Gene Expression Shifts Drive Pufferfish Pigmentation Differences Across Shared Gene Pools

A targeted gene expression analysis of aquacultured tiger pufferfish, Takifugu rubripes, has revealed that striking variations in skin and fin pigmentation correlate with altered transcript activity rather than fixed genomic divergence. Marine biologists and molecular geneticists investigating morphological ambiguities among commercially valuable pufferfish examined whether regulatory shifts at the transcriptional level account for distinct physical traits seen in cultured specimens. Pufferfish classified within the genus Takifugu represent an evolutionary lineage known for rapid speciation across East Asian marine waters, including the Yellow Sea, the East China Sea, the Sea of Japan, and the Pacific coast of Japan, alongside South Korean coastal basins and river systems such as the Geum, Han, and Imjin. Despite diversifying over the last 1.8 to 5.3 million years into 25 recognized species, members of this genus maintain an exceptionally high degree of genetic similarity. This tight genetic coherence frequently confounds standard molecular authentication methods because conventional genetic markers demonstrate minimal interspecific divergence across closely related taxa.

The regulatory and commercial necessity of resolving these morphological ambiguities stems directly from seafood safety frameworks and market monitoring programs. Under Article 2 of South Korea’s Food Code, Takifugu rubripes and the closely related Takifugu chinensis are formally designated as edible pufferfish species approved for trade and human consumption, whereas the morphologically similar Takifugu pseudommus is excluded from the authorized listing. Despite these strict statutory classifications, market surveillance revealed an estimated mislabeling rate of approximately 70 percent among pufferfish items distributed in South Korean commercial retail channels, where specimens of T. rubripes, T. pseudommus, and T. chinensis are routinely traded interchangeably under the common vernacular designations Jajubok and Chambok. Traditional taxonomic keys distinguish these three taxa using subtle anatomical criteria, including the presence, density, and robustness of defensive skin spines, as well as distinct coloration patterns across the dorsal dermis and fins. T. rubripes classically presents a white dorsal surface overlaid with irregular black spotting paired with uniformly pink anal fins. In contrast, T. pseudommus displays a black dorsal surface patterned with small white speckles alongside either pink or black anal fins, while T. chinensis displays a solid black dorsal mantle that typically lacks spots outside of a single prominent black mark and terminates in black anal fins.

Previous whole-genome analyses established that despite their pronounced external differences, T. rubripes, T. chinensis, and T. pseudommus share an identical gene pool and represent a single biologically unified species capable of producing viable and fertile hybrids. Earlier comparative genomic surveys evaluating the T. pseudommus assembly against the T. rubripes reference genome, designated fTakRub1.2, identified 226 large deletions spanning lengths greater than one kilobase in T. pseudommus. When screening 50 individual pufferfish specimens across all three nominal species, researchers documented 12 specific genomic deletions that yielded polymorphic, variable amplification patterns across samples. However, these structural deletions failed to segregate consistently with the dorsal dot patterns or anal fin coloration defining the nominal species boundaries. Because structural genomic variations did not align with external color traits, investigators formed the working hypothesis that phenotypic divergence is governed instead by differential post-transcriptional expression of the transcripts residing within or flanking these deletion regions, driving diverse pigmentation patterns through regulatory plasticity rather than hardwired chromosomal sequence differences.

To rigorously test this transcriptional hypothesis while minimizing confounding background genetic noise, the research team selected six five-year-old cultured T. rubripes individuals, designated cTR1 through cTR6, from an aquaculture facility operated by Aquatopia International in Tongyeong, South Korea. This commercial facility represents the nation’s sole enterprise executing artificial fertilization, juvenile propagation, and commercial dissemination of T. rubripes for marine aquaculture, thereby supplying a study cohort characterized by minimal baseline genetic heterogeneity compared to wild marine populations. The sampled fish measured between 44.5 and 50.0 centimeters in total length, averaging 47.7 ± 2.1 centimeters, and weighed between 1.55 and 2.05 kilograms, with a cohort mean of 2.1 ± 0.2 kilograms. Working under animal care approvals granted by Seoul Women’s University Institutional Animal Care and Use Committee under protocol SWU IACUC 2024 A-15, the investigators grouped the specimens into two explicit phenotypic cohorts based on fin coloration. The experimental test cohort comprised four individuals, cTR1 through cTR4, displaying mixed pink-and-black anal fins, while the control cohort consisted of two fish, cTR5 and cTR6, exhibiting uniformly pink anal fins characteristic of standard T. rubripes descriptions.

Experimental procedures began with standardized euthanasia by immersing each specimen in an aqueous solution of 250 milligrams per liter of tricaine methanesulfonate until complete cessation of gill movement was observed. Technicians dissected approximately 10 grams of spotted dorsal skin and 10 grams of anal fin tissue from each specimen, immediately immersing the harvest in 10 milliliters of RNAprotect Tissue Reagent to preserve intracellular ribonucleic acid structures. Genomic DNA was isolated from skeletal muscle tissue using the classical phenol-chloroform-isoamyl alcohol extraction method at a 25:24:1 ratio and evaluated spectrophotometrically at 260 nanometers, confirming high purity through absorbance ratios. Total cellular RNA was extracted from preserved skin and fin tissues utilizing TRIzol Reagent, followed by enzymatic digestion of trace genomic DNA with one unit of DNase I per microgram of total RNA at 25 degrees Celsius for 30 minutes. Spectrophotometric readings confirmed RNA purity, while intact 28S and 18S ribosomal RNA bands visualized on one percent formaldehyde agarose gels confirmed structural integrity. Three micrograms of purified total RNA per sample was subsequently reverse-transcribed into complementary DNA templates using a commercial synthesis kit and preserved at minus 20 degrees Celsius for downstream quantitative real-time polymerase chain reaction assays.

The investigation targeted eight predicted transcript sequences mapped to six distinct genomic deletions that fell within transcribed chromosomal regions, annotated via the ENSEMBL repository against the fTakRub1.2 reference genome. These candidate loci comprised deletion Ch1-5, located within the intron of the 5-hydroxytryptamine 3A receptor gene, known as 5-HT3, and si: dkey-49c17.4; deletion Ch3-15, situated in the intron of sterile alpha motif histidine-aspartate domain-containing protein 1, or SAMHD1; deletion Ch5-11, containing the uncharacterized transcript ENSTRUG30845; deletion Ch7-19, harboring uncharacterized transcript ENSTRUG29592; deletion Ch8-7, mapping to the intronic sequences of fibronectin type III domain-containing 4b, or FNDC, and mitochondrial glycerol-3-phosphate dehydrogenase 2, known as GPD2; and deletion Ch20-4, containing uncharacterized transcript ENSTRUG28536. Initial conventional PCR assays spanning 30 amplification cycles across the six cultured specimens validated variable amplification patterns for these deletions but confirmed an absence of correlation between deletion presence and the mixed pink-and-black or uniformly pink anal fin phenotypes, demonstrating that the structural deletions themselves do not determine fin coloration.

Quantitative real-time PCR primers were synthesized to generate amplicons spanning 150 to 200 base pairs with an optimal annealing temperature of 54 degrees Celsius, utilizing the beta-actin gene as a reliable endogenous normalization control. Assays were conducted using SsoFast EvaGreen Supermix across serial tenfold template dilutions from 0.01 to 10 nanograms of pufferfish DNA. Standard curve parameters verified high precision, showing correlation coefficients exceeding 0.98 and amplification efficiencies ranging from 108.78 percent to 119.64 percent across all primer pairs. Melting curve evaluations revealed clean unimodal thermal dissociation profiles with average melting points extending from 78.34 ± 0.07 degrees Celsius to 88.76 ± 0.22 degrees Celsius, confirming exceptional assay specificity. At the lowest template concentration of 0.01 nanograms, primers for ENSTRUG28536 exhibited an auxiliary dissociation peak at 74.09 degrees Celsius indicating primer-dimer artifacts past 30 cycles, prompting researchers to restrict quantitative determinations to cycle quantification values below 30 cycles to maintain absolute analytical stringency.

Quantitative expression profiling using the comparative threshold cycle 2−ΔΔCq method demonstrated statistically significant upregulation of select metabolic and uncharacterized transcripts in individuals bearing darker fin pigmentation. Three candidate transcripts failed to register below the 30-cycle threshold, with 5-HT3 receptor, SAMHD1, and FNDC yielding undetectable signals in both tissues, suggesting that these predicted gene models may be virtually nonexistent in these anatomical structures. Uncharacterized transcript ENSTRUG29592 appeared sporadically in the anal fins of only three individuals, cTR2, cTR4, and cTR6, representing individual-level biological variation unconnected to coloration cohorts, while cDNA from the dorsal skin of specimen cTR3 failed amplification across all markers including beta-actin and was excluded. Among consistently detected loci, anal fin tissues of the mixed pink-and-black cohort exhibited a statistically significant 2.82-fold upregulation of ENSTRUG30845, a 1.75-fold upregulation of GPD2, and a 1.60-fold upregulation of ENSTRUG28536 relative to uniformly pink control fins. Parallel evaluations in spotted dorsal skin demonstrated that ENSTRUG30845 was upregulated 3.31-fold and GPD2 was upregulated 1.29-fold in the mixed coloration group, confirming that altered transcriptional regulation operates across multiple anatomical skin regions.

The significant upregulation of GPD2 provides a functional metabolic explanation for darker pigment accumulation across teleost dermal tissues. Melanogenesis is an energetically demanding physiological process carried out in specialized neural crest-derived chromatophores known as melanophores, which operate alongside xanthophores, erythrophores, iridophores, leucophores, and cyanophores to establish fish skin patterns. The enzymatic cascades of melanin production require substantial intracellular adenosine triphosphate reserves to sustain tyrosinase catalytic activity, melanosome biogenesis, and vesicle trafficking along the cytoskeleton. As an integral inner mitochondrial enzyme critical for the glycerol phosphate shuttle, cellular electron transport, and redox homeostasis, GPD2 modulates the cytosolic ratio of oxidized to reduced nicotinamide adenine dinucleotide. Through these redox-sensitive checkpoints, elevated GPD2 expression provides energetic and metabolic support that promotes tyrosinase function, accelerating melanogenesis and yielding mixed pink-and-black fin coloration. Conversely, reduced GPD2 activity limits cellular redox support and ATP availability, constraining melanin output and maintaining paler pink coloration. The concomitant elevation of ENSTRUG30845 and ENSTRUG28536 indicates that additional uncharacterized regulatory sequences participate in this pigmentary modulation, warranting future sequence characterization and cDNA cloning.

These transcriptional discoveries demonstrate that the morphological diversity characterizing T. rubripes, T. chinensis, and T. pseudommus reflects environmentally and developmentally driven phenotypic plasticity within an interbreeding genetic population rather than speciation-level evolutionary divergence. Although the authors note that the small cohort of six cultured specimens limits statistical power, the documented shifts in transcript expression confirm that substantial color variations can emerge from identical genomic backgrounds under metabolic and physiological influences. Because external anatomical markers such as dorsal spot arrangements and anal fin coloration vary continuously across individuals, traditional morphological criteria fail to provide a robust scientific foundation for regulatory enforcement or food authentication under national food safety statutes. Demonstrating that these pufferfish share an undivided gene pool whose morphological traits respond dynamically to transcriptional regulation supports the conclusion that T. rubripes, T. chinensis, T. pseudommus, and their fertile hybrids belong to a single biological taxon, offering an empirical foundation to revise seafood safety standards and harmonize commercial market regulations.

Subject of Research: Transcriptional regulation of pigmentation variation in cultured Takifugu rubripes

Article Title: Differential transcript expression associated with pigmentation variation in cultured Takifugu rubripes

Article References: Lee, J. Y., Park, J., Kang, H., & Kang, T. S. (2026). Differential transcript expression associated with pigmentation variation in cultured Takifugu rubripes. Applied Biological Chemistry, 69(1), Article 40. https://doi.org/10.1186/s13765-026-01109-x

Image Credits: AI Generated

DOI: 10.1186/s13765-026-01109-x

Keywords: Takifugu rubripes, pufferfish, gene expression, GPD2, pigmentation, aquaculture, transcriptomics, phenotypic plasticity, Differential, transcript, expression, associated