Transcriptome Assembly and Comparative Analysis of the Superoxide Dismutase (SOD) Gene Family in Three Hyotissa Species

TL;DR

This study explores the SOD gene family in three Hyotissa bivalve species, revealing diverse and specialized genes that may help them survive in harsh reef environments.

Contribution

The study provides the first comprehensive transcriptomic and comparative analysis of SOD genes in Hyotissa species.

Findings

46 SOD genes were identified, showing both conserved and diversified features.

Specialized SODs like Dominin and CSRPs suggest functional divergence and adaptation.

The SOD family in Hyotissa appears to have evolved to cope with high-oxygen and high-ultraviolet reef conditions.

Abstract

The marine bivalves of the genus Hyotissa (family Gryphaeidae) are important for ocean ecosystems and fisheries; however, their molecular biology information is notably scarce. Our study aimed to specifically explore crucial defense genes called superoxide dismutases (SODs), which act as the body’s natural defenders against cellular damage. We discovered 46 diverse SOD genes in three Hyotissa species. Our analysis revealed that these genes possess both ancient, conserved features and newer, diversified characteristics. Intriguingly, we found specialized SODs like Dominin and copper-only SOD repeat proteins (CSRPs), exhibiting structural changes hinting at new roles. These variations suggest Hyotissa’s protective genes have adapted to perform various tasks, potentially enabling them to thrive in demanding reef environments characterized by high ultraviolet radiation and high oxygen levels. This study provides foundational transcriptomic resources for Hyotissa and offers new insights into the evolution and environmental adaptation of SOD genes in marine bivalves.

The genus Hyotissa (family Gryphaeidae) comprises ecologically and economically important marine bivalves, yet their molecular biology remains poorly characterized. This study presents de novo transcriptome sequencing of three Hyotissa species—H. sinensis, H. inaequivalvis, and Hyotissa sp.—to systematically identify and characterize the superoxide dismutase (SOD) gene family, a crucial component of the antioxidant defense system. We identified 46 SOD genes, including both Cu/Zn-SOD and Fe/Mn-SOD types, which exhibited considerable variation in molecular properties, domain architecture, and potential phosphorylation sites. Phylogenetic analysis revealed both evolutionary conservation and diversification of SODs across species. Notably, we identified homologs of two specialized SOD types: Dominin, which showed mutations in metal-binding sites suggestive of functional divergence, and copper-only SOD repeat proteins (CSRPs), which retained copper-binding residues but lost zinc-binding capacity. These findings suggest that the SOD family in Hyotissa has undergone significant functional diversification, potentially as an adaptive response to their high-oxygen, high-ultraviolet reef habitats. This study provides foundational transcriptomic resources for Hyotissa and offers new insights into the evolution and environmental adaptation of SOD genes in marine bivalves.

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1. Introduction

The superfamily Ostreoidea, which includes the families Gryphaeidae and Ostreidae, represents a major clade of marine bivalves [1]. While Ostreidae has been relatively well-studied, Gryphaeidae diverged earlier, during the Permian–Triassic period [2]. Within Gryphaeidae, the genus Hyotissa has notably rich species [3], making it a valuable group for understanding evolutionary trajectories and adaptive diversification in Ostreoidea. Hyotissa species are widely distributed in tropical and subtropical waters of the Western Atlantic, Indo-Pacific, and Eastern Pacific [4,5]. They are typically large, with thick shells, prominent radial ribs, and unique vesicular microstructures [6,7]. The complex three-dimensional structure of their shells provides habitat for diverse microfauna, underscoring their ecological importance [8]. Additionally, the adductor muscle of Hyotissa species—comprising over half of their soft tissue—is highly valued for its quality, giving these species significant economic value and aquaculture potential [9].

Superoxide dismutase (SOD) is a key antioxidant enzyme that catalyzes the dismutation of superoxide anion (O_2_^−^) into oxygen and hydrogen peroxide (H_2_O_2_), thereby mitigating reactive oxygen species (ROS) and reducing oxidative damage [10,11,12]. SODs are classified based on their metal cofactors into Cu/Zn-SOD, Fe/Mn-SOD, and Ni-SOD types [13]. In eukaryotes, Cu/Zn-SODs are further divided into intracellular (icCu/Zn-SOD) and extracellular (ecCu/Zn-SOD) forms, which differ in subcellular localization and function [14]. Recent studies have revealed considerable expansion and functional diversification of the SOD gene family in some invertebrates [15,16,17]. For instance, the extracellular Cu/Zn-SOD homolog Dominin in oysters appears to have lost canonical SOD activity, acquiring new roles in metal binding and immune regulation [18,19,20]. Similarly, the recently discovered copper-only SOD (Cu-only SOD) and its multi-domain form, copper-only SOD repeat proteins (CSRPs), retain conserved copper-binding sites for catalysis but have mutated zinc-binding sites, further enriching the functional diversity of the SOD family [21,22].

Unlike Ostreidae species such as the Pacific oyster (Crassostrea gigas), which inhabit estuarine, intertidal, or shallow marine environments with muddy or sandy substrates, Hyotissa species typically attach to coral reefs or other hard substrates [23,24]. These habitats are characterized by clear water, stable high salinity, warm temperatures, and intense ultraviolet radiation, leading to elevated oxidative stress [23]. Such conditions, particularly high UV exposure and temperature, promote persistent ROS production [25]. As SODs constitute the first line of defense in the antioxidant system, the pronounced oxidative stress in Hyotissa habitats may have driven adaptive evolution in their SOD gene family, resulting in a more robust antioxidant defense system.

Advances in high-throughput sequencing have revolutionized biological research, enabling large-scale genomics and transcriptomics projects [26]. However, despite the recent completion of the first chromosome-level genome for H. hyotis [27], genomic and transcriptomic data for Hyotissa remain scarce in public databases like NCBI. This data gap has led to species misidentification (e.g., H. hyotis being mislabeled as H. sinensis) and has hindered related research. To address this, we performed de novo transcriptome sequencing of three Hyotissa species and systematically identified and characterized their SOD genes. Our analysis of molecular characteristics, conservation, and specificity provides a foundation for understanding environmental adaptation and phenotypic evolution in Gryphaeidae oysters.

2. Materials and Methods

2.1. Sample Collection

Five wild Hyotissa individuals were collected from Weizhou Island, Beihai City, Guangxi Zhuang Autonomous Region, China (21°02′4″ N, 109°06′6″ E). Based on mitochondrial 16S rRNA gene fragment sequencing, three species were identified: individuals 1 and 3 as Hyotissa inaequivalvis, individual 2 as Hyotissa sp. and individuals 4 and 5 as Hyotissa sinensis [28]. After collection, each oyster was dissected to separate gill, mantle, smooth muscle, and striated muscle tissues (Figure 1).

2.2. Total RNA Extraction and Quality Control

Total RNA was extracted from four tissues using the TRNzol Universal Reagent Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RNA integrity (RIN) and quality were assessed using an Agilent 5400 Bioanalyzer (Agilent, Santa Clara, CA, USA). Only RNA samples with a concentration ≥ 50 ng/µL, a RIN value > 6.0, and no detectable protein or organic solvent contamination were used for cDNA library construction.

2.3. Library Construction and Sequencing

Libraries were constructed and sequenced by Novogene Corporation (Beijing, China). The Fast RNA-seq Lib Prep Kit V2 (Abclonal, Wuhan, China) was used for mRNA enrichment, fragmentation, and cDNA library construction. Libraries were preliminarily quantified with a Qubit 2.0 Fluorometer (Thermo, Waltham, MA, USA), diluted to 1.5 ng/μL, and insert size was assessed with an Agilent 5400 Bioanalyzer. After confirming expected insert size, the effective concentration was quantified via qRT-PCR to ensure ≥1.5 nM. Qualified libraries were sequenced on the Illumina NovaSeq X Plus platform (Illumina, San Diego, CA, USA), generating 150 bp paired-end reads, with a target data output of 6 G for each library.

2.4. RNA-Seq Data Processing and Quality Control

Raw read quality was assessed with FastQC (v0.12.1) for base quality, GC content, duplication rate, overrepresented k-mers, and quality score distribution [29]. MultiQC (v1.30) aggregated quality metrics across samples [30]. Raw reads were processed with fastp (v0.19.7) [31] to remove adapter-containing reads, poly-N sequences, and low-quality reads, yielding clean reads. Q20 scores, Q30 scores, and GC content of the clean data were calculated.

2.5. Transcriptome Assembly

In this study, we performed independent de novo transcriptome assembly and annotation for five Hyotissa individuals. For each oyster, high-quality sequencing data were assembled de novo using Trinity (v2.13.2), yielding five separate transcriptomes [32]. The assembly was performed using default parameters optimized for transcriptome data. The completeness of the transcriptome assembly was assessed using Benchmarking Universal Single-Copy Orthologs (BUSCO) software (v5.5.0) with default parameters. Each assembly was compared with the mollusca_odb10 database (http://busco.ezlab.org/) (accessed on 10 December 2025) to evaluate its completeness [33]. Then, TransDecoder (v5.5.0) was used to predict the open reading frames (ORFs) of the filtered nucleotide sequences and translate them into protein sequences [34]. The longest protein sequence from the resulting protein sequence file was extracted as the input file for subsequent analysis.

2.6. Transcriptome Assembly and SOD Gene Identification

SOD genes were identified from transcriptomic data of five Hyotissa individuals. Gene IDs were labeled with individual-specific prefixes (“Hsi1-2”, “Hin1-2”, “Hsp1”) to denote species origin. Candidate SOD sequences were obtained using BLAST (v2.12.0) and HMMER (v3.2.1) with significant hits (E-value ≤ 1.00 × 10^−10^) and SOD domain matches (PF00080, PF00081, PF02777; E-value ≤ 1.00 × 10^−10^). Domain presence was verified with SMART (https://smart.embl.de/) (accessed on 15 May 2025). Genes with partial coding sequences were excluded from further analysis.

2.7. Molecular Characterization of SOD Family Members

Amino acid length, molecular weight (kDa), isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY) were calculated using Expasy ProtParam (https://web.expasy.org/protparam/) (accessed on 15 May 2025) with default parameters [35]. Subcellular localization was predicted with WoLFPSORT (https://wolfpsort.hgc.jp/) (accessed on 15 May 2025) with default parameters [36]. Phosphorylation sites were predicted using NetPhos (v3.1) (https://services.healthtech.dtu.dk/services/NetPhos-3.1/) (accessed on 15 May 2025) [37]. The prediction score ranges from 0.000 to 1.000, and sites with a score above the threshold of 0.500 were considered positive predictions. Signal peptides and transmembrane domains were analyzed using SignalP 6.0 with default settings (https://services.healthtech.dtu.dk/services/SignalP-6.0/) (accessed on 15 May 2025) [38]. Conserved motifs were identified with MEME (https://meme-suite.org/meme/tools/meme) (accessed on 15 May 2025), with the maximum number set to 10 and other parameters as default [39].

2.8. Sequence Alignment and Phylogenetic Analysis

The SOD genes identified from the transcriptome data of Hyotissa were subjected to multiple sequence alignment with the SOD protein sequences retrieved from the NCBI database for Pacific oyster (Crassostrea gigas), human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), and nematode (Caenorhabditis elegans). The alignment was performed using MAFFT software (v7.526) with the BLOSUM62 amino acid substitution matrix [40]. The best substitution model (WAG+G) was selected using MEGA 11, and a phylogenetic tree was constructed using the Maximum Likelihood (ML) method in MEGA 11, with branch support assessed by the bootstrap method with 1000 replicates.

To further analyze the evolutionary relationships of the two specialized SOD types in oysters, Dominin and CSRP, the Dominin3 (XP_011439797.1) and CSRP (XP_034330263.1) sequences from C. gigas were used as a reference to search for homologous sequences in transcriptomic data from five individuals of the Hyotissa genus, with an E-value threshold of less than 1 × 10^−10^ and a query coverage greater than 40% as screening criteria. The homologous Dominin sequences identified from the Hyotissa genus were subjected to multiple amino acid sequence alignment with the intracellular Cg-Cu/Zn-SOD (XP_034334952.2) from C. gigas [20], Cg-Dominin3, human extracellular SOD (HuEcSOD, P08294), and human cytoplasmic SOD (HuCySOD, P00441) to analyze their key active sites, and a phylogenetic tree was constructed. Additionally, the individual SOD domains of Hyotissa CSRP were aligned with HuEcSOD (P08294), HuCySOD (P00441), and C. gigas intracellular Cu/Zn-SOD (XP_034334952.2) using multiple sequence alignment to analyze the conserved amino acid residues essential for copper ion coordination in their active sites.

3. Results

3.1. Technical Validation of Transcriptome Sequencing

Illumina sequencing generated 464,824,286 paired-end reads (Table 1). The number of raw reads per sample ranged from 21,172,853 to 24,393,919. MultiQC reports confirmed high base quality (Phred score > 35) and high mean quality scores across sequences. After processing with fastp, 456,109,722 high-quality clean read pairs were obtained. This corresponded to a per-sample clean read count range of 20,776,969 to 25,021,203 reads. Q20 and Q30 scores were ≥98.07% and ≥94.23%, respectively, and GC contents were within expected ranges (Table 1). After assembly, the number of transcripts across the five samples ranges from 327,624 to 379,502, with assembly continuity, as measured by N50, varying between 1324 and 1595 bp (Table S1). Additionally, BUSCO analysis revealed high completeness (Table S2). These findings show that a high-quality RNA-seq dataset was successfully generated. Raw transcriptome data are available in the NCBI SRA under accession number PRJNA1356962.

3.2. Identification of the SOD Gene Family

A total of 46 SOD family genes were identified from the five Hyotissa transcriptomes: six each from Hin1 and Hsp1, ten from Hin2, sixteen from Hsi1, and eight from Hsi2 (Table 2 and Table S3). Domain analysis classified five sequences as Fe/Mn-SODs, based on the presence of N-terminal Fe/Mn-SOD α-hairpin (PF00081) and C-terminal Fe/Mn-SOD (PF02777) domains; one sequence contained only the C-terminal domain. The remaining sequences contained one to four Sod_Cu domains (PF00080) and were identified as Cu/Zn-SOD homologs. Six of these also contained a heavy metal-associated (HMA) domain (PF00403) (Figure 2).

Physicochemical properties varied widely: amino acid lengths ranged from 95 to 1035 residues, molecular weights from 11.06 to 112.32 kDa, and pI from 5.10 to 9.75 (mostly 5.5–7.8). Instability indices ranged from 14.29 to 54.06, with many exceeding 40, indicating potential instability. Aliphatic indices were generally high (>70), suggesting thermostability. All proteins had negative GRAVY values, indicating hydrophilicity. Twelve proteins possessed signal peptides, suggesting extracellular secretion or organelle localization. Phosphorylation sites ranged from 5 to 130, with Hin1-DN1483c1g1i7.p1 having the highest number, indicating potential for functional diversification.

MEME analysis identified ten conserved motifs (Figure 2B). Motif 1, associated with the Sod_Cu domain, was present in all Cu/Zn-SODs. Except for Hsi1-DN51085c0g1i2.p1, all Fe/Mn-SODs contained Motif 2 (N-terminal Fe/Mn-SOD α-hairpin) and Motif 5 (C-terminal Fe/Mn-SOD domain).

3.3. Phylogenetic Analysis of Hyotissa SOD

A phylogenetic tree was constructed using SOD sequences from Hyotissa and six other species. Due to significant expansion in C. gigas, its SODs were designated SOD1a-b, SOD2, and SOD3a-l based on relationships with intracellular Cu/Zn-SOD (SOD1), Mn-SOD (SOD2), and extracellular Cu/Zn-SOD (SOD3). The tree revealed three major clades: Cu/Zn-SOD (48% bootstrap), Mn-SOD (99% bootstrap), and copper chaperone for SOD (94% bootstrap) (Figure 3).

3.4. Sequence Analysis of the ecSOD Homolog Dominin in Hyotissa

Seven Dominin homologs were identified in the Hyotissa transcriptomes. Sequence alignment with Cg-Cu/Zn-SOD, Cg-Dominin3, HuEcSOD, and HuCySOD showed that Hsi1-DN129556c0g1i2.p1 shared five metal-binding site mutations with Cg-Dominin3, while Hsi2-DN4644c0g2i2.p1 had only one mutation. The other five homologs were identical to Cg-Cu/Zn-SOD at all seven metal-binding sites (Figure 4). Phylogenetic analysis indicated divergent evolution: one group was orthologous to C. gigas Dominin3, while the other formed a distinct clade (Figure 5).

3.5. Sequence and Domain Comparison of Hyotissa CSRPs

Eleven CSRPs were identified among the 46 SOD genes. Pairwise amino acid identity ranged from 40.35% to 99.31% (Figure 6). The amino acid coordinates for all Cu-SOD domains are listed; two CSRPs were found to contain only three such domains, while the remaining nine contained the typical four-domain architecture (Table S4). Except for two sequences with three Cu-SOD domains, all others had four. Alignment showed that Hyotissa CSRP domains retained the five conserved residues for Cu^2+^ coordination but had mutations in two histidine residues critical for Zn^2+^ binding (Figure 7).

4. Discussion

Despite extensive omics data for Ostreidae (e.g., C. gigas, C. hongkongensis) [24,41], Hyotissa (Gryphaeidae) remains underrepresented in public databases—with only H. hyotis’ genome recently published [27]. This scarcity has hindered studies of trait-associated genes (e.g., stress resistance, biomineralization) and molecular mechanisms of adaptation, and our de novo transcriptomes of three Hyotissa species address this gap, providing a resource for functional gene mining and evolutionary research.

The 46 identified SOD genes (Cu/Zn-SOD, Fe/Mn-SOD) exhibit broad variation in domains, physicochemical properties, and phosphorylation sites—suggesting functional flexibility. Phylogenetic alignment with model organisms confirms their evolutionary conservation while highlighting Hyotissa-specific diversification, likely driven by coral reef stressors (high UV, oxidative stress). Since such stress results in elevated ROS levels, antioxidant enzymes are upregulated in response to stress stimuli in order to protect the organism from cellular damage [42,43]. The Hyotissa-specific SOD lineages may facilitate a more robust and tiered defense system, allowing these bivalves to thrive in highly fluctuating reef habitats.

Dominin, a major plasma protein in Crassostrea virginica, constitutes over 40% of plasma and pallial cavity fluid protein, indicating a critical physiological role. Despite having a Cu/Zn-SOD domain, mutations in five metal-coordinating histidines abolish SOD activity [18,19,20]. Among the seven Dominin homologs identified in Hyotissa, phylogenetic analysis revealed two groups: one orthologous to C. gigas Dominin3 and another distinct clade. Beyond descriptive sequence alignment—which showed varying mutations in metal-binding sites—we infer potential functional divergences. Sequence alignment showed varying metal-binding site mutations: Hsi1-DN129556c0g1i2.p1 shared five mutations with Cg-Dominin3, likely losing SOD activity, while Hsi2-DN4644c0g2i2.p1 had only one mutation, potentially preserving SOD activity [20]. The other five homologs contain fully conserved metal-binding sites, indicating they may function as active extracellular SODs (EC-SODs). This contrasts sharply with Ostreidae oysters, which appear to have evolved Dominin primarily as a non-enzymatic protein, possibly leaving them without an EC-SOD-active form. These differences suggest divergent evolutionary paths in immune-related protein function between oyster families, which may reflect adaptation to distinct physiological or environmental challenges.

CSRPs, multi-domain copper-only antioxidant enzymes, have been identified in various oysters [17]. They possess SOD activity and are upregulated under stress, indicating a role in extracellular antioxidant defense [22]. We identified several CSRP homologs, four with Cu-SOD domains and two with three domains. The latter deviates from the typical four-domain architecture in Ostreidae, possibly due to incomplete assembly. One domain in Hsi1-DN2474c0g1i4.p1 lacked a full set of conserved residues. These are tentatively classified as “candidate CSRP genes with three Cu-SOD domains,” requiring full-length cDNA validation. If confirmed, a three-domain CSRP in Hyotissa would suggest that the four-domain structure is not essential for antioxidant defense under certain evolutionary pressures. From an evolutionary perspective, the reduction in repeated domains often correlates with functional specialization, structural stability adjustment, and adaptive optimization [44]. The three-domain architecture in Hyotissa may therefore not represent a simple loss but a refined adaptation to its unique habitat. This streamlined form could alter protein–protein interactions or ROS-scavenging kinetics, representing an evolutionary trade-off that balances antioxidant capacity with the specific environmental pressures characteristic of its niche, such as thermal fluctuations, hypoxia, or osmotic stress.

Finally, this study has limitations that should be addressed in future research: the small sample size (n = 5 individuals) may limit the representativeness and generalizability of the findings, the three-domain CSRP structure requires full-length cDNA cloning to rule out assembly gaps, SOD activity assays (e.g., for Dominin and CSRPs) are needed to confirm enzymatic capacity, and integrating additional Hyotissa genomes will clarify SOD family evolution across Gryphaeidae.

5. Conclusions

In summary, this study provides high-quality transcriptomic resources for three Hyotissa species, addressing a significant data gap. We systematically identified the SOD gene family and revealed its evolutionary relationships through cross-species comparison. Further analysis of Dominin and CSRP homologs uncovered potential diversity in antioxidant defense mechanisms within this genus. Crucially, the next critical step involves rigorous functional validation of the identified SOD genes, for example, through gene expression analysis under oxidative stress, protein activity assays, or RNA interference experiments. These future functional assays, combined with genomic data from additional species, will not only validate our current findings but also further elucidate the evolutionary history of the SOD family in bivalves and the molecular basis of their environmental adaptation.