The experimental animal treatments in this study were consistent with animal protection, animal welfare and ethical principles, and in accordance with the relevant provisions of the national experimental animal ethical welfare and approved by the Institutional Animal Care and Use Committee of Shanxi Agricultural University.

A total of 36 normal crayfish (N), white crayfish (W), red-orange crayfish (R), and blue crayfish (B) were bred and cultured in our laboratory (Figures 1a-e). The shrimp and crab were fed twice a day (9:00 and 18:00); the total daily feed amount was 5% of their body weight, with 30% provided in the morning and 70% in the afternoon. The feed residues and excreta were cleaned before each feeding. Nine crayfish from each of the four body-color groups were randomly selected (three replicates of each body color) after a growth period of 1 year for analysis (average adult weight: 26.6 ± 6.3 g). Culture water was continuously aerated (24 h) at a temperature of 20–23°C, pH 7.9, salinity 5.3 ‰, ammonia−nitrogen 0.2 mg L^-1, nitrite 0.005–0.01 mg L^-1, and dissolved oxygen 6.0–10.0 mg L^-. The photoperiod was 12 h light / 12 h dark, with a light intensity of 500–600 lx. The crayfish were anesthetized on ice for 40 seconds before sample collection. The hepatopancreases and endocarps of each crayfish were removed using scissors and forceps, immediately frozen in liquid nitrogen and stored in a −80°C refrigerator until further analysis.

Corresponding color parameters and carotenoids were measured in portions of the endocarps and hepatopancreases from each crayfish species. Body-color measurements on the shield, ventral-dorsa, and pygopod of each crayfish carapace were taken using a precision colorimeter (3nh, NR110, China). The International Commission on Illumination (CIE) uniform chromaticity spatial system CIE L*a*b* (CIE1931) was adopted, accounting for parameters such as the determination of luminance (L*), redness (a*), and yellowness (b*). The endocarps and hepatopancreases were harvested from each crayfish for to determine of carotenoids after body-color measurements were taken. Carotenoid values were measured, and pigment cell types, morphologies, and sizes were observed under a continuous-magnification stereomicroscope (Cnoptec, SZ680, China) (2.0×).

Endothelial and hepatopancreatic total RNA was extracted using a Trizol kit (Invitrogen), following to the manufacturer’s instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified using RNase-free agarose gel electrophoresis. After extracting total RNA eukaryotic mRNA was enriched using Oligo (dT) beads. The library was constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were end repaired; subsequently a base was added and ligated to Illumina sequencing adapters. The ligation reaction was purified using AMPure XP Beads (1.0X). Meanwhile, polymerase chain reaction (PCR) was used to amplify target gene sequences. The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Reads obtained from the sequencing machines included raw reads containing adapters or low-quality bases, which could negatively impact the assembly and analysis. Thus, to obtain high-quality clean reads, the raw reads were further filtered using fastp software (version 0.18.0) (Chen et al., 2018). An index of the reference genome was established, and paired-end clean reads were mapped to the reference genome using HISAT2 (version 2.4) with default parameter settings (Kim et al., 2015). Subsequently, the read alignments were sorted using Samtools (version 1.17) (Danecek et al., 2021), and transcripts were reconstructed using genome-guided de novo assembly approaches using Trinity (version 2.12.0) (Haas et al., 2013). Principal component analysis (PCA) was performed using the gmodels package (http://www.r-project.org/) for R (v2.18.1). RNA differential expression analysis between the two groups was conducted using DESeq2 software (Love et al., 2014). Genes with a false discovery rate (FDR) of < 0.05 and absolute fold change (FC) of ≥ 2 were considered differentially expressed. The genes were annotated using the KEGG ontology database. Meanwhile, functional annotation and pathway enrichment analyses were performed for DEGs using KEGG gene IDs (Wu et al., 2021).

Harvested endothelia and hepatopancreas were quickly frozen in liquid nitrogen after dissection. Tissues (~80 mg) were cut on dry ice and placed in an Eppendorf tubes (2 mL). Subsequently, tissue samples were then homogenized with 200 μL of H₂O and five ceramic beads using the homogenizer (MP, FastPrep-24 5G). Afterward, 800 μL of methanol/acetonitrile (1:1, v/v) was added to the homogenized solution for metabolite extraction, after which the mixture was centrifuged for 15 min (14,000×g, 4°C). The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were redissolved in 100 μL acetonitrile/water (1:1, v/v) solvent. Quality control samples were prepared by pooling 10μL of each sample to monitor the stability and repeatability of the instrument analysis, which were analyzed alongside the other samples.

For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7µm column (Waters, Ireland). In ESI-positive and ESI-negative modes, the mobile phase contained A = 25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B = acetonitrile. The gradient was 85% B for 1 min, linearly reduced to 65% in 11 min, then 40% in 0.1 min and kept for 4 min, and then increased to 85% in 0.1 min, with a 5-min re-equilibration period.

An AB TripleTOF 6600 mass spectrometer was used for the analysis. The ESI source conditions after HILIC chromatographic separation were as follows: in ESI-positive and ESI-negative modes, ion source gas 1 (60), ion source gas 2 (60), and curtain gas. The secondary mass spectrum was obtained via information-dependent acquisition (IDA) using the high-sensitivity mode. In ESI-positive and ESI-negative modes, the declustering potential is ± 60 V, collision energy is 35 ± 15 eV, and IDA was set as follows: excluding intrusion within 4 Da and 10 candidate ions to monitor per cycle.

The raw MS data were converted to mzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. Orthogonal partial least squares discriminant analysis (OPLS-DA) was conducted using the packages gmodels and ropls (R v2.18.1) (Warnes et al., 2018). Subsequently, we combined the variable importance in projection (VIP) value of the OPLS-DA and the P value of the univariate statistical analysis t-test to screen for differential metabolites between the comparison groups (Saccenti et al., 2014). The threshold value of the difference was VIP ≥ 1, and significance threshold was P < 0.05.

For KEGG annotation and enrichment analyses, the identified metabolites were annotated using the KEGG compound database (http://www.kegg.jp/kegg/compound/). Subsequently, the annotated metabolites were mapped to the KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html). Pathways with differential metabolites were fed into metabolite sets for enrichment analysis, and the significance of such enrichment was determined by the resulting P value.

For the joint analysis of the histology, we took the mean values of the six sets of samples in the metabolic group two by two to correspond with the transcriptome data. Two-way orthogonal partial least squares (O2PLS) analysis was conducted using the OmicShare online tool (https://www.omicshare.com/tools/). Differentially expressed genes (FDR < 0.05 and |log2FC| > 1) and differential metabolites (VIP > 1 and P < 0.05) were analyzed in the following pairwise groups: NP vs. WP, NP vs. RP, NP vs. BP, and NH vs. WH, NH vs. RH, and NH vs. BH. O2PLS was conducted using the integral quantitative values of DEGs and differential metabolites from transcriptome analysis to screen for associated genes and metabolites.

Three crayfish from the same tank were set up as one biological replicate for a total of three biological replicates for the three tanks. The samples used for targeted carotenoid omics analysis were the same as that for the not-target analysis. Approximately 1 mL of methanol and 0.1% BHT were added to the accurately weighed endocarps and hepatopancreases (0.1–0.5 g). Subsequently, the sample was homogenized and left to stand overnight at 4°C. The next day, the samples were centrifuged at 3000 rpm for 5 min. The methanol layer was transferred into a 2mL volumetric flask. Afterward, 1mL of n-hexane was added to the remaining sample in the vial, vortexed for 30 s, and then centrifuged at 3000 rpm for 5 min. The n-hexane layer was transferred into a fresh test tube. This procedure was repeated one more time using 1mL n-hexane, followed by evaporation to dryness under a gentle flow of nitrogen at 30°C. The residue was reconstituted with 0.2 of mL methanolic solution.

The sample extracts were analyzed at 450 nm using a UPLC system equipped with a DAD detector (UPLC, U3000; Thermo). The analytical conditions were as follows: UPLC column; YMC Carotenoid S-3um (150 mm×4.6 mm); column temperature, 40°C; flow rate, 1.0 mL/min; injection volume, 2 μL; solvent system, MeOH (MeOH:MTBE:H2O=20: 75: 5); and gradient program, 100:0 V/V at 0 min, 39:61 V/V at 15 min, 0:100 V/V at 25 min, 100:0 V/V at 25.1 min, and 100:0 V/V at 30 min. Concentrations of 0.1–17.5 μg/mL of standard samples with astaxanthin, lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, neoxanthin, violaxanthin, capsanthin, xanthophyll, and lycopene were used in the analysis.

Genes related to body color were selected from endothelial and hepatopancreatic transcriptome analyses for quantitative real-time PCR (qRT-PCR), to investigate the body color regulatory mechanisms of crayfish. Targeted carotenoid-related genes were CRA2, CRC1, and BCO1; melanin-related genes were SLC2A3 and mitf; and pteridine-related genes were ABCG21 and XDH. Total RNA extracted from endothelia and hepatopancreas (1 μg) was reverse transcribed into cDNA. The system was supplemented to 10 μL with 1 μg of RNA, 2 μL of 5× gDNA eraser buffer, 1 μL of gDNA eraser, and RNase-free dH₂O. Subsequently, the solution was incubated at 42°C for 2 min. Afterward, 1 μL of PrimeScript RT Enzyme Mix I, 1 μLof RT Primer Mix, 4 μL of 5× PrimeScript Buffer 2, and 4 μL of RNase-free dH₂O were added to the reaction solution on ice. The mixture was reacted at 37°C for 15 min, and then again at 85°C for 5 s. Primer Premier 5.0 software was used to design qRT-PCR primers using 18s rRNA as the internal reference gene (Yu et al., 2022). Supplementary Table 1 presents primer amplification sizes. Subsequently, 10 μl of total qRT-PCR mix, comprising 1 μL cDNA template, 5 mL 2×Universal SYBR Green qPCR Supermix, 0.5 μL upstream and downstream primers, and 3 μL ddH₂O, was used for qRT-PCR, which was conducted on a QuantStudio 6 Real-Time PCR system (Applied Biosystems, USA). Data were statistically analyzed using the 2^-ΔΔCt method.

We analyzed the color parameters of the carapaces and hepatopancreases of crayfish to identify notable differences in color among the four body colors. The crayfish shield, ventral-dorsal, and pygopod of the three parts of the carapaces and hepatopancreases were positive in all four body colors, as shown in Figures 1a–h. In particular, the L* values of the hepatopancreases and carapaces exhibited a consistency pattern (N < B < R < W, Figures 1e, h); however, the a* and b* of the carapaces were different, following the order of B < N < W < R (Figures 1f, g). The consistency pattern of the hepatopancreas a* and b* values was opposite (R < W < N < B, Figure 1h) to that of the carapace values. The carapace L* of the white crayfish was significantly higher (P < 0.05) than those of the other three crayfish, and the carapace L* of the normal crayfish was significantly lower (P < 0.05) than those of the other three crayfish (Figure 1e). In addition, the carapace a* and b* values of red–orange crayfish were significantly higher (P < 0.05) than those of normal, white, and blue crayfish (Figures 1f, g).

Stereomicroscope observations indicated that the crayfish carapace had four body colors resulting from xanthophores, erythrophores, and blue pigment cells. The normal body color of crayfish carapaces is dominated by erythrophores, where pigment cells are widely dispersed and dark red, mostly with punctate and dendritic distribution (Supplementary Figure A). In the carapaces of white crayfish, only xanthophores were found, and they were primarily scattered in dots with a less dense, yellowish tint (Supplementary Figure B). The carapace of red–orange crayfish primarily comprised erythrophores; however, there were also a few visible xanthophores, which were dense, less cellular, and mostly found as extended dendrites (Supplementary Figure C). Erythrophores and blue pigment cells predominated in the carapaces of blue crayfish and they were more densely distributed than the blue pigment cells. However, their density was still smaller than that in normal crayfish coloration, and they were primarily distributed as dots. Moreover, the carapace in blue crayfish contained a mixture of dark red erythrophores and blue pigment cells (Supplementary Figure D).

We investigated the carotenoid concentrations in the endodermises and hepatopancreases of the four color variations of crayfish species to explore carotenoid variations across and between tissues. By measuring the target metabolites, the astaxanthin, neoxanthin, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, carotene and β-carotene levels were established (Supplementary Table 2). The findings indicate that astaxanthin existed in the hepatopancreases and endodermis of white, red-orange, and blue crayfish although the absolute values of the content significantly differed. The hepatopancreases and endodermis of the normal crayfish had the highest absolute values and the greatest number of the seven carotenoid types. There were only three types of carotenoids found in the endodermises and hepatopancreases of white crayfish: lutein, β-carotene, and astaxanthin. In addition, the absolute values of the content were the lowest in white crayfish. β-cryptoxanthin was not detected in the endothelium of any crayfish, regardless of body color. Instead, β-cryptoxanthin was found to be specific to the hepatopancreas and was not detected in the WH group. Moreover, zeaxanthin was also not detected in the white crayfish endodermis. The carotenoid content in the hepatopancreas of the normal crayfish was 2 to 12 times higher than that of the other color variations. A comparison of the total carotenoid content of the endodermises of the four body colors of crayfish resulted in a consistency pattern of NP > RP > BP > WP. BH > NH > RH > WH was the order of total carotenoid concentration in the hepatopancreatic samples, because blue crayfish hepatopancreases had the highest amount of β-carotene (35.3322 μg/mL). The four color variations of crayfish clearly differ according to carotenoid species and content, which regulate variances in body color. Within the metabolic pathway of secondary metabolites, we identified three carotenoid-related metabolites (β-cryptoxanthin, fucoxanthin, and zeaxanthin) and two differential metabolites (astaxanthin and canthaxanthin) in the endothelia, however, only astaxanthin and canthaxanthin were found in the hepatopancreas.

To explore the differences in gene expression among the four body colors of crayfish, RNA-seq was conducted in three replicates. Q30 and Q20 were >92% and >97% for every group in the endothelium and hepatopancreas, respectively, indicating that all of the sequencing data could be further analyzed (Table 1). In PCA, the FPKM value for each gene was determined. In biological duplicates, the PCA distribution structure indicated comparable expression patterns (Figures 2i, j).

The distribution of fold change and the P value of the genes were determined to validate the differentially expressed genes (FC ≥ 2; P value < 0.05), as shown in Figures 2a–f. In the endothelial samples, a total of 828 differentially expressed genes were found between NP and WP: 513 and 315 upregulated and downregulated genes, respectively. Moreover, there were 951 differentially expressed genes between NP and RP: 698 and 253 upregulated and downregulated genes. A total of 4,402 differentially expressed genes were measured in NP vs. BP: with 1,303 and 3,099 upregulated and downregulated genes, respectively (Figure 2g). Hepatopancreatic samples revealed 2,972 differentially expressed genes in NH vs. WH (1698 and 1274 downregulated and upregulated, respectively), 2270 differentially expressed genes between NH and RH (1285 and 985 downregulated and upregulated, respectively), 4768 differentially expressed genes between NH and BH(3006 and 1762 downregulated and upregulated, respectively)(Figure 2h).

We conducted KEGG annotation analysis of differentially expressed genes to further explore the biological functions of differentially expressed genes. The differentially expressed genes in the endothelium and hepatopancreas of four body colors of crayfish were annotated into six KEGG databases: metabolic processes, human diseases, organismal systems, cellular processes, genetic information processes, and environmental information processes. Differentially expressed genes were enriched in 294, 291, and 345 KEGG pathways in the NP and WP, NP and RP, as well as NP and BP groups, respectively. The pathways of the top 10 were selected for display based on the P value (Supplementary Table 3). The results indicate that the differentially expressed genes in the NP and WP groups were significantly enriched in the pathways of metabolic, pantothenate and CoA biosynthesis, and the IL-17 signaling pathway. Differentially expressed genes in the NP and RP groups were significantly enriched in pathways such as amebiasis, renin–angiotensin system, and phenylalanine metabolism. Meanwhile, differently expressed genes in the NP and BP groups were in metabolic pathways such as cardiac muscle contraction, ribosome biogenesis in eukaryotes, and hypertrophic cardiomyopathy. Differently expressed genes were involved in 344, 341, and 348 KEGG pathway enrichments in the NH/WH, NH/RH, and NH/BH groups, respectively. Differential genes were significantly enriched in metabolic pathways such as endocytosis, metabolic pathways, pentose and glucuronate interconversions, and other metabolic pathways in NH and WH, coronavirus disease-COVID-19, ribosome, metabolic pathways, and other pathways.

We selected genes related to body color, including CRA2, CRC1, BCO1, ABCG21, XDH, SLC2A3, and mitf, in our transcriptomics analyses to further investigate the metabolism of body color–related pigments in the four body colors of crayfish. Among the four body colors, NP and NH exhibited high expression of CRA2 and CRC1, (Figures 3b, i) whereas WP, RP, BP, WH, RH, and BH exhibited low or no expression (Figures 3a, h). Moreover, BCO1 expression was higher in the endothelia than in the hepatopancreases in the crayfish, regardless of body color. BCO1 expression was low in NP but high in BP and BH, and expression in NH was significantly higher than that in WH compared with RH (Figures 3c, j). In WP, ABCG21 and XDH exhibited high expression levels; however, in NP, their expression levels were significantly low, and in RP and BP, expression levels were either nonexistent or significantly low. In RH, ABCG21 and XDH were substantially expressed; in NH, XDH was less expressed than in WH; and ABCG21 was more expressed than in WH, whereas neither gene was expressed in BH (Figures 3d, e, k, l). The expression levels of the melanin-related genes SLC2A3 and mitf were high in WP, low in NP and RP, and not present in BP. In WH, RH, and NH, SLC2A3 and mitf were expressed; however, in BH, they were either nonexistent or significantly low. SLC2A3 expression was higher in WH than in NH, RH, and BH, and mitf was expressed higher in NH than in WH and RH (Figures 3f, g, m, n).

Six biological replicates for each of the four body colors of crayfish were used for the metabolic analyses of the endothelia and hepatopancreas (each biological replicate contained three individuals). There were 11,611 and 12,218 metabolites in total in the POS and NEG modes, respectively, across the endothelial samples. Meanwhile, 12,395 and 11,202 metabolites in total in the POS and NEG modes were identified in the hepatopancreatic samples (Table 2).

OPLS-DA VIP values and t-test P values (VIP ≥ 1, P < 0.05) were used to evaluate metabolites that exhibited significant differences between groups. The differential metabolites from the NEG and POS models were integrated and analyzed. Six comparison groups were created from the endothelia and hepatopancreas of crayfish with four different body colors (NP vs. WP, NP vs. RP, and NP vs. BP as well as NH vs. WH, NH vs. RH, and NH vs. BH; Figures 4h, g). In the endothelial samples (Figures 4a–c), there were 441 differential metabolites in NP vs. BP (346 and 95 upregulated and down-regulated metabolites, respectively), 370 differential metabolites in NP vs. RP (120 and 250 upregulated and downregulated metabolites, respectively), and 367 differential metabolites in NP vs. WP, (162 and 205 upregulated and downregulated metabolites, respectively). In hepatopancreatic samples (Figures 4d–f), 455 upregulated and 93 downregulated metabolites were identified in 548 differential metabolites in NH vs. WH; 454 upregulated and 118 downregulated metabolites were obtained in 572 differential metabolites in NH vs. RH; and 315 upregulated and 189 downregulated metabolites were identified in 504 differential metabolites in NH vs. BH (Figures 4d–f).

We annotated differential metabolites in KEGG databases for analysis to further explore the role of differential metabolites in metabolic pathways. A total of seven categories of KEGG databases were annotated for differential metabolites in the endothelium and hepatopancreas of the four body colors crayfish: metabolic processes, organismal systems, human diseases, drug processes, environmentally informative processes, genetically informative processes, and cellular processes. Differential metabolites were involved in the metabolic processes of 129, 97, and 128 KEGG pathways in the NP vs. WP, NP vs. RP and NP vs. BP groups, respectively. The top 10 pathways were selected for display based on the P value (Supplementary Table 4). The results indicate that differential metabolites were significantly enriched in metabolic pathways such as vitamin B6 metabolism, histidine metabolism, ubiquinone and other terpenoid–quinone biosynthesis. In the NP and RP groups, differential metabolites were significantly enriched in ubiquinone and other terpenoid–quinone biosynthesis and other metabolic pathways. Differential metabolites in the NP and RP groups were significantly enriched in ubiquinone and other terpenoid–quinone biosyntheses, steroid hormone biosynthesis, steroid hormone biosynthesis, and prostate cancer. Differential metabolism was significantly enriched in the NP and BP groups for insulin resistance, sphingolipid signaling pathway, metabolic pathways, and other pathways. sphingolipid signaling pathway, metabolic pathways and other pathways. Differential metabolites were enriched in 110, 163, and 136 KEGG-enriched enrichment pathways in the NH vs. WH, NH vs. RH, and NH vs. BH groups, respectively. Differential metabolites in the NH and WH groups were enriched in enrichment pathways such as tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and metabolic pathways. Differential metabolites in the NH and RH groups were enriched in metabolic pathways such as metabolic pathways, pyrimidine metabolism and ABC transporters. Differential metabolites in the NH and BH groups were enriched in the metabolism of taurine and hypotaurine metabolism, neuroactive ligand-receptor interaction, and pentose and glucoronate interconversion. Differential metabolites in the NH and BH groups were enriched in metabolic pathways such as taurine and hypotaurine, metabolism, neuroactive ligand–receptor interaction, pentose and glucuronate interconversion, and other enrichment pathways.

KEGG enrichment analysis of differentially expressed endothelial and hepatopancreatic genes with differential metabolites revealed that the NP vs. WP, NP vs. RP, and NP vs. BP groups were enriched in 99, 73, and 110 pathways, respectively. Similarly, the NH vs. WH, NH vs. RH, and NH vs. BH groups were enriched in 94, 101, and 114 pathways, respectively. Among the pathways associated with body color were vitamin digestion and absorption, ABC transporters, glycolysis and gluconeogenesis, arachidonic acid metabolism, melanogenesis, and purine metabolism. There were a total of 131 differentially expressed genes and 50 differentially expressed metabolites in the coanalyzed pathways in the endothelial comparator group. In addition, 187 differentially expressed genes and 105 differential metabolites were found in the coanalyzed pathways in the hepatopancreatic comparator group. These included genes such as Scarb1, XDH, and ABCB and ABCG family members along with differential metabolites such as xanthine, urea, and carotenoid (Supplementary Table 5).

The economic worth of crayfish depends on its color, which is regulated by complex molecular mechanisms and interactions. For instance, carotenoids and pteridine-like pigments in Neocaridina denticulata sinensis are associated with red and yellow body coloration in crustaceans (Lin, 2017). In addition, teleost fish body-color studies support this idea (Fang et al., 2022; Johnson and Fuller, 2015). This study detected the type and content of carotenoid in the hepatopancreases and carapaces of the four body colors of crayfish. The carotenoid composition was found to be associated with body color. The carapace and hepatopancreas L* of white crayfish were significantly higher than those of the normal, red-orange, and blue crayfish. This can be attributed to the presence of the less carotenoid content within the tissues of white crayfish. In a previous study, the carapace and hepatopancreas L*, a*, and b* of Chinese mitten crabs were highly correlated with carotenoid content (Li et al., 2021). In the study of body-color regulation in fish and other crustaceans, the body color was determined by the type and content of the carotenoid (Yagiz et al., 2010). In this study, the astaxanthin content of white crayfish carapaces and hepatopancreases was 12.1 and 4.25 times lower than that of the normal crayfish, respectively. This finding is consistent with a previous study on lobsters, in which the pigment content in red lobster is 2.4 times that of the white lobster (Wade et al., 2005).

Combining metabolomics, astaxanthinin, a carotenoid biological metabolic pathway (map00906) of white crayfish endothelia, was transformed by β-carotene using canthaxanthin. In the study of Carcinus maenas carotenoid metabolism, the No.II metabolic pathway in Figure 5 was consistent with the speculation, which was converted from β-carotene to astaxanthin. The type and content of carotenoids in normal-body-color crayfish are greatest in carapace and hepatopancreas, particularly astaxanthin. Combined with the carotenoid synthesis pathways of transcriptome (map00906) and metabolome (map01062) (Figure 5), four astaxanthin synthesis pathways may exist in the endothelia of normal crayfish. The metabolic end products of pathways I, II, III and IV are astaxanthin; therefore, considerable amount of astaxanthin is deposited in the endothelia and hepatopancreas of normal crayfish. For the biological metabolism of ketocarotenoids, β-carotene was used as the substrate, and two hydroxyl and ketone groups were added to each β-ionone ring through a series of reactions. Under the action of hydroxylase and ketolase, it was metabolized to astaxanthin and deposited to the target organ. The difference in the composition and content of carotenoids in the hepatopancreas is likely to be the internal cause of the difference in the shell color phenotype of different strains of crayfish, which aligns with the results of the study of the shell color and embryo of the Chinese mitten crab having green and white shell lines (Zhang et al., 2022, 2023).

BCO1, a crucial gene in carotenoid metabolism, cleaves pertinent carotenoids (Lindqvist and Andersson, 2002; Redmond et al., 2001; Von Lintig and Vogt, 2000; Wyss et al., 2000). The BCO family genes are correlated to the body color of aquatic animals. In our study, NP had the highest concentrations of β-carotene (0.8551 μg/mL); however, BP had a lesser amount (0.2625 μg/mL) than NP. Nevertheless, NP expressed less BCO1 than BP. WP expressed more BCO1 expression than NP, although NP had a greater β-carotene concentration. Therefore, we suggest that high BCO1 expression promotes the conversion of β-carotene into other carotenoids. As shown in Figure 5, β-carotene deposition in the endothelium may be limited, because it could undergo conversion into astaxanthin. Meanwhile, the RP adheres to this pattern. However, all four body colors, endothelium BCO1 expression was higher than in the hepatopancreas. Consistent with the results of this study, the carotenoid accumulation of the triangle sailing mussel was decreased by the high expression of HcBCO1 in the triangular pearl mussel (Yan, 2023). Thus, high BCO1 expression may promote the cleavage of β-cryptoxanthin in the endothelium into other carotenoids. For instance, the expression level of BCO-like in the white Patino scallop was significantly higher than in the orange counterparts (Li et al., 2019). Exopalaemon carinicauda deepened after knocking out BCO (Sun et al., 2020a, b). These observations indicate that BCO plays an important role in body-color regulation.

Crustacyanin (CRCN) is a protein-binding molecule that binds to ingested astaxanthin, thereby altering the energy level structure of astaxanthin and determining the pattern and color of the carapace (Cianci et al., 2002; Yan, 2023; Zhao et al., 2021). The endothelium and hepatopancreas of crayfish contain two isoforms of the CRCN family genes, CRA2 and CRC1. Scholars (Ertl et al., 2013) believed that the expression of the CRCN-A and CRCN-C genes of the albino Metapenaeus penaeus was significantly lower than that of bright and dark varieties. In this study, these two genes were highly expressed in NP and NH. Moreover, CRA2 and CRC1 were expressed in WP, although both at lower levels than in NP. CRC1 was not expressed in RP, RH, or WH, although it was expressed at low levels in BP and BH. According to research, a lower expression level of the CRCN may limit the binding and subsequent utilization of free astaxanthin, which could be one of the reasons why normal crayfish have a dark body color (Jin et al., 2021).

Body-color regulation can be improved by ABCG, a member of the G subfamily of the ABC transporter protein family. The results of this investigation indicate that the expression of ABCG21 in WP was greater than that in NP, RP, and BP and that the expression of its homologous gene, ABCG14, in WP was higher than that of the strains of Marsupenaeus species with dark colors (Wang et al., 2023). Notably, the previously mentioned information is consistent with the results of this study. Although most ABC transporter protein family G subfamily members are involved in body-color regulation, their individual methods might differ, necessitating additional research. For instance, a previous study indicates that the white gene in Drosophila and Bombyx mori was homologous to the ABCG homolog in the Neocaridina denticulata sinensis body color, and both mutant Drosophila bodies containing the white gene and Bombyx mori eggs were white (Lin, 2017). In the present study, in WP, RP, and WH, the pteridine metabolism related gene XDH was strongly expressed; in BP and BH, it was either nonexistent or considerably low. The nontargeted metabolome contained uric acid and xanthine as differentially abundant metabolites. Therefore, we analyzed this possible mechanism of xanthine synthesis of uric acid in white crayfish by combining the expression pattern of XDH with untargeted metabolomic results. Our findings are in accordance with a previous study on Metapeneaus peneaus, in which the XDH gene produced uric acid from xanthine as a defense mechanism against UV damage. When the body is transparent, the expression of XDH (Hu et al., 2013), a gene found in the bodies of Marsupenaeus species, is high (Lin, 2017).

In crustaceans, body color is regulated in part by genes related to melanin. mitf is a crucial regulator of melanocyte formation and plays a role in cell survival, differentiation, and growth. In our study, mitf was not expressed in BP but was expressed at high levels in WP, RP, and NP. This finding corresponds to the expression pattern of the mitfa gene in all-red Oujiang-colored carp, which have no melanocytes and an all-red phenotype (Yu et al., 2020). The study concluded that although mitfa is highly expressed, it does not participate in melanocyte synthesis. In the same way, we only discovered the xanthophores in WP. There were only a few xanthophores found in RP but many erythrophores, many of which covered the NP xanthophores. Melanocytes were absent from WP and RP. mitf might promote the xanthophore formation when combined with the discoveries of the study that zebrafish mitf is expressed by melanocytes (melanocyte stem cells) and xanthophore precursors (Parichy et al., 2000; Rawls et al., 2001). Meanwhile, a member of the soluble carrier (SLC) family is SLC2A3. Another member, Slc45a2, plays a crucial role in melanin deposition in mammals (Graf et al., 2005), and low melanin concentration and misshapen melanocytes are caused by gene mutations in mice and Oryzias latipes (Du and Fisher, 2002; Fukamachi et al., 2001). However, SLC family members can also function as carotenoid transporter proteins, particularly for vitamins. Tsukaguchi et al. have identified the SLC23a1 member as a vitamin C carrier (Tsukaguchi et al., 1999). Investigating the significance of melanin-related genes in the control of body color in crustaceans is vital, because they account for a smaller portion of the body color of crayfish, particularly mitf.

In this study, KEGG enrichment analysis was conducted for differentially expressed genes and metabolites in the transcriptome and metabolome. The pathways related to body color regulation that were present in the endothelial and hepatopancreatic comparative groups were vitamin digestion and absorption, retinol digestion and absorption, and purine metabolism. Pigments in crustaceans not only regulate body color but also maintain balanced physiological metabolic functions, such as oxidative metabolism, visual maintenance, and defense mechanisms.

In this study, Scavenger receptor class B type I (Scarb1) of vitamin digestion and absorption was significantly upregulated in NP vs. RP. However, there were no significant differences in the expression in hepatopancreas. Because Scarb1 is an HDL receptor (high-density lipoprotein) that binds to cholesterol transport particles and promotes cellular uptake of carotenoids, it is highly focused on by scholars (Toomey et al., 2017). A study by Kanika et al. found that Scarb1 expression in the skin of white mutant Oujiang carp was lower than that in wild red and mutant red Oujiang carp (Kanika et al., 2023). Tian et al. have shown that the use of miR-430b to limit Scarb1 expression disturbs carotenoid metabolism and decreases chromatophore distribution density, thereby weakening the skin redness value of koi carp (Tian et al., 2022). This study found that Scarb1 expression in white crayfish (WP) is higher than in normal crayfish (NP) possibly owing to the low carotenoid content, resulting in decreased oxidation capacity. Scarb1 is used to maintain antioxidant capacity by converting β-carotene to retinol.

The retinol metabolism pathway plays an important role in the growth, development, and body-color regulation of aquatic animals (Bi et al., 2024; Wiseman et al., 2017). Retinol is a form of vitamin A, which can be transformed through metabolic pathways in organisms into active forms such as retinol ester and retinol acid. It is involved in regulating the growth, differentiation, antioxidant and immune functions, reproduction and embryonic development of almost all cells (Das et al., 2014). Retinol deficiency can cause visual system disorders (Saleh et al., 1995) such as, exophthalmos (Yang et al., 2008), small eyes or retinopathy, and retinal development disorders. After adding an appropriate amount of retinol to the shrimp feed, the vision became sensitive, the function became normal, and the eye tissue is not distorted (Chen and Li, 1994). Carotenoid metabolism indirectly promotes retinol metabolism in the digestive system, of which β-carotene is metabolized into retinol via the regulation of Scarb1, which participates in pigmentation, antioxidation, and development. In this study, Scarb1 expression in the hepatopancreas of normal crayfish was upregulated and promoted pigmentation. In the study of crap, when Scarb1 was knocked out, the red skin turned white, and the type and content of astaxanthin decreased. Scarb1 converts the part of β-carotene to retinol (vitamin A) to maintain a balanced physiological function in the body. White crayfish use retinol to compensate for the deficiencies in antioxidant capacity, reproductive capacity and visual system.

The purine metabolic pathway is an important in regulating the balance of ammonia nitrogen in insects and crustaceans. Crustaceans use xanthine as a substrate to decompose urea into ammonia and CO₂ under the action of urease. Because ammonia has certain toxicity in animals, it must be promptly removed (Liu et al., 2005). In the metabolic process of silkworm pigment, XDH oxidizes xanthine to urate, and the accumulation of urate increases the transparency of the body and strengthens the its defenses against ultraviolet light (Hu et al., 2013). XDH is highly expressed in the stomach and heart of Pacific shrimp (Marsupenaeus japonicus), which helps to scavenge free radicals and resist Vibrio, and controls the occurrence of white spot syndrome (Lin, 2017; Okamura et al., 2018). In this study, XDH was significantly upregulated in WP, and uric acid was downregulated, xanthine and uric acid accumulation increased. The body transparency of white crayfish was increased, which could strengthen their defense mechanism against ultraviolet light.