Introduction
Understanding the genetic regulation of muscle growth is of great interest in aquaculture, as it directly impacts fish quality and production efficiency. Myostatin, also referred to as Growth Differentiation Factor-8 (GDF-8), functions as a negative regulator of skeletal muscle formation, making it a significant target for genetic improvement in farmed fish. Originally identified in mice (McPherron et al., Reference McPherron, Lawler and Lee1997), myostatin has been shown to influence muscle growth in various species. Over time, genome duplication events have led to the presence of two copies of the myostatin gene (mstn1 and mstn2) in most fish species and four copies in salmonids, with their expression patterns differing across tissues and developmental stages (Maccatrozzo et al., Reference Maccatrozzo, Bargelloni, Cardazzo, Rizzo and Patarnello2001a). Given the increasing global demand for sustainable aquaculture practices, enhancing fish muscle growth has become a priority for improving production and quality. Consequently, modulating myostatin expression has garnered significant interest due to its potential to optimize growth rates and muscle yield in farmed species.
Myostatin gene identification and characterization has been completed in numerous commercially significant fish species, including rainbow trout (Garikipati et al., Reference Garikipati, Gahr, Roalson and Rodgers2007, Reference Garikipati, Gahr and Rodgers2006; Rescan et al., Reference Rescan, Jutel and Rallière2001), tilapia (Rodgers and Weber Reference Rodgers and Weber2001), Atlantic salmon (Østbye et al., Reference Østbye, Galloway, Nielsen, Gabestad, Bardal and Andersen2001), striped bass (Rodgers et al., Reference Rodgers, Weber, Sullivan and Levine2001), gilthead sea-bream (Maccatrozzo et al., Reference Maccatrozzo, Bargelloni, Radaelli, Mascarello and Patarnello2001b), shi drum (Maccatrozzo et al., Reference Maccatrozzo, Bargelloni, Patarnello, Radaelli, Mascarello and Patarnello2002), channel catfish (Kocabas et al., Reference Kocabas, Kucuktas, Dunham and Liu2002), zebrafish (Xu et al., Reference Xu, Wu, Zohar and Du2003), Croceine croaker (Xue et al., Reference Xue, Qian, Qian, Li, Yang and Li2006), European sea bass (Terova et al., Reference Terova, Bernardini, Binelli, Gornati and Saroglia2006), orange spotted grouper (Ko et al., Reference Ko, Chiou, Chen, Wu, Chen and Lu2007), Japanese sea bass (Ye et al., Reference Ye, Chen, Sha and Liu2007), Japanese flounder (Zhong et al., Reference Zhong, Zhang, Chen, Sun, Qi, Wang, Li, Li and Lan2008), Barramundi (De Santis et al., Reference De Santis, Evans, Smith-Keune and Jerry2008), Medaka (Chisada et al., Reference Chisada, Okamoto, Taniguchi, Kimori, Toyoda, Sakaki, Takeda and Yoshiura2011), spotted halibut (Terova et al., Reference Terova, Rimoldi, Bernardini and Saroglia2013), large yellow croaker (Liu et al., Reference Liu, Xue, Sun, Xu and Yu2014), puffer fish (Sheng et al., Reference Sheng, Sun, Zhang, Wan, Yao, Liang, Li, Liu, Zhong, Zhang and Wang2020), and Pacu (Lattanzi et al., Reference Lattanzi, Dias, Hashimoto, Costa, Neto, Pazo, Diaz, Villanova and Reis Neto2024). Of the two copies of myostatin genes, mstn1 is widely recognized for its role in negatively regulating muscle development and growth, and its polymorphic nature makes it an important candidate gene for improving growth performance and productivity in cultured fish. mstn1 inactivation or mutation has significantly increased muscle mass in species such as zebrafish (Capilla et al., Reference Capilla, Gabillard, Garcia De La Serrana, Lou Louqiyong, Yin, Gao, Dai, Shi, Zhai, Jin, He and Lou2016), yellow catfish (Zhang et al., Reference Zhang, Wang, Dong, Dong, Chi, Chen, Zhao and Li2020), and common carp (Shahi et al., Reference Shahi, Mallik and Sarma2022). Meanwhile, mstn2 is primarily associated with neural functions, being abundantly expressed in the brain while still playing a role in myogenesis (de Santis et al., Reference de Santis, Gomes and Jerry2012). The transcriptional regulation of myostatin promoters in teleosts also remains largely unexplored compared to higher vertebrates. Comparative studies on myostatin within closely related species can provide insights into species-specific regulatory mechanisms, which are essential for developing genetic and nutritional interventions to optimize muscle growth in aquaculture.
Pompano species, belonging to the Carangidae family, are distributed across the Indian and Western Central Pacific Oceans. In India, they are found along both western and eastern coastal regions. Trachinotus mookalee (Indian pompano) and Trachinotus blochii (silver pompano/snubnose pompano) are two promising marine aquaculture species due to their rapid growth rate, adaptability to artificial feed, high market demand and suitability for captive rearing in marine and estuarine environments (Chavez et al., Reference Chavez, Fang and Carandang2011; Gopakumar et al., Reference Gopakumar, Abdul Nazar, Jayakumar, Tamilmani, Kalidas, Sakthivel, Rameshkumar, Rao, Premjothi, Balamurugan, Ramkumar, Jayasingh and Rao2012; Sekar et al., Reference Sekar, Ranjan, Xavier, Ghosh, Pankyamma, Ignatius, Joseph and Achamveetil2021). Despite similarities in morphological development, the performance of both species differs, and higher muscle growth was reported for Indian pompano than that reported for silver pompano, T. blochii (Abdul Nazar et al., Reference Abdul Nazar, Jayakumar, Tamilmani, Sakthivel, Kalidas, Ramesh Kumar, Anbarasu, Sirajudeen, Balamurugan, Jayasingh and Gopakumar2012; Sekar et al., Reference Sekar, Ranjan, Xavier, Ghosh, Pankyamma, Ignatius, Joseph and Achamveetil2021). Studies indicate that T. mookalee attains a greater body weight within the same culture period with higher muscle fibre density and size, contributing to improved fillet yield. When the performance of T. mookalee and T. blochii was compared under similar cage culture conditions in two separate experiments conducted in 2021 and 2023 by ICAR-Central Marine Fisheries Research Institute (ICAR-CMFRI), T. mookalee consistently outperformed T. blochii, reaching an average weight of 452 g in 10 months and 1235 g in 16 months, compared to 401.2 and 865 g, respectively, for T. blochii, from an initial stocking size of 11 g (unpublished data). However, differences in growth performance among these two closely related species remain poorly understood at the molecular level. Investigating the molecular architecture and expression dynamics of growth-regulating genes such as myostatin can provide insights into the genetic basis of such variation (Carnac et al., Reference Carnac, Ricaud, Vernus and Bonnieu2006; Wang et al., Reference Wang, Cheng, Su and Dunham2024). Song et al. (Reference Song, Ye, Shi, Ouyang, Sun and Luo2022) identified and characterized the complete cDNA sequences of mstn1 and mstn2 genes from T. blochii (may correspond to Trachinotus anak or its hybrids) along with their expression profiles. However, it is important to note that the species name of cultured pompano of China remains under taxonomic review, as recent genomic analyses suggest that farmed populations previously identified as T. blochii may in fact correspond to T. anak or its hybrids rather than T. blochii (Guo et al., Reference Guo, Wang, Wei, Peng, Shen, Zhou and Wu2026). Besides, the information on the genomic organization of these two genes remains unknown in any Trachinotus spp. Further, no information is available on the myostatin genes of T. mookalee. The data on the molecular structure, regulatory elements, and expression patterns of myostatin genes in various pompano species remain poorly understood despite the developments in broodstock development, captive breeding and aquaculture practices. Addressing this knowledge gap is critical for leveraging genetic improvements that can enhance the growth and productivity of these two economically important pompano species. In this study, for the first time, we identify and report the complete genetic organization and phylogenetic relationships of mstn1 and mstn2 genes in T. mookalee, along with a detailed comparative analysis of the corresponding genes in T. blochii. Notably, the paper forms the first report on the genetic organization of mstn1 and mstn2 genes in T. blochii. A detailed comparative analysis of 5’ upstream and 3’ downstream regions of mstn1 was also conducted in both species to have crucial first insights on its regulation at the molecular level. We further examined the tissue-specific expression patterns of both paralogs in ten different tissues of both species to understand their role beyond muscle regulation.
Materials and methods
Experimental animals
Three healthy adult T. mookalee (mean length: 47 ± 1.3 cm; mean weight: 1387 ± 13.4 g) and T. blochii (mean length: 40 ± 0.7 cm; mean weight: 1080 ± 13 g) of the same age (two years) and gender under similar culture conditions were collected from the marine finfish hatchery at the Indian Council of Agricultural Research-Central Marine Fisheries Research Institute (ICAR-CMFRI), India. Ten tissues, viz. skeletal muscle, gill, brain, heart, liver, kidney, intestine, spleen, stomach and gonad, were dissected and immediately preserved in RNA later at −20℃ for RNA extraction and tissue expression analysis. Additionally, muscle tissue samples from one individual of each species were preserved in 100% ethanol for subsequent DNA extraction.
All live animals were handled following the guidelines of the U.K. Animals (Scientific Procedures) Act (1986) and the E.U. Directive 2010/63/E.U. for animal experiments (2019). The live animal experiments complied with the ARRIVE guidelines (du Sert et al., Reference du Sert2020), and the experimental protocols were approved by ICAR-CMFRI, Kochi, India (MBT/GNM/25).
DNA isolation, primer designing and PCR amplification
Eight oligonucleotide primers were designed based on the conserved areas of the reported mstn1 sequences in Perciformes, while six specific primers were designed for the mstn2 sequence. Primer3 (v.0.4.0) (https://bioinfo.ut.ee/primer3-0.4.0/) was used to design the primers that cover the exons and introns in the genomic DNA. Table 1 lists the primers that were created.
Specific primers designed and used in this study
Total genomic DNA was extracted using the phenol-chloroform method, and its quality was assessed via 0.8% agarose gel electrophoresis and quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). PCR amplification was performed under the following conditions: an initial denaturation at 94℃ for 4 minutes, followed by 35 cycles of denaturation at 94℃ for 30 sec, annealing at the temperatures specified in Table 1 for 30 sec, and extension at 72℃ for 1 min. A final extension was conducted at 72℃ for 10 min. The 25 µL PCR reaction mixture contained 2.5 µL of 10× Taq buffer, 0.2 mM dNTP mix, 0.2 µM of both forward and reverse primers, 1.5 IU of Taq polymerase (Sigma), and 1 µL of template DNA (1000 ng/µL). The amplified PCR products were analysed on a 1.2% agarose gel to verify the expected amplicon sizes. The purified amplicons were subsequently sequenced using the Sanger sequencing method.
Total RNA isolation and cDNA synthesis
The TRI Reagent® (Sigma-Aldrich, USA) was used to isolate total RNA from samples preserved in RNA later, as per the manufacturer’s protocol. The quality check of the isolated RNA was done in 1% agarose gel, followed by quantification and integrity check using Nanodrop 2000 (Thermo Fisher Scientific, USA) at OD 260/280. Samples with a proper absorbance at the 260/280 ratio were selected for cDNA synthesis. The PrimeScript™ 1st strand cDNA synthesis kit (Takara, Otsu, Japan) was used for first-strand cDNA synthesis in a volume of 20 μl with 2 μg of total RNA. The cDNA samples synthesized were sequenced to get the coding region of the genes. The cDNA and genomic DNA sequences were aligned to get the exon-intron boundaries of the genes characterized. Additionally, the cDNA was kept at −20°C until it was subjected to further quantitative real-time PCR (qRT-PCR) analysis.
In silico sequence analysis of mstn1 and mstn2 genes in T. mookalee and T. blochii
The complete consensus sequences of mstn1 and mstn2 were obtained by merging overlapping PCR fragments. The full gene sequences of mstn1 and mstn2 in T. mookalee and T. blochii were submitted to NCBI via BankIt, and accession numbers were received. The Open Reading Frames (ORFs) were evaluated with ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi). Blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) performed the homology analysis of the amino acid sequence.
The physico-chemical properties of mstn1 and mstn2 protein sequences were calculated using Expasy ProtParam Tool (http://web.expasy.org/protparam/). Signal sequences, N-glycosylation sites, and phosphorylation sites were predicted using SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP), NetNGlyc1.0 Server (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/), and NetPhos 2.0 Server (https://services.healthtech.dtu.dk/services/NetPhos-3.1/), respectively. The tandem repeats in the target protein sequences were predicted using Tandem Repeat Finder (https://tandem.bu.edu/trf/trf.html), and protein domains and motifs were predicted by InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/).
Analysis of partial 5’ upstream and 3’ downstream regions of mstn1
The 5’ upstream of the mstn1 genes from the genomic DNA were carefully checked for the promoter region. The transcription factors (TFs) and the transcription factor binding sites (TFBS) within the promoter region were predicted using Tfsitescan (http://www.ifti.org/cgi-bin/ifti/Tfsitescan.pl) and analysed using the software CiiiDER, version 0.9 (Gearing et al., Reference Gearing, Cumming, Chapman, Finkel, Woodhouse, Luu, Gould, Forster and Hertzog2019). The Jaspar 2020 core vertebrate sequences were used to find perfect matches and a deficit threshold score of 0.1 to restrict the number of false positive sites.
The 3’ downstream region of mstn1 in both Trachinotus sp. was assessed for the microRNA (miRNA) target sites. The complete list of Danio rerio miRNA-mstnb binding target sites was taken from TargetScanFish 6.2 (https://www.targetscan.org/fish_62/). The predicted regulatory miRNA target sites were hybridized with the 3’ downstream region of mstn1 in T. mookalee and T. blochii using RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) (Rehmsmeier et al., Reference Rehmsmeier, Steffen, Höchsmann and Giegerich2004). Based on the stringent seed types: 8mer, 7mer-m8, 7mer-A1, 6mer, moderate stringent seed types: GUM, GUT, BM, BT, LP (Gaidatzis et al., Reference Gaidatzis, van Nimwegen, Hausser and Zavolan2007; Riolo et al., Reference Riolo, Cantara, Marzocchi and Ricci2021) and minimum free energy of hybridization, the miRNA targets were shortlisted.
Phylogenetic analysis
CLUSTAL-W was used for multiple amino acid sequence alignment, followed by phylogenetic analysis, which was done using MEGA 7.0 software (http://www.megasoftware.net). The best-fit model for amino acid substitution of the target genes was performed in MEGA 7.0. The maximum likelihood tree was constructed using the Jones–Taylor–Thornton model with gamma distribution rates. Reliability of the analysis was evaluated by 1000 bootstrap replicates.
Quantitative real-time PCR
Primers for qRT-PCR were designed across an exon: intron border sequence to rule out false positive results caused by genomic DNA contamination (Table 1). The relative quantification of mstn1 and mstn2 mRNA levels in different tissues of T. mookalee and T. blochii was measured using the qRT-PCR method; β-actin and β-2-microglobulin were selected as the internal reference genes. An aliquot of 1 µl cDNA (400 ng/µl) was used as a template in a 20 µl RT-PCR reaction system (TB GreenTM Premix Ex Taq II) with 0.8 µl of each primer (10 µM). RT-PCR was performed in triplicate via the ABI QuantStudio™ 5 real-time PCR system (Applied Biosystems). The PCR conditions were as follows: initial denaturation at 95°C for 4 min, followed by 40 cycles of 94°C for 30 sec, 62°C for 30 sec and 72°C for 25 sec, and finally terminated the reaction at 4°C. The relative quantification of mstn1 and mstn2 genes within different tissues was determined using the efficiency calibrated Pfaffl method (Pfaffl Reference Pfaffl2001; Pfaffl et al., Reference Pfaffl, Horgan and Dempfle2002). Two-way Analysis of Variance (two-way ANOVA) was employed to assess the variability of mstn1 and mstn2 expressions among different targets of fish species, and between the two fish species. A pairwise multiple comparison with Bonferroni correction was used to understand the variability of mstn1 and mstn2 expressions among different targets, keeping fish groups as the factor. Similarly, a pairwise comparison with Bonferroni correction was also used to understand the target-wise variability in the mstn1 and mstn2 expressions between the two fish species. All statistical analysis was done at a 95% significance level using SPSS 21.0 (SPSS, Inc., Chicago, Illinois).
Results
Genetic organization of mstn1 in T. mookalee and T. blochii
The complete consensus sequence of mstn1 was 3777 bp in T. mookalee and 3836 bp in T. blochii. The sequences of mstn1 were submitted to GenBank and assigned the following accession numbers: MK430047 (Tm-mstn1, T. mookalee) and MK430048 (Tb-mstn1, T. blochii).
The Tm-mstn1 sequence (3777 bp) comprised three exons (379, 371, and 381 bp) and two introns (419 and 881 bp). Similarly, the Tb-mstn1 sequence (3836 bp) contained three exons (378, 370, and 380 bp) and two introns (418 and 906 bp). The splicing GT-AG rule was consistently observed in both genes. The ORF in Tm-mstn1 and Tb-mstn1 measured 1131 bp, encoding a 376-amino acid polypeptide. The 5’ upstream region was 840 bp in Tm-mstn1 and 837 bp in Tb-mstn1, while the 3’ downstream region measured 537 bp and 552 bp, respectively. Notably, a tandem repeat of the CA consensus pattern-35 bp in Tm-mstn1 and 47 bp in Tb-mstn1-was found in the second intron, serving as potential microsatellite markers tightly linked to the mstn1 gene.
Genetic organization of mstn2 in T. mookalee and T. blochii
The complete mstn2 sequences in T. mookalee and T. blochii were 2075 bp and 2147 bp, respectively. These sequences were submitted to GenBank under the accession numbers ON246337 (Tm-mstn2, T. mookalee) and ON246336 (Tb-mstn2, T. blochii). The mstn2 gene in both Trachinotus species contained three exons, measuring 327, 370, and 380 bp in length. There were two introns in Tm-mstn2, with 603 and 272 bp in size. Further, a 93 bp 5’ upstream region and a 25 bp 3’ downstream region were also identified in Tm-mstn2. In Tb-mstn2, the introns measured 621 and 299 bp, with an 89 bp 5’ upstream region and a 55 bp 3’ downstream region. The splicing GT-AG rule was maintained in the mstn2 genes as well. The ORF for Tm-mstn2 and Tb-mstn2 was 1080 bp, encoding a 359-amino acid polypeptide.
Comparative in silico analysis of the mstn1 gene from T. mookalee and T. blochii
The predicted molecular weight of the mstn1 protein in both Trachinotus species was 42.7 kDa, with an isoelectric point (pI) of 5.52. SignalP predicted an N-terminal signal peptide of 22 residues between Met1 and Ser22 in Tm-mstn1 and Tb-mstn1. InterProScan analysis showed that mstn1 genes belonged to the GDF-8 protein family. The non-cytoplasmic domain of mstn1 was divided into two regions: the TGF-β propeptide domain (Val46 to Met254) and the TGF-β domain (Gly270 to Ser376). A conserved RXRR proteolytic cleavage site at Arg264 and a cysteine knot cytokine domain, containing nine conserved cysteine residues (positions 273–375), were identified in both Tm-mstn1 and Tb-mstn1 (Figure 1).
The cDNA sequences and deduced amino acid sequences of T. mookalee and T. blochii mstn1.
The mstn1 protein in both Trachinotus species featured a single N-glycosylation site at Asp73 and eight O-glycosylation sites at Thr26, Ser31, Thr33, Ser34, Thr38, Thr43, Ser259, and Thr269. Additionally, 37 phosphorylation sites were detected, including 21 serine, 14 threonine, and 2 tyrosine sites. Six variable sites were identified in the ORF of mstn1 at positions 372 (A/G) in exon 1, and positions 404 (G/C), 484 (T/C), 498 (G/A), 637 (A/T), and 735 (C/A) in exon 2 of T. mookalee and T. blochii (Table 2). Two of these variations resulted in non-synonymous amino acid substitutions: at position 135, glycine (G) in T. mookalee was replaced by alanine (A) in T. blochii, while at position 213, methionine (M) in T. mookalee was replaced by leucine (L) in T. blochii (Figure 1, highlighted in green).
Variable sites observed in ORF of mstn1 and mstn2
Bold values indicate positions corresponding to non-synonymous substitution sites in mstn1 and mstn2.
Comparative in silico analysis of the mstn2 gene from T. mookalee and T. blochii
The Tm-mstn2 protein had a molecular weight of 40.58 kDa and a pI of 6.17, while Tb-mstn2 had a molecular weight of 40.48 kDa and a pI of 6.03. SignalP analysis predicted the presence of an N-terminal signal peptide consisting of 16 residues (Met1 to Ser16) in both Tm-mstn2 and Tb-mstn2. Similar to mstn1, InterProScan classified mstn2 as part of the GDF-8 protein family, with a propeptide domain spanning 198 amino acids (Phe38 to Phe236) and a functional domain covering 112 amino acids (Arg247 to Ser359). The conserved RXRR proteolytic cleavage site was identified at Arg247, along with nine conserved cysteine residues positioned between 255 and 358 (Figure 2).
The cDNA sequences and deduced amino acid sequences of T. mookalee and T. blochii mstn2.
The mstn2 protein of both species contained three N-glycosylation sites (Asp65, Asp167, and Asp206), eight O-glycosylation sites (Ser29, Ser67, Thr100, Thr102, Thr105, Thr108, Ser248, and Ser252), and 32 phosphorylation sites, including 21 serine, 8 threonine, and 3 tyrosine residues. In the mstn2 ORF, eight variable sites were identified: position 136 (A/G) in exon 1, positions 438 (G/A), 465 (G/A), 534 (A/C), 546 (C/T), and 570 (C/T) in exon 2, and positions 724 (A/C) and 1047 (T/G) in exon 3 of T. mookalee and T. blochii (Table 2). Two of these variations led to non-synonymous amino acid substitutions: at position 46, arginine (R) in T. mookalee was replaced by glycine (G) in T. blochii, and at position 242, isoleucine (I) in T. mookalee was substituted by leucine (L) in T. blochii (Figure 2, highlighted in green).
Analysis for TFBS in mstn1 of T. mookalee and T. blochii
Approximately 840 and 837 bp of upstream sequences from the translation start codon of mstn1 were obtained from T. mookalee and T. blochii, respectively. This partial 5’ upstream sequence of mstn1 from both species was aligned and exhibited 93% homology. The proximal promoter region approx. −400 bp was conserved when compared to distal sequences. A list of 15 major TFs predicted with Tfsitescan, their sequence and positions in the 5’ upstream region of Tm-mstn1 and Tb-mstn1 is given in Table 3. The conserved regulatory sites included the TATA-box binding protein, CAAT box, Octamer (OCT-3), E box, putative elements responsive to growth hormone signalling, cAMP response element binding protein, POU class 1 homeobox 1(POU1F1a) or Pit-1a, growth factor independent 1B (Gfi-1B) and transforming growth factor-β2 and fork head factor (TGFβ2-FKH). Four and two E boxes were present in the analysed 5’ upstream region of T. blochii and T. mookalee, respectively.
List of major transcription factor binding sites predicted using Tfsitescan on the partial promoter region of Tm- mstn1 and Tb-mstn1
a Position (−1) given to the first nucleotide 5’ of the start codon ATG, and (R) indicates site on the reverse strand.
The partial promoter region obtained from all the samples collected was analysed in CIIIDER, and the binding sites of 25 significant TFs associated with mstn1 regulation are represented in Figure 3. TFBS, were found to be greater in the mstn1 5′ flanking region of T. blochii (47 in numbers) than in T. mookalee, which had 43 TFBS. The TFBS analysed and identified were: homeobox protein Meis1 (MEIS1), nuclear transcription factor Y Subunit C (NFYC), homeobox protein Hox-A1 (HOXA1), SRY-box transcription factor-3 (SOX3), Smad 4, basic helix-loop-helix-Per-Arnt-Superfamily(bHLH-PAS) – ARNT-2, and Zinc finger protein 384 (ZNF384).
Transcription factor binding sites (TFBS) analysed using CIIIDER in the 5’ upstream region of Tm-mstn1 and Tb-mstn1.
Analysis for miRNA target sites in mstn1 of T. mookalee and T. blochii
A total of 80 zebrafish miRNAs were computationally predicted to have potential binding sites within the downstream of coding sequence of mstn1 sequences (537 and 552 bp) of Tm-mstn1 and Tb-mstn1, respectively, using in silico alignment-based target prediction methods. Of these 80 miRNAs, 16 had potential target sites in Tm-mstn1 and 15 in Tb-mstn1 (Table 4). There were 14 common miRNA target sites in Tm-mstn1 and Tb-mstn1, while two miRNA target sites: dre-miR-27b-5p and dre-miR-30e-5p were found only in Tm-mstn1 and the miRNA target site: dre-miR-301c-5p was found only in Tb-mstn1.
List of potential miRNA target sites in the 3’ downstream region of T.Mookalee (Tm-mstn1) and T.Blochii (Tb-mstn1)
Abbreviations used: MFE: Minimum free energy; TM: T. mookale; TB: T. blochii.
Sequence alignment and phylogenetic analysis of MSTN1 and MSTN2
A comparison of MSTN1 and MSTN2 amino acid sequences within the same species revealed approximately 53% identity. Whereas MSTN1 and MSNT2 amino acid sequences of T. blochii and T. mookalee were nearly identical, with only two variable locations in the amino acid sequences brought on by non-synonymous substitutions in the ORF. Additionally, Tm-MSTN1 and Tb-MSTN1 displayed 99% similarity with the previously reported T. blochii MSTN1 (AYA21607.1) and 98% identity with Seriola lalandi MSTN1 (AZQ19980.1) (Figure 4). The protein sequences of Tm-MSTN2 and Tb-MSTN2 exhibited 96% similarity with Lates calcarifer MSTN2 (ADD91333.3) and Pampus argenteus MSTN2 (ALC79589.1) (Figure 5).
Protein multiple alignment of MSTN-1 in T. mookalee (TM_MSTN-1*) and T. blochii (TB_MSTN-1*).
Protein multiple alignment of MSTN-2 in T. mookalee (TM_MSTN-2*) and T. blochii (TB_MSTN-2*).
The phylogenetic trees for MSTN1 and MSTN2 are shown in Figures 6 and 7. The Tm-MSTN1 and Tb-MSTN1 sequences clustered with T. blochii MSTN1 (AYA21607.1) and S. lalandi MSTN1 (AZQ19980.1), both belonging to the Carangidae family, followed by L. calcarifer MSTN1 (ABS19667.1) and Lates niloticus MSTN1 (ABS19668.1) from the Latidae family. Similarly, Tm-MSTN2 and Tb-MSTN2 clustered with T. blochii MSTN2 (AXR95004.1), followed by L. calcarifer MSTN2 (ADD91333.3) and Toxotes jaculatrix MSTN2 (XP_040888291.1), representing the Carangidae, Latidae, and Toxotidae families, respectively. Both Trachinotus species in this study exhibited the closest genetic distance and highest sequence homology within the Perciformes order. Additionally, MSTN1 and MSTN2 from birds and mammals formed a distinct outgroup in both evolutionary trees.
Maximum likelihood phylogenetic tree of MSTN-1 amino acid sequences deduced from T. mookalee (TM-MSTN-1*) and T. blochii (TB-MSTN-1*).
Maximum likelihood phylogenetic tree of MSTN-2 protein sequences deduced from T. mookale (TM-MSTN-2*) and T. blochii (TB-MSTN-2*).
Quantitative expression analysis of mstn1 and mstn2
The test of between-effects revealed statistically significant variation in both mstn expressions across different target tissues within the same species (F (9, 80) = 22.357, p < 0.05) and between species (F (3, 80) = 7.626, p < 0.05). Additionally, a significant interaction effect was observed (F (27, 80) = 13.367, p < 0.05).
The expression of mstn1 was significantly higher (p < 0.05) in the muscle tissue of T. mookalee and T. blochii compared to other tissues. In T. mookalee, the second-highest mstn1 expression was observed in the kidney, whereas in T. blochii, it was in the gonads. The lowest expression levels of mstn1 were detected in the gill and intestine of both Trachinotus species. Notably, mstn1 expression in muscle tissue was significantly higher in T. blochii than in T. mookalee (p < 0.05) (Figure 8).
The expression pattern of mstn1 was determined by qRT-PCR.
For mstn2, the brain exhibited the highest expression in both T. mookalee and T. blochii (p < 0.05), followed by the gonads (Figure 9). Compared to mstn1, mstn2 showed lower expression in muscle tissue in both species. Overall, mstn1 expression was significantly higher than mstn2 (p < 0.05).
The expression pattern of mstn2 was determined by qRT-PCR.
Discussion
T. mookalee and T. blochii are two promising tropical mariculture species. Previous studies (Abdul Nazar et al., Reference Abdul Nazar, Jayakumar, Tamilmani, Sakthivel, Kalidas, Ramesh Kumar, Anbarasu, Sirajudeen, Balamurugan, Jayasingh and Gopakumar2012; Kalidas et al., Reference Kalidas, Ramesh Kumar, Linga Prabu, Tamilmani, Anbarasu, Rajendran and Thiagu2022; Ranjan et al., Reference Ranjan, Megarajan, Xavier, Ghosh, Santhosh and Gopalakrishnan2018; Sekar et al., Reference Sekar, Ranjan, Xavier, Ghosh, Pankyamma, Ignatius, Joseph and Achamveetil2021) and personal observations have consistently recorded superior growth and better fillet characteristics in T. mookalee compared to T. blochii. Similar observations were made in the present study, where two-year-old T. mookalee (1387 ± 13.4 g) showed a higher body weight than T. blochii (1080 ± 13 g). Despite these clear differences, the molecular mechanisms behind this variation remain largely unexplored. Myostatin (mstn), a key growth-regulating gene in the TGF-β superfamily, has emerged as a crucial candidate for studying growth differences in teleosts. This study, therefore, aimed to comparatively analyse mstn1 and mstn2 genes in T. mookalee and T. blochii, along with their expression profiles, as the initial step to gain insights into their potential differential muscle growth.
Comparative protein sequence analysis revealed high similarity between Tm-mstn1 and Tb-mstn1 (99.5%), as well as between Tm-mstn2 and Tb-mstn2 (99.4%), indicating strong evolutionary conservation within the Trachinotus genus. This conservation was further reflected in the N-terminal signal peptides, two conserved TGF-β domains, and nine cysteine residues critical for dimerization and functional activity. Additionally, a conserved RXRR proteolytic cleavage site was also observed in other vertebrates, highlighting the functional integrity of these proteins. Notably, the highly conserved C-terminal TGF-β domain reinforces the evolutionary stability of this region across vertebrate lineages. Phylogenetic analysis supported these observations, clustering mstn1 and mstn2 sequences of Trachinotus species closely with those of other Carangidae family members. The distinct separation of avian and mammalian mstn sequences as outgroups further emphasizes the evolutionary divergence of myostatin genes in teleosts. Comparative analyses across multiple teleost species support the idea that, while mstn genes share a common ancestral origin, adaptive evolution has contributed to their diversification in response to species-specific environmental and physiological demands. These findings lay the groundwork for future investigations into the evolutionary and functional mechanisms influencing growth-related traits in aquaculture species.
The mstn1 and mstn2 genes in both Trachinotus species encode proteins comprising 376 and 359 amino acids, respectively, with only 53% amino acid sequence identity between the two paralogs within the same species. This divergence is indicative of gene duplication events commonly observed in teleosts. Similar patterns of divergence between mstn1 and mstn2 have been reported in grass carp and pufferfish (Yan et al., Reference Yan, Lu, Kong, Meng, Luan, Dai, Chen, Cao and Luo2020; Zheng et al., Reference Zheng, Sun, Pu, Chen, Jiang and Zou2015).
Genomic analysis revealed that both mstn1 and mstn2 in T. mookalee and T. blochii possess a conserved three-exon, two-intron structure, consistent with previous findings in other teleosts (Chisada et al., Reference Chisada, Okamoto, Taniguchi, Kimori, Toyoda, Sakaki, Takeda and Yoshiura2011; Garikipati et al., Reference Garikipati, Gahr, Roalson and Rodgers2007, Reference Garikipati, Gahr and Rodgers2006; Ko et al., Reference Ko, Chiou, Chen, Wu, Chen and Lu2007; Kocabas et al., Reference Kocabas, Kucuktas, Dunham and Liu2002; Lattanzi et al., Reference Lattanzi, Dias, Hashimoto, Costa, Neto, Pazo, Diaz, Villanova and Reis Neto2024; Liu et al., Reference Liu, Xue, Sun, Xu and Yu2014; Maccatrozzo et al., Reference Maccatrozzo, Bargelloni, Patarnello, Radaelli, Mascarello and Patarnello2002, 2001b; Østbye et al., Reference Østbye, Galloway, Nielsen, Gabestad, Bardal and Andersen2001; Rescan et al., Reference Rescan, Jutel and Rallière2001; Rodgers et al., Reference Rodgers, Weber, Sullivan and Levine2001; Rodgers and Weber Reference Rodgers and Weber2001; Sheng et al., Reference Sheng, Sun, Zhang, Wan, Yao, Liang, Li, Liu, Zhong, Zhang and Wang2020; Song et al., Reference Song, Ye, Shi, Ouyang, Sun and Luo2022; Terova et al., Reference Terova, Rimoldi, Bernardini and Saroglia2013, Reference Terova, Bernardini, Binelli, Gornati and Saroglia2006; Xu et al., Reference Xu, Wu, Zohar and Du2003; Xue et al., Reference Xue, Qian, Qian, Li, Yang and Li2006; Ye et al., Reference Ye, Chen, Sha and Liu2007; Zhong et al., Reference Zhong, Zhang, Chen, Sun, Qi, Wang, Li, Li and Lan2008). Despite this conserved architecture, notable differences were observed in the intron lengths of both mstn genes between the two species. Specifically, the second intron of mstn1 measured 881 bp in T. mookalee and 906 bp in T. blochii. For mstn2, the introns were 603 bp and 272 bp in T. mookalee, and 621 bp and 299 bp in T. blochii. Despite the overall structural conservation, Takifugu spp. also exhibited a comparable pattern of intron length variation (Sheng et al., Reference Sheng, Sun, Zhang, Wan, Yao, Liang, Li, Liu, Zhong, Zhang and Wang2020). Although introns are non-coding, they often harbour regulatory elements such as enhancers, silencers, and TFBS. Therefore, the observed intronic variations may influence transcriptional efficiency and protein expression, potentially contributing to differences in muscle growth between species. These variations may also be valuable for designing species-specific primers or probes for the mstn gene studies.
A notable finding was the presence of a CA tandem repeat in the second intron of mstn1 (35 bp in T. mookalee and 47 bp in T. blochii), which could serve as a potential microsatellite marker closely linked to the mstn1 gene. This repeat was absent in mstn2 of both species. The presence and species-specific length variation of this repeat may reflect underlying genomic divergence. Given that mstn1 is a negative regulator of muscle growth, the observed variation might have functional implications and can be correlated with growth performance differences between T. mookalee and T. blochii and warrants future research.
In the next phase of the study, a systematic analysis of the 5′ upstream region of mstn1 was conducted in both Trachinotus species. The identification of a TATA box within the proximal promoter region, along with binding sites for key TFs such as CEBPs, OCT-3, putative elements responsive to Growth Hormone signalling, POU1F1a, Gfi-1B, MEIS1, and TGF-β2-FKH, suggests a complex regulatory network orchestrating mstn1 transcription in both species. These findings are consistent with previous reports in teleosts, where TFBS, including TATA boxes and MEF2 elements, are known to regulate MSTN expression (Grade et al., Reference Grade, Mantovani and Alvares2019). Further, given the well-established antagonistic roles of growth hormone and myostatin in muscle growth, the presence of GH-responsive elements may propose an inverse regulatory relationship by which GH signalling could suppress myostatin expression (Liu et al., Reference Liu, Thomas, Asa, Gonzalez-Cadavid, Bhasin and Ezzat2003). The promoter region also included a predicted binding site for POU1F1a, a TF primarily expressed in the anterior pituitary to regulate the expression of hormones like growth hormone and prolactin (Ben-Batalla et al., Reference Ben-Batalla, Seoane, Macia, Garcia-Caballero, Gonzalez, Vizoso and Perez-Fernandez2010). While POU1F1 is not known to be expressed in skeletal muscle, its binding motif in the myostatin promoter may suggest a potential regulatory role in pituitary-specific expression of myostatin, where POU1F1 is active. Although mstn1 is primarily expressed in skeletal muscle, reports of its expression in the pituitary tissues (Taketa et al., Reference Taketa, Nagai, Ogasawara, Hayashi, Miyake, Tanaka, Watanabe, Ohwada, Aso and Yamaguchi2008) raise the possibility of tissue-specific regulatory interactions that warrant further investigation. Accordingly, studies are needed to validate whether POU1F1 directly regulates myostatin transcription in the pituitary and its potential involvement in endocrine regulation of muscle growth. Notably, several major TFBS were identified, including homeobox protein MEIS1, Nuclear Transcription Factor Y Subunit C (NFYC), homeobox protein HOXA1, SRY-box TF SOX3, SMAD4, the bHLH-PAS family member ARNT-2, and Zinc Finger Protein ZNF384, all of which are involved in developmental and signalling pathways (Grade et al., Reference Grade, Mantovani, Fontoura, Yusuf, Brand-Saberi and Alvares2017; Haldin and LaBonne Reference Haldin and LaBonne2010). Importantly, T. blochii exhibited more TFBS (47 vs 43) and E-box elements (4 vs. 2) than T. mookalee, suggesting a potentially more complex transcriptional regulation of mstn1 in T. blochii. These interspecies differences may reflect divergent regulatory evolution of the mstn1 gene and underpin phenotypic differences such as muscle mass, growth rate, or physiological responses to environmental stressors reported between T. blochii and T. mookalee. However, these TFBS and E-box motifs predictions are computational only, and should be experimentally validated to confirm their biological roles. Further, future investigations focusing on the functional role of these additional TFBS and E-box motifs in T. blochii will be valuable in elucidating their roles as activators or repressors of mstn1. Such insights may guide the development of CRISPR/Cas-based regulatory editing strategies for enhancing growth traits in these economically important species.
Next, we identified potential miRNA target sites within the downstream of the coding sequence of mstn1 to gain insights into the post-transcriptional regulatory mechanisms modulating gene expression. A total of 14 miRNA target sites were conserved between Tm-mstn1 and Tb-mstn1. In contrast, two miRNA target sites (dre-miR-27b-5p and dre-miR-30e-5p) were unique to Tm-mstn1, while dre-miR-301c-5p was exclusive to Tb-mstn1. The shared miRNAs suggest a conserved regulatory mechanism of mstn1 across species, whereas the differential sites indicate species-specific post-transcriptional control of mstn1 expression. The miRNAs can regulate gene expression by binding to complementary sequences in target mRNAs, resulting in mRNA degradation or inhibition of translation (Wilczynska and Bushell Reference Wilczynska and Bushell2015). Several miRNAs targeting mstn1 have been implicated in modulating muscle growth and development in teleosts. For example, in Larimichthys crocea, miR-2014-5p and miR-1231-5p were reported to suppress mstn1 expression, enhancing muscle development (Lou et al., Reference Lou, Zhao, Zhang, Zheng, Feng, Hosain and Xue2021). Among the differentially identified miRNA target sites, dre-miR-27b-5p and dre-miR-30e-5p have been shown to repress mstn1 expression in cattle and mice, respectively, facilitating increased muscle growth (Jia et al., Reference Jia, Zhao, Li, Zhang and Zhu2017; Miretti et al., Reference Miretti, Martignani, Accornero and Baratta2013). However, the role of dre-miR-301c-5p in mstn1 regulation remains uncharacterized. Understanding miRNA-mediated regulation of mstn1 offers valuable opportunities for optimizing muscle growth in aquaculture species. Specifically, dre-miR-27b-5p and dre-miR-30e-5p may serve as promising candidates for enhancing growth performance in T. mookalee, while dre-miR-301c-5p could be targeted in T. blochii to achieve similar outcomes. However, we have not conducted any wet experiments on these identified miRNAs, forming a limitation of the study. Future studies involving functional validation through targeted knockdown experiments are essential to substantiate these findings and advance selective breeding efforts to improve growth traits in pompano species.
Overall, the comparative analysis of molecular features revealed a high degree of similarity in the protein sequence and exon-intron organization of the mstn genes between T. mookalee and T. blochii. In contrast, notable differences were observed in the intron lengths, the length variation of the CA tandem repeat within intron 2, the number of TFBS and E-box motifs in the 5′ upstream region, and the miRNA target sites of mstn1. These variations in the non-coding regions suggest the presence of potential regulatory elements that may contribute to species-specific differences in mstn1 gene expression. It is important to note that this study examined only partial segments of the regulatory regions, approximately 840 bp of the 5′ upstream and 550 bp of the 3′ downstream sequences of mstn1. In teleosts, however, the 5′ upstream region typically spans 1–2 kb (Grade et al., Reference Grade, Mantovani and Alvares2019), and the 3′ downstream region ranges from 800 bp to 1.4 kb (De Santis et al., Reference De Santis, Evans, Smith-Keune and Jerry2008; Garikipati et al., Reference Garikipati, Gahr, Roalson and Rodgers2007, Reference Garikipati, Gahr and Rodgers2006). Despite this limitation, the analysed segments revealed significant regulatory differences. These findings point to substantial divergence in regulatory control, which may result in differing mstn1 expression profiles between the two species.
To evaluate whether these molecular differences influence mstn1 expression, we performed quantitative PCR. Both mstn1 and mstn2 genes exhibited ubiquitous expression across all analysed tissues, suggesting their involvement in a broad range of physiological processes beyond muscle regulation, including metabolism, development, immune response, and reproduction, depending on the tissue. These findings align with observations in other teleosts (Helterline et al., Reference Helterline, Garikipati, Stenkamp and Rodgers2007; Sheng et al., Reference Sheng, Sun, Zhang, Wan, Yao, Liang, Li, Liu, Zhong, Zhang and Wang2020; Zhong et al., Reference Zhong, Zhang, Chen, Sun, Qi, Wang, Li, Li and Lan2008). For example, mstn1 and mstn2 expression in the spleen of rainbow trout was proposed to be associated with immune function (Garikipati et al., Reference Garikipati, Gahr and Rodgers2006), and Helterline et al., (Reference Helterline, Garikipati, Stenkamp and Rodgers2007) reported elevated mstn1 expression in the spleen under stocking stress, supporting its immunomodulatory role. Further analysis revealed distinct expression profiles for mstn1 and mstn2, indicating functional divergence between these paralogs. Expression of mstn1 was significantly higher (p < 0.05) in muscle tissue of both species compared to other tissues, approximately 10-fold in T. mookalee and 40-fold in T. blochii, consistent with its established role as a negative regulator of muscle growth. This result corroborates previous findings in teleosts, where mstn1 is predominantly expressed in skeletal muscle to modulate myogenesis (De Santis et al., Reference De Santis, Evans, Smith-Keune and Jerry2008; Garikipati et al., Reference Garikipati, Gahr and Rodgers2006; Ko et al., Reference Ko, Chiou, Chen, Wu, Chen and Lu2007; Liu et al., Reference Liu, Yu and Tong2012; Østbye et al., Reference Østbye, Galloway, Nielsen, Gabestad, Bardal and Andersen2001; Sheng et al., Reference Sheng, Sun, Zhang, Wan, Yao, Liang, Li, Liu, Zhong, Zhang and Wang2020; Xu et al., Reference Xu, Wu, Zohar and Du2003). Conversely, mstn2 displayed the highest expression levels in the brain, followed by the gonads, indicating its likely involvement in neuroendocrine regulation and reproductive processes. This pattern, together with its relatively lower expression in muscle tissues, reinforces the notion that mstn2 functions beyond muscle development, in agreement with reports from other fish species (Garikipati et al., Reference Garikipati, Gahr and Rodgers2006; Schuelke et al., Reference Schuelke, Wagner, Stolz, Hübner, Riebel, Kömen, Braun, Tobin and Lee2004). In summary, the observed expression patterns suggest that mstn1 primarily contributes to myogenesis, while mstn2 is implicated in neuroendocrine regulation, followed by reproductive regulation. At the same time, their widespread and tissue-specific expression highlights the complexity of their roles, which may vary across tissues and species. Further investigations into the effects of environmental factors, nutritional inputs, and hormonal regulation on mstn1 and mstn2 expression will be crucial for refining growth enhancement strategies in aquaculture.
Comparative analysis between the two Trachinotus species revealed that mstn1 exhibited the lowest expression levels in the gill and intestine of both T. mookalee and T. blochii. Interestingly, the second-highest expression of mstn1 was observed in the kidney of T. mookalee, whereas in T. blochii, it was found in the gonads. These findings suggest a potential functional diversification of the mstn1 gene beyond its canonical role in muscle development, possibly shaped by species-specific ecological, physiological, or evolutionary pressures. Notably, mstn1 expression in muscle tissue was significantly higher in T. blochii compared to T. mookalee. This observed variation may reflect differences in promoter architecture, transcriptional activity, or post-transcriptional regulation between the two species, supporting the regulatory disparities identified in the earlier molecular feature comparison. These results also reinforce the growing consensus that mstn1 expression patterns are not conserved even among closely related species, underscoring the importance of species-specific investigations rather than relying on generalized assumptions.
Farming trials conducted by ICAR-CMFRI have shown that T. mookalee achieves approximately 30% faster growth than T. blochii under comparable pond and cage culture conditions (personal communication). The higher expression of mstn1 in T. blochii than in T. mookalee may underlie the relatively slower growth observed in T. blochii. Myostatin (mstn) is a well-established negative regulator of muscle growth, and decreased expression has been associated with increased muscle mass and growth performance in fish (Acosta et al., Reference Acosta, Carpio, Borroto, Gonzalez and Estrada2005; Tao et al., Reference Tao, Tan, Chen, Xu, Liao, Li and Hu2021; Zhang et al., Reference Zhang, Wang, Dong, Dong, Chi, Chen, Zhao and Li2020). Hence, the observed variation in mstn1 expression has certain practical implications for selective breeding, as individuals with naturally lower mstn1 activity may display superior growth traits and thus represent preferable candidates for broodstock selection. Furthermore, targeted gene-editing approaches such as CRISPR/Cas9-mediated mstn knockout have demonstrated significant enhancement of growth performance in aquaculture species, including channel catfish (Ictalurus punctatus) (Khalil et al., Reference Khalil, Elayat, Khalifa, Daghash, Elaswad, Miller and Dunham2017), red sea bream (Pagrus major) (Kishimoto et al., Reference Kishimoto, Washio, Yoshiura, Toyoda, Ueno, Fukuyama and Kinoshita2018), and loach, Misgurnus anguillicaudatus (Tao et al., Reference Tao, Tan, Chen, Xu, Liao, Li and Hu2021). Such interventions could be explored in T. blochii to mitigate growth limitations imposed by high mstn1 expression and accelerate genetic improvement programs. Nevertheless, further research at the protein level, including functional assays such as gene knockdown or CRISPR-mediated knockout experiments, is essential to confirm the functional relevance of these findings. Additionally, a deeper exploration of the regulatory networks and upstream modulators of mstn1 expression, such as nutritional inputs, hormonal cues, and environmental stressors, could identify novel targets for precision-based growth enhancement strategies. Such insights may enable the development of tailored biotechnological interventions to optimize growth and productivity in Trachinotus species, advancing sustainable aquaculture practices.
Conclusion
This study presents the first complete genetic characterization of the myostatin genes (mstn1 and mstn2) in T. mookalee, along with the first report on the genomic DNA organization of these genes in T. blochii. Comparative analysis of mstn1 and mstn2 within each species revealed up to 53% sequence similarity, indicating divergence in sequence and potentially in function. A notable finding was the presence of a tandem CA repeat motif in intron 2 of mstn1 (35 bp in T. mookalee and 47 bp in T. blochii), which could serve as a potential microsatellite marker tightly linked to the mstn1 gene. Gene expression profiling supported distinct functional roles for the two paralogs: mstn1 is primarily involved in myogenesis, while mstn2 appears to function mainly in neuroendocrine regulation, followed by roles in reproductive processes. Despite strong conservation in protein sequences and exon-intron organization, significant interspecies differences were observed in the non-coding regions of mstn1, including intron lengths, the length of CA tandem repeats in intron 2, the number of TFBS and E-box elements in the 5′ upstream region, and miRNA target sites. These differences highlight the presence of divergent regulatory elements that may contribute to species-specific expression patterns and regulatory mechanisms. This hypothesis was validated by differential mstn1 expression profiles observed between the two species. In particular, mstn1 expression in muscle tissue was significantly higher in T. blochii compared to T. mookalee, which could partially explain the differences in muscle mass or growth rates previously reported for these species. Such findings point to a stronger inhibitory control of muscle development in T. blochii, consistent with its higher mstn1 expression. Overall, the findings offer the first insights into the molecular basis of differential muscle growth between these two commercially important mariculture species. Further exploration of the regulatory pathways controlling mstn1 expression holds a strong potential for developing species-specific strategies to enhance growth performance in sustainable aquaculture systems.
Acknowledgements
The authors are thankful for the financial support rendered by the Indian Council of Agriculture Research, Department of Agriculture, Research and Education, Government of India, New Delhi. The authors express their gratitude to the Director and employees of ICAR-CMFRI for providing the necessary resources to conduct this study and for their unwavering support during the study period.
Ethical standards
All live animals were handled following the guidelines of the U.K. Animals (Scientific Procedures) Act (1986) and the E.U. Directive 2010/63/E.U. for animal experiments (2019). The live animal experiments complied with the ARRIVE guidelines (du Sert et al., Reference du Sert2020), and the experimental protocols were approved by ICAR-CMFRI, Kochi, India (MBT/GNM/25).
Data availability statement
This manuscript includes every piece of data created or analysed during this investigation. The NCBI-GenBank (https://www.ncbi.nlm.nih.gov/genbank/) has the sequence data generated during this study under accession numbers MK430047, MK430048, ON246337, and ON246336.
Author contributions
C.L.V., A.K., S.T.G. and G.G.: conceptualization and funding acquisition. C.L.V., W.S., and R.P.: experimental work, data collection and analysis and original draft preparation. C.L.V., A.K., G.G., S.S., and S.T.G.: data curation, formal analysis and investigation; all authors: writing, review and editing.
Funding
The work was supported by the Indian Council of Agriculture Research, Department of Agriculture, Research and Education, Government of India, New Delhi (MBT/GNM/25).
Competing interests
The authors declare no conflict of interest.
Declaration of AI-assisted technologies
During the preparation of this work, the authors used ChatGPT in order to improve the language and readability in certain selected portions. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.
