TORC1 Regulates Thermotolerance via Modulating Metabolic Rate and Antioxidant Capacity in Scallop Argopecten irradians irradians

Abstract

Target of rapamycin complex 1 (TORC1) is a key regulator of metabolism in eukaryotes across multiple pathways. Although TORC1 has been extensively studied in vertebrates and some invertebrates, research on this complex in scallops is limited. In this study, we identified the genes encoding TORC1 complex subunits in the scallop Argopecten irradians irradians through genome-wide in silico scanning. Five genes, including TOR, RAPTOR, LST8, DEPTOR, and PRAS40, that encode the subunits of TORC1 complex were identified in the bay scallop. We then conducted structural characterization and phylogenetic analysis of the A. i. irradians TORC1 (AiTORC1) subunits to determine their structural features and evolutionary relationships. Next, we analyzed the spatiotemporal expressions of AiTORC1-coding genes during various embryo/larvae developmental stages and across different tissues in healthy adult scallops. The results revealed stage- and tissue-specific expression patterns, suggesting diverse roles in development and growth. Furthermore, the regulation of AiTORC1-coding genes was examined in temperature-sensitive tissues (the mantle, gill, hemocyte, and heart) of bay scallops exposed to high-temperature (32 °C) stress over different durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d, and 10 d). The expression of AiTORC1-coding genes was predominantly suppressed in the hemocyte but was generally activated in the mantle, gill, and heart, indicating a tissue-specific response to heat stress. Finally, functional validation was performed using the TOR inhibitor rapamycin to suppress AiTORC1, leading to an enhanced catabolism, a decreased antioxidant capacity, and a significant reduction in thermotolerance in bay scallops. Collectively, this study elucidates the presence, structural features, evolutional relationships, expression profiles, and roles in antioxidant capacity and metabolism regulation of AiTORC1 in the bay scallop, providing a preliminary understanding of its versatile functions in response to high-temperature challenges in marine mollusks.

Keywords: Argopecten irradians irradians; TORC1; expression regulation; functional allocation; genome-wide identification; thermotolerance.

Figure 1

Genomic structure of AiTORC1-coding genes. Arrows indicate the gene locations on chromosomes. Exons within the ORF are shown as colored boxes, whereas 5′ and 3′ UTRs are represented by uncolored boxes, with introns represented by dashed lines. The number above each exon presents the exon length (bp).

Figure 2

Structure and predicted protein domains of AiTORC1 by SMART analysis. Various domains are shown in boxes with different colors and shapes, with their names provided in boxes.

Figure 3

Secondary and tertiary structures of AiTORC1 subunits. Alpha helices are shown in pink cylinders, beta strands in yellow arrows, represent beta turns are in blue arrows, and random coils are in gray wavy lines.

Figure 4

Multiple sequence alignment of AiTORC1 subunits, AiTOR (A), AiRAPTOR (B), AiLST8 (C), AiDEPTOR (D), and AiPRAS40 (E), amino acid sequences with homologous sequences from other species. Conserved amino acid are shaded in black. The gray-shaded regions represent similar amino acid residues. Sequences with low homology were not colored. Gaps are represented by dashes to improve the alignment. The symbols indicate various domains: The represent HEAT domains, the represents DUF3385 domain, the represents FAT domain, the represents Rapamycin bind domain, the represents PI3Kc domain, the represents FATC domain, the represents Raptor N domain, the represent WD40 domains, the represent DEP domains, the represents PDZ domain, and the represents PRAS domain. Hs: H. sapiens, Mm: M. musculus, Gg: G. gallus, Xl: X. laevis, Dr: D. rerio, Ai: A. i. irradians, Pm: P. maximus, My: M. yessoensis, Cg: C. gigas, Ca: C. angulata. Accession numbers for these TORC1 subunits in other species are included in Supplementary Table S2.

Figure 4

Multiple sequence alignment of AiTORC1 subunits, AiTOR (A), AiRAPTOR (B), AiLST8 (C), AiDEPTOR (D), and AiPRAS40 (E), amino acid sequences with homologous sequences from other species. Conserved amino acid are shaded in black. The gray-shaded regions represent similar amino acid residues. Sequences with low homology were not colored. Gaps are represented by dashes to improve the alignment. The symbols indicate various domains: The represent HEAT domains, the represents DUF3385 domain, the represents FAT domain, the represents Rapamycin bind domain, the represents PI3Kc domain, the represents FATC domain, the represents Raptor N domain, the represent WD40 domains, the represent DEP domains, the represents PDZ domain, and the represents PRAS domain. Hs: H. sapiens, Mm: M. musculus, Gg: G. gallus, Xl: X. laevis, Dr: D. rerio, Ai: A. i. irradians, Pm: P. maximus, My: M. yessoensis, Cg: C. gigas, Ca: C. angulata. Accession numbers for these TORC1 subunits in other species are included in Supplementary Table S2.

Figure 4

Multiple sequence alignment of AiTORC1 subunits, AiTOR (A), AiRAPTOR (B), AiLST8 (C), AiDEPTOR (D), and AiPRAS40 (E), amino acid sequences with homologous sequences from other species. Conserved amino acid are shaded in black. The gray-shaded regions represent similar amino acid residues. Sequences with low homology were not colored. Gaps are represented by dashes to improve the alignment. The symbols indicate various domains: The represent HEAT domains, the represents DUF3385 domain, the represents FAT domain, the represents Rapamycin bind domain, the represents PI3Kc domain, the represents FATC domain, the represents Raptor N domain, the represent WD40 domains, the represent DEP domains, the represents PDZ domain, and the represents PRAS domain. Hs: H. sapiens, Mm: M. musculus, Gg: G. gallus, Xl: X. laevis, Dr: D. rerio, Ai: A. i. irradians, Pm: P. maximus, My: M. yessoensis, Cg: C. gigas, Ca: C. angulata. Accession numbers for these TORC1 subunits in other species are included in Supplementary Table S2.

Figure 5

Phylogenetic tree of TORC1 based on the amino acid sequences of AiTORC1 subunits and homologous sequences from other species, constructed using the Neighbor-Joining method. AiTOR, AiRAPTOR, AiLST8, AiDEPTOR, and AiPRAS40 are represented by red, green, blue, yellow, and purple circles, respectively. Hs: H. sapiens, Mm: M. musculus, It: I. tridecemlineatus, Vu: V. ursinus, Bt: B. taurus, Xl: X. laevis, Xt: X. tropicalis, Dr: D. rerio, St: S. tigrinum, Sk: S. kowalevskii, Dm: D. melanogaster, Ap: A. planci, Sp: S. purpuratus, Cg: C. gigas, Ca: C. angulata, Pm: P. maximus, Py: P. yessoensis, Ai: A. i. irradians, Ac: A. californica, Dg: D. gigantean, The accession numbers of TORC1 subunits in other species are summarized in Supplementary Table S2.

Figure 6

Spatio-temporal expression profiles of AiTORC1-coding genes. (A) The expression profiles of AiTORC1-coding genes from embryos or larvae across eight developmental stages (zygotes, 2–8 cells, blastula, gastrula, trochophores, D-shaped larvae, umbo larvae, and juvenile scallops) based on log₂TPM after laterally homogenizing. (B) The expression profiles of AiTORC1 from 10 types of adult tissues (hepatopancreas, foot, mantle, gill, gonad, kidney, smooth muscle, striated muscle, hemocyte, and heart) based on log₂TPM after laterally homogenizing. Significance (p < 0.05) is indicated through “*”.

Figure 7

Expression regulations of AiTORC1-coding genes to high-temperature (32 °C) stimulation assessed in four types adult tissues (mantle, hemocyte, gill, and heart) and expressed as log2FC. Significance (p < 0.05) is indicated through “*”.

Figure 8

Effects of TOR inhibitor rapamycin on metabolism indicators in bay scallops following 24 h treatment. (A) Changes in oxygen consumption and ammonia excretion in bay scallops 24 h post treatment. Values that lack a single conserved letter differ significantly (p < 0.05) from each other. (B) Heart rate variations in bay scallops with temperature rising from 22 °C to 36 °C 24 h post treatment. (C) RAP variations in bay scallops with temperature from 22 °C to 34 °C 24 h post treatment.

Figure 9

Effects of TOR inhibitor rapamycin on antioxidant ability assessment in bay scallops following 24 h treatment. (A) SOD activity. (B) CAT activity. (C) ROS content. Values that lack a single conserved letter differ significantly (p < 0.05) from each other.