Research paperA potential negative regulation of myostatin in muscle growth during the intermolt stage in Exopalaemon carinicauda
Introduction
Molting, the cyclic exchange of exoskeleton, is a growth‐related phenomenon in crustaceans. The periodic molting is a special growth and development mode of crustaceans. Like most other crustaceans, the growth and development of shrimp primarily depend on muscle restoration and net muscle gain during molting. Abdominal muscle buildup occurs mainly during intermolt, a cell proliferating metabolic phase (Lemos and Weissman, 2021). Therefore, it is essential to explore the target genes regulating muscle buildup in crustaceans during intermolt. Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and is a potent negative regulator of skeletal muscle differentiation and growth. MSTN plays important roles in regulating embryonic development and maintaining tissue homeostasis in mammals (Martinez-Hackert et al., 2021). Initial studies found that mutations in MSTN can cause a significant increase in muscle mass and stronger musculature in mammals (Gao et al., 2014, Hu et al., 2013), leading to the double-muscling phenotype of some cattle and sheep (Huang et al., 2011). Similarly, MSTN-deficient medaka showed a significant increase in body weight from the post-juvenile to the adult stage (Chisada et al., 2011), and fast-growing loaches were obtained through the targeted mutation of MSTN (Tao et al., 2021).
In invertebrates, MSTN has been cloned and identified from insects, molluscs (Li et al., 2016) and crustaceans (Kim et al., 2009, Nguyen et al., 2016, Qian et al., 2013). Similar to that in mammal, MSTN restricts muscle growth in some crustaceans, for instance, the preliminary histological analysis following MSTN silencing of Macrobrachium rosenbergii favors muscle regeneration, supporting its functional role as a negative growth regulator (Easwvaran et al., 2019). Reduced MSTN levels after RNA interference in Fenneropenaeus chinensis result in dramatically faster growth rates than those of a control group of shrimp (Yan et al., 2020), and knockdown of MSTN accelerated growth and shortened molt interval in Eroicheir sinensis (Yue et al., 2020). Mstn of Homarus americanus is an important regulator of protein turnover in molt-induced claw muscle atrophy (MacLea et al., 2010). The mTOR plays a role in regulating protein turnover in molting (Medler and Mykles, 2015, Wittmann et al., 2018), and MSTN inhibits protein synthesis in Gecarcinus lateralis through the mTOR pathway (Covi et al., 2010, MacLea et al., 2012). These data suggested that MSTN might negatively regulate muscle growth in crustaceans. However, some other studies indicated that MSTN might positively regulate muscle growth in crustaceans. For example, MSTN knockdown in Penaeus monodon adversely affected its growth trait (De Santis et al., 2011), muscle growth was restricted in Litopenaeus vannamei after MSTN knockdown (Lee et al., 2015). It is still inconclusive that whether MSTN is a positive or negative regulator of muscle growth in crustaceans. Therefore, it is crucial to clarify the function of the MSTN gene and its molecular mechanism of regulating growth traits in crustaceans.
Growth is one of the most critical factors for the commercial success of aquatic species. Most commercial traits are quantitative, influenced by many genes and environmental factors. Association analysis based on SNP markers is a common strategy to obtain major genes that affect quantitative traits in crustaceans (Jung et al., 2013). MSTN provides more functional molecular markers for the genetic improvement of aquatic species, including fish (Wang et al., 2016), echinoderms (Li et al., 2016), molluscs (Fan et al., 2017, Niu et al., 2015) and crustaceans (Zhuo et al., 2017). Accordingly, MSTN is considered as a potential candidate gene for improving growth-related trait in aquatic animals.
The ridgetail white shrimp Exopalaemon carinicauda, belonging to the Palaemonidae family of crustaceans, is a major commercial mariculture species naturally distributed on the coasts of the Yellow Sea and Bohai Sea (Xu et al., 2010). Similar to other aquaculture species, one of the main focuses of the E. carinicauda farming industry is improving the growth rate by genetic breeding (Wang et al., 2020a). Since MSTN in aquatic animals plays a vital role in regulating of growth traits and its manipulation may significantly impact aquaculture and domestication, we aimed to elucidate its function in E. carinicauda. The main purpose of the current study was to identify and characterize the MSTN gene from E. carinicauda and investigate the possible function of MSTN during the intermolt stage in Exopalaemon carinicauda and provide effective genetic markers from MSTN associated with growth traits for the genetic improvement of E. carinicauda.
Section snippets
Isolation of full-length cDNA and gDNA of EcMSTN
Based on the partial sequences from transcriptome database in E. carinicauda cDNA library of our laboratory, some specific primers were designed for 3′ RACE and 5′ RACE (Table 1). The template of 3′ and 5′ ends were obtained using abdominal muscle tissue RNA by SMART™ RACE cDNA Amplification Kit (Clontech, USA). PCR reactions were performed with 3′ and 5′ RACE template using the gene specific primers and the anchor primer UPM (Table 1).
The genomic sequence of the EcMSTN gene was subsequently
Molecular characterization of EcMSTN
The E. carinicauda MSTN was deposited in the GenBank database under accession number MG545149.1 and named EcMSTN. The nucleotide and deduced amino acid sequences are shown in Fig. 1. The complete EcMSTN cDNA was 1,518 bp in length and included a 5′ untranslated region (UTR) of 134 bp, an open reading frame (ORF) of 1,287 bp, and a 3′ UTR of 97 bp including a polyA signal sequence. The complete gDNA sequences from the ATG start codon to the TGA stop codon of EcMSTN are 1,851 bp in length and
Discussion
The MSTN is a pivotal negative regulator of skeletal muscle growth and differentiation in mammals. Muscle growth in crustaceans is a complicated process where the muscle grows and develops through muscle restoration during molting. The growth rate depends on the net muscle gain during each molt (Hyde et al., 2019, Tian et al., 2020). Therefore, the function of MSTN in crustaceans is probably different from that in mammals. The first MSTN gene identified in a crustacean species was cloned from
CRediT authorship contribution statement
Jiajia Wang: Conceptualization, Investigation, Data curation, Writing - original draft. Jitao Li: Methodology, Writing - review & editing. Qianqian Ge: Software, Resources. Jian Li: Supervision, Validation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This project was financially supported by the earmarked fund for National Natural Science Foundation of China (No. 31902368), National Key R & D Program of China (No. 2019YFD0900403), China Agriculture Research System of MOF and MARA (No. CARS-48), Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO. 2020TD46).
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