Vitamin C Promoted Feeding and Growth Rate of Apostichopus japonicus by Increasing Its Digestive Capacity Through a 5-Hydroxytryptamine-Mediated Signaling Pathway

Simple Summary

Sea cucumbers (Apostichopus japonicus) are economically valuable marine organisms, but their growth in artificial farming is often limited by feeding and digestive efficiency. To address this issue, we conducted a 60-day feeding study to explore how different levels of dietary Vitamin C (VC) affect the growth and physiological performance of A. japonicus. We found that a 5000 mg/kg VC supplement significantly improved A. japonicus growth, as shown by faster weight gain and more active feeding behavior. This improvement was linked to enhanced digestive enzyme activity, increased mucus secretion for better food capture, and healthier intestinal structures. Additionally, 5000 mg/kg VC intake promoted the production of 5-hydroxytryptamine, a key substance that regulates feeding and digestion. In contrast, either VC deficiency or excess failed to provide such benefits and even hindered growth. Our findings offer practical guidance for optimizing A. japonicus aquaculture practices, helping to improve its productivity and sustainability.

Abstract

To investigate the effects of dietary vitamin C (VC) supplementation at different concentrations (0, 5000, and 15,000 mg/kg) on the growth and feeding performance of sea cucumbers (Apostichopus japonicus), a 60-day feeding trial was conducted. The results demonstrated that supplementation with 5000 mg/kg VC significantly enhanced growth performance, evidenced by a higher specific growth rate and weight gain, along with a shorter feeding initiation time and increased tentacle feeding frequency. Furthermore, the activities of digestive enzymes—including amylase, lipase, and pepsin—were markedly elevated in the intestines of A. japonicus fed 5000 mg/kg VC, accompanied by increased mucus secretion in the oral tentacles. Correspondingly, intestinal 5-hydroxytryptamine (5-HT) levels and the expression of tryptophan hydroxylase (AjTPH) and 5-HT receptor (Aj5-HT4R) genes were significantly upregulated in the same group. In contrast, relatively lower growth performance was observed in the control (0 mg/kg) and high-dose (15,000 mg/kg) VC groups. Histological analysis further revealed that 5000 mg/kg VC increased villus height and width and thickened the intestinal muscle layer. Overall, dietary supplementation with 5000 mg/kg VC enhanced feeding activity, digestive function, and intestinal morphology, thereby promoting growth. However, excessive supplementation (15,000 mg/kg) failed to provide similar benefits and was associated with impaired performance.

1. Introduction

Renowned for its abundant nutritional composition and considerable medicinal properties, sea cucumber (Apostichopus japonicus) holds significant economic and medicinal importance in aquaculture, making it a popular tonic in East Asia and a key product in the aquatic product market [1]. However, in artificial aquaculture settings, the growth performance and health status of A. japonicus are often constrained by its feeding capacity and digestive efficiency [2,3]. These two core factors directly influence aquaculture outcomes and economic value. Therefore, enhancing feeding capacity and optimizing digestive efficiency have become key technological priorities in the development of sustainable A. japonicus aquaculture.

Feeding and digestion are fundamental physiological processes through which organisms acquire energy from the environment, initiate its conversion, and sustain various life activities. The efficiency of these processes directly reflects an organism’s growth and health status. As a typical benthic organism, A. japonicus uses its oral tentacles as the primary feeding organ to obtain nutrients by adhering to organic particles on the seabed [4,5]. The feeding process consists of four key stages: extension of the tentacles to the sediment surface, contraction of the tentacles after food adhesion, retraction from the sediment, and transfer of food-laden tentacles to the buccal cavity [6]. The frequency of tentacle movement has been identified as a direct indicator for assessing feeding capacity. Existing studies indicate that mucus released from the oral tentacles is essential for enhancing the attachment of organic particles onto the tentacle surface, thereby improving the efficiency of the feeding process [7]. As the main digestive organ of A. japonicus, the intestine is responsible for food digestion through peristalsis and the secretion of digestive enzymes [8,9].

Vitamin C (VC) is an essential dietary nutrient for aquatic animals [10,11], widely recognized for its classical functions in promoting growth, enhancing antioxidant defense, and supporting tissue repair [12,13,14]. There are also some existing studies which suggest that VC can enhance digestive capacity by improving intestinal function. Earlier studies demonstrated that supplementing the diet with 80 mg/kg of VC markedly enhances digestive enzyme activities and promotes growth in discus fish (Symphysodon haraldi) [15]. Furthermore, supplementation with 100 mg/kg VC has been found to significantly enhance digestive enzyme activity in the giant river prawn (Macrobrachium malcolmsonii) [12]. However, the effects of VC on the feeding capacity and digestibility of A. japonicus remain insufficiently explored. Furthermore, accumulating research evidence has demonstrated that VC is also involved in modulating the synthesis of 5-hydroxytryptamine (5-HT) in specific animal species [16,17]. In vertebrates, adequate VC supply aids 5-HT synthesis [18,19]. In crustaceans, VC acts as an essential cofactor for tyramine β-hydroxylase (TBH), which catalyzes tyramine conversion to octopamine and ensures their normal synthesis [20]. In arthropods, VC significantly upregulates the expression of the dopamine 1-like receptor (Dop1R1) gene in Drosophila tissues [21]. 5-HT, an important neurotransmitter, serves a crucial regulatory function in feeding behavior, locomotion, and digestive functions. Studies have demonstrated that increased 5-HT levels can markedly enhance intestinal digestive enzyme activity, elevate the feeding frequency of oral tentacles, and promote general locomotor activity of sea cucumbers [22].

These results emphasize the crucial role of 5-HT signaling in regulating feeding behavior and physiological functions in A. japonicus. Therefore, the present study was aimed to systematically investigate how dietary VC supplementation affects the feeding behavior and digestive capacity of juvenile A. japonicus. The primary objectives of this study are: (1) to assess the impact of VC on feeding behavior and digestive capacity, and (2) to explore the role of VC in regulating these processes, with a focus on its interaction with the 5-HT signaling pathways.

2. Materials and Methods

2.1. Experimental Dietary Formulations

Detailed in

Table 1. Experimental diet composition (dry matter, g/kg).

Ingredient	Diets with Different Levels of Vitamin C
	0 mg/kg	5000 mg/kg	15,000 mg/kg
Fish meal 1	50	50	50
Soybean meal 2	40	40	40
Alfalfa meal 3	300	300	300
Vitamin premix 4	5	5	5
Mineral premix 5	5	5	5
Sea mud	600	599.5	598.5
Vitamin C	0	0.5	1.5
Proximate analysis
Crude protein (%)	9.46 ± 0.23	9.52 ± 0.11	9.55 ± 0.20
Crude lipid (%)	0.42 ± 0.01	0.41 ± 0.01	0.42 ± 0.01

¹ Fish meal: crude protein 67.6%, crude lipid 8.9% DM (Dalian Xinyulong Marine Biological Seed Technology Co., Ltd., Dalian, China). ² Fermented soybean meal: crude protein 51.56%, crude lipid 0.9% DM (same as above). ³ Alfalfa meal: crude protein 16.8% DM (same as above). ⁴ Vitamin A, 32 mg; Vitamin D, 5 mg; Vitamin K, 10 mg; Vitamin B1, 25 mg; Vitamin B2, 45 mg; Vitamin B6, 20 mg; Vitamin B12, 10 mg; folic acid, 20 mg; pantothenic acid, 60 mg; biotin, 60 mg; niacin, 200 mg; inositol, 800 mg; ascorbic acid, 2000 mg; microcrystalline cellulose, 16.71 g. ⁵ MgSO₄·7H₂O, 1200 mg; Ca(IO₃)₂, 180 mg; FeSO₄·H₂O, 80 mg; MnSO₄·H₂O, 60 mg; CuSO₄·5H₂O, 50 mg; CoCl₂·6H₂O (1%), 50 mg; ZnSO₄·H₂O, 10 mg; Na₂SeO₃ (1%), 25 mg; zeolite, 18.35 g. Notes: DM, dry matter; values represent the composition of the basal diet prior to vitamin C supplementation.

are the composition and formulation of the basal diet. As the sea mud used in the control diet was mainly inorganic and presumed to contain negligible vitamin C, no additional VC was supplemented (0 mg/kg). Wherein alfalfa meal, fermented soybean meal, and fish meal served as the principal protein sources. VC (L-ascorbic acid, ≥99% purity, supplied by Nanjing Dulai Biotechnology Co., Ltd., Nanjing, China) was incorporated at 0, 5000, and 15,000 mg/kg. All raw materials were passed through an 80-mesh sieve prior to subsequent processing, thoroughly blended, and mixed with 30% distilled water before being formed into 1 cm diameter pellets using a pelleting machine (Haiyang Huatong Feed Machinery Co., Ltd., Haiyang, China). The prepared pellets were oven-dried at 65 °C, followed by cooling, hermetic sealing, and storage at −20 °C until subsequent utilization.

2.2. Feed Composition Analysis

The experimental diet’s proximate composition consists of key components, including crude lipid, crude protein, and moisture, which are fundamental indicators for evaluating dietary nutritional quality. The aforementioned nutritional components were quantitatively analyzed employing the standardized analytical protocols delineated by the Association of Official Analytical Chemists (AOAC). These standardized procedures ensured accurate and reliable determination of the key components in the diet [23]. In summary, the Kjeldahl method (nitrogen-to-protein conversion factor: 6.25) was employed for crude protein quantification, and ether extraction with a Soxhlet apparatus was used to determine crude lipid content. To assess moisture content, test samples were subjected to drying at 105 °C in a hot air oven until a constant weight was attained. Each experiment was repeated three times to validate the consistency and accuracy of the results. And the resultant values are presented on a dry weight basis to ensure consistency and comparability across the entire dataset.

2.3. Feeding Experiment

At the Ministry of Agriculture’s Key Laboratory of Marine Aquaculture (Dalian Ocean University), a 60-day feeding experiment was performed, from April to June 2025. Juvenile. Sourced from Dalian Xinyulong Marine Biotechnology Co., Ltd. (Dalian, China), all A. japonicus individuals were subjected to a 7-day acclimation period before the experiment during which they were provided with a basal diet. Tank maintenance involved daily removal of uneaten feed and feces at 08:00, along with a 50% seawater exchange to maintain water quality. After acclimation, A. japonicus with comparable initial body weights (7.50 ± 0.50 g) were randomly allocated to nine 30 L experimental tanks, with 14 individuals stocked per tank. To guarantee the reliability of the trial outcomes, three independent tank replicates were set up for each experimental treatment. Throughout the experimental period, to minimize VC leaching in seawater, we adopted a feeding strategy of reducing single feeding amount and increasing feeding frequency. The A. japonicus were fed three times daily at 07:30, 14:00, and 19:30, respectively. Feed was administered at 1.5% of body weight at 07:30 and 14:00, and at 3.0% at 19:30. Feeding amounts were adjusted daily based on actual feed intake. Water exchange was performed daily at 13:00 using a siphon to replace two-thirds of the tank water. Uneaten feed and feces were carefully removed from the tanks three times daily, at 07:30, 13:00 and 19:00, to maintain water quality. The culture tanks were kept under low-intensity lighting conditions, and the temperature of the natural seawater was gradually increased over the course of the experiment, ranging from 9 °C to 17 °C. Throughout the study, concentrations of ammonia nitrogen and nitrite were closely monitored throughout the experimental period, with their levels strictly maintained below 0.04 mg/L to secure optimal rearing water quality. Meanwhile, the pH value was stably regulated within the range of 7.7–8.2, and dissolved oxygen (DO) concentrations were sustained above 6.5 mg/L, which collectively supported the physiological health and normal activity of the A. japonicus.

2.4. Feeding Frequency Observation

At the termination of the 60-day feeding experiment, random selection of nine A. japonicus, was conducted from each experimental group, and their feeding behaviors were observed and recorded for 30 min using a digital video camera (model: Legria HF20; manufacturer: Canon Inc., Tokyo, Japan). Feeding events were quantified by counting the number of times the A. japonicus tentacles grasped food during the 30 min observation period, with each tentacle grasp counted as one feeding event.

2.5. Measurement of Feeding Latency

To evaluate the effect of dietary VC supplementation on the feeding response of A. japonicus, feeding latency was measured as a key indicator. (i.e., the time from feed delivery to the initiation of feeding behavior) was recorded. Individual A. japonicus were placed in a rectangular rearing frame (40 × 30 × 20 cm) within the experimental tanks and acclimated for 10 min prior to testing. A pre-weighed quantity of the experimental formulated feed was then gently placed on one side of the observation frame, whereas the individual A. japonicus was positioned on the opposite side to ensure a consistent initial distance between the A. japonicus and the feed. The timing began immediately upon feed placement. The feeding latency was defined as the time elapsed from the moment of feed delivery until the A. japonicus moved toward the feed and initiated feeding behavior, characterized by the tentacles grasping and bringing feed to the mouth. Each individual A. japonicus was continuously observed for 30 min via the same digital video camera (model: Legria HF20; manufacturer: Canon Inc., Tokyo, Japan), ensuring no external disturbance during the behavioral recording process, and the measurements were analyzed using playback to ensure accuracy. Three replicate measurements were conducted per treatment group, and the mean value was used for statistical analysis.

2.6. Collection of Experimental Samples

Upon completion of the 60-day feeding trial, all A. japonicus were fasted for 48 h prior to subsequent procedures. Subsequently, nine A. japonicus specimens were randomly sampled from each experimental tank for morphological measurement and body weight measurement; these sampled individuals were further subjected to digestive enzyme activity analysis, 5-HT content determination, and quantitative real-time PCR (qPCR) analysis. Strict biological replicates were set up in the experiment, with 3 independent biological replicates per treatment group; intestinal tissues from three individuals were pooled into one sample for subsequent analysis. These collected data were subsequently utilized to compute the WGR, SGR, FCR, and SR. For histological analysis, oral tentacles and intestines were carefully dissected from each specimen using sterile scalpels. After rinsing with sterile saline, the samples were immediately placed in 4% paraformaldehyde for fixation. The collected tissue samples were later used for detailed histological observation. Additionally, oral tentacle and intestinal tissue samples were stored at −80 °C for later analysis of digestive enzyme activities, along with 5-HT gene expression and content. Meanwhile, Mucus secreted from each experimental tank’s A. japonicus was collected and weighed, with samples stored at −80 °C for further biochemical testing.

2.7. Growth Performance and Feed Conversion Rate

To assess growth performance and feed utilization, the initial and final body weights of each tank’s A. japonicus were quantified. The following parameters were calculated:

$$\mathrm{Specific} \mathrm{growth} \mathrm{rate} (\mathrm{SGR}, \%) = \left\{\left[\ln \left(W_i\right)-\ln \left(W_f\right)\right]/T\right\}\times 100$$

$$\mathrm{Feed} \mathrm{Conversion} \mathrm{Ratio} (\mathrm{FCR}, \%)=P_W/\left(W_i-W_f\right)\times 100$$

$$\mathrm{Survival} \mathrm{rate} (\mathrm{SR}, \%)=N_i/N_f\times 100$$

$$\mathrm{Weight} \mathrm{growth} \mathrm{rate} (\mathrm{WGR}, \%)=\left(W_i-f_f\right)/W_f\times 100$$

where $N_i$ and $N_f$ represent the final and initial number of A. japonicus, respectively $W_f$, and $W_i$ denote the initial and final average body weights in each tank, respectively; $P_W$ and $W$ represent the wet mass of the Total feed intake, and whole-body weight of A. japonicus, respectively; and $T$ represents the feeding period.

2.8. Digestive Enzyme Activity Analysis

Oral tentacle, intestinal, and mucus samples, initially frozen, were thawed on ice before being homogenized in 0.1 M phosphate buffer (pH 7.0) kept on ice. After centrifuging the homogenates at 12,000× g for 10 min at 4 °C, the supernatants were carefully harvested to determine digestive enzyme activity. The activities of three key digestive enzymes, namely amylase, pepsin, and lipase, were quantified using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The commercial kits applied were as follows: amylase assay kit (Catalog No.: C016-1-1), pepsin assay kit (Catalog No.: A080-1-1) and lipase assay kit (Catalog No.: A054-1-1). Following the manufacturer’s standardized protocols [24,25]. All enzyme activities were expressed as units per milligram of protein (U/mg protein). Each assay was performed in triplicate for each tank to ensure reproducibility.

2.9. Histological Analysis of Intestine

Intestinal tissue samples fixed in 4% paraformaldehyde solution were initially rinsed with phosphate-buffered saline (PBS, pH 7.4) to eliminate residual fixative, ensuring no interference with subsequent histological processing. The intestinal tissue samples were subsequently dehydrated via a graded ethanol series to eliminate residual water, thereby preparing the tissues for paraffin embedding. Following dehydration, the samples were embedded in paraffin to preserve their structure for sectioning. The paraffin-embedded tissues were sectioned at a thickness of 5 μm using a rotary microtome (Leica RM2245; Leica Biosystems, Nussloch, Germany).and subsequently mounted on poly-L-lysine-coated glass slides to prevent section detachment. Hematoxylin and eosin (HE) staining was performed on the tissue sections to highlight intestinal general morphology and tissue structure; following staining, the sections were examined to assess structural details. The HE-stained tissue sections were observed under a light microscope (Olympus Corporation, Tokyo, Japan) at 100× and 400× magnifications, and representative microscopic images were captured using a digital camera (Nikon Eclipse E100, Nikon, Tokyo, Japan) attached to the microscope for subsequent detailed morphological and histological analysis.

2.10. Determination of Intestinal 5-HT Content

Approximately 0.5 g of intestinal tissue, which had been stored at −80 °C to preserve its integrity, was carefully homogenized in 1 mL of ice-cold PBS-EDTA buffer containing 1 mM EDTA to protect against protease activity. The tissue homogenate was then centrifuged at 10,000× g for 10 min at 4 °C to separate the cellular debris from the soluble components. The resulting supernatant, containing the dissolved substances, was carefully collected for further analysis. To determine the levels of 5-HT in the intestinal tissue, a commercial enzyme-linked immunosorbent assay (ELISA) kit (Catalog No.: H104-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was utilized following the manufacturer’s standardized protocols. The absorbance of each sample was determined at a wavelength of 450 nm using a microplate reader (Synergy H1; BioTek Instruments, Winooski, VT, USA), and the resultant data were expressed as nanograms of 5-HT per gram of wet tissue (ng/g). All samples were analyzed in triplicate with three technical replicates per sample to ensure result reliability, reproducibility, and accuracy.

2.11. Quantitative Real-Time PCR

Using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA), total RNA was purified from the intestinal tissues of A. japonicus in strict accordance with the manufacturer’s protocols. For the assessment of RNA quality (including purity and concentration), the extracted RNA samples were subjected to analysis using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was evaluated based on the A260/A280 absorbance ratio, with a range of 1.8–2.0 considered indicative of high-quality RNA, while the concentration was directly quantified from the spectrophotometric measurements. Agarose gel electrophoresis was employed to validate RNA integrity. Agarose gel electrophoresis was used to confirm the integrity of the RNA, which was performed using a 1× TAE buffer and analytical-grade agarose (Sangon Biotech, Shanghai, China). The quality of the RNA was assessed by examining the clear separation of ribosomal RNA bands on the gel, ensuring that only high-quality RNA was used for subsequent cDNA synthesis

cDNA Synthesis: First-strand cDNA was synthesized from the qualified total RNA (A260/A280 = 1.8–2.0) using the PrimeScript™ RT Reagent Kit (Takara Bio Inc., Otsu, Japan), which includes gDNA Eraser to eliminate genomic DNA contamination. Reverse transcription was conducted at 37 °C for 15 min, followed by immediate inactivation of the reverse transcriptase at 85 °C for 5 s to terminate the reaction. The synthesized cDNA products were then stored at −20 °C in aliquots to avoid repeated freeze–thaw cycles, and reserved for subsequent quantitative real-time qPCR analysis. Primer design Gene-specific primers for AjTPH and Aj5-HT4R were designed using NCBI Primer-BLAST (National Center for Biotechnology Information, Bethesda, MD, USA; accessed on 24 August 2025), based on coding sequences obtained from the A. japonicus transcriptome database. β-actin has been widely used as a reference gene in A. japonicus qPCR analyses [26,27]. In the present study, the Ct values of β-actin showed no apparent systematic variation among VC treatments, and thus β-actin was used for normalization. With primer sequences obtained from previously published literature. All primer pairs were validated for specificity by conventional PCR and agarose gel electrophoresis, and the amplification efficiency of each primer set was confirmed to fall within the acceptable range for RT-qPCR analysis. Primer sequences are listed in

Table 2. Real-time quantitative PCR primers applied in this study.

Genes	Primer Sequences (5′-3′)	Genes ID/Reference
β-actin	F:5′-CCATTCAACCCTAAAGCCAACA	[28]
	R:5′-ACACACCGTCTCCTGAGTCCAT
AjTPH	F:5′-AGACCATTCTTGCCCTCCTG	BSL78_17977
	R:5′-TGCGAGTGTTTTATGCTGCG
Aj5-HT4R	F:5′-TTCAGCTGACTCAACTCACCCTCTG	[29]
	R:5′-CTGATTGACCATTGACTTTGTTGTC

β-actin, beta-actin. AjTPH, putative tryptophan 5-hydroxylase 1. Aj5-HT4R, Apostichopus japonicus 5-Hydroxytryptamine 4 receptor.

. Relative Gene Expression Analysis: Relative expression levels were determined by the 2^−∆∆CT method, with the means ± standard error of the mean (SEM) (n = 3).

2.12. Date Analysis

All collected data were processed using SPSS 27.0 (IBM, Armonk, NY, USA). The normality of the dataset was evaluated with the Shapiro–Wilk test, and variance homogeneity was verified using Levene’s test to ensure that parametric analyses were appropriate. One-way analysis of variance (ANOVA) was performed to assess differences among dietary treatments. When significant differences were observed (p < 0.05), Duncan’s multiple range test was used for post hoc pairwise comparisons. Results are expressed as mean ± standard error of the mean (SEM, n = 3).

3. Results

3.1. Effects of Dietary Vitamin C on Growth Performance and Feed Conversion Ratio of A. japonicus

Survival rates of A. japonicus remained at 100% across all VC dietary treatments, with no significant differences observed (p > 0.05). However, dietary VC significantly affected both WGR and SGR (p < 0.05). The 5000 mg/kg VC-supplemented group exhibited the highest growth performance, while the control and 15,000 mg/kg groups showed similar outcomes (p > 0.05). VC supplementation also influenced FCR. FCR in the 5000 mg/kg group demonstrated significantly better feed efficiency than that in the control group (p < 0.05), whereas the 15,000 mg/kg group showed no significant improvement relative to the control (p > 0.05) (

Table 3. Growth response of A. japonicus to dietary vitamin C supplementation.

VC Levels (mg/kg)	0 mg/kg	5000 mg/kg	15,000 mg/kg
Survival Rate (%)	100	100	100
Weight Growth Rate (%)	80.14 ± 4.11 b	114.28 ± 3.12 a	75.33 ± 2.35 b
Specific Growth Rate (%/d)	0.96 ± 0.03 b	1.23 ± 0.02 a	0.92 ± 0.03 b
Feed conversion ratio	2.16 ± 0.14 b	1.81 ± 0.09 a	2.1 ± 0.09 b

Note: Data are expressed as the mean ± standard error of the mean (SEM), with n = 3 for each treatment group. Statistically significant differences among experimental treatments are denoted by different lowercase superscript letters within the same row (p < 0.05). This style of annotation is consistently applied throughout the manuscript to highlight significant differences across the experimental groups.

).

3.2. Effects of Dietary Vitamin C on Tentacle Feeding Frequency in A. japonicus

Dietary VC supplementation exerted a marked regulatory effect on the tentacle feeding frequency of A. japonicus, with distinct differences observed among VC-supplemented groups relative to the control (Figure 1). The 5000 mg/kg VC-supplemented group displayed a notably elevated tentacle feeding frequency relative to both the control and 15,000 mg/kg groups (p < 0.05), whereas no appreciable difference was detected in this behavioral trait between the control and 15,000 mg/kg groups (p > 0.05). These results indicate that moderate VC supplementation can substantially enhance tentacle feeding capacity of A. japonicus, while higher doses do not produce a comparable stimulatory effect.

3.3. Feeding Latency

Dietary VC levels significantly affected the feeding latency of A. japonicus (Figure 2). The 5000 mg/kg VC-supplemented group showed a significantly shorter feeding latency compared to both the control (0 mg/kg) and the 15,000 mg/kg VC group (p < 0.05), indicating a quicker response to feed stimulation. However, no significant difference in feeding latency was observed between the 15,000 mg/kg VC group and the control (p > 0.05). These findings suggest that moderate VC supplementation (5000 mg/kg) most effectively enhances feeding motivation and responsiveness, while higher VC levels (15,000 mg/kg) offer no additional benefits for feeding latency.

3.4. Effect of Dietary Vitamin C on Mucus Secretion in A. japonicus

Dietary VC supplementation significantly affected mucus secretion in the tentacles of A. japonicus (Figure 3). The 5000 mg/kg VC-supplemented group showed significantly higher mucus secretion compared to the control (0 mg/kg) and the 15,000 mg/kg VC group (p < 0.05). However, no significant difference in mucus output was observed between the latter two groups (p > 0.05), suggesting that moderate VC supplementation (5000 mg/kg) effectively promotes mucus secretion, while the highest VC dose (15,000 mg/kg) does not result in further increases.

3.5. Effect of Vitamin C on Intestinal Digestive Enzymes and Villus Morphology in A. japonicus

3.5.1. Effects of Dietary Vitamin C on Intestinal Digestive Enzyme Activities in A. japonicus

Dietary VC supplementation significantly influenced the activities of intestinal digestive enzymes in A. japonicus. The 5000 mg/kg VC group exhibited significantly higher activities of digestive enzymes, including amylase, pepsin, and lipase, compared to the control group (p < 0.05). However, no significant differences in digestive enzyme activities were observed between the 5000 mg/kg and 15,000 mg/kg VC groups (p > 0.05) (Figure 4A–C).

3.5.2. Effects of Dietary Vitamin C on Intestinal Morphology of A. japonicus

Dietary VC supplementation significantly influenced the intestinal morphology of A. japonicus (Figure 5). The 5000 mg/kg VC-supplemented group exhibited significantly greater villus height compared to the control group (0 mg/kg) (p < 0.05). No significant difference in villus height was observed between the 5000 mg/kg and 15,000 mg/kg groups, indicating that higher VC doses do not further increase this parameter (p > 0.05). Similarly, the 5000 mg/kg group showed significantly increased villus width compared to both the control and 15,000 mg/kg groups (p < 0.05). Moreover, intestinal muscle layer thickness was significantly greater in the 5000 mg/kg group than in the control group (p < 0.05), with no significant difference between the 5000 mg/kg and 15,000 mg/kg groups (p > 0.05).

3.6. Effects of Dietary Vitamin C on Intestinal 5-HT Content in A. japonicus

Dietary VC supplementation significantly affected intestinal (5-HT) levels in A. japonicus (Figure 6). The 5000 mg/kg VC-supplemented group showed a significant increase in 5-HT content compared to both the control (0 mg/kg) and 15,000 mg/kg VC groups (p < 0.05). However, no significant difference in 5-HT levels was observed between the control and the 15,000 mg/kg VC groups, indicating that the highest VC dose did not provide additional benefits over the control (p > 0.05).

3.7. Effects of Dietary Vitamin C on the Expression of AjTPH and Aj5-HT4R Genes in A. japonicus

Dietary VC supplementation significantly influenced the intestinal expression of 5-HT-related genes in A. japonicus (Figure 7). Specifically, AjTPH expression was significantly higher in the 5000 mg/kg VC group compared to the control group (0 mg/kg), indicating that this level of supplementation promotes 5-HT synthesis (p < 0.05). However, no significant difference in AjTPH expression was observed between the 5000 mg/kg and 15,000 mg/kg VC groups (p > 0.05), suggesting that the higher dose does not further upregulate AjTPH expression. Similarly, the 5000 mg/kg group showed significantly higher Aj5-HT4R expression compared to both the VC-free control and the 15,000 mg/kg VC group (p < 0.05), with no significant difference between the latter two groups (p > 0.05). These results suggest that 5000 mg/kg VC supplementation enhances the expression of both the 5-HT synthesis enzyme (AjTPH) and the receptor (Aj5-HT4R), while higher VC doses (15,000 mg/kg) do not further increase gene expression. This indicates a dose-dependent effect of VC on the regulation of 5-HT-related genes in A. japonicus.

4. Discussion

The results of this study showed that dietary VC supplementation markedly enhanced the growth performance of A. japonicus. Individuals fed 5000 mg/kg VC displayed increased WGR and SGR, along with a lower FCR. As VC is a water-soluble vitamin, its high leaching rate in seawater resulted in an actual ingested dose lower than 5000 mg/kg. However, existing studies have demonstrated that dissolved VC in water still confers beneficial effects on aquatic animals; for instance, waterborne VC supplementation can enhance the antioxidant capacity of zebrafish (Danio rerio) and improve the pesticide toxicity resistance of (crucian carp) [30,31]. In the present study, such dissolved VC compensated for the dietary VC loss caused by leaching. More importantly, leached VC exerts effects analogous to dietary VC not only via cutaneous absorption but also through the digestive tract. As a benthic species, A. japonicus continuously takes in ambient seawater through its anus, and dissolved VC can enter the digestive system along with the inhaled water, thereby participating in key physiological processes including neurotransmitter synthesis. These results are consistent with earlier studies conducted in fish, crustaceans, and other aquaculture species, in which VC supplementation was shown to enhance growth by promoting tissue development, improving nutrient utilization, and reducing metabolic losses [32,33,34]. For example, juvenile walleye pollock (Gadus chalcogrammus) exhibited optimal growth performance and feed conversion efficiency when fed a diet supplemented with 145.88 mg/kg VC [10], whereas 3000 mg/kg VC markedly improved gonadal texture and collagen content in sea urchins (Mesocentrotus nudus) [11]. As benthic foragers, A. japonicus lack the locomotor agility of vertebrates and can only feed on the dissolved diet via oral tentacles, which results in substantial loss of water-soluble VC during the dissolution process [6]. Despite our multiple efforts (e.g., increasing feeding frequency to shorten the exposure time of diet per feeding), this issue could not be completely eliminated. The high VC requirement observed in the present study may be attributed to partial VC leaching, thus necessitating a relatively high dietary VC supplementation level. Additionally, the unique dietary composition of sea cucumbers, such as the large proportion of sea mud in the diet, may contain certain harmful substances that impair immunity and elevate disease susceptibility. Moreover, sea cucumbers are prone to evisceration in response to environmental stimuli, and all these factors require a high dose of VC to enhance the immunity and anti-stress capacity of A. japonicus [32]. In addition, previous studies have demonstrated that VC plays an essential role in collagen synthesis, helping maintain the structural stability of A. japonicus tissues. Therefore, appropriate VC supplementation supports connective tissue integrity and contributes indirectly to growth [35,36]. Furthermore, the antioxidant and immune-enhancing effects of VC also provide additional benefits to overall growth performance [37,38]. The body wall of A. japonicus mainly consists of collagen, and prior studies have shown that collagen content correlates positively with the growth of A. japonicus. However, excessive VC has been shown to inhibit the synthesis of hydroxyproline (Hyp), a key amino acid in collagen, in human skin fibroblasts [39]. Therefore, excessively high dietary VC supplementation may inhibit collagen synthesis and fail to support optimal growth of A. japonicus.

The current study demonstrates that dietary VC plays a pivotal role in promoting the feeding latency of A. japonicus. First, 5000 mg/kg VC effectively shortened the feeding latency time, enabling individuals to enter an active feeding state more rapidly after feed delivery. This phenomenon is associated with the regulatory function of VC in neurotransmitter biosynthesis, particularly through its involvement in 5-HT [40], which is known to enhance feeding drive [41,42]. Furthermore, 5000 mg/kg VC significantly increased the feeding frequency of oral tentacles, indicating that VC directly enhances tentacle sensory capacity and motor function, thereby improving feeding efficiency. Beyond behavioral responses, VC strongly influenced digestive physiology associated with feeding. Results indicated that dietary VC supplementation increased the activities of key digestive enzymes, including lipase, amylase, and protease, in the oral tentacles, enhancing feed utilization through strengthened local digestive capacity. This effect is attributed to the antioxidant properties of VC, which maintain cellular homeostasis and protect digestive enzymes from oxidative inactivation [15,43,44]. Moreover, histological observations showed that VC supplementation led to a significant increase in the number of mucus cells in the oral tentacles. Since mucus cells are key sites for digestive enzyme secretion and the initial processing of feed particles [45], their proliferation provides a stronger cellular basis for enhanced feeding and digestion.

This study demonstrated that dietary VC significantly influences the intestinal structure and digestive physiology of A. japonicus. Morphologically, supplementation with appropriate levels of VC markedly increased both intestinal length and intestinal weight. This finding suggests that VC promotes intestinal tissue growth and development, thereby expanding the potential surface area available for digestion and absorption [46,47]. Comparable results have also been observed in fish and crustaceans, where VC enhances collagen synthesis and maintains the integrity of the mucosa and extracellular matrix, ultimately improving tissue strength and extensibility [11,48]. In A. japonicus, elongation and increased mass of the intestine significantly enhance the residence time of ingested food, thereby improving digestive efficiency, which directly accounts for the observed increases in feeding performance and nutrient utilization.

In terms of digestive physiology, our findings demonstrated that VC supplementation significantly enhanced the activity of key digestive enzymes, such as amylase and protease. This finding demonstrates that VC effectively supports the functional performance of the digestive enzyme system. Beyond its well-documented antioxidative role, VC has been found to relieve oxidative stress in the intestinal milieu and protect digestive enzymes from oxidative injury, according to earlier research [43,49]. In addition, VC stimulates epithelial cell metabolism and enhances glandular secretion in the intestine, contributing directly to the upregulation of digestive enzyme production. Collectively, these effects strengthen the digestive efficiency and energy conversion capacity of A. japonicus.

The shortened feeding latency and increased tentacle feeding frequency observed in the present study are likely mediated by elevated 5-HT levels, suggesting that VC indirectly enhances feeding drive and digestive performance by stimulating 5-HT pathways. This mechanism parallels findings in vertebrates, where VC-mediated regulation of 5-HT synthesis is closely linked to appetite and gastrointestinal physiology, but has not previously been documented in echinoderms. Moreover, 5-HT has been implicated in modulating intestinal morphology and enzyme 5-HT, thereby improving nutrient absorption efficiency [42]. When integrated with the enhanced digestive enzyme activity and improved villus architecture observed in the VC-supplemented group, the present results support the conclusion that VC enhances both behavioral and physiological aspects of feeding performance via 5-HT-mediated pathways. Previous research on VC in A. japonicus has largely focused on its antioxidant and immunomodulatory functions [33,50], whereas its involvement in neurotransmitter-based feeding regulation has received little attention. Therefore, this study provides the first evidence for a VC–5-HT regulatory axis in A. japonicus, offering a new perspective on nutrient-driven control of feeding behavior in invertebrates.

Although intermediate VC gradients such as 2500 or 10,000 mg/kg were not set in this study, existing studies have indicated that VC generally exhibits a dose-dependent pattern accompanied by a plateau effect in aquatic animals. From a practical aquaculture perspective, increasing VC supplementation beyond the effective dose will directly elevate feed costs with limited physiological gains, while reducing VC levels below 5000 mg/kg may attenuate the promoting effects observed in the present study.

Taken together, these findings demonstrate that VC improves the feeding performance of A. japonicus through two coordinated mechanisms: (1) by enhancing serotonergic regulation—elevating intestinal 5-HT concentrations and upregulating the expression of AjTPH and Aj5-HT4R, which accelerates feeding latency and strengthens tentacle feeding activity; and (2) by improving digestive capacity—optimizing intestinal morphology, increasing digestive enzyme activities, and reinforcing the structural basis for nutrient absorption. This integrated regulatory effect establishes VC as a key nutritional factor governing both neurochemical and intestinal physiological pathways.

5. Conclusions

In conclusion, dietary VC supplementation significantly enhances the growth performance, feeding behavior, and digestive physiological functions of A. japonicus. Optimal VC levels (5000 mg/kg) shorten feeding latency, increase tentacle feeding frequency, and promote the secretion of digestive enzymes (amylase, lipase, pepsin) and mucus in the oral tentacles. VC also improves intestinal morphology, including villus height and width, as well as muscle layer thickness, thereby facilitating nutrient absorption. On a molecular scale, VC supplementation enhances the expression of genes involved in 5-HT synthesis (AjTPH) and its receptors (Aj5-HT4R), resulting in elevated 5-HT concentrations in the gut. These results demonstrate that VC promotes feeding and digestive efficiency in A. japonicus through direct effects on intestinal structure and serotonergic signaling, providing a robust basis for optimizing dietary strategies in A. japonicus aquaculture.