Decoding color variation: genome-wide characterization and expression analysis of pigment pathways in Neocaridina denticulata

Abstract

Body coloration in crustaceans is a fascinating ecological and evolutionary trait, yet its genetic basis remains poorly understood. The freshwater shrimp Neocaridina denticulata, which possesses diverse color variants, serves as an ideal model for dissecting the molecular mechanisms of crustacean pigmentation. Here, we systematically identified and characterized the core gene repertoire for the carotenoid, melanin and pteridine pigment pathways in N. denticulata and analyzed their expression patterns across strains of different color (red, yellow, blue). Most key genes involved in these pigment pathways were found in N. denticulata, including BCO-like for carotenoid cleavage, GCH1 and PTPS for pteridine synthesis, and TH for melanogenesis, suggesting conserved pigment metabolism and regulation in crustaceans. Furthermore, expression profiling of these key pigment genes across tissues in various color strains revealed distinct color-specific patterns. Within the carotenoid pathway, BCO-like1 showed dominant hepatopancreatic expression, with levels highest in the yellow strain and lowest in the blue strain. BCO-like2 and BCO-like3 were expressed across tissues, with strain-specific high expression in the intestine (red/yellow strain) or epidermis (blue strain). Correspondingly, the abundance of the astaxanthin-binding protein CRCN was significantly lower in the red and yellow strains than in the blue strain. In the pteridine pathway, GCH1 and PTPS exhibited a similar tissue distribution pattern in different strains, with high levels in the hepatopancreas, intestines, and eyestalks. However, their expression levels and pteridine contents were significantly higher in the red and yellow strains than in the blue strains. The melanin pathway gene TH was strongly and specifically expressed in eyestalks, and its level was significantly lower in the blue strain. Together, our findings provide the first comprehensive characterization of pigment pathway components in N. denticulata and demonstrate that the carotenoid and pteridine pigment pathways are major contributors to color variation, particularly distinguishing the blue strain from the red and yellow strains. This study establishes a crucial genetic foundation for understanding the evolution and regulation of crustacean coloration, and offers direct implications for selective breeding in aquaculture.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12880-y.

Introduction

Coloration is one of the important phenotypes of animals and is significant for species formation and evolution. Because color traits can be assessed and quantified relatively easily compared to other traits, the biology of color has been studied extensively in the context of animal behavior, ecology, genetics, developmental biology, and evolutionary biology [1]. The complexity and diversity of colors and patterns are largely due to the type, number and distribution pattern of pigment cells [2]. The three main biological pigments of animal body color include melanin, carotenoids and pteridin pigments, of which melanin and pteridin are endogenous pigments that can be synthesized by the animal itself, whereas carotenoids can only be obtained from external sources [3]. Compared with plants, vertebrates and insects, the synthesis pathways of these pigments and related key genes in crustaceans have not yet been investigated. Characterizing the pathway genes of these pigments and elucidating their functions in crustaceans will enhance our understanding of body coloration diversity in this group.

Melanin production requires the involvement of several metabolic enzymes. In insects, tyrosine is catalyzed by tyrosine hydroxylase (TH) to form dopa which can be converted into dopamelanin and dopamine melanin via two pathways. One is the conversion of dopa to dopa melanin catalyzed by dopaquinone convertase (DCE) and phenol oxidase (PO). The other is the conversion of dopa to dopamine catalyzed by dopa decarboxylase (DDC) [4]. Subsequently, dopamine is further converted to dopamine melanin by PO, which has a brown coloration [5]. In addition to these synthetic enzyme genes involved in melanin production, other factors and signaling pathways have been found to regulate melanin formation. These include α-MSH/MC1R/cAMP, Wnt/β-catenin, PI3K/Akt/GSK3β, MAPK, and NO signaling pathways [6]. MITF (Microphthalmia-associated transcription factor) also plays a central role in the regulation of melanocyte differentiation and melanin synthesis [7]. Therefore, identifying and characterizing the melanin synthesis pathway and its related regulators in crustaceans will help elucidate the role of melanin in their body coloration.

Most aquatic animals are rich in carotenoids, which are the basis of their pigmentation [8]. Common carotenoids found in crustaceans include astaxanthin, β-carotene, zeaxanthin and lutein, with astaxanthin being the most abundant. Normally, absorbed carotenoids can be metabolized via symmetric or asymmetric cleavage. To date, two types of carotene oxygenases are identified in vertebrates, which are 15, 15′-beta-carotene oxygenase (BCO1) and 9′,10′-beta-carotene oxygenase (BCO2) [9, 10]. BCO1 is localized in cytoplasm and mainly catalyzes β-carotene, whereas BCO2 is found in mitochondria and catalyzes most of the carotenoids with a high substrate specificity [11]. In the shrimp Exopalaemon carinicauda, a total of seven BCO-like genes and two carotenoid isooxygenase genes were identified. BCO-like 1 gene is essential for the survival and may play an important role in the basal metabolism of the shrimp, and BCO-like 6 assumes an important role in the metabolism of β-carotene [12]. Some crustaceans might have the ability to convert β-carotene to astaxanthin [13, 14]. Conversion of β-carotene to astaxanthin proceeds via a series of ketonylation and hydroxylation reactions, with echinenone and canthaxanthin as key intermediates [15], and the resulting astaxanthin is then stored in the body [16, 17]. However, detailed characterization of the astaxanthin metabolic pathway components in crustaceans is limited. Only a high-throughput transcriptome library of the copepod Acartia fossae was screened for differentially expressed β-carotene hydroxylase genes in metabolic transformation pathways by bioinformatics analysis [16]. Besides, crustaceans possess a specific lipid transport protein known as crustacyanin (CRCN) which is a multimeric protein widely distributed in the exoskeleton of shrimp and crab species. CRCN is usually composed of A and C subunits and plays a key role in binding and transporting carotenoids such as astaxanthin, contributing to the characteristic coloration of crustaceans [18].

Pteridine pigment, an endogenous pigment, is similar to melanin and can be synthesized by animals themselves. Despite its ubiquity in nature, pteridine-based pigmentation has received little attention compared to other major pigment classes. Studies of pteridine metabolic pathways have focused on model animals such as zebrafish and fruit fly, where a complete pteridine pathway has been identified [19], but have been less extensively studied in crustaceans like shrimps and crabs. It is now widely accepted that pteridine pigments are synthesized from tetrahydrofolate using guanosine triphosphate (GTP) as the biosynthetic precursor and then converted into biopterin [20, 21]. GTP cyclisation hydrolase (GCH1), 6-pyruvoyltetrahydrobiopterin synthase (PTPS) and murine pterin reductase (SPR) are the synthetic enzymes of pteridine pigments [22, 23]. For example, tetrahydrobiopterin (BH4) is synthesized from GTP by the sequential action of GCH1, PTPS and SPR. GCH1 is the first key rate-limiting enzyme gene of pterin metabolism pathway, and its high expression leads to the elevation of the pterin pigmentation [24]. The expression level of GCH1 in the skin of red crucian carp is significantly higher than that in the skin of white crucian carp [25]. In addition, the developmental expression pattern of GCH1 is also correlated with the differentiation of yellow pigment cells PTPS, as a key enzyme in the second step of the BH4 de novo synthesis pathway, also plays an important role in the production of pigments such as Xanthopterin and Drosophila pteridines. A PTPS mutant of silkworm failed to translate PTPS properly, resulting in lethal albinism while supplementing the larvae with BH4 was able to rescue the lethality, confirming that abnormal PTPS leads to impaired BH4 synthesis [26].

Neocaridina denticulata, a freshwater shrimp species widely distributed in East Asia, is an excellent crustacean model for understanding the molecular mechanisms of various biological and physiological processes due to its ontogenic characteristics, availability, and ease of culture and genetic manipulation [27]. Furthermore, N. denticulata possesses diverse and visually distinct color variants, a transparent exoskeleton to facilitate pigment observation, and a previous transcriptome study on the integuments of three color strains (red, yellow, and transparent) revealed several candidate genes associated with pigmentation [28], making N. denticulata an ideal model for crustacean body coloration research. This study aimed to systematically identify and analyze the expression patterns of genes involved in the melanin, pteridine and carotenoid related pathways in N. denticulata, and to provide a theoretical basis and direction for the study of body coloration in crustaceans.

Materials and methods

Animal culture and sample collection

Various color strains of N. denticulata were purchased from Jinghua Aquatic Ecosystem Museum, and domesticated in the Laboratory of Tropical Marine Germplasm Resources and Breeding Engineering, Sanya Oceanographic Institution, Ocean University of China. The shrimps were reared in a recirculation system (29 cm × 18 cm × 15 cm, transparent tank) at a temperature of 26 ± 2 °C, pH 7.2–7.5, and fed twice daily (at 9:00 and 17:00) with a commercial diet (Sera shrimps natural) at a total of 5% of their body weight per day. Adult shrimps with a body length of 1.8–2.2 cm were used for tissue collection. Juvenile shrimps (10–15 days post-hatching) with a body length of 0.4–0.6 cm were also collected. The eyestalks (Es), epidermis (Ep), hepatopancreas (Hp), appendages (Lg), intestine (In), and muscles (Ms) dissected from adult shrimps and whole individuals of juvenile shrimps were immediately frozen in liquid nitrogen for total RNA extraction and tissue expression testing.

Gene homologue identification, sequence features and phylogenetic analysis

To identify gene homologs of N. denticulata involved in pigment pathways, we first retrieved known pigment pathway genes of other organisms from NCBI and KEGG databases. The genomic and transcriptomic datasets used in this study include unpublished data generated by our research group, as well as publicly available datasets, including the N. denticulata genome sequencing data (BioProject: PRJNA224755) [27] and transcriptome data from different color strains (BioProject: PRJNA382065) [28]. The native tblastN tool in TBtools (with parameters: e-value < 1e-5 and score > 100) was used to search for gene homologs with the highest identity in both genomic and transcriptomic data (unpublished) [29]. Subsequently, candidate homologs were validated using the blastX to search against the non-redundant protein sequence database of NCBI. The complete open reading frame (ORF) regions and amino acid sequences of the pigment pathway-related genes were deduced using SnapGene software [30]. Amino acid sequences of relevant genes from different species used for phylogenetic analysis were obtained from the NCBI database (Supplementary Table S1). For each target pigment pathway gene, independent phylogenetic analyses were performed to validate sequence orthology. Species were selected based on decapod taxonomic representativeness and availability of well-annotated homologs. Phylogenetic analysis was then performed by MEGA 7.0 [31] and phylogenetic trees were constructed by the neighbor-joining distance algorithm [32]. The robustness of the trees was assessed through a bootstrapping test with 1,000 replicates.

Total RNA isolation and synthesis of cDNA

Total RNA was extracted separately from different tissues by Triziol reagent (Invitrogen) per the manufacturer’s instructions. The quality of the extracted RNA was assessed by electrophoresis on a 1% agarose gel and quantified by a NanoDrop 2000 spectrophotometer. Total RNA was used to generate complementary DNA (cDNA) using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). Genomic DNA (gDNA) was first removed using the gDNA Eraser according to the manufacturer’s instructions, and then first-strand cDNA was synthesized.

Real-time quantitative PCR

The real-time quantitative PCR (qPCR) analyses were performed to detect the expression profiles of the pigment pathway related genes in N. denticulata. The amplification cycle threshold (Ct value) were determined using the ChamQ Universal SYBR qPCR Master Mix (Vazyme). The 18 S rRNA gene was used as an internal reference, with primers adopted from the previous study [33]. Primers used for qPCR detection were listed in Supplementary Table S2. Primers for target genes were designed using Primer Premier 6.0 based on the specific regions of the obtained sequences, and the amplicon lengths were also provided in Supplementary Table S2. The amplification procedure was as follows: denaturation at 95 °C for 2 min, followed by 40 cycles: 95 °C for 15 s, 59–60 °C for 15 s, and 72 °C for 20 s. For each gene, three biological replicates (each with technical triplicates) were detected, and no-template controls (NTC) were included in each run. Relative gene expression levels were calculated by the 2^−ΔΔCT method [34].

Western blot

Firstly, the eyestalk, hepatopancreas and intestinal tissues of whole shrimp were removed. Then the total protein of whole shrimp was extracted using RIPA protein lysate. Protein concentration was quantified using BCA protein assay kit (Beyotime, China). Subsequently, the samples were diluted to the same concentration and mixed with 5×up-sample buffer (Beyotime, China). The mixture was boiled at 99 °C for 10 min. Equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane and blocked with 5% skimmed milk powder for 1 h at room temperature. Subsequently, the sample was detected using a specific anti-CRCN primary antibody (dilution 1:5000) and an HRP-labeled secondary antibody (dilution 1:8000, ServiceBio). For each sample, Tubulin was also detected as the internal control using the anti-Tubulin antibody (dilution 1:5000, ServiceBio). The expected molecular weights are 22 kDa for CRCN and 70 kDa for Tubulin. After visualization with an ECL kit (EpiZyme), band intensities were quantified in ImageJ (n = 3). Relative CRCN expression was calculated as (CRCN intensity/Tubulin intensity)×100%.

Pteridine quantification

To compare the contribution of pteridine contents to external body coloration among different color strains (red, yellow, and blue) of N. denticulata, the hepatopancreas and intestines were removed to avoid potential interference from tissues not directly associated with external body coloration. Pteridine content was then extracted and measured separately from the cephalothorax and abdomen of each strain as described previously [35], with three biological replicates per strain. Each sample was homogenized for 1 min in 400 µL of 1 M Tris-HCl buffer (pH 8.0). After centrifugation at 6000 × g for 5 min at 4 °C, 150 µL of the supernatant was transferred to a 96-well plate, covered with aluminum foil to protect from light. Fluorescence intensity was quantified using a microplate reader with excitation/emission wavelengths of 380/450 nm [35]. All pteridine levels were presented as relative fluorescence units (RFU) [36].

Statistical analysis

All data were presented as mean ± SEM. Normality was assessed using the Shapiro-Wilk test, and homogeneity of variances was assessed using the Brown-Forsythe test. For data that met both assumptions of normality (p > 0.05) and homogeneity of variances (p > 0.05), one-way ANOVA followed by least significant difference (LSD) post hoc test was used for multiple comparisons. For data that met normality but violated homogeneity of variances (p < 0.05), Welch’s ANOVA followed by Games-Howell post hoc test was applied. For data that did not meet the normality assumption, the Kruskal-Wallis test was used. All statistical analyses were performed using GraphPad Prism 9.5, and statistical significance was set at p < 0.05. Different letters indicate significant differences between groups (p < 0.05), whereas the same letters indicate no significant difference.

Results

To better understand the mechanisms of body coloration in crustacean, genes involved in major pigmentation pathways including carotenoid, pteridine and melanin pathways were systematically identified in N. denticulata. To verify the evolutionary conservation of the identified genes and their relationships with homologs from other species, we constructed phylogenetic trees for each of these pigment pathway-related genes (Figs. 1B, 2B and 3B, and Supplementary Fig. S1-23).

Fig. 1.

Carotenoids pathway in N. denticulata. A Schematic diagram showing the absorption and metabolism of carotenoids. Related genes identified in N. denticulata are marked in red. Abbreviations: NPC1L1, Niemann-Pick C1-like protein 1; CD36, cluster of differentiation 36; SR-B1, scavenger receptor class B type 1; LPCAT1, lysophosphatidylcholine acyltransferase 1; APOD, apolipoprotein D; STAR, steroidogenic acute regulatory protein; BCO1, β-carotene 15,15′-oxygenase; BCO2, β-carotene 9′,10′-oxygenase; BKT, β-carotene ketolase; BCH, β-carotene hydroxylase; CRCN, crustacyanin. B Evolutionary relationships of BCO-like proteins from N. denticulata and other species. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates. The pentagram (★) denotes the BCO-like sequences of N. denticulata. Colors indicate different taxonomic/functional clades: green, vertebrate BCO1 clade; yellow, vertebrate BCO2 clade; blue, decapod BCO1-like/BCO2-like homolog clade; orange, an independent lineage formed by two BCO-like paralogs (BCO-like2 and BCO-like3) from N. denticulata. Bootstrap values are labeled at the nodes

Fig. 2.

Pteridine pigment synthesis-related genes in N. denticulata. A Schematic diagram of the biosynthetic pathway for pteridine pigment. Related genes identified in N. denticulata are marked in red. Abbreviations: GCH1, GTP cyclohydrolase 1; PTPS, 6-pyruvoyltetrahydropterin synthase; SPR, sepiapterin reductase; DHPR, dihydropteridine reductase; XDH, xanthine dehydrogenase. B Evolutionary relationships of GCH1 from N. denticulata and other species. The phylogenetic tree was constructed by the Neighbor-Joining distance algorithm, and bootstrap value was set at 1,000. N. denticulata GCH1 is marked with Pentagram

Fig. 3.

Biosynthetic pathway of melanin in N. denticulata. A Schematic diagram showing the biosynthetic pathway of melanin. Related genes identified in N. denticulata are marked in red. Abbreviations: TH, tyrosine hydroxylase; DDC, dopa decarboxylase; aaNAT, arylalkylamine N-acetyltransferase; NADA, N-acetyldopamine; NBAD, N-β-alanyldopamine. B Evolutionary relationships of TH from N. denticulata and other species. The phylogenetic tree was constructed by the Neighbor-Joining distance algorithm, and bootstrap value was set at 1,000. N. denticulata TH is marked with Pentagram

Carotenoid pathway genes

Various carotenoid metabolism, absorption and transport-related gene homologues were identified in N. denticulata, such as ATP-binding cassette sub-family B member 6 (ABCB6), ATP-binding cassette sub-family G member 5 (ABCG5), Apolipoprotein D (APOD), Beta-carotene oxygenase (BCO like), Carotenoid isomerooxygenase (NinaB), Lysophosphatidylcholine acyltransferase 1 (LPCAT1), Steroidogenic acute regulatory protein (STAR), Scavenger receptor class B type 1 (SR-B1) and Stearoyl-CoA Desaturase (SCD) (Fig. 1; Table 1, Supplementary Table S3, Fig. S1-S9). Enzymatic degradation of carotenoids is a key step in animal carotenoid metabolism. Three BCO-like genes (BCO-like1, BCO-like2, and BCO-like3) and two NinaB-like genes were identified.

Table 1.

Carotenoid pathway genes identified in N. denticulata

Gene	Function	ORF (bp)	Amino acids (aa)
BCO-like1	Cleavage [12]	1416	471
BCO-like2	Cleavage [12]	1575	524
BCO-like3	Cleavage [12]	1575	524
STAR	Transport [37]	1032	343
APOD	Transport [38]	615 / 576 / 783 / 615	204 / 191 / 260 / 204
SR-B1	Transport [39]	2256	751
LPCAT1	Metabolism [40]	1545	514
NinaB-like1	Isomerization [41]	2934	977
NinaB-like2	Isomerization [41]	1569	522
ABCG5	Transport [28]	1287	428
ABCB6	Transport [28]	2565	854
SCD	Desaturation [42]	1038	345

Phylogenetic analysis showed that the BCO1 and BCO2 in vertebrates were separately clustered. For the invertebrate species, the reported BCO1-like genes and BCO2-like genes were not clustered. The BCO-like genes in N. denticulata completely separated from carotenoid oxygenases in vertebrates, indicating that BCO-like genes were different from BCO1 and BCO2 in vertebrates. Among these BCO-like genes, the BCO-like2 and BCO-like3 showed a closer evolutional relationship (Fig. 1B). SR-B1 is a transmembrane protein receptor that plays an extremely critical role in carotenoid absorption and transport. The ORF of the identified SR-B1 gene was 2256 bp, encoding 751 aa. Phylogenetic analyses showed that SR-B1 genes clustered with other crustaceans into one major clade and other vertebrates into one major clade (Supplementary Fig. S8). It is assumed that the evolutionary relationship between SR-B1 genes and the evolutionary classification of species is basically the same, indicating that this gene is relatively conservative. In addition, we identified a total of fourteen CRCN genes, which were divided into seven A subunits and seven C subunits (Supplementary Table S3). Phylogenetic analyses showed a clear segregation between the A and C subunit clades (Supplementary Fig. S5).

Pteridine pathway genes

We identified seven pteridine pigment synthesis-related genes of N. denticulata, including Dihydropteridine Reductase (DHPR), GTP Cyclohydrolase 1 (GCH1), Pterin-4α-carbinolamine Dehydratase (PCBD), Pyrimidodiazepine synthase (PDAS), 6-Pyruvoyltetrahydropterin Synthase (PTPS), Sepiapterin Reductase (SPR) and Xanthine Dehydrogenase (XDH) (Fig. 2; Table 2, Supplementary Table S3, Fig. S10-15). To assess their evolutionary relationships, neighbor-joining phylogenetic trees were constructed using homologous sequences from representative decapods and other arthropods. Phylogenetic analyses showed that these genes cluster with their respective homologs from Macrobrachium species, within a larger clade shared with other crustaceans (Fig. 2B, Fig. S10-S15).

Table 2.

Pteridine pathway genes identified in N. denticulata

Gene	Function	ORF (bp)	Amino acids (aa)
GCH1	Synthesis [43]	804	267
PTPS	Synthesis [44]	447	148
DHPR	Synthesis [19]	708	235
PCBD	Synthesis [19]	516	171
PDAS	Synthesis [13]	732	243
SPR	Synthesis [45]	795	264
XDH	Synthesis [46]	4125	1374

Melanin pathway genes

A total of ten melanin pathway related genes were identified in N. denticulata, including Tyrosine Hydroxylase (TH), Aralkylamine N-Acetyltransferase (AANAT), Dopa Decarboxylase (DDC), Metallophosphoesterase Domain-Containing Protein 2 (MPPED2), N-β-alanyl-dopamine synthase (Ebony), Glycogen Synthase Kinase 3 Beta (GSK3β), Tan-like1, Tan-like2 and Wnt Family Member 5B (Wnt5B) (Fig. 3; Table 3, Supplementary Table S3, Fig. S16-23).

Table 3.

Melanin pathway genes identified in N. denticulata

Gene	Function	ORF (bp)	Amino acids (aa)
TH	Synthesis [47]	1503	500
AANAT	Synthesis [48]	708	235
DDC	Synthesis [49]	1428	475
MPPED2	Synthesis [50]	915	304
Ebony	Conjugation [51]	2610	869
GSK3β	Regulation [52]	1197	398
Tan-like1	Synthesis [20]	1161	386
Tan-like2	Synthesis	1164	387
GSTM3	Metabolism [53]	654	217
Wnt5B	Regulation [54]	1119	372

TH is a key gene for the first step in melanin synthesis, its phylogenetic analyses showed that N. denticulata is closely related to E. carinicauda and clustered into a clade with three other freshwater shrimps (Fig. 3B). Phylogenetic reconstruction revealed that Tan-like1 formed a strongly supported clade with Litopenaeus vannamei and Penaeus indicus (Supplementary Fig. S22), indicating close evolutionary relationships. In contrast, Tan-like2 exhibited substantial phylogenetic divergence from Tan-like1, occupying a basal position within the decapod lineage despite clustering within this group. The phylogenetic analysis revealed a consistent pattern for the remaining melanin pathway genes in N. denticulata, all of which formed a clade with close affinities to E. carinicauda, Macrobrachium rosenbergii or M. nipponense (Supplementary Fig. S16-23).

Expression profiles of pigmentation pathways in different N. denticulata color variants

To understand the relationship between various pigmentation pathways with the body coloration, we have examined the expression profiles of representative genes in different pigment pathways in various N. denticulata color strains (red, yellow and blue). Photographs of these color strains were shown in Supplementary Fig. S24. Tissue expression profiles of these genes in each strain were examined and shown in Supplementary Fig. S25-27. BCO-like1 expression was highest in the hepatopancreas, with significant differences compared to other tissues (p < 0.05, Supplementary Figure S25). Among various color strains, its expression was lowest in the blue strain and highest in the yellow strain. Notably, the yellow strain also showed expression in the leg and muscle (Fig. 4A). BCO-like2 was primarily expressed in the intestine in the red and yellow strains, while it is mainly expressed in the eyestalks and epidermis in the blue strain (Fig. 4B). While BCO-like3 expression was localized primarily to the epidermis and appendages in all strains examined, significantly higher intestinal expression was observed specifically in the red and yellow strains (Fig. 4C). In all three strains, GCH1 and PTPS showed relatively high expression in the eyestalks and hepatopancreas, with lower levels in muscle and appendages (Fig. 4D-E, Supplementary Figure S26). TH was mainly expressed in the eyestalks (Supplementary Fig. S27), with the blue strain showing significantly lower expression levels compared to the red and yellow strains (Fig. 4F). Further, we examined and compared the expression patterns of these key pigment pathway genes among various color variants at the juvenile stage. The expression level of BCO-like1 was similar across all three strains. BCO-like2 was highly expressed in red and blue juveniles, whereas BCO-like3 expression was significantly higher in red juveniles than in the blue or yellow strains (Fig. 4G). GCH1 and PTPS expressions were consistent across strains (Fig. 4H-I), while TH was most highly expressed in the red strain (Fig. 4J). Moreover, the content of CRCN proteins, the major astaxanthin-binding proteins, was significantly lower in the red and yellow strains than in the blue strain (Fig. 5A-B). Pteridine content also differed among these color strains. It was significantly higher in the cephalothorax of the red and yellow strains than in the blue strain, and abdominal levels were highest in the yellow strain (Fig. 5C-D). Together, our results demonstrate that the carotenoid and pteridine pigment pathways are the major contributors to the color differences between the blue strain and the red/yellow strains.

Fig. 4.

Expression of BCO-like1, BCO-like2, BCO-like3, GCH1, PTPS and TH in the red, yellow and blue varieties of N. denticulata. A-E Relative expression levels of BCO-like1, BCO-like2, BCO-like3, GCH1 and PTPS in the red, yellow and blue strains across various tissues. F) Expression of the TH in the eyestalk. G-J) Relative expression levels of BCO-like1, BCO-like2, BCO-like3, GCH1, PTPS and TH in the juvenile of red, yellow and blue strains. Different letters denote significant differences between groups (p < 0.05). Es, eyestalk; Ep, epidermis; Hp, hepatopancreas; In, intestine; Lg, appendages; Ms, muscle. Bar colors represent relevant strain: red, red strain; yellow, yellow strain; blue, blue strain

Fig. 5.

CRCN protein abundance and pteridine content across different color strains. A Western blot detection of CRCN and tubulin (internal control) proteins in red, yellow, and blue color strains. Uncropped Western blot images with molecular weight markers are provided in Supplementary Figure S28. B Relative band intensity of CRCN normalized to tubulin. Relative pteridine content in the (C) cephalothorax and (D) abdomen. Data are presented as mean ± S.E. Different letters indicate significant differences between groups (p < 0.05)

Discussion

Among invertebrates, melanin synthesis has been most thoroughly studied in Drosophila melanogaster and Bombyx mori [55, 56]. TH is the first key rate-limiting enzyme in melanin synthesis, converting tyrosine to dopa [4]. DDC is classified in the family of α-aminotransferases, also known as aromatic L-amino acid decarboxylases, and can effectively catalyze the decarboxylation of L-dopa to produce dopamine [49]. TH has multiple functions in insects. It is involved in epidermal hardening in Tribolium castaneum [47], in epidermal/pupal shell tanning and immune melanization processes in Manduca sexta [57] and Plutella xylostella [58, 59]. Key melanin synthetic genes, such as TH and DDC, have been identified in some crustaceans. However, their functional characterization has focused primarily on immunity rather than pigmentation [49, 60]. Unlike well-studied insect pigment regulators such as ebony and tan, characterization and functional roles of these genes in crustacean coloration remain unclear. In this study, we identified the conserved TH and other melanin-associated genes (e.g. Ebony, Tan and AANAT) in the shrimp N. denticulata. The N-β-alanyldopamine (NBAD) synthase encoded by the ebony catalyzes the conjugation of dopamine and β-alanine to form NBAD. This function links the ebony gene to light coloration, and homologs have been found in D. melanogaster, Papilio appalachiensis, P. xuthus, and B. mori [51, 61, 62]. In contrast to ebony, Tan promotes the darkening of markings, as demonstrated in D. melanogaster and P. xuthus [51, 61]. AANAT acetylates dopamine to produce NADA, which is involved in melanin synthesis [63]. Knockdown of the AANAT in B. mori and T. castaneum resulted in increased concentrations of dopamine and other biogenic alkylamines, and excessive melanin production and deposition [48, 64, 65]. GSTM3 is involved in glutathione metabolism and has been shown to be highly expressed during phaeomelanogenesis in developing feathers, where it may influence pigmentation by regulating the availability of glutathione [53]. In melanin synthesis, the balance between eumelanin (dark) and phaeomelanin (yellow/red) is influenced by the availability of glutathione and cysteine. When glutathione levels are high, dopamine is preferentially converted to phaeomelanin, resulting in yellow to red hues [37]. Thus, GSTM3, by regulating glutathione availability, may indirectly influence the type of melanin produced and contribute to color variation in N. denticulata. Furthermore, this study has identified certain members of signaling pathways such as PI3K/Akt/GSK3β, which have been demonstrated in both mammals and mollusks (such as Sinohyriopsis cumingii) to influence melanin production and deposition by regulating core transcription factors including MITF [50, 52, 66, 67]. We found that TH expression was markedly higher in the eyestalk than in other tissues (e.g. hepatopancreas, epidermis and muscle), where levels were negligible. This suggests that TH and melanin are primarily involved in visual or neuro-specific functions, rather than body coloration in N. denticulata.

Pteridine pigments, first discovered and studied in butterfly wings [68], are involved in brighter pigmentation. These pigments are commonly shared across different animal groups and are often deposited as pigment granules in the stratum corneum or epidermis. Although pteridines have been biochemically detected in some crustaceans, their biosynthetic genes and functional roles in body coloration remain uncharacterized. This study identified seven genes associated with the pteridine biosynthetic pathway in N. denticulata, including the rate-limiting enzyme gene GCH1, as well as PTPS, SPR, XDH, DHPR, PCAD, and PDAS. Comparisons with relevant studies in insects (such as Drosophila and B. mori) and fish reveal a highly conserved core set of genes in this pathway across the animal kingdom. In Vespa orientalise, xanthopterin granules are deposited in the cuticle and are involved in the formation of the yellow coloration of the body surface [46]. XDH is a key enzyme in the regulation of purine metabolism and the synthesis of pteridine and its analogues. In Nothobranchius fish, RNA-seq analysis revealed that the expression of XDH was significantly higher in red fins than in yellow fins [69]. SPR is a key enzyme in the synthesis of BH4, one of the important pigments in the formation of insect body color. Researchers characterized the GCH1, PTPS and SPR genes in carp Cyprinus carpio L. and investigated their influence on red coloration, establishing that pteridine pigments are significant contributors to red body coloration [43–45]. In N. denticulata, we found the tissue expression patterns of GCH1 and PTPS were highly similar across all strains, but their expression levels and pteridine contents were higher in red and yellow strains than in blue strains. Together, these findings indicate the involvement of pteridine pigments in the body coloration of red and yellow strains, providing a new theoretical basis for understanding the function of pteridine in crustaceans. In juvenile shrimp, GCH1 and PTPS expression did not differ significantly among the three strains, whereas BCO-like expressions varied. This differential pattern indicates a potential role for carotenoid metabolism in juvenile coloration, while the pteridine pathway may function in other developmental processes rather than in color formation.

Aquatic animals obtain carotenoids from their diet and modify them through metabolic reactions. These carotenoids include metabolites such as β-carotene, fucoxanthin, polymethalin, diatom xanthophylls, tetraxanthin and astaxanthin [8, 13, 70]. This study identified multiple key genes involved in carotenoid metabolism (e.g. three BCO-like genes), transport (e.g. SR-B1 and APOD) and storage in N. denticulata. Cleavage of carotenoids is a key step in carotenoid utilization, BCO1 and BCO2 are two important carotenoid oxygenases. In invertebrates, the function of the carotenoid oxygenase gene has rarely been studied. In Drosophila, only a single carotenoid isomerooxygenase, encoded by ninaB, has been identified [71]. Study in Eriocheir sinensis identified NinaBl and BCO1l, each exhibiting β-carotene degradation activity. Larval knockdown experiments demonstrated that silencing either gene elevated hepatopancreatic redness. Importantly, NinaBl knockdown resulted in significant β-carotene accumulation in the hepatopancreas, providing in vivo evidence for its role in cleavage [41]. In E. carinicauda, seven BCO-like genes were identified, and RNA interference targeting three of them revealed that knockdown of BCO-like 6 induced color changes in the hepatopancreas [12]. This study identified multiple oxygenase genes in N. denticulata, which are more similar to those in E. carinicauda but fewer in number. This suggests that BCO like genes undergo expansion and functional differentiation within decapods, providing key candidates for investigating the metabolism of dietary carotenoids. The coloration of carotenoids depends on the integrity of their conjugated double-bond system, which serves as the chromophore. BCOs cleave this system, thereby disrupting the chromophore and causing loss of color [41]. We found the differential expression of various BCO-like genes in different color strains of N. denticulata (Fig. 4A-C) may directly affect carotenoid accumulation and coloration. The process of carotenoid uptake and transport involves many genes, including APOD, SR-B1, STAR, LPCAT1 and the ABC family of transporter proteins. SR-B1 acts as an HDL receptor, and mutations or deletions in the gene encoding this protein cause carotenoid defects in zebrafish and canaries [72, 73]. In N. denticulata, the SR-B1 has been identified as a key factor in body color regulation. Previous expression profiling and RNAi studies have shown that this gene is significantly upregulated in red populations and pre-nauplius stages. Knockdown in post-nauplius stages significantly increased pigment cell number and advanced their development, whereas earlier stages showed no phenotypic change [39]. APOD has been found to be associated with carotenoid coloration in Chrysolophus pictus feathers [38]. LPCAT1 is an endoplasmic reticulum membrane protein that supports the formation of phosphatidylcholines that are key components in the formation of lipid droplets, the main site of intracellular carotenoid storage [40]. STAR belongs to the START domain family, and its domain has been implicated in carotenoid binding and trafficking in other species [37]. Although APOD, LPCAT1 and STAR were identified in N. denticulata, their functional roles in body coloration need further validation.

The tissue expression profiles of BCO varies greatly among different species. The BCO genes are highly expressed in the liver of fish and rat [74, 75]. In the E. carinicauda, most BCO-like genes are highly expressed in the hepatopancreas and stomach, with the exception of BCO-like 6, which is highly expressed in the heart [12]. In this study, we found that BCO-like1 was highly expressed in the hepatopancreas across all three N. denticulata strains, whereas BCO-like2 and BCO-like3 were most highly expressed in the intestines of the yellow and red strains. In the blue strain, BCO-like2 was predominantly expressed in the epidermis, while both BCO-like2 and BCO-like3 showed lower intestinal expression. Our results indicate that carotenoid pathway plays an important role in the body color difference between the blue and the red/yellow strains. In addition, our Western blot results showed elevated CRCN protein content in the blue strain compared to the other two strains. Since CRCN is an astaxanthin-binding protein, this finding further supports the contribution of the carotenoid pathway to blue coloration. This is consistent with known mechanisms of CRCN-mediated coloration in crustaceans. CRCN binds astaxanthin and shifts its absorbance spectrum from red to blue, a protein-pigment interaction responsible for the blue coloration of the exoskeleton in crustaceans [76]. For example, heating induced thermal denaturation of CRCN in Procambarus clarkii, causing protein unfolding and astaxanthin release, which restored its red color [77]. Similarly, RNAi knockdown of CRCN in E. carinicauda led to the loss of the blue subepidermal protein complex, resulting in the body color shift from transparent to red [78].

In summary, this study systematically identified and characterized key genes from melanin, carotenoid and pteridine pigment pathways in N. denticulata, and revealed their distinct color-specific expression patterns. Our findings advance the understanding of the molecular mechanisms underlying body color variation in this species, providing the foundation for future research on crustacean pigmentation and offering potential applications in aquaculture and selective breeding.

Authors’ contributions

Shuo Bai: Investigation, Formal analysis, Validation, Writing – original draft, Writing – review & editing. Zhihui Ma: Investigation. Ruixue Gu: Formal analysis. Xiaoliu Yang: Investigation. Yue Sun: Resources, Writing – review & editing. Jingjie Hu: Resources, Supervision. Zhenmin Bao: Resources, Supervision. Zhe Qu: Conceptualization, Funding acquisition, Project administration, Resources, Writing – original draft, Writing – review & editing.

Funding

This work was supported by Hainan Province Science and Technology Talent Innovation Project (KJRC2025B14), Hainan Seed Industry Laboratory (project of B25H10CI9), Shandong Excellent Young Scientists Fund Program (Overseas, 2023HWYQ-055) and Fundamental Research Funds for the Central Universities (202212022).

Data availability

The datasets supporting this article are included within the article and its supplementary information. Other details will be made available on request.

Declarations

Ethics approval and consent to participate

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Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.