The body color of animal plays a vital role in camouflage, mimicry, socialization, and protection against the harmful effects of solar radiation (Slominski et al., 2004). As the natural habitats, sea cucumbers prefer to reside under reefs and may occasionally bury themselves in sand or mud (Purcell et al., 2016). Melanin is the primary pigment that determines the body color of sea cucumber Apostichopus japonicus (Xing et al., 2017). A. japonicus have melanin covering their backs or entire bodies, which helps to protect against UV rays and provide camouflage (Xing et al., 2018). Therefore, pigmentation is crucial for the survival of sea cucumbers juveniles. In this study, white juveniles gradually turned black as they grew up (Fig. 1D). However, the rate of pigmentation varies among different individuals, with sea cucumbers of the same size exhibiting different depths of coloration (Fig. 1B). Therefore, the rate of pigmentation may be influenced by specific factors. In this study, a combined analysis of the metabolome and transcriptome was employed to investigate this issue in the tropical sea cucumber H. leucospilota.
The pigment-related metabolites include astaxanthin (red), melanin (black), and beta-carotene (yellow)(Wade et al., 2017, Xing et al., 2017). Insufficient diet provided raw materials for pigment synthesis can result in the loss or absence of hyperpigmentation in some animals (Wade et al., 2017). However, our current study found no significant differences in melanin content or the activity of melanin synthesis-related TYR enzymes between white and black H. leucospilota individuals (Fig. 4). This suggests that the black coloration in H. leucospilota may not be directly linked to melanin content, but rather to its presentation. Other factors, such as nutritional status, as indicated by our analysis of metabolomics and transcriptomics associations (Fig. 5–7), may play roles instead.
The differences in metabolites and gene expression between the white and black H. leucospilota juveniles are mainly related to nutrition, including pathways related to lipid metabolism, such as linoleic acid decomposition (Fig. 5C), enzymes related to carbohydrate digestion, such as AMY, MGA and TREA (Fig. 6A), and gene expression related to protein storage, such as MYP (Fig. 7A & 7B). The pigmentation phase of H. leucospilota juveniles requires a substantial amount of energy for the synthesis of pigments and other substances. Insufficient nutrient reserves during larval development can have a negative impact on larval metamorphosis (Yu et al., 2022). In our study, the black individuals catabolized linoleic acid into downstream 16(R)-HETE, 12-KETE and 9(S)-HPODE, while the white individuals exhibited higher expression of digestive enzymes. This suggests that during the pigmentation stage, the initial individuals (white) primarily obtain energy for life activities through the digestion of carbohydrates. After accumulating energy from exogenous carbohydrates, the sea cucumbers enter a hypometabolic phase where fatty acid oxidation gradually increases to meet their energy needs, as has been reported in A. japonicus (Zhao et al., 2022).
Remarkably, the white H. leucospilota juveniles exhibit high contents of two sphingolipid derivatives, namely sphingosine and sphingosine 1-phosphate (Fig. 2E & 5A). Sphingolipids are bioactive lipids that play crucial roles in various cellular processes, including cell growth, survival, apoptosis, proliferation, inflammation, and normal metabolism (Quinville et al., 2021, Parveen et al., 2019, Shaw et al., 2018). Sphingosine 1-phosphate-like molecules can also contribute to morphogenesis, development, reproduction, and survival through alternative mechanisms (Oskouian and Saba, 2004). In this study, the elevated levels of sphingosine 1-phosphate in the pigmentation phase of H. leucospilota juveniles may facilitate the completion of pigmentation by promoting cell growth.
Interestingly, apart from variations in lipids and digestive enzymes, our study identified two transcript products that were annotated as major yolk proteins (MYP), which play a crucial role in sea cucumber embryonic development. MYP is a distinctive protein found in echinoderms and belongs to the transferrin family, responsible for binding iron ions (Brooks and Wessel, 2002). In general, during embryonic development in oviparous animals, nutrients stored in the yolk are utilized for growth before hatching and feeding. The sources of yolk differ between various oviparous species. For instance, in birds and fish (Hiramatsu et al., 2015, Tian et al., 2010), yolk is derived from the liver (Hiramatsu et al., 2015), while in insects, it is synthesized by the fat body (Tufail and Takeda, 2008). Molluscs produce yolk directly in the ovary (Ni et al., 2014), whereas crustaceans primarily synthesize it in the hepatopancreas (Li et al., 2022, Ruan et al., 2020). Interestingly, the MYP gene is expressed in both male and female sea urchins (Shyu et al., 1986). During the embryonic phase of sea urchins, MYP products are synthesized in the gut and transported to the epidermis (Unuma et al., 2009). On the other hand, MYP in sea cucumbers was initially associated with ovarian development (Fujiwara et al., 2010). Our study, for the first time, reveals a link between MYP transcript expression and pigmentation (Fig. 7). The significantly lower expression of both MYPs in black H. leucospilota individuals suggests a decrease in the energy-consuming process during the late pigmentation stage, with no excessive nutrient supply required. This finding correlates with the observed trend in lipid metabolism (Fig. 5C) and further emphasizes the importance of lipid and nutrient changes in sea cucumber pigmentation.
In this study, we specifically chose white and black H. leucospilota juveniles to conduct a comprehensive analysis of their metabolomics and transcriptomics, aiming to explore the mechanism of pigmentation in echinoderms. Based on the results, it is indicated that the pigmentation in H. leucospilota may differ from the classical melanin pathway, and it is more likely to be a pathway associated with nutritional status, including changes in linoleic acid-based lipid metabolism, enzyme-based carbohydrate digestion, and MYP-based accumulation of protein nutrient sources.