a faba bean diet, offering novel insights into the molecular
   mechanisms underlying muscle hardening in fish.

   Keywords: extrachromosomal circular DNA, fillet quality improvement,
   muscle texture
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   We provide a new and basic eccDNA database for the molecular regulation
   of muscle firmness in grass carp. The eccDNA abundance in crisp grass
   carp was significantly higher than that in ordinary grass carp. EccDNAs
   likely play important roles in the muscle‐hardening of crisp grass
   carp.

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1. Introduction

   Muscle texture is a critical determinant of fish quality, directly
   influencing the production of high‐quality fish products (Hyldig and
   Nielsen [40]2001). Understanding the molecular mechanisms regulating
   fish muscle texture could significantly advance the development of
   premium fish products and enhance profitability in the aquaculture
   industry (Elvevoll et al. [41]1996). Among muscle characteristics,
   hardness, or firmness, is particularly important in shaping consumer
   preferences (Veland and Torrissen [42]1999). Grass carp