Phylogenetic analysis of mstn nucleotide sequences. The mstn genes cluster into five main clades including an invertebrate clade, a clade of reptiles and avians, a mammalian clade that also includes the ancient coelacanth fish, a clade of piscine mstn1 and a clade for piscine mstn2. Sequences used in this analysis are: Mozambique tilapia, Oreochromis mossambicus, AF197193; Lanzhou catfish, Silurus lanzhouensis, KU302769; Nile tilapia 1, Oreochromis niloticus, XM_003458832; Nile tilapia 2, Oreochromis niloticus, MN708486; Silver pomfret 1, Pampus argenteus, KM259900; Lake Qinghai scale-less carp 1, Gymnocypris przewalskii, KJ607138; Japanese grenadier anchovy, Coilia nasus, KF638401; Macrobrachium nipponense, KF530847; Roughskin sculpin, Trachidermus fasciatus, GU198192; goldfish, Carassius auratus, KC851952; Chinese rare minnow, Gobiocypris rarus, FJ482232; starlet sea anemone, Nematostella vectensis, AGL96595; common quail, Coturnix coturnix, AF407340; spotted rose snapper, Lutjanus guttatus, JX987064; spotted halibut, Verasper variegatus, JN226745; Barbel chub, Squaliobarbus curriculus, JN230816; Barramundi perch, Lates calcarifer HQ731440; cow, Bos taurus, GQ184147; yellowcheek carp, Elopichthys bambusa, HM461971; white perch, Morone Americana, AF290911; striped sea-bass, Morone saxatilis, AF290910; gilthead seabream 1, Sparus aurata, AF258448; white bass, Morone chrysops, AF197194; turkey, Meleagris gallopavo, AF019625; human, Homo sapiens, AF104922; Norway rat, Rattus norvegicus, AF019624; sheep, Ovis aries, AF019622; common carp 1a, Cyprinus carpio, GU014395; common carp 1b, Cyprinus carpio, GU014396; common carp 2a, Cyprinus carpio, GU014397; common carp 2b, Cyprinus carpio, GU0143958; yellow catfish 1, Tachysurus fulvidraco, DQ767966; Alpine ibex, Capra ibex, AY629305; Schlegel's black rockfish, Sebastes schlegelii, DQ423474; blackback land crab, Gecarcinus lateralis, EU432218; black porgy, Acanthopagrus schlegelii, DQ303480; white seabass, Atractoscion nobilis, AY966401; stinging catfish, Heteropneustes fossilis, HQ003245; North African catfish, Clarias gariepinus, KJ372760; swamp eel, Monopterus albus, KM103284; Akoya pearl oyster, Pinctada martensii, KJ579132; Schizopygopsis pylzovi, JX088635; bighead catfish, Clarias microcephalus, JX456396; Mandarin fish, Siniperca chuatsi, JF896453; blue catfish, Ictalurus furcatus, AY540992; largemouth bass, Micropterus salmoides, DQ666527; Florida lancelet, Branchiostoma floridae, XM_002599415; domestic yak, Bos grunniens, EU926669; zebrafish a, Danio rerio, NM_001004122; zebrafish b, Danio rerio NM_131019; fugu 2, Takifugu rubripes, NM_001032672; Lake Qinghai scale-less carp 2, Gymnocypris przewalskii, KP277103; grass carp 2, Ctenopharyngodon idella, KM874827; grass carp 1, Ctenopharyngodon idella, KM874826; Japanese medaka, Oryzias latipes, NM_001201499; yellow catfish 2, Tachysurus fulvidraco KF537384; shi drum 2, Umbrina cirrosa, JX002683; Wuchang bream 2, Megalobrama amblycephala, JQ065337; Wuchang bream 1, Megalobrama amblycephala, JQ065336; fugu 1, Takifugu rubripes, AY445322; fine flounder 1, Paralichthys adspersus, EU443627; Australian saltwater crocodile, Crocodylus porosus, XM_019554060; large yellow croaker 1, Larimichthys crocea, AY842933; red-bellied piranha, Pygocentrus nattereri, XM_017713514; gecko, Gekko japonicas, XM_015405581; Mexican tetra, Astyanax mexicanus, XM_007253246; coelacanth, Latimeria chalumnae, XM_005996542; Chinese tree shrew, Tupaia chinensis, XM_006147825; channel catfish, Ictalurus punctatus, XM_017469117; southern platyfish, Xiphophorus maculatus XM_014468464; gilthead seabream 2, Sparus aurata, AY046314; large yellow croaker 2, Larimichthys crocea, JF304776; Pacific white shrimp, Litopenaeus vannamei, JQ045427; northern pike, Esox Lucius, XM_010879812; killer whale, Orcinus orca, XM_004276934; shi drum 1, Umbrina cirrosa, AF316881; silver pomfret 2, Pampus argenteus, KT726407; rainbow trout 1a, Oncorhynchus mykiss,NM_001124282; rainbow trout 1b, Oncorhynchus mykiss,NM_001124283; rainbow trout 2a, Oncorhynchus mykiss,DQ417326; rainbow trout 2b (pseudogene), Oncorhynchus mykiss, DQ417327; Atlantic salmon 1a, Salmo salar, NM_001123634; Atlantic salmon 1b, Salmo salar, NM_001123549; Atlantic salmon 2a, Salmo salar, JN990763; Atlantic salmon 2b (pseudogene), Salmo salar, JN990773. Phylogenetic analysis was performed using MEGA7 software (37). The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (44). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (45). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 85 nucleotide sequences.

Conservation of tilapia mstn2. (A) Chromosomal synteny of Nile tilapia mstn1 and mstn2. Genes adjacent to mstn in the Nile tilapia, zebrafish and human genomes were manually identified using both USCS and ensembl genome browsers (https://genome.ucsc.edu/ and https://www.ensembl.org/index.html, respectively). The genes are named according to their annotation in the human genome. While Nab1 and MFSD6a genes were found to be syntenic in both piscine mstn genomic regions, most of the human MSTN neighboring genes were syntenic only to mstn1 or mstn2. (B) Pairwise alignment of tilapia Mstn1 and Mstn2 amino acid sequences illustrates the high similarity of the tilapia Mstn proteins. Red asterisk indicates conserved cysteine residues, which are important for Mstn peptide activity. The conserved proteolytic RXRR motif is indicated by a red rectangle.

Localization of timstn1 and timstn2 mRNA in various tissues of Nile tilapia using real-time PCR. Expression levels of timstn1 and timstn2 were determined by real-time PCR in juvenile male (A), juvenile females (B), adult males (C), and adult females (D). The data are presented as mean ± SD.

Environmental challenges affect the expression of timstn1 and timstn2 in Nile tilapia brain. Expression levels of timstn1(A–C) and timstn2(D–F) were determined by real-time PCR. Expression was analyzed in the fish forebrain (A,D), midbrain (B,E), and hindbrain (C,F). The data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ^#p = 0.053.

Environmental challenges affect the expression of timstn1 in Nile tilapia muscle tissues. Expression levels of timstn1 mRNA in the white muscle (A) and red muscle (B) were determined by real-time PCR. The data are presented as mean ± SD. *p < 0.05; ****p < 0.0001.

Metabolic parameters in plasma of Nile tilapia remained unchanged in response to various environmental conditions. Total protein (A) and triglyceride (B) levels were determined by photometric measurement. Plasma cortisol levels (C) were analyzed using a specific ELISA. The data are presented as mean ± SD. ^#p = 0.098.