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Journal of Marine Life Science Vol.10 No.2 pp.132-145
DOI : https://doi.org/10.23005/ksmls.2025.10.2.132

Identification of Sexual Maturation Genes in Octopus minor Using Transcriptome Analysis

Duk-Young Kang, Jinik Hwang*
West Sea Fisheries Institute, National Institute of Fisheries Science, Incheon 22383, Republic of Korea
Corresponding Author Jinik Hwang E-mail : jinike12@korea.kr
September 24, 2025 ; November 10, 2025 ; November 10, 2025

Abstract


Octopus minor is an important seafood species inhabiting Korean coastal mudflats and holds considerable industrial value. However, the restricted distribution of mudflats and increasing environmental pollution have highlighted the need for stock restoration and the development of superior strains. In this study, we analyzed the transcriptomic profiles of O. minor across sexes, tissues, and developmental stages to identify genes associated with sexual maturation. A de novo transcriptome assembly yielded 993,121 transcripts, and after redundancy removal and expression refinement, 40,688 transcripts were retained. Differential expression analysis identified 8,104 DEGs in females (gonad: 7,068; optic lobe: 1,036) and 5,285 in males (gonad: 4,765; optic lobe: 520). Gene expression changes related to sexual maturation were more pronounced in females and in gonadal tissues than in males and optic lobes. These findings provide essential genomic insights into the molecular mechanisms underlying sexual maturation and offer valuable resources for selective breeding and sustainable aquaculture of O. minor.


Ctopus minor , De novo transcriptome assembly , Sexual maturation , Gonad , Optic lobe

초록

    Introduction

    The octopus (Octopus minor), a molluscan species distributed across Korea, Japan, and China, is an important seafood resource in the coastal mudflats of southwestern Korea and holds considerable industrial value (Oh et al., 2012). Ecologically, octopuses act as key energy transmitters within marine food webs, preying on lower-trophic organisms such as crustaceans and mollusks (Vaz-Pires et al., 2004;Bo et al., 2020). As a low-calorie and taurine-rich food, O. minor is abundant in protein, phosphorus, iron, and vitamins, contributing to cholesterol reduction and anemia prevention, and is often recommended for convalescent patients and pregnant women (Lee et al., 2019).

    Despite its ecological and nutritional importance, O. minor faces persistent challenges in reproduction, spawning, and aquaculture due to its restricted habitat distribution, pollution of mudflats, short lifespan (1–1.5 years), and low fecundity. Therefore, artificial breeding and selective breeding technologies are urgently required to develop superior traits and ensure sustainable resource management. Sexual maturation plays a crucial role in reproduction, requiring complex physiological and molecular processes associated with growth and gonadal development. Previous studies on O. minor have mainly focused on morphological and biological characteristics in Gyeonggi Bay (Moon et al., 1989), behavioral observations (Jong et al., 2003), and growth and spawning patterns in southern coastal regions (Chung et al., 1999). Research on reproduction has primarily addressed spawning and hatching mechanisms (Zheng et al., 2014).

    Recent advances in molecular biology have facilitated genomic and transcriptomic studies using next-generation sequencing (NGS) technologies (Shendure et al., 2008). Although microarrays have traditionally been used to investigate gene expression patterns, their application is limited in non-model organisms lacking reference genome sequences. In contrast, RNA sequencing (RNA-seq) provides a powerful alternative, enabling the analysis of gene expression levels and differential expression without prior genomic information. Moreover, RNA-seq offers higher sensitivity and a broader dynamic range than microarray-based approaches (Roh et al., 2010).

    In the present study, RNA-seq was employed to identify genes and differentially expressed genes (DEGs) associated with sexual maturation in O. minor. Transcriptomic data were obtained from gonadal and optic lobe tissues across different sexes and developmental stages, with triplicate samples to ensure reproducibility. De novo transcriptome assembly was performed to construct reference sequences, which were subsequently normalized for comparative analysis. Identified DEGs were then subjected to functional annotation and enrichment analyses to elucidate molecular mechanisms underlying sexual maturation in O. minor.

    Materials and Methods

    1. Sample Collection and RNA Extraction

    Samples of the gonads and optic lobes were collected from female and male O. minor during the developmental, spawning, and degenerative stages. A total of 36 samples were analyzed in triplicate to ensure reproducibility. Total RNA was extracted using the TruSeq RNA Sample Preparation Kit (Illumina, USA) following the manufacturer’s protocol. RNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was evaluated with an Agilent 2100 BioAnalyzer (Agilent Technologies, USA) (Table 1).

    RNA integrity number (RIN) values ranged from 5 to 6.6 among the analyzed samples. Although RIN values above 8.0 are generally preferred for RNA-seq library construction, samples with RIN ≥5.0 were included in this study based on previous cephalopod transcriptomic research (Castellanos-Martínez and Gestal, 2021;da Fonseca et al., 2022). This threshold was adopted because molluscan tissues are known to exhibit rapid RNA degradation due to high endogenous RNase activity and mucus content. All RNA samples were visually inspected for intact 18S and 28S rRNA peaks, and sequencing quality metrics (e.g., read mapping rate and duplication rate) confirmed that library quality was not compromised by the slightly lower RIN values.

    2. Library Construction and Reference Transcriptome Assembly

    Sequencing libraries were prepared by attaching adapters to 200–300 bp fragments, followed by sequencing using the NextSeq 500 platform. De novo assembly was conducted to construct the reference transcriptome sequence of O. minor (Subramanian, A. et. al., 2005;Haas, B.J. et. al., 2013). The assembly process used Trinity (v2.2.0) software (Grabherr, M.G. et. al., 2011), which operates in three steps: Inchworm, Chrysalis, and Butterfly. During the Inchworm step, overlapping sequences were grouped into subunits. In the Chrysalis step, contigs produced during the Inchworm step were clustered and detailed de Bruijn graphs (Compeau, P.E. et. al., 2011) were created within each cluster. Finally, the Butterfly step interpreted these graphs to predict the transcript sequences. For the assembled reference transcriptome of O. minor, statistics such as contig count, GC content, and N50 length were examined to evaluate assembly quality. Additionally, the presence of conserved orthologs was verified by identifying essential genes across different taxa. These assessments were conducted using the Trinity tool and BUSCO program (Simão, F.A. et. al., 2015). TransDecoder (Haas, B.J. et. al., 2013) was used to identify the longest Open Reading Frames (ORFs) in each transcript. ORFs were predicted in six frames and the most probable sequences were extracted. These predicted ORFs were annotated through homology searches against the NCBI NR database, which contains non-redundant protein sequences. Functional category analysis was further performed using Blast2GO to assign Gene Ontology and KEGG pathway annotations (Ashburner, M. et. al., 2000;Conesa, A. et. al., 2008;Kanehisa, M. et. al., 2016).

    3. Identification of O. minor-specific Sexual Maturation Genes

    To identify O. minor-specific candidate sexual maturation genes, a comparative analysis was conducted with the full gene sequences of ten other species: humans (Homo sapiens), mice (Mus musculus), lancelets (Branchiostoma floridae), zebrafish (Danio rerio), pufferfish (Takifugu rubripes), limpets (Lottia gigantea), oysters (Crassostrea gigas), sea hares (Aplysia californica), sea urchins (Strongylocentrotus purpuratus), and octopuses (O. bimaculoides). Transcripts without homologous sequences were classified as O. minorspecific. Of these, transcripts showing differential expression across maturation stages, tissues, and sexes were identified as candidate sexual maturationspecific transcripts.

    4. Gene Expression Patterns and DEG Identification

    The gene expression levels for each sample were calculated based on the constructed reference transcriptomes. Bowtie and RSEM were used for reference mapping and normalization. Expression levels, measured as the expected read counts of mapped sequencing reads, were used to analyze differential expression (Li, P. et. al., 2015;Robinson, M.D et. al., 2010;Lee, P. 1995). DEGs based on maturation stages were identified using analysis of variance (p-value <0.01) in R; tissue- and sex-specific DEGs were identified using the edgeR package in R, with a log fold change (LogFC) threshold of ≥6 and a false discovery rate (FDR) threshold of ≤0.01. The results were visualized using heat maps to represent expression profiles (Fig. 5).

    Results

    1. Reference Transcriptome Construction

    Using the NextSeq 500 platform, an average of 7.7– 12.45 GB of sequencing data were generated (Table 2). A total of 993,121 transcripts were obtained from the octopus samples based on sex, tissue, and maturation stage. Of these, 102,120 transcripts with ORFs (open reading frames) were identified, showing improvements in N50, length, and GC content (Fig. 1A). Using BUSCO, the proportion of essential genes was calculated for each taxonomic group, indicating high coverage rates of 97.15% for metazoans and 90.44% for eukaryotes (Fig. 1B). To analyze the functional distribution of the sequences, BLASTX searches were performed against the NCBI NR database. The results showed the highest homology with cephalopods such as octopuses, followed by sea anemones, oysters, and sea snails (Fig. 1C). Non-cephalopod species contributed less than 10%, highlighting the close evolutionary relationships within cephalopods. Additionally, Gene Ontology analysis was conducted to categorize the functions into cellular components, molecular functions, and biological processes. The integral component of the membrane was highly represented in cellular components, with prominently expressed nuclear and membrane-related functions. Molecular functions were dominated by binding-related categories, such as ATP and metal ion binding, whereas biological processes included the regulation of transcription and DNAtemplated and oxidation-reduction processes. G-protein receptor activity, which is critical for physiological regulation, was also observed (Fig. 2A–C). Sequencing produced 7.7–12.45 GB of data per group. From the 993,121 transcripts, 102,120 ORF-predictable transcripts with improved N50, length, and GC content were identified (Fig. 1A). BUSCO indicated essential gene content rates of 97.15% and 90.44% in metazoans and eukaryotes, respectively (Fig. 1B). Functional annotation indicated a high homology with cephalopods, including octopuses, sea anemones, and oysters (Fig. 1C). Gene Ontology analysis indicated important categories, such as ATP binding and transcription regulation (Fig. 2).

    2. Principal Component Analysis (PCA) of Expression Patterns

    The 102,120 transcripts were annotated using the NCBI NR database and indicated reproduction-related genes. PCA (principal component analysis) was conducted on the normalized expression data to identify any potential experimental biases, including variations in experimental timing, personnel, and platform. Tissue type (gonad and optic lobe) exhibited the clearest separation (Fig. 3A–C). Within the optic lobe, differences in expression patterns by sex and stage were minimal, whereas in the gonads, males displayed pronounced differences across stages compared to females (Fig. 4A–D). Overall, variations in expression were most distinct according to tissue type, followed by sex, and stage. In the female gonads, the developmental and spawning stages showed similar patterns, whereas the degeneration stage was substantially different. In males, the developmental stage was the most distinct, with sustained expression levels across stages, in contrast to the sharp changes observed in female degenerative stages. Further examination of sex-specific differences indicated substantial variability in the gonads but minimal differences in the optic lobes.

    3. Identification of Differentially Expressed Genes (DEGs)

    The number of differentially expressed transcripts per condition ranged from 3 to 11,855 (Table 3A). Tissue-specific differences were the most notable, with the gonads showing more DEGs than the optic lobes. Heatmap analysis indicated that transcripts in the degenerative stage were predominantly upregulated, whereas male gonads showed gradual increases in expression across the developmental, spawning, and degenerative stages (Table 3B). The expression patterns in the optic lobes of males and females were relatively uniform compared to those in the gonads. Sex-specific DEG analysis showed similar transcript numbers in male and female optic lobes, whereas male gonads had markedly fewer DEGs than female gonads (Table 3A). This discrepancy was particularly notable during the developmental and spawning stages. Across all stages, male gonads demonstrated increased gene expression compared to females. Tissue-specific DEG analysis indicated that both sexes had more DEGs in the gonads than in the optical lobes. Female gonads exhibited an approximately 100-fold higher number of DEGs than the optic lobes during the developmental and spawning stages (Table 3A).

    4. Identification of Octopus-Specific Sexual Maturation Genes

    Of the 102,120 transcripts expressed in O. minor, 9,068 were identified as species-specific, with no homologous sequences in other species. Of these, transcripts showing differential expression according to sex, tissue, and maturation stage were identified as candidate octopus-specific sexual maturation genes. In female gonads, 7,068 genes were differentially expressed, with the highest expression observed during the degenerative stage (S3) (Fig. 5A). Male gonads exhibited 4,766 differentially expressed genes, with a similar peak during the degeneration stage (Fig. 5B). In contrast, the optic lobe exhibited fewer DEGs, with females expressing 1,036 genes and males expressing 520 genes (Fig. 5C, D). Gonadal tissues showed markedly increased expression during the degenerative stage than in the optic lobe, with females exhibiting much higher gene expression levels than males (Fig. 5). The top 10 DEGs by developmental stage in female O. minor are shown in Table 4. In the gonads of females, the NRID gene exhibited the highest expression, with expression levels changing from 11.6 in the developmental stage (S1) to 35.2 in the spawning stage (S2) and further increasing to 126.2 in the degeneration stage (S3) (Table 4A). In the optic lobes, the HSD11 gene showed the highest expression, followed by MAPK, CTTN, and HCRT in descending order (Table 4B). Similarly, the top 10 differentially expressed genes by developmental stage were identified in male O. minor. In male gonads, CK showed the highest expression changes (Table 5A), whereas in the optic lobes, MBT and CDPK exhibited the highest expression levels (Table 5B).

    Discussion

    Octopus minor is an economically important cephalopod species in Korea, yet transcriptomic studies on sexual maturation remain limited. Identifying and characterizing maturation stages are essential for artificial breeding and resource management, and the use of transcriptome analysis to predict sexual maturation provides a valuable tool for aquaculture and stock enhancement programs. In this study, developmental stages were divided into three phases by sex, and differentially expressed genes (DEGs) were identified in the gonads and optic lobes.

    Like other cephalopods, O. minor exhibits rapid growth and a short lifespan (Lee et al., 1995;Moltschaniwskyj, 2004). Proteins serve as the main energy reserves in muscle tissues, whereas lipids function as critical energy sources in the ovaries during reproduction (Rosa et al., 2004;Kilada, 2008). Previous studies have reported substantial decreases in protein and lipid contents in gonadal and neural tissues during spawning (Morillo‐Velarde et al., 2013;Nilsen et al., 2003). However, the molecular and transcriptomic mechanisms underlying these metabolic shifts in O. minor remain largely unexplored.

    Our RNA-seq data revealed significant expression changes in genes associated with oxidative phosphorylation, carbon metabolism, and glycolysis during sexual maturation (Tables 5 and 6). These findings suggest that energy metabolism is finely regulated to meet the high energy demands of gonadal development. The differential expression of oxidative phosphorylation– related genes such as LDLR, HSD11, CASK, and CK may influence sperm quality and quantity by modulating mitochondrial activity in reproductive cells (Wang et al., 2022). Heat shock protein (HSP) genes were also differentially expressed, particularly in male gonads and optic lobes, suggesting their involvement in protecting germ cells from oxidative stress and apoptosis during spawning (Tang et al., 2021).

    The de novo transcriptome assembly of O. minor yielded approximately 993,000 contigs, from which about 102,000 open reading frames (ORFs) were predicted. The markedly higher number of contigs compared with ORFs suggests potential redundancy and fragmentation in the assembly. Such imbalance is commonly observed in molluscan and other non-model invertebrate transcriptomes due to biological and technical factors, including high transcript isoform diversity, variable tissue expression levels, and the absence of a reference genome. Nevertheless, this contig-to-ORF ratio exceeds the expected range of 20,000–40,000 functional genes typically found in eukaryotic species (Albertin et al., 2020;Castellanos- Martínez et al., 2021), indicating that many assembled contigs likely represent partial or overlapping fragments of the same genes.

    This excessive fragmentation likely reflects the transcriptomic complexity of O. minor and the challenges of short-read assembly in cephalopods, which possess large and repetitive genomes. Although redundancy may not directly compromise DEG identification, it can influence expression quantification and downstream enrichment analyses by inflating transcript counts or underestimating isoform-specific expression. To minimize these effects, stringent clustering and redundancy filtering were applied before DEG analysis, and expression levels were normalized across biological replicates. Future studies incorporating long-read sequencing platforms (e.g., PacBio or Oxford Nanopore) and hybrid assembly approaches are recommended to enhance transcriptome completeness and gene annotation accuracy, thereby improving resolution in gene structure and pathway analyses.

    Our Gene Ontology (GO) enrichment results further highlighted the roles of DEGs in cell cycle control, meiosis, and membrane-related molecular functions. Genes involved in ATP, metal ion, and nucleic acid binding were highly expressed, suggesting their potential as molecular biomarkers for monitoring physiological status across maturation stages.

    Recent omics research has expanded our understanding of cephalopod development and adaptation. For instance, transcriptomic analysis of Octopus sinensis paralarvae revealed dynamic regulation of lipid metabolism and neuropeptide signaling during early development (Jin et al., 2024). Similarly, de novo transcriptome assembly of O. vulgaris early life stages identified stage-specific gene expression patterns linked to growth and stress responses (Sicuro et al., 2022). Comparative genomic studies have also shown that cephalopods possess unique regulatory mechanisms, including extensive RNA editing and gene family expansions, which may underlie their complex developmental and behavioral traits (Albertin et al., 2022). Furthermore, epigenomic studies indicate that DNA methylation and histone modification systems are functionally conserved in cephalopods, suggesting additional layers of gene regulation during reproduction and development (Palumbo et al., 2022).

    Collectively, these findings indicate that O. minor sexual maturation is governed by intricate interactions among energy metabolism, stress response, and possibly epigenetic regulation. The present study identified candidate DEGs, including NR1D, CK, and HSP, that likely play sex-specific roles during gonadal development and optic lobe differentiation. These genes may serve as molecular markers for assessing maturation status and improving broodstock selection in O. minor aquaculture. Future multi-omics approaches integrating transcriptomic, epigenetic, and proteomic data will further elucidate the molecular mechanisms driving sexual maturation and reproductive success in cephalopods.

    Conclusion

    This study provides the first comprehensive transcriptomic characterization of Octopus minor across sexes and developmental stages, identifying key genes involved in sexual maturation. Differential expression analyses revealed that genes such as NR1D, CK, and HSPs play pivotal roles in the reproductive physiology of O. minor. NR1D (nuclear receptor subfamily 1 group D) is known to regulate circadian and metabolic pathways that influence gonadal development and reproductive timing. Its upregulation in mature gonads suggests a critical role in coordinating energy metabolism with reproductive readiness. CK (creatine kinase), a central enzyme in ATP buffering and energy transfer, showed increased expression during gonadal maturation, indicating elevated energy demands for gametogenesis. Meanwhile, the upregulation of HSP (heat shock protein) genes highlights their involvement in maintaining protein homeostasis and protecting reproductive cells from stress-induced apoptosis during spawning.

    Collectively, these findings provide molecular evidence that energy metabolism and stress-response pathways are tightly linked to the reproductive cycle of O. minor. The identified genes represent promising molecular biomarkers for monitoring reproductive status and hold potential as targets for selective breeding programs aimed at improving fecundity and spawning success. Furthermore, understanding the molecular mechanisms governing sexual maturation can inform resource management strategies, aiding in the restoration and sustainable aquaculture of O. minor populations.

    Acknowledgments

    This study was supported by the project ‘Fisheries by-product valuation and quality improvement research’ (R2025057) of the National Institute of Fisheries Science (NIFS), Incheon, Republic of Korea

    Figures

    JMLS-10-2-132_F1.jpg

    Statistical analysis of the assembled octopus reference transcriptome and open reading frame (ORF) sequences. (A)All transcripts and ORF-predicted transcripts, (B) BUSCO statistics for essential gene identification, (C) Homology search results using BLAST.

    JMLS-10-2-132_F2.jpg

    Gene Ontology analysis for the top 15 categories in Octopus minor open reading frame (ORF) transcripts. (A) cellular components, (B) molecular function, (C) biological processes.

    JMLS-10-2-132_F3.jpg

    Principal component analysis (PCA) of 102,120 octopus transcripts based on developmental stage, tissue, and sex. (A) Stage: Developmental S1 (Orange dot), Spawning S2 (Red dot), Degeneration S3 (Black dot), (B) Tissue: Gonad G (Purple dot), Optic lobe OL (Green dot), (C) Sex: Female F (Red dot), Male M (Blue dot).

    JMLS-10-2-132_F4.jpg

    Principal component analysis (PCA) results for expression similarity by sexual maturation stage across tissues (gonads and optic lobes) in males and females. (A) PCA analysis by developmental stage for female gonads, (B) PCA analysis by developmental stage for female optic lobes, (C) PCA analysis by developmental stage for male gonads, (D) PCA analysis by developmental stage for male optic lobes.

    JMLS-10-2-132_F5.jpg

    Heat-map analysis of differentially expressed genes by developmental stage: (A) 7,068 genes in female gonads, (B) 4,766 genes in male gonads, (C) 1,036 genes in female optic lobes, (D) 520 genes in male optic lobe.

    Tables

    Quality control results for Octopus minor samples based on sex, tissue, and developmental stage using Bioanalyzer

    Transcriptome data production results for Octopus minor (Gbp)

    Number of differentially expressed genes by sex, tissue, and developmental stage. (A) Upregulated and downregulated genes across three developmental stages, (B) Number of differentially expressed genes by sex and tissue at each developmental stage.

    Top 10 differentially expressed genes by developmental stage in female Octopus minor. (A) female gonads, (B) female optic lobes

    Top 10 differentially expressed genes by developmental stage in female Octopus minor. (A) male gonads, (B) male optic lobes

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