Abstract:Fish phenomics is the science that systematically studies fish phenotypes at the genomic level. Through the integrated analysis of fish phenotypic characteristics, genomes, and environmental conditions, fish phenomics can decipher the response and adaptation mechanisms of fish to the environment, which could provide valuable information and indicators for fish farming. Combining fish phenomics and genomic analyses has significant potential for increasing the efficiency of fish breeding. Nevertheless, compared with the rapid development of genomics research, the development of fish phenomics is relatively slow. Owing to the aquatic environment, high breeding density, and swimming ability of fish, it is difficult to determine their phenotypes accurately. In addition, with the expansion of the research population, high-throughput and accurate acquisition and analysis of fish phenotypes on a large scale have become more challenging, becoming one of the major bottlenecks hindering fish phenomics research. Recent advances in novel technologies, including sensor technology and artificial intelligence, have provided potential solutions to resolve this bottleneck and improve the efficiency of fish phenotyping by using high-throughput, noninvasive, and accurate methods. This study provides an overview of the history of phenomic research. We then propose strategies, introduce high-throughput phenotyping approaches, and discuss the status quo of fish phenomics research. Finally, we discuss challenges and provide perspectives on fish phenomics research. This review considers fish as the main research object, covers the current mainstream methods and progress in fish phenomics research, and aims to enrich the understanding of fish phenomics research. This review provides aquaculture researchers and fish farmers with updated information and valuable references for subsequent research and practices in fish phenomics.

Abstract:Grouper aquaculture constitutes an important industry within marine aquaculture in China. In recent years, inter-specific hybridization has emerged as a prevalent method for grouper breeding, with novel hybrid species, such as the Hulong and Yunlong groupers accounting for more than 70% of domestic grouper production. Although inter-specific hybrid selection has greatly advanced the development of the grouper industry, it has concurrently brought about challenges in species identification and germplasm preservation. In addition, introducing hybrid fish can contaminate the gene pool of species. Hybrid fish possess morphological characteristics of both parents, complicating morphological identification. Therefore, an urgent need exists for precise identification and authentication of hybrid groupers and their parents to ensure the healthy and sustainable development of the grouper industry. This study compared the morphological differences between F1 hybrids and their parents and used the mitochondrial gene CO Ⅰ and nuclear gene RYR3 as dual molecular markers to trace the parents of five common hybrid species: Yunlong, Hulong, Jinhu grouper, Shanhu and Longshu groupers. The results showed the following: (1) The body shape of the hybrid resembled that of the maternal parent, with species, such as Yunlong, Hulong, Jinhu and Shanhu groupers, exhibiting an oval-shaped form, Longshu grouper had a pointed head and humpback. The color and pattern of hybrid groupers combined the characteristics of both parents, among which the Jinhu and Shanhu groupers exhibited high similarity with the color and pattern of their parents, complicating the differentiation between the hybrid offspring from parents based solely on color; conversely, the Yunlong, Hulong, and Longshu groupers exhibited new color patterns through the blending of parental colors. (2) The genetic distance between the five hybrid species and their maternal parents based on the CO Ⅰ gene was 0.000–0.002, all less than 0.020; in their phylogenetic tree, hybrid species were clustered with their maternal parents. The CO Ⅰ gene could accurately identify the maternal parent of hybrid species. (3) Given that nuclear genes transmitted genetic information from both parents, calculating the genetic distance between parental RYR3 genes of hybrids (range: 0.006–0.023) and constructing a phylogenetic tree could effectively distinguish each purebred grouper, indicating that RYR3 was suitable as a molecular marker to assist the CO Ⅰ gene in identifying the parents of hybrid groupers. (4) Based on these results, a method for identifying the parents of hybrid groupers using CO Ⅰ and RYR3 dual molecular markers was established. Initially, the determination of a sample as a hybrid was based on the heterozygosity of its RYR3 gene sequence; subsequent sequence comparison confirmed its parental information; if classified as a hybrid, the parents were identified using CO Ⅰ results. (5) This method was utilized to identify the parents of 16 hybrid grouper samples, and the identification outcomes were consistent with their recorded parental information, demonstrating that the hybrid parent identification method was feasible and effective. These findings indicate that COⅠ and RYR3 dual molecular marker technology can accurately identify the parents of hybrid groupers, providing important insights for the conservation of stingray germplasm resources and genetic breeding.

Abstract:Turbot (Scophthalmus maximus) belongs to the Scophthalmidae family and is one of the most economically valuable flatfish aquaculture species worldwide. It is widely distributed in the Mediterranean, Black, and Baltic Seas. China has made significant progress in introducing turbot as an aquaculture species over the past 30 years, but key challenges remain in advancing its industrial aquaculture. Because turbot are cold-water fish with strict environmental temperature requirements, they are particularly susceptible to temperature stress. In the turbot aquaculture area in North China, the natural seawater temperature exceeds 26 ℃ throughout the summer (May to September), rendering it unsuitable for turbot aquaculture during this period. Genetically improving the heat tolerance of turbot to overcome this limitation is critical to promoting the sustainable and stable development of the turbot industry. In this study, we estimated the genetic parameters of heat resistance and turbot growth traits. Thirty full-sib families were constructed by male-female pairing with equal weights of approximately 25 g, and heat resistance experiments were carried out. Thirty turbot were selected from each of the 30 families, total 900 individuals, for the large-scale high-temperature stress experiment evaluating the genetic parameters of high-temperature tolerance traits in turbot. Four models [linear animal model (LAM), cross-sectional linear animal model (CLAM), cross-sectional threshold animal model–variant 1 (CTAM1), and cross-sectional threshold animal model with probit link function (CTAMp)] were used to fit two high-temperature tolerance traits (upper limit trait of heat tolerance, UTT, and binary death survival trait, BTS). The variance components were estimated by the restricted maximum likelihood method. The heritability of the high-temperature tolerance traits in turbot was 0.110–0.208, which was a medium–low heritability trait. Among them, the heritability estimated by linear models (LAM and CLAM) was 0.110±0.074 and 0.155±0.082, respectively, and the heritability estimated by threshold models (CTAMl and CTAMp) was 0.214±0.072 and 0.208±0.074, respectively. This indicates that turbot high-temperature tolerance can be improved through genetic selection. The genetic correlations of the two heat-resistant phenotypic traits with body weight were –0.07±0.40 and –0.13±0.33, respectively, and the phenotypic correlations were –0.04±0.05 and –0.08±0.11, respectively, both of which were extremely low correlations. The correlation analysis of the estimated breeding values (EBVs) by different models showed that when different models fitted the same heat-resistant phenotype, the correlation coefficient between EBVs was ＞0.97. That is a high-intensity positive correlation, indicating that when the same phenotypic definition was used, the linear or threshold model had little effect on the ranking of EBVs. The correlation analysis of EBVs estimated by different models and phenotypes revealed key differences. The correlation coefficient between EBVs estimated using threshold models (CTAMl and CTAMp) and phenotypic BTS was higher than that of the linear models (LAM and CLAM). This suggests that phenotypic BTS is a more suitable heat-resistant trait than phenotypic UTT. In addition, the correlation coefficient between EBVs estimated by UTT and BTS in the linear model was ＜0.50. This indicates that the EBV rankings based on these two phenotypic definitions for heat tolerance in turbot were inconsistent. Therefore, using phenotypic BTS and cross-sectional threshold animal models (CLAMl or CTAMp) is more advantageous for estimating the genetic parameters of heat tolerance in turbot. The results of this study supplement the research on genetic parameters of heat tolerance in turbot and provide a theoretical basis for the formulation of breeding plans for heat tolerance traits in cold-water fish.

Abstract:Circadian rhythms are a prevalent physiological phenomenon in organisms, referring to the adaptation and regulation of the internal clock to a 24-h cycle. This biological clock governs various physiological processes, such as the sleep-wake cycle, hormone secretion, and metabolic rate, enabling organisms to synchronize with external environmental changes between day and night. Although circadian rhythms have been thoroughly researched in terrestrial animals, their importance in aquatic animals has gradually gained attention in recent years. Fish and other aquatic animals rely on circadian rhythms to regulate their daily physiological and behavioral activities, such as feeding, swimming, and reproduction. The stability of circadian rhythms is essential for fish health. If the rhythm is imbalanced, the physiological activities of fish are disrupted, potentially leading to decreased digestive and absorptive capacity, weakened immune responses, and increased infection risk. Therefore, maintaining stable circadian rhythms indicates healthy aquaculture practices that promote overall fish health and aquaculture efficiency. The stable operation of circadian rhythms is influenced by various environmental factors, including light, temperature, salinity, dissolved oxygen, and density. These factors not only influence the formation of circadian rhythms but can also alter the metabolism and behavior of fish by disrupting physiological homeostasis. Despite environmental stress causing temporary imbalances in circadian rhythms, fish generally possess the capacity to readjust their physiological rhythms through adaptive changes and establish a new steady state suited to the new environment, thereby allowing their physiological rhythms to resynchronize with the external cycle and potentially approximate the rhythmic state of standard aquaculture environments. This phenomenon can be considered an expression of adaptability in fish. Therefore, stress resistance can be effectively assessed by monitoring changes in the stability of circadian rhythms in groups or families, thereby establishing a scientific basis for the genetic selection of stress-resistant traits in aquaculture. Circadian rhythms encompass various aspects, such as digestive and metabolic, immune, and endocrine rhythms. Among these, digestive and metabolic rhythms, fundamental to life activities, are particularly important for fish and other aquatic animals because they directly affect the efficiency of energy utilization by the body and subsequently influence the growth rate and health status. Digestive and metabolic rhythms are primarily monitored by detecting a series of key physiological and biochemical indicators, including digestive enzyme activity, glucose levels, cortisol concentrations, and triglyceride and cholesterol contents. These indicators can reflect the digestive function and stress status of fish at various time points. Despite applying these biochemical indicators in the health assessment of farmed fish, research remains limited regarding the impact of various aquaculture environments on the circadian rhythms of digestion and metabolism. Furthermore, there is even less investigation into how these rhythms are altered by different environmental pressures and their correlation with the stress resistance of fish, which requires further study. In this study, we aimed to investigate the circadian rhythm of digestive metabolism in turbot (Scophthalmus maximus) juveniles under various culture conditions. The experiment had three experimental groups, each representing distinct aquaculture environments: high-temperature group (23 °C, salinity 30), control group (16 °C, salinity 30), and low-salinity group (16 °C, salinity 10). The water temperature was controlled using a chiller, and salinity was regulated by adjusting seawater and freshwater flow rates. The light cycle was regulated by an automatic timer, with a light period occurring from 6:00 to 20:00 and a dark period occurring from 20:00 to 6:00 the next day. Feeding was conducted daily at 6:00 and 18:00, with each feeding session amounting to 1% of the body weight of the fish. Residual feed and feces were expeditiously cleaned to maintain water quality. The experiment lasted 30 days. After the experiment was concluded, sampling was performed every 4 h for 72 h (at 08:00, 12:00, 16:00, 20:00, 24:00, and 04:00) to analyze the weight gain of juvenile turbot in different aquaculture environments and changes in key digestive enzymes (trypsin, lipase, and amylase) and serum metabolites (cortisol, glucose, cholesterol, and triglycerides). The results showed that the average weight of the high-temperature group was significantly lower than that of the control and low-salinity groups (P ＜ 0.05), whereas no significant difference was observed between the low-salinity and control groups. The rhythmicity of trypsin, lipase, and amylase did not reach significant levels in any of the groups. However, the digestive enzyme activity in the control and low-salinity groups demonstrated regular fluctuations during the first 48 h, whereas the high-temperature group lacked clear regularity, resulting in earlier peaks and more drastic variations. The cortisol analysis indicated that the 72-h and 48-h rhythms in the control group were significant (P ＜ 0.05), whereas the low-salinity and high-temperature groups did not show significant rhythmicity. Cortisol levels in the high-temperature group were significantly higher than those in the other two groups at multiple measurement points (P ＜ 0.05), whereas no significant difference was detected in cortisol levels between the low-salinity and control groups. Glucose concentrations did not show significant rhythmicity in any group. However, the control and low-salinity groups demonstrated marked periodic changes in glucose levels during the first 48 h, whereas the high-temperature group showed irregular fluctuations. The analysis of triglyceride and cholesterol levels revealed significant rhythmicity in both the control and low-salinity groups over 48 h (P ＜ 0.05), whereas the high-temperature group did not show significant rhythmicity. The triglyceride levels in the low-salinity group were slightly lower than those in the control group at most measurement points, whereas the triglyceride and cholesterol levels in the high-temperature group were significantly higher than those in the control group (P ＜ 0.05). Our findings indicate that high temperature has a greater impact on the metabolism and digestive functions of juvenile turbot than low salinity, manifesting as significant stress responses and abnormal digestive enzyme activities. Serum cortisol and triglyceride levels can accurately reflect the health status and stress response of turbot under different environmental stress conditions and may serve as indicators for evaluating resilience breeding. This study elucidates the dynamic changes in the digestive metabolic rhythms of turbot across different aquaculture environments, providing novel insights into the metabolic regulation mechanisms affected by environmental stress. The results not only enhance the health management of turbot but also provide potential indicators and practical references for resilience breeding.

Abstract:Low-temperature acclimation technology is necessary for transporting live fish because it induces a state of dormancy, reducing injuries and mortality during transportation. To explore low-temperature acclimation technology for Lateolabrax maculatus, this study investigated the effects of gradient cooling on the physiological and biochemical properties, muscle texture characteristics, and structural changes in the liver and gill tissues of L. maculatus. Gradient cooling allows for gradual adjustment of water temperature, preventing stress in fish caused by sudden temperature changes and minimizing damage while preserving live circulation. The fish were cooled at a rate of 1–2 ℃/h, and their behavioral characteristics at different temperature ranges were observed to determine their dormancy and critical temperatures. The fish were cooled using a gradient cooling method (when T > 20 ℃, the cooling rate was 3 ℃/h; for 10 ℃ < T < 20 ℃, the cooling rate was 2 ℃/h (for T < 10 ℃, the cooling rate was 1 ℃/h). Samples were taken after holding the fish at 24, 20, 16, 12, and 8 ℃ for two hours each. Serum biochemical, antioxidant, lipid metabolism, and cardiac enzyme indicators were measured, and muscle texture characteristics were assessed. Additionally, the microscopic structural changes in the liver and gill tissues of the fish were observed using an optical microscope. The respiratory rate of L. maculatus gradually decreased as the temperature dropped, with 16 ℃ being the dormancy temperature and 8 ℃ the critical temperature for L. maculatus. During the gradient cooling process (24–8 ℃), the malondialdehyde, lactate dehydrogenase, and creatine kinase contents significantly decreased at 16 ℃, whereas the catalase content significantly increased. The aspartate transaminase and alanine transaminase contents significantly increased at 8 ℃, and the triglyceride, total cholesterol, and high- and low-density lipoprotein cholesterol levels remained relatively stable during the later stages of cooling (16–8 ℃). After gradient cooling, the taste and quality of L. maculatus muscle improved; the degree of lipid peroxidation and cellular membrane damage was reduced, metabolic activity slowed down, and antioxidant capacity was enhanced at 16 ℃. After low temperature (8℃) stress, round vacuoles appeared in the cytoplasm of L. maculatus hepatocytes, which were irregularly arranged, and some nuclei were deviated or lysed. The gill filaments on the same side were neatly arranged, lengthened, and spaced, and a few epithelial cells of gill filaments were detached from the gill vesicles, and the number of chlorine-secreting cells was increased. Low-temperature stress causes changes in the structure of the liver and gill tissues of L. maculatus. This study serves as a reference for researching and applying gradient cooling technology in long-distance fish transportation and stress prevention.

Abstract:Population aging has become a universal phenomenon with the rapid development of the global economy and medical technology. China, which has the largest number of older adult people, has one of the highest aging rates in the world. Despite the large elderly population, the market lacks food designed to meet their safety and nutritional needs. Therefore, food development for older adults has become a popular research topic. In 2018, the National Health Commission of China released the “National Food Safety Standard–General Rules for Elderly Food” (draft for soliciting opinions), aiming to regulate food production and quality standards for older adults. It classifies food for older adults into easily consumable, nutritional-formula, and nutritional-supplement foods and sets specific nutritional requirements for them. Experts have proposed principles for developing food for older adults, including providing sufficient high-quality protein, dietary fiber, vitamins, and trace elements, while emphasizing low-fat, low-sugar, low-calorie, and low-cholesterol formula designs. Additionally, texture, sensory properties, and packaging suitability during food processing are considered to meet the physiological and psychological needs of older adults. Compared with the international market, the food market for older individuals in China is still in its infancy and sells mainly health products. The variety of foods suitable for older adults is relatively limited and includes milk powder, pastries, and biscuits. Although these foods are easy to chew and swallow, they often lack sufficient nutritional value and do not fully meet the nutritional needs of the elderly population. Research has shown that vitamin and folic acid content in domestic pre-packaged food for older individuals is insufficient, and the carbohydrate and energy content is often high, increasing the risk of obesity and cardiovascular and cerebrovascular diseases in older individuals with a low metabolic rate. Leveraging the high economic and nutritional value, as well as the delicious taste of seabass (Lateolabrax japonicus), its meat serves as an excellent source of high-quality protein with a low fat content, soft texture, and easy chewability, making it an ideal ingredient in food designed for older adults. Based on the dietary needs of older adults, this study developed an easily chewable and digestible seabass paste by adding carrot powder, celery powder, and inulin. Single-factor experiments and response surface methodology were used to determine the optimal ratios of the three nutrient powders, using hardness and sensory scores as key evaluation criteria. Textural characteristics and nutritional indicators of the products were also analyzed. The study also assessed the effect of different packaging methods (regular packaging and vacuum packaging) and refrigeration temperatures (4 ℃ and –2 ℃) on the quality of the sea bass product. Quality indicators included textural characteristics, sensory aspects, volatile basic nitrogen content (TVB-N), thiobarbituric acid value (TBA), and total colony count. The optimal amounts of nutrients for sea bass paste were 1.15% carrot powder, 1.10% celery powder, and 1.00% inulin. The paste contained 15.24 g protein and 5.73 g fat per 100 g. The amino acid profile aligned with the ideal protein pattern recommended by the Food and Agriculture Organization/World Health Organization. Moreover, the sea bass paste contained 18 types of fatty acids, with the relative unsaturated fatty acid content reaching 72.1% and the total EPA and DHA content accounting for 11.12%. The sea bass paste contained the highest amount of vitamin E (3.54 mg/100 g), followed by niacin (3.48 mg/100 g). It exhibits moderate texture, good water retention, and is easily chewable, making it suitable for consumption by older adults. During the first 20 days of storage, the hardness of sea bass paste gradually increased, but there was no significant difference between the two refrigeration temperature groups (4 and –2 ℃). However, the hardness of the vacuum-packed sea bass products continued to increase for up to 30 days of storage, but it decreased sharply in the regularly packaged product. Storage experiments demonstrated that vacuum packaging could effectively delay the increase in the TVB-N, TBA value, and total colony count, maintaining the quality of the sea bass paste. The TVB-N of the regularly packaged product was close to the 30 mg/100 g limit after 30 days, whereas vacuum-packed sea bass paste stored at –2 °C was still below 13 mg/100 g (within the range of Class Ⅰ products). Fat oxidation was higher in the regularly packaged fish paste than in the vacuum-packed group. This indicates that vacuum packing was effective in slowing down the fat-oxidation process. Vacuum-packed sea bass paste can be stored for 7 days at 4 ℃ and 15 days at –2 ℃, showing a significantly superior preservation effect compared to regular packaging. In conclusion, this study not only introduces a nutritious and healthy food option for older adults but also presents novel insights into the processing and high-value utilization of sea bass, offering technical support and a reference for the diversification and development of marine fish products.

Abstract:Hybrid groupers have emerged as a significant aquaculture species owing to their high market value and growing demand. However, carbohydrate-rich diets pose a challenge for efficient glucose metabolism in this species. This phenomenon stems from the limited capacity of fish to effectively metabolize glucose in response to high-carbohydrate diets, resulting in persistent hyperglycemia that adversely affects growth performance. Impaired insulin sensitivity and dysfunction in insulin signaling, characterized by compromised insulin receptor function, blocked insulin cascades, and failure to activate critical signaling pathways, such as the PI3K/AKT pathway, have been identified as key factors contributing to this metabolic disorder. Therefore, this study investigated the application of metformin, a biguanide with recognized hypoglycemic properties, to ameliorate the adverse effects of high-carbohydrate diets in hybrid groupers (Epinephelus lanceolatus × E. fuscoguttatus). A total of 540 pearl gentian grouper, averaging (10.44±0.01) g, were selected and randomly distributed into six treatment groups, each consisting of three replicates with 30 fish per replicate. Six iso-nitrogenous and iso-lipid diets were formulated: A positive control (20% carbohydrate, PC), one negative control (30% carbohydrate, T0), and four experimental groups supplemented with metformin at concentrations of 0.2% (T2), 0.4% (T4), 0.6% (T6), and 0.8% (T8) relative to the negative control. The results show that the carbohydrate and metformin levels in the feed had no statistically significant effect (P＞0.05) on the feed coefficient ratio (FCR) and survival rate (SR). Notably, a high-carbohydrate diet reduced growth performance (WGR, SGR) in hybrid groupers and altered serum biochemical parameters, increasing glucose, triglycerides, and LDL. Meanwhile, decreasing insulin and HDL. Metformin supplementation mitigated these effects, improving lipid metabolism markers and partially restoring HDL levels, with optimal outcomes at higher doses. Growth and organ indices demonstrated linear or quadratic trends in response to metformin, though condition factor (CF) and alpha diversity of gut microbiota were unaffected. Metformin also modulated gut microbial composition, decreasing Proteobacteria and Photobacterium while increasing Firmicutes and Brevibacillus. In the present study, a high-carbohydrate diet inhibited growth performance, decreased insulin sensitivity, and increased the abundance of pathogenic intestinal bacteria in hybrid groupers. In the present study, the administration of a high-sugar diet augmented with 0.2% metformin altered the composition of intestinal microorganisms, thereby enhancing the disruption of glucose metabolism in the intestinal flora of groupers. This disruption is induced by a high-carbohydrate diet that promotes glycolysis and inhibits gluconeogenesis, thereby regulating glucose homeostasis. This, in turn, facilitates an improvement in the utilization rate of carbohydrates by the pearl gentian grouper.

Abstract:With the continuous expansion of the aquaculture industry and the advancement of technology, the production model of the industry has gradually shifted toward greater modernization, mechanization, and automation. This transformation has become the primary trend in industry development, signaling the movement from traditional farming methods to intelligent and automated approaches. Fish fry, as a crucial link in the aquaculture supply chain, play an important role in the entire industry. Accurate fish fry counting is essential for managing the industry effectively, conducting scientific feeding practices, controlling stocking density, and ensuring fair pricing and transparent transactions in the sale of fry. In traditional aquaculture practices, fish fry counting mainly relies on manual methods, which are not only time consuming and labor intensive, but also prone to significant errors. Many farms still use the "pushing method" and "bowl method" for fry counting. The pushing method involves estimating the number of fry in a pile manually, whereas the bowl method estimates this number based on a sample. Both methods are subject to human error and often lead to inaccurate counts. Moreover, these manual methods are not only inefficient but can also harm the fry. During counting, the fry are often handled repeatedly, which can negatively impact their growth and survival, causing stress and affecting fry quality. With the advancement of technology and the development of computer systems, automated devices have been gradually introduced into the aquaculture industry. The advent of automatic fish fry counters has effectively addressed the inefficiencies of manual counting while ensuring accurate and transparent counting. These automated devices use sensors, image recognition, and machine learning technologies to automatically detect and track fry, efficiently completing counting with real-time data collection, high accuracy, and reliability. These tools provide aquaculture operators with a more scientific and convenient way of management, allowing for more precise feeding practices and reducing overfeeding or underfeeding, thus improving farm efficiency. However, despite the widespread use of automated counting technology in aquaculture, several challenges remain. In particular, when dealing with large volumes of fry, existing counting technologies face limitations in efficiency, accuracy, and handling fry overlap. As the number of fry increases, counting accuracy tends to decrease, especially when dealing with smaller fry, where detection systems can make errors. Additionally, the complex environment of aquaculture farms, such as light conditions, bubbles, and debris in the water, can interfere with counting accuracy, making the process complicated for automatic counting systems. Therefore, enhancing the accuracy of automated counting, especially in large volumes of fry or in complex environments, is still a technical issue that needs to be addressed. To tackle these issues, researchers have made significant improvements and innovations in automatic counting technology. The accuracy and efficiency of automatic counting systems have been significantly enhanced by incorporating advanced image recognition algorithms, deep learning techniques, and multisensor fusion technologies. These improved algorithms are effective at separating and tracking targets, achieving precise counting. Furthermore, with the development of simulation technology, virtual testing and simulations have played a crucial role in optimizing and designing automatic counting devices. Simulation allows device performance to be predicted under different working conditions, reducing the need for testing with live fry and minimizing potential losses. It also improves design efficiency and ensures that the stability, safety, and durability of the devices are thoroughly validated before practical application, providing reliable technical support for their implementation. Nevertheless, when handling large volumes of fry counting, challenges related to efficiency and dealing with overlapping fry remain. For instance, when designing automatic fry counting devices, design parameters are often difficult to calculate accurately because of the limitations of the fry and the aquaculture environment. Therefore, machine operating parameters must be adjusted to test counting effectiveness. However, this method wastes both human and material resources, and repeated testing can cause stress and harm to the fry. Simulation allows for testing the operational performance of prototype designs, reducing the need for physical testing and saving costs. This study introduces an improved YOLOV8-ByteTrack algorithm to achieve high-precision detection and tracking of fry. This algorithm focuses on real-time performance and efficiency by combining the lightweight YOLOV8 model for object detection with the precise multi-target tracking capabilities of ByteTrack. YOLOV8, being a lightweight model, reduces computational load while maintaining high detection accuracy, offering fast and stable performance in resource-limited environments. Once the fry are detected, ByteTrack uses an efficient data association strategy to track multiple targets and maintain identity consistency, significantly reducing issues such as ID switching and target loss caused by rapid movement, overlap, or environmental changes. Unlike traditional algorithms that rely solely on high-confidence detections, ByteTrack incorporates low-confidence results by utilizing motion consistency and appearance features, thus improving counting accuracy and continuity. To verify the performance and stability of the proposed fish fry automatic counting device in practical applications, a series of accuracy testing experiments was conducted on fish fry of different sizes. The experiments tested 3–5, 6–8, and 9–12 cm-sized grouper fry, with 200, 250, and 300 fry per group, respectively. The test results showed that the counting accuracy of the device was 99.1% and 99.6% for the 6–8 and 9–12 cm fry, respectively, with a slightly lower accuracy of 98.5% for the 3–5 cm fry. The algorithm achieved an average frame rate of 155 FPS, with a single-frame processing time of approximately 6.5 ms. Moreover, the processing speed at different frame rates demonstrated high real-time stability, with a minimum processing speed of 6.3 ms (158 FPS) and a maximum of 6.6 ms (152 FPS). The lower accuracy for the 3–5 cm fry can be attributed to their smaller size, which makes them more susceptible to background complexity and rapid movement, leading to a slight decrease in detection precision. As fry size increases, their features become more distinct, and their movements become more stable, resulting in higher detection accuracy. These results validate the excellent real-time and efficient performance of the algorithm even with limited hardware resources, meeting the practical needs of speed and accuracy in fish fry counting scenarios. The fish fry automatic counting device proposed in this study offers an innovative solution to improve fry counting precision and efficiency, providing valuable insights for theoretical research and practical applications in aquaculture.

Abstract:Odontobutis yaluensis is a small benthic fish endemic to northeastern China. In its natural habitat, the fish breeds within a temperature range of 8 ℃ to 20 ℃, with optimal embryo hatching occurring at 16–20 ℃. The development and utilization of O. yaluensis fishery resources have faced significant challenges due to the vulnerability of their fertilized eggs to external factors, leading to low hatching rates. In previous artificial breeding experiments, we observed that excessively high water temperatures caused substantial mortality before the embryos reached the gastrula stage, contributing to low hatching rates. Elevated temperatures can impair tissue differentiation and organogenesis in a fish’s early embryonic developmental stages, affecting physiological activity and development. When organisms are subjected to environmental stress, excessive production of reactive oxygen species can surpass the capacity of the antioxidant defense system, resulting in oxidative stress. This induces lipid peroxidation and DNA damage. In vertebrates, oxidative stress has been implicated in embryonic damage and is potentially associated with developmental arrest. To further investigate the mechanisms underlying high-temperature stress-induced mass mortality of early embryos in O. yaluensis, we examined the causes of high embryonic mortality by observing abnormal development in early embryos under high-temperature stress and analyzing changes in antioxidant enzyme activities and growth-related gene expression. The experiment was conducted using three water temperatures (15 ℃, 20 ℃, and 25 ℃). Embryos at the 2-cell stage were selected for a 24-hour temperature treatment. After the experiment, the embryonic development was monitored microscopically for abnormalities, survival rates were recorded, and antioxidant-related enzyme activities were measured. Additionally, the mRNA levels of key growth-related genes, including the eukaryotic translation initiation factor 4E binding protein 1 (4e-bp1), MYC proto-oncogene (c-myc), ribosomal protein S6 kinase B1 (s6k1), and insulin-like growth factor 1 receptor (igf-1r), were quantified. We found that O. yaluensis embryos were more prone to abnormal development at 25 ℃ than at the other temperatures. The observed abnormalities included uneven blastomere size, cytoplasmic extrusion into the perivitelline space, blastomere detachment from the yolk, and cytoplasm filling the perivitelline space as the eggs gradually turned opaque. Disruption of membrane permeability led to water absorption and egg swelling, followed by cytoplasmic extrusion. These abnormally developed embryos failed to progress to the blastocyst stage. The survival rate of O. yaluensis embryos was significantly higher at 15 and 20 ℃ than at 25 ℃ (P<0.05). Embryos at 15 ℃ and 20 ℃ had higher total antioxidant capacity and lower malondialdehyde content and superoxide dismutase activity than those at 25 ℃ (P<0.05). The catalase activity was in the order of 15 ℃>25 ℃>20 ℃ (P<0.05). Glutathione peroxidase activity did not differ significantly among the three groups (P>0.05). Temperatures of 15–20 ℃ favored early embryonic development in O. yaluensis. However, as temperature increased, oxidative stress occurred, activating the embryonic antioxidant system and mitigating the damage caused by oxidative stress. The expression level of 4e-bp1 was significantly lower in embryos at 15 and 20 ℃ than at 25 ℃, whereas c-myc expression was highest at 15 ℃, followed by the 20 ℃ group, both significantly higher than at 25 ℃ (P<0.05). The expression levels of s6k1 and igf-1r were significantly higher in embryos at 20 ℃ than in the other two groups (P<0.05). The genes s6k1 and igf-1r are associated with accelerated development and improved protein synthesis efficiency. Identified as a rapid-response gene during early embryogenesis, c-myc has low expression levels that may lead to developmental arrest. Additionally, the increased expression of 4e-bp1 has been shown to inhibit the initiation of protein translation in embryos. In summary, at 15–20 ℃ water temperatures, O. yaluensis embryos exhibited higher survival rates and enhanced antioxidant capacity to regulate oxidative stress levels than at 25 ℃. Oxidative stress induced by high temperatures has been identified as a key factor causing oxidative damage to embryonic cells and leading to abnormal development and significant mortality in O. yaluensis embryos. Additionally, the expression of genes related to embryonic development and protein synthesis is downregulated to prevent the progression of abnormal development in damaged embryos, thereby slowing the developmental process and suppressing further abnormalities.

Abstract:The common carp (Cyprinus carpio) is an economically important freshwater fish species for aquaculture, and the FFRC No.2 strain common carp has recently become widespread in China. A differentiation of skin color exists during the breeding practice of FFRC No.2 strain common carp, where most individuals are gray, occasionally accompanied by a small number of red individuals. To further clarify the growth and morphological variation between individuals with different skin colors and the effects of phenotypic measurements on body weight, six growth-related traits (body weight BW, total length TL, body length BL, head length HL, body thickness BT, and body height BH) and 19 truss measurements (pairwise linear distances between nine landmarks, such as Dis12, Dis13, and Dis14) were measured for 50 individuals randomly selected with different skin colors at three and six months of age, respectively. The mean comparisons, principal component analysis (PCA) and path analysis were further performed using morphological data. The results showed that the gray population was significantly higher than the red population in six growth traits at three and six months of age (P＜0.05). Meanwhile, 17 and 13 phenotypic parameters (ratio of distance measurements to BL) significantly differed between gray and red populations at three and six months of age, respectively. Among these, four head-related phenotypic parameters (HL/BL, Dis12/BL, Dis14/BL, and Dis24/BL) and six trunk-related phenotypic parameters (BH/BL, Dis16/BL, Dis23/BL, Dis34/BL, Dis36/BL, and Dis46/BL) in the gray population were significantly lower than those in the red population (P＜0.05). In contrast, Dis68/BL in the gray population was significantly higher than that in red population (P＜0.05) at three and six months of age. PCA was performed based on phenotypic parameters, those with significant differences were observed between the gray and red populations at three and six months of age. Scatter plots of PC1 and PC2 showed that most individuals with different skin colors were distinguishable in distribution, with a small number of overlapping individuals at three and six months of age. In addition, significantly positive correlations (P＜0.01) were detected between the growth traits of the different populations at three and six months of age, with correlation coefficients (r) ranging from 0.434 to 0.984. The path analysis was performed using a stepwise linear regression algorithm. The results showed that TL, BT, and BH of the gray population at three months of age, BL, BT, and BH of the gray population at three months of age and the red population at six months of age, and BL and BT of the red population at six months of age had significant effects on BW, with determinant coefficients (R² ) ranging from 0.935 to 0.967. Differences were observed in the path coefficients of morphological traits on BW for different populations at different stages. The highest direct path coefficients on BW were found in TL of the gray population at three month of age (0.611), and BL of the gray population at six months of age (0.633), and BL of the red population at three and six months of age (0.654 and 0.764, respectively). The study revealed significant differences in growth and morphological characteristics between individuals with different skin colors of FFRC No. 2 strain common carp; the specific selection programs of growth traits in the subsequent breeding process for different skin color populations should be considered.

Abstract:This study investigated the effects of different selenium sources (sodium selenite and yeast selenium) and selenium levels on growth performance, tissue selenium content, antioxidant capacity, and serum biochemical indices of Carassius auratus var. Pengze. A 2 × 3 factorial design was used for the feeding trial: Selenium source (sodium selenite and yeast selenium), selenium supplementation level (0, 0.5, and 5 mg/kg), and five diets were prepared. The control group (CON), sodium selenite group (LS and HS), and yeast selenium group (LY and HY) were established. A total of 240 C. auratus var. Pengze with an initial body weight of (210.28±1.06) g were randomly divided into five groups, with three replicates and 16 fish in each replicate. The experimental diets were fed, and the experimental period was 56 days. The results showed no significant differences in survival rate, weight gain rate, specific growth rate, and feed conversion ratio among all groups (P＞0.05). The highest muscle selenium content was observed in the high selenium yeast group, followed by the high sodium selenite group; this value was significantly higher than that of the control group (P＜0.05), and selenium sources and selenium levels had a significant interaction on muscle selenium content (P＜0.05). For liver selenium content, the highest levels were found in the high sodium selenite group, followed by the high selenium yeast group; these levels were significantly higher than those observed in the control and low selenium groups (P＜0.05). In addition, serum catalase activity was significantly increased in the high selenium yeast group compared to the control group (P＜0.05). The serum glutathione peroxidase activity in the low sodium selenite and the selenium yeast groups was significantly higher than that of the control group (P＜0.05). Furthermore, selenium sources and levels showed significant interactions that affected serum glutathione peroxidase and catalase activities (P＜0.05). In addition, serum lysozyme activity and blood glucose levels were significantly increased in the high selenium yeast group compared to the other groups (P＜0.05). Blood glucose levels in the high selenium group were also significantly higher than those in the control group (P＜0.05), with significant interactions between selenium sources and levels affecting low-density lipoprotein levels (P＜0.05). Intestinal amylase activity in the high selenium group was notably higher than that in the control and low selenium groups (P＜0.05). Moreover, trypsin activity in the selenium yeast and high sodium selenite groups was significantly increased relative to that of the control group (P＜0.05), with significant interactions between selenium sources and levels affecting trypsin activity (P＜0.05). In conclusion, utilizing high levels of selenium yeast increases tissue selenium deposition, and the administration of two levels of selenium yeast improves antioxidant capacity and intestinal enzyme activity in C. auratus var. Pengze. Similarly, using high levels of sodium selenite increases tissue selenium deposition and intestinal enzyme activity within this species, whereas employing low sodium selenite levels improves antioxidant capacity in C. auratus var. Pengze.

Abstract:Paralichthys olivaceus is an economically important target species in marine aquaculture because of its large size, rapid growth rate, excellent meat quality, rich nutritional value, and short-distance migratory habits. Industrial farming models have been widely used owing to their high degree of intensification and environmental controllability. Artificial lighting is commonly used in factory farming to meet the farming requirements. Compared to the traditional artificial lighting of straight-tube fluorescent lamps or compact fluorescent lamps, LED lamps have the advantages of energy saving and environmental protection, long service life, low heat generation, and high photoelectric conversion efficiency. Additionally, LEDs can accurately regulate the spectrum and intensity of light according to demand, which has been rapidly promoted in factory farming. Different light-colored environments have a certain degree of inhibitory or promotional effects on the growth, physiological and biochemical processes, and stress responses of aquatic organisms. The study aimed to provide theoretical support for the selection of light colors for juvenile P. olivaceus in factory farming. In this study, a total of 450 individuals with good body condition and uniform body size were selected, with a mean body mass of (309.66±32.73) g and an initial total length of (28.67±2.66) cm. Five LED spectral parameters were set, which were red (λ 625–630 nm), yellow (λ 570–575 nm), blue (λ 450–455 nm), and green (λ 525–530 nm), and full spectrum (λ 380–780 nm) as the control group. Water temperature was controlled at (18.0±1.0 ℃), photoperiod was 12L:12D. The light intensity was set at (250±20) mW/m2 . The effects of five different light colors on the growth, enzyme activity, hormone levels, and gene expression of juvenile P. olivaceus were studied. The enzyme activities included two antioxidant enzymes, SOD and CAT, and two digestive enzymes, amylase and fibrillase; whereas, the hormones included growth hormone and cortisol, and the genes were mainly SOD and CAT. The results showed that the weight gain and specific growth rates of juvenile P. olivaceus in the blue and green light groups were significantly higher than those in the other groups (P < 0.05). The growth hormone content of juvenile P. olivaceus in the blue light group was the highest at (20.74±1.52) ng/mL, which was significantly higher than that of other groups (P<0.05). The growth hormone content of juvenile P. olivaceus in the red light group was the lowest at (10.68±0.61) ng/mL, which was significantly lower than that of other groups (P<0.05), while the cortisol content was the highest at (1 487.44±54.42) pg/mL, which was significantly higher than that of other groups (P<0.05). The gastric amylase activity of juvenile P. olivaceus in the green light group was significantly higher than those in the red light and control groups (P<0.05). The gastric cellulase activity of juvenile P. olivaceus in the red light group was significantly lower than that in the control group (P<0.05). The intestinal cellulase activity of juvenile P. olivaceus in the red light group was significantly lower than that in the other groups (P<0.05). CAT activity in the serum of juvenile P. olivaceus in the red light group was significantly lower than that in the other groups (P<0.05), and liver CAT activity was significantly lower than that of the control group (P<0.05). The differences in the liver SOD activity of juvenile P. olivaceus in each light-colored group were not significant (P>0.05). The relative expression of liver SOD gene of juvenile P. olivaceus in the red light group was significantly lower than that in the yellow light group (P<0.05), but the difference with other groups was not significant; the relative expression of CAT genes in the liver of juvenile P. olivaceus in the red light group was the lowest and significantly lower than that in the other groups (P<0.05), the relative expression of liver CAT gene of juvenile P. olivaceus in the yellow light group was the highest, and significantly higher than that in the other groups (P<0.05), and the relative expression of CAT gene of juvenile P. olivaceus in the blue light group, green light group and control group was not significant different. These results show that blue and green light cultures significantly affected the growth of juvenile P. olivaceus, whereas red light continuously stressed juvenile P. olivaceus weakening their antioxidant and digestive capacities. Furthermore, these results provide a theoretical basis for improving the indoor factory aquaculture efficacy of juvenile P. olivaceus and promoting green healthy factory aquaculture of juvenile P. olivaceus.

Abstract:Leptin, a 16 kDa protein hormone encoded by the obesity gene (ob), is secreted by adipose tissue and essential for regulating various physiological processes, such as fat metabolism, feeding, reproduction, and immunity. Seriola aureovittata, prized for its flavor and high nutritional content, often experiences excessive fat accumulation under artificial farming conditions because of spatial constraints in farming facilities and the provision of fresh fish as feed. This fat accumulation can adversely impact their growth and quality. A recombinant leptin protein for S. aureovittata was constructed using a prokaryotic expression vector to investigate the multiple physiological functions of leptin, and its biological activity was verified via intraperitoneal injection. This study provides technical support for further investigation of the physiological regulatory role of leptin in the growth and fat metabolism of S. aureovittata and for developing specialized products for growth and quality control. Total RNA was isolated from the brain tissue of S. aureovittata, and the first strand of cDNA was synthesized. Mature peptide fragments were synthesized based on amino acid sequences encoded by lepa and lepb in S. aureovittata. Using the prokaryotic expression vector pQE30, recombinant plasmids lepa/pQE30 and lepb/pQE30 were constructed and transformed into Escherichia coli M15. Following induction with 0.5 mmol/L IPTG at 37 ℃ for 4 h, the expressed proteins were validated for their expected sizes via western blot analysis, revealing clear bands at approximately 17.9 and 17.3 kDa. This confirmed that the recombinant proteins exhibited antigenic activity and were specifically recognized by 6×His antibodies. Further purification of the target proteins via a Ni²⁺-NTA affinity chromatography column yielded purified LepA and LepB recombinant proteins, with purity verified at ＞90% and endotoxin levels ≤ 1 EU/μg. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins showed distinct bands at approximately 17.9 kDa and 17.3 kDa, aligning with the expected size of the recombinant proteins, thereby confirming the effective purification of the LepA and LepB fusion recombinant proteins. The concentrations of the purified LepA and LepB recombinant proteins were measured at 0.2 and 0.3 mg/mL, respectively, using a protein quantification kit. In the intraperitoneal injection experiment, four groups were established: a control group (0.9% saline solution) and experimental groups with concentrations of 0.05, 0.1, and 0.2 μg/kg. Each group consisted of 18 fish reared in a 1 m³ water tank. Each fish received an intraperitoneal injection of recombinant leptin protein at a dose of 1 μL/g body weight. At 6, 12, and 24 h after the first injection, brain tissues from six randomly selected fish were used to detect biological activity. After injecting three concentration groups (0.05, 0.1, and 0.2 μg/kg) of LepA and LepB proteins at different time points (6, 12, and 24 h), the expression of lepa and lepb genes in the brain was either upregulated or downregulated, indicating that the recombinant proteins LepA and LepB possessed biological activity. These results provide technical support for further research into the physiological functions and regulatory mechanisms of leptin in the growth and development of S. aureovittata

Abstract:The Conger myriaster is commercially important in fisheries and aquaculture. The artificial culture of C. myriaster is presently conducted by catching wild fry. Recently, this resource has notably declined, highlighting an urgent need to develop seedling production for its aquaculture. C. myriaster gonads cannot mature naturally in artificial culture owing to insufficient synthesis of endogenous gonadotropins. However, their gonads can be induced to mature by injecting hormones in vivo. The follicle-stimulating hormone (FSH) gene is crucial in fish sexual development and reproduction, and its regulatory mechanisms have been extensively studied. In this study, the coding sequence (CDS) of fshβ was cloned by PCR techniques. Bioinformatical analysis revealed that the CDS of FSH comprises 378 bp, encoding 125 amino acids. By analyzing the physical and chemical properties of the protein, the fshβ protein has a molecular weight of 13.19 kDa. The aliphatic index was 71.04, and the theoretical pI was 6.00. An instability index value of 51.57 indicates a state of instability. The functional domain analysis showed that the fshβ protein has a highly conserved GHB functional domain (comprising 104 amino acids at 19–122). The hydrophilicity and hydrophobicity analysis revealed that the fshβ protein is a hydrophilic protein. The 6th and 7th amino acids (Ala) and 8th amino acid (Leu) exhibited the strongest hydrophobicity (2.178), whereas the 106th amino acid (Asn) exhibited the strongest hydrophilicity (–1.722). The leader peptide and transmembrane domain analyses of fshβ protein revealed that the 1–19 amino acids are signal peptides without any transmembrane domains. The analysis of protein glycosylation sites showed that the fshβ protein contains two N-glycosylation sites and three O-glycosylation sites. The analysis of protein phosphorylation sites demonstrated that the fshβ protein contains 14 potential phosphorylation sites. Specifically, seven serine (Ser) and seven tyrosine (Tyr) phosphorylation sites exist. Subcellular localization analysis revealed that the fshβ protein was mainly localized in the nucleus. The secondary structure of the fshβ protein mainly comprises an alpha helix (12.0%), extended strand (24.8%), and random coil (63.2%). The tertiary structure of C. myriaster fshβ protein was compared with that of Anguilla japonica, A. rostrata, A. Anguilla, A. marmorata and Homo sapiens. The C. myriaster fshβ protein tertiary structure was observed to be similar to that of A. Japanica, A. rostrata, A. Anguilla, A. marmorata. However, differences were observed with the tertiary structure of H. sapiens. Based on the sequence alignment and phylogenetic analysis, the C. myriaster fshβ exhibited a closer evolutionary relationship with A. japonica, A. rostrata, A. anguilla and A. marmorata. The fshβ amino acid sequence of C. myriaster compared with A. japonica, A. rostrata, A. anguilla, and A. marmorata revealed similarities of 70.08%, 70.08%, 70.08% and 68.50%, respectively. The fshβ of C. myriaster exhibits high homology with other eels but low homology with other bony fish and mammals, indicating that fshβ has evolutionary differences during evolution. This study presents the successful production of recombinant C. myriaster fshβ protein using the pET-28a(+) expression system. NdeⅠ and XhoⅠ cleavage sites were inserted at both ends of the primers, and then fshβ was amplified by PCR. Subsequently, the fragment was conjugated to the pEASY-T1 vector to obtain the recombined plasmids, named pEASY-T1-fshβ. The recombinant plasmid pEASY-T1-fshβ and the pET-28a(+) vector were double-digested using restriction endonuclease NdeⅠ/XhoⅠ. After double-digested, the fragment was ligated into the pET-28a(+) vector to obtain the recombined plasmids named pET-28a-fshβ. To investigate the biological activities and physiological significance of pET-28a-fshβ, we used the pET protein fusion and purification system to produce plasmids pET-28a-fshβ in Trans1-T1 competent cells. Recombinant plasmids pET-28a-fshβ were transferred into Rosetta (DE3) cells, which were cultured under different IPTG induction conditions (0.01, 0.1, 0.5, and 1 mmol/L) at a temperature of 16 ℃ for 16 h. fshβ was efficiently expressed at different IPTG concentrations and under the above induction conditions. Therefore, the following optimized parameters were used in subsequent experiments: IPTG concentration of 0.5 mmol/L, incubated at 16 ℃ for 16 h. The supernatant and precipitate were analyzed using SDS-PAGE following induction of expression. SDS-PAGE analysis showed an evident thickened band at 26.0 kDa. It was also present in the supernatant as a soluble protein. The protein was purified using a His-tagged nickel affinity chromatography column. Western blot analysis confirmed the presence of the purified protein, demonstrating that the recombinant protein was specifically recognized by antibodies. The molecular mass of the protein was approximately 26.0 kDa, which was consistent with the expected size. This result indicates that the fshβ recombinant protein was successfully expressed. The results of this study revealed the preparation of recombinant fshβ protein in the C. myriaster to provide a theoretical reference for the subsequent use of exogenous recombinant fshβ protein to induce sexual maturation in Parent fish.

Abstract:Shellfish inhabiting mudflats constitute an important component of the mariculture industry in China, with an annual output accounting for approximately one-third of shellfish breeding output. The Manila clam Ruditapes philippinarum, one of the main species of mudflat mollusks cultivated in China, is characterized by a short cultivation cycle and strong adaptability, the culture of which requires low investment and is highly profitable. Moreover, it is a suitable species for artificial high-density cultivation, with an annual output exceeding 3 million tons, accounting for more than 90% of the world's cultured production. R. philippinarum is one of the four major mollusk species traditionally cultured in China that is highly valued for its delicate taste and rich nutrition, and is particularly popular among the general public. The current domestic Manila clam aquaculture industry is based on a mainstream pattern of southern (Fujian Province) breeding and northern (Liaoning and Shandong Province) rearing of seedlings. During the entire Manila clam cultivation process, the intermediate stage of cultivation (cultivating juveniles to suitable sizes for bottom-seeding cultivation) is an important link connecting the two stages of factory-based seed production and bottom seedling cultivation. With the annually increasing market demand for Manila clams, the scale of cultivation scale has also expanded, making the intermediate cultivation stage particularly important. Currently, the southern intermediary stage of Manila clam juvenile cultivation is mainly carried out in natural marine areas, whereas the intermediate cultivation of northern Manila clam is mainly conducted in ponds. Although pond cultivation is conducive to artificial control and management, with juveniles being less affected by predators and more efficiently harvested, in northern regions this type of cultivation has the disadvantages of low temperatures in winter and insufficient food supplies. Consequently, the intermediate cultivation of juvenile shellfish is typically one of the key factors currently constraining the further development of the industry. Studies in this regard have shown that the accumulation of nutrients in juvenile shellfish plays a decisive role in the survival of juveniles for enhanced breeding success, and this requirement is accordingly gaining increasing attention from breeding enterprises and researchers, given that ensuring a sufficient food supply during the intermediate stage of juvenile cultivation is a key factor contributing to the success of the entire cultivation process. In this study, to address the problems of feed shortages or nutritional deficiencies during Manila clam cultivation, we sought to identify effective or alternative feed supplements by assessing the effects of the addition of four nutritionally rich and readily obtainable feed sources, namely, spirulina powder, yeast, plasma protein powder, and soybean peptides. Soybean peptides represent a high-protein source that is obtained by harnessing biological enzymatic technology to degrade large soybean protein molecules to smaller molecular fragments. This process yields a rich array of amino acids, thereby making soybean peptides not only a concentrated source of protein but also a potentially beneficial component for enhancing the growth and immune function of mollusks in aquaculture. Plasma is the liquid fraction of blood that contains a diverse range of proteins, minerals, hormones, and spectrum of essential nutrients. For the purposes of aquaculture, it can be converted to a plasma protein powder and used as a protein supplement, providing a source of essential nutrients for mollusks. Yeast is a nutrient-rich source of microbial proteins, B-vitamins, minerals, and dietary fiber, which can be used as a nutritional supplement that may contribute to enhancing the nutritional value and growth performance of young Manila clams. Spirulina is a high-quality plant-based source of nutrients that is rich in proteins, vitamins, minerals, and essential amino acids, which may have a significant influence on the fat content of Manila clams and also contributes to enhancing immunity. To investigate the effects of soybean peptide, plasma protein powder, and yeast and spirulina powder on the growth, survival, and nutrient accumulation of R. philippinarum juveniles, in this study, we adopted a physiological ecology approach. Specifically, we compared two substrate conditions (with and without sand) for each of the following four supplemental nutrient groups: H1 (soybean peptide), H2 (plasma protein powder), H3 (yeast), and H4 (spirulina powder). As a control group, shellfish were fed solely on microalgae, without nutrient supplementation. The results revealed that among the assessed feed supplements, the survival of juveniles cultivated on a sandy substrate and receiving yeast supplementation was notably higher at (79.60±0.86)%, which was significantly higher than that of juveniles reared in the absence of a sandy substrate (P＜0.05). On the tenth day of observations, a maximum shell length specific growth rate of (1.09±0.09)%/d was recorded in the H4 group cultivated with sand, whereas a maximum wet weight specific growth rate of (3.11±0.62)%/d was recorded for H3 group clams cultivated with sand. And the effect of substratum on growth rate was found to be significant (P＜0.05). Furthermore, the glycogen content of H4 group R. philippinarum juveniles cultivated with sand (79.03±18.60 mg/g) was significantly higher than that in all other groups (P＜0.05), with this difference between the groups with and without sand also being statistically significant (P＜0.05). The carbon content of juveniles in the H3 group with sand (45.23%±0.33%) was significantly higher than that observed in the other groups (P＜0.05), whereas the nitrogen content of juveniles in the H4 group with sand (12.25%±0.22%) was significantly elevated compared with that detected in the other groups (P＜0.05). However, whereas with respect to carbon content, we detected no significant differences between the groups cultivated with and without a sand substratum (P＞0.05), the presence of a substratum did have a significant effect on nitrogen content (P＜0.05). In conclusion, our findings in this study provide evidence to indicate that nutrient supplementation can contribute to a significant alteration in the nutrient content of R. philippinarum juveniles, and that these effects are partially influenced by the presence of sand. The valuable insights gained in this study will contribute to developing systems for the efficient cultivation of healthy R. philippinarum juveniles.

Abstract:Microplastics (MPs) are emerging global pollutants that have attracted considerable attention because of their widespread contamination and profound ecological impacts. Fish, as one of the most diverse groups of aquatic organisms, is a sensitive indicator of MP contamination in aquatic ecosystems. Hainan Island, which is located in the tropical and subtropical zone, is rich in freshwater fishery resources. However, freshwater fishes in Hainan Island are facing the threat of MP pollution caused by human activities. Wanquan River, the third largest river on Hainan Island, is facing eutrophication, heavy metal pollution, organic pollution, and MP pollution. However, the contamination status of MPs in the gastrointestinal tract (GI) of fish from the Wanquan River and the effects of feeding habits and spatial distribution on the abundance and characteristics of MPs in the GI tract of fish have not been reported. In this study, 11 representative sampling sites along the upstream, midstream, and downstream sections of the Wanquan River, covering urban residential areas and natural watersheds, were selected. Fish samples were collected using trawl nets from July to September 2021. All samples were immediately transported to the laboratory and stored in a freezer for subsequent analyses. In the laboratory, the surface of the fish was rinsed, and the GI tracts were carefully eviscerated and weighed. The GI tract samples were digested with potassium hydroxide, and the supernatant was filtered through 0.45 µm glass fiber filters. The insoluble materials such as sediment in the digestion solution were added with saturated zinc chloride solution for flotation, and the flotation supernatant was filtered through 0.45 µm glass fiber filters. Fourier transform infrared spectroscopy (FTIR) was used to identify the composition of the MPs. A total of 213 fishes spread over 34 species were collected, and MPs were detected in 85.0% of the samples. The average abundance of MPs was (3.15±1.70) ind./fish, which was consistent with the economic development level in Wanquan River basin. The MPs in the samples were mainly ＜1.0 mm (78.7%) transparent (46.0%) fibers (74.6%), which are widely present in the environment, easy to be ingested, and difficult to be excreted. FTIR results showed that most of the MPs were composed of polypropylene (PP, 35.8%) and polyethylene (PE, 25.4%). Given their good temperature resistance and corrosion resistance, PP and PE are the main components of carpets, industrial fabrics, fishing gear, shipping ropes, etc. Thus, PP and PE are widely detected in aquatic ecosystems because of their durability and widespread usage. Principal component analysis showed that transparent/blue fibers with 0.5–1.0 mm contributed the most to the abundance of MPs in the samples. The control of urban waste, fishing supplies, and agricultural waste must be strengthened to reduce the MP pollution in the Wanquan River. The abundance of MPs varied with feeding fish and followed the order molluscivorous (7.33 ind./fish) ＞ pelagic carnivorous (6.83 ind./fish) ＞ benthic carnivorous (4.33 ind./fish) ＞ detritivorous (3.36 ind./fish) ＞ crustaceavorous (3.15 ind./fish) ＞ insectivorous (2.40 ind./fish) ＞ epiphytivovrous (2.30 ind./fish) ＞ herbivorous (1.89 ind./fish). The abundance of MPs in the samples was affected by several factors, such as the status of MP pollution in foraging areas or habitats, the abundance of MPs in fish prey, frequency of fish predation, factors of the fish itself (e.g. body weight, body length, body shape, gut structure, etc.), retention time of MPs in the GI tract of fish, and excretion rate of MPs by the GI tract. The abundance of MPs in the samples gradually increased from upstream (2.67±0.43 ind./fish) to downstream (3.81±0.39 ind./fish). This result was related to the increasing urbanization and industrialization in the downstream areas and thus the increased input of MPs downstream. The proportion of red/yellow/blue fragments and thin film MPs with size ＞1.0 mm increased, whereas the proportion of black fiber MPs with ＜1.0 mm decreased from upstream to downstream. This result was related to the increase in shipyard, shipping, and tourism activities downstream. For individual fish species at the different sampling sites, the abundance of MPs in the samples did not show a continuous increasing trend. This result was because the MPs in the samples were mainly derived from passive ingestion, which is affected by many factors (e.g., feeding habits, intestinal structure, excretion ability, food intake, and the status of MP contamination in the environment). The results of this study provide data support for the risk assessment and control of MPs in fish in the Wanquan River and serve as a scientific basis for the prevention and control of MP pollution in freshwater rivers on Hainan Island.

Abstract:The aquaponics system, an integrated ecological agriculture model, effectively combines aquaculture and the cultivation of aquatic plants. This system not only utilizes the nutrients in aquaculture water to promote plant growth but also purifies water quality through plant absorption, reduces the discharge of aquaculture waste, and provides novel ideas for the green transformation of the aquaculture industry. Microorganisms are crucial in the aquaponics system for elemental cycling, water quality purification, ecological balance maintenance, and healthy growth regulation of flora and fauna. The co-occurrence of bacterial and microeukaryotic species in aquaculture water is a common ecological phenomenon; however, research on the interactions between bacterial and microeukaryotic communities remains relatively limited. Currently, insufficient research exists on the complexity and structural characteristics of microbial community diversity in aquaculture waters of aquaponic systems, and the interactive relationship between bacteria and microeukaryotic communities remains unclear, thereby limiting the optimization and regulation of system operations via microbial methods. In this study, we constructed an aquaponic system as the experimental group and a recirculating water system as the control group. We conducted a 78-day aquaculture experiment, collected water samples from aquaculture ponds,and employed high-throughput sequencing technology targeting the 16S rRNA and 18S rRNA genes to assess the diversity and structural composition of microbial communities in aquaculture waters from different treatment systems. We also revealed the differences in microbial community composition, bacterial networks, microeukaryotic networks, and bacterial-microeukaryotic interaction network structures between different systems, with the objectives of clarifying the differences in bacterial and microeukaryotic community diversity, structure, and composition between the aquaponic and recirculating water aquaculture systems and understanding the structural differences in the microbial community co-occurrence networks between systems. This provided a theoretical basis for establishing aquaponic systems from a microbial mechanism perspective. The observed species, Chao1, and Pdfaith indices of the bacterial community in the control group were significantly higher than those in the aquaponic group (P＜0.05), and a significant difference was observed in the microeukaryotic community structure (ANOSIM R = 0.082, P= 0.036). Significant differences were noted in the diversity of the bacterial communities and the structure of the microeukaryotic communities among the different treatment groups. At the phylum level, Bacteroidota and Fusobacteriota exhibited increased relative abundances in the aquaponic group, whereas the relative abundance of Chlorophyta was lower than that in the control group. The relative abundance of Chlorophyta in the aquaponic group continuously decreased over time, whereas it consistently increased in the recirculating aquaculture group. At the genus level, Flavobacterium, Cetobacterium, and Anurofeca were more abundant in the aquaponics group than in the control group. Significant differences were observed between the aquaponic and control groups in the compositions and relative abundances of the dominant bacterial and microeukaryotic groups. Analysis of the microbial community co-occurrence network showed that the bacterial and microeukaryotic communities in the aquaponic group had more complex interactions and network structures than those in the control group, with more pronounced competitive relationships and greater stability among microbial communities. Additionally, the network structures of the microeukaryotic communities in both the aquaponic and control groups were more complex than those of the bacterial communities, with more intense internal competition and greater stability. Bacterial-microeukaryotic interaction network analysis indicated that, in the aquaponic group, Bacteroidota and Chlorophyta were the core nodes of the bacterial-microeukaryotic interaction network. In the control group, Proteobacteria and Fusobacteriota were the core nodes of the bacterial-microeukaryotic interaction network. Substantial differences existed in the interaction relationships between bacterial and microeukaryotic communities and the proportions of various groups between the aquaponic and control groups. The interaction relationships between bacterial and microeukaryotic communities in the control group were more complex and closer than those in the aquaponic group, which exhibited more antagonistic relationships. This study analyzed the differences in diversity, compositional structure, and co-occurrence networks of bacterial and microeukaryotic communities between aquaponic and recirculating water aquaculture systems. The growth performance, immunity, and digestive function of fish in the aquaponic system were significantly better than those in the recirculating water aquaculture system, highlighting the advantages of aquaponic systems. This study elucidated the microbial community characteristics of aquaponic and recirculating water aquaculture systems and revealed the interactive relationships between bacterial and microeukaryotic communities in both aquaculture modalities to provide a theoretical microbial basis for the stable operation of aquaponic systems.

Abstract:Fishery production surveys constitute the basis for assessing and managing fishery resources. A well-defined and reasonable sample size is essential for the accuracy and precision of survey outcomes. In this study, we aggregated production surveys from major economic fishing ports in the northern South China Sea from 2008 to 2018, totaling 36 499 forms. It was assumed that these data accurately reflected the catch per unit effort (CPUE) of Decapterus maruadsi employing various fishing gear. We focused on optimizing the investigations by analyzing the CPUE of D. maruadsi across five distinct fishing operations: otter trawl, twin trawl, light purse seine, gillnet, and light falling net. We organized the survey data by fishing type and stratified them according to engine power and survey time. We used a proportional allocation for the sample sizes and stratified random sampling without replacement for the simulations. We utilized computer simulations to resample the CPUE of D. maruadsi derived from five different fishing operation types, employing the relative estimation error (REE) and relative bias (RB) as evaluation metrics. We aimed to analyze the relationship between the CPUE of D. maruadsi and sample size in the northern South China Sea. The port catch sampling survey yielded production information for different fishing operation types, with each survey form reflecting the CPUE data for a single voyage. Because of the variability of the CPUE for D. maruadsi among different fishing operation types and across seasons within the same operation type, this study categorized the survey forms by operation type and season. We calculated the CPUE for each operation type in the different seasons and used these values as the "true values" for comparison. We consolidated the survey data from various fishing gears across different power ranges and computed the CPUE for these forms. Furthermore, we employed CPUE as a metric to compare the fishing capacity and efficiency of the different fishing gear targeting the species of interest. We observed seasonal variations in the CPUE estimates for D. maruadsi across different fishing operation types. By averaging the CPUE estimates over the four quarters, we discovered that the light purse seine method had the highest CPUE estimate at 3.577 kg/(kW·d), whereas the gillnet method had the lowest CPUE estimate at 0.143 kg/(kW·d). The results of this study revealed differences in the distribution range of REE values for catch rate estimates among different fishing operation types; however, the overall trend of change was similar. Particularly, with an increase in sample size, the boxplot of REE values for CPUE estimates of each fishing gear showed a gradually decreasing trend, whereas the RB values exhibited decreasing dispersion and tended to stabilize. Notably, the distribution range of REE values for the light purse seine and gill net methods was relatively smaller than that of other fishing gear. We found that the minimum sample sizes required for estimating CPUE varied among different fishing operation types, and the rules for determining these minimum sample sizes also differed. Otter trawl, pair trawl, and light purse seine determined the minimum sample size based on REE ≤ 10%, whereas gillnets and light falling nets (except in winter) determined the minimum sample size based on REE ≤ 5%. We also found that, as the sample size reached a specific threshold, the impact of increasing the number of survey forms on the estimation accuracy of the average catch rates gradually decreased. In the summer, when the sample size reached 600, the REE values for twin trawl, light purse seine, and light falling net were below 10%; when the sample size reached 800, the REE values for the otter trawl decreased to within 10%; and when the sample size increased to 1 200, the REE value for the gill net decreased to within 10%, whereas the REE values for other operation types remained below 5%. As the sample size continued to expand, the impact on sampling accuracy became increasingly minimal. In general, when the sample size reached a certain threshold, the changes in REE and RB tended to stabilize, and the redundant portion of the sample size could be optimized. Even with a reduced sample size, estimation accuracy could be ensured to a certain extent. In this study, the minimum acceptable sample size for CPUE estimation varied across fishing operation types. Assuming that the survey data from 2008 to 2018 accurately represented fishery production and considering an REE of less than 10%, the minimum number of survey trips required for CPUE estimation of D. maruadsi by operation type and season were as follows: otter trawl (91, 68, 59, and 86), twin trawl (41, 41, 82, and 52), light purse seine (164, 87, 95, and 57), gillnet (218, 218, 245, and 191), and light falling net with attractors (100, 81, 64, and 43). On average, these corresponded to 76 trips for the otter trawl, 54 for the twin trawl, 218 for the gillnet, 101 for the light purse seine, and 72 for the light falling net with attractors. In this study, we optimized sample size using the mean CPUE of D. maruadsi as the survey target, and the evaluation results may serve as a reference for catch surveys in northern South China Sea fishing ports.

Abstract:With the expansion of aquaculture and deterioration of farming environments, diseases are a major issue in Portunus trituberculatus farming, among which Vibrio parahaemolyticus is the primary pathogen causing mortality. Rab GTPases, which belong to the Rab protein family within the small GTPase Ras superfamily, play critical roles in regulating intracellular membrane trafficking in eukaryotic cells. Studies have shown that Rab enhances immunity in invertebrates against pathogenic infection by regulating phagocytosis in hemolymph tissues. DNA methylation, a common epigenetic regulatory mechanism, is involved in controlling the expression of key genes. However, the molecular mechanism by which DNA methylation regulates gene expression and thus influences the immune response of P. trituberculatus upon pathogen infection remains unclear. To explore the epigenetic regulatory mechanism involving DNA methylation in response to V. parahaemolyticus infections in P. trituberculatus, the gene structure, sequence similarity, and phylogenetic analysis of PtRab2A were characterized. In addition, the PtRab2A expression and its DNA methylation patterns in response to V. parahaemolyticus infection were investigated. Preliminary experiments were conducted with 90 randomly selected P. trituberculatus specimens, weighing approximately (30±5) g. Python was used to calculate the median lethal concentration (LC50) of V. parahaemolyticus infection at 72 h post-infection, which was determined to be 5×106 CFU/mL. This concentration was then applied in the formal experiment. A total of 40 temporarily cultured crabs were selected for the formal infection test, and hemolymph tissues were collected at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h after infection for subsequent DNA methylation and gene expression analysis. The RNAi experiment was divided into two groups, each consisting of 80 individuals. At 12 and 24 h post-injection, hemolymph tissues were collected from five crabs each in both the blank control and experimental groups. Following 24 h of RNAi treatment, surviving crabs were infected with V. parahaemolyticus (5×106 CFU/mL). Mortality rates were recorded at 1-h intervals until complete mortality occurred. The results showed that the full length of PtRab2A was 1,824 bp, including a 15 bp 5′ UTR, an 822 bp ORF region, and a 987 bp 3′ UTR. Two conserved cysteine residues were identified at the C-terminus of PtRab2A. The predicted molecular formula was C1322H2084N382O399S12, with a molecular weight of 30.10 kDa and a theoretical isoelectric point (pI) of 7.05. The gene encodes a total of 274 amino acids. Homology analysis revealed that the conserved amino acid sequence of PtRab2A showed the highest similarity with that of Scylla paramamosain. SMART analysis identified a RAB domain within PtRab2A. Phylogenetic analysis using MEGA11 indicated that P. trituberculatus PtRab2A clustered most closely with S. paramamosain, and then grouped with Eriocheir sinensis. These crabs subsequently clustered with Penaeus monodon, Penaeus chinensis, and Drosophila melanogaster. Meanwhile, Homo sapiens, Mus musculus, Danio rerio, Oncorhynchus mykiss, Larimichthys crocea, Ruditapes philippinarum, and Crassostrea gigas formed another large cluster. Notably, D. melanogaster and crustaceans were grouped together, while Rab2A genes of invertebrates (excluding mollusks) such as P. trituberculatus were distinctly separated from those of vertebrates. The methylation profile of the PtRab2A gene following V. parahaemolyticus infection was analyzed using IGV visualization software. The monoclonal sequencing results were further analyzed using the QUMA online platform. After 72 h of V. parahaemolyticus challenge, the average methylation rate of PtRab2A was 40%, compared to 69% at 0 h, indicating a significant reduction in PtRab2A methylation levels. This trend was consistent with the IGV visualization results. Pearson correlation analysis revealed a correlation between PtRab2A methylation levels and gene expression levels in hemolymph tissue after V. parahaemolyticus infection, with a correlation coefficient of −1.000. This indicates a significant negative correlation between PtRab2A methylation and its gene expression levels. The RNAi results showed that the expression level of PtRab2A was significantly reduced at 12 h after injection of the interfering RNA (P＜0.05), and knockdown efficiency reached 80% at 24 h. After 24 h of interference, V. parahaemolyticus was injected into both the experimental and control groups. Mortality statistics indicated that in the experimental group, the mortality rate began to increase 2–3 h after infection, rising from 34.3% to 78.1%, representing a 2.27-fold increase, with all individuals dying by 4 h post-infection. In contrast, the mortality rate in the control group did not begin to rise until 4–5 h after infection, increasing from 15.0% to 62.5%, a 4.17-fold increase, with complete mortality occurring at 6 h. These results indicate that the mortality rate in the experimental group was significantly higher than that in the control group at the same time points, and peak mortality occurred earlier in the experimental group. Therefore, under V. parahaemolyticus infection, P. trituberculatus may enhance the expression of immune-related genes by reducing DNA methylation levels, thereby improving its immune capacity. These findings enrich the understanding of the molecular regulatory mechanisms involved in the defense against pathogen invasion in P. trituberculatus.

Abstract:Viral covert mortality disease (VCMD), caused by the covert mortality nodavirus (CMNV), is a highly prevalent viral infection affecting the majority of cultured shrimp. Shrimp with this disease exhibit clinical soft shells, hepatopancreatic atrophy and necrosis, empty stomachs and guts, and slow growth. During the acute infection stage, infected shrimp display opaque abdominal muscles. Farmers have observed daily mortalities in the diseased population, with mortality rates increasing between 60 and 80 days post-stocking and reaching cumulative mortalities up to 80%. CMNV can infect major cultured crustaceans, including Penaeus vannamei, P. chinensis, Marsupenaeus japonicus, P. monodon, and Macrobrachium rosenbergii, as well as fish, sea cucumber. The prevalence of VCMD has resulted in considerable economic losses within the shrimp farming industry over the past decade. Recognizing its threat to the aquaculture animals and substantial economic impact on Asian shrimp farming operations, the World Organization for Animal Health has recently issued CMNV infection as an emerging disease in aquaculture. The occurrence of VCMD in shrimp is closely related to factors such as positive CMNV infection, culture density, and culture environment. After shrimp are infected with CMNV, the disease usually occurs when the water temperature is high and the environment changes sharply. However, accurately determining the extent to which each factor is associated with VCMD occurrence remains challenging. Therefore, this study aimed to assess the risk of VCMD occurrence during shrimp culture by establishing a comprehensive risk assessment framework using the Delphi method, analytic hierarchy process, and multi-objective comprehensive evaluation. The evaluation index system consisted of 1 target level (risk assessment and early warning of VCMD occurrence), 5 criterion layers (including health status, water quality and temperature, culture mode, culture management, and environmental status), and 20 indicator layers (including CMNV infection, soft shell, empty stomach and guts,, opaque abdominal muscle, dissolved oxygen, ammonia nitrogen, nitrites, pH value, water temperature, factory culture, greenhouse culture, high-altitude tank culture, soil pond culture, breeding density, compound feed, frozen bait, fresh food, surrounding disease, facility isolation, and water treatment). The relative weight and absolute weight of each risk indicator in the risk assessment framework of VCMD occurrence were calculated based on expert scoring results. The total matrix consistency ratio was CR ＜ 0.1, which passed the overall consistency test. The weight values of five criterion-level risk factors were W= {0.420, 0.127, 0.094, 0.214, 0.146}. The high weight values of risk factors, including CMNV infection (0.173), culture density (0.095), opaque abdominal muscle (0.086), and soft shell (0.084), indicated that these factors might be crucial for the incidence of VCMD in shrimp culture. Then, the results were preliminarily validated by studying the case of three shrimp farms in Shandong Province with methods of multiple objective comprehensive evaluation. The risk values of VCMD occurrence in these three shrimp farms were determined to be 0.550, 0.508, and 0.466, all indicating a high risk of VCMD outbreak. Consistently, within 4 days after monitoring and sampling activities, severe cases of VCMD occurred in all three farms with a mortality rate exceeding 80%. These findings demonstrate that the risk assessment results align with the monitoring results and confirm the accurate early warning capabilities of the model for assessing VCMD occurrence. In summary, this study established a convenient and feasible risk assessment model for VCMD occurrence in shrimp culture. The risk assessment model comprehensively considers various factors that contribute to VCMD occurrence during the culture process, ensuring accurate assessment results. This research suggests that the proposed risk assessment model could be used to estimate the risk of VCMD occurrence in shrimp culture and could serve as an early warning tool for prevention and control, thereby ensuring the healthy and sustainable development of shrimp farming.

Abstract:Alzheimer's disease (AD) is a neurodegenerative disease characterized primarily by progressive memory decline, cognitive impairment, and disturbances in language and psychomotor functions. With the aging of the global population and the deterioration of the living environment, the number of patients is also increasing. The World Alzheimer's Disease Report estimates that the global population of Alzheimer's patients is expected to increase to 115 million by 2050, which could notably affect the world economy and society. The cholinergic hypothesis is a widely accepted theory describing AD pathology, which considers acetylcholine to be an important neurotransmitter involved in learning and memory. The lack of acetylcholine results in insufficient cholinergic signal transmission, consequently contributing to the development of AD. Acetylcholinesterase (AChE) inhibitors can reduce the decomposition of acetylcholine by inhibiting the activity of AChE, making them one of the primary drugs for the treatment of AD. The AChE inhibitor drugs approved by the FDA include donepezil and galantamine. However, the micro-molecule drugs have strong side effects; therefore, finding new safe and efficient AChE inhibitors is necessary. Pathological protein aggregation, oxidative stress, and metal ion homeostasis imbalance exacerbate disease progression. The correlation between different pathogenic factors has shifted drug research from single to multiple targets. Bioactive peptides are peptide compounds with biological activity, which have the advantages of high selectivity, high specificity, multi-target, high safety, and low immunogenicity. In particular, bioactive peptides from marine sources have many specific structures and functions owing to their unique growth environment. Researchers have prepared and isolated peptides from marine organisms with antioxidants, anti-inflammatory, high blood pressure, uric acid, immune regulation, and other effects. Trachinotus ovatus has the advantages of high yield, fast growth, strong disease resistance, high nutritional value, high protein and essential amino acid content, and is a good raw material for preparing bioactive peptides. Enzymatic hydrolysis is the most commonly used method for preparing peptides owing to its mildness, controllability and low cost. In this study, the AChE inhibitory peptide was prepared by enzymolysis of T. ovatus. Enzymolysis was performed under five proteases (neutral protease, papain, alkaline protease, protamines, and trypsin), and different enzymolysis times (1, 2, 4, 6, and 8 h), and the optimal enzymolysis time were screened based on AChE inhibition activity and antioxidant capacity. The hydrolysis degree, molecular weight distribution, amino acid composition and chelating ability of metal ions were determined, and the structure of the products was determined by ultraviolet and Fourier transform infrared. The results showed that the 4 h hydrolysate of papain had the highest AChE inhibition rate (18.02±0.78)%, which was significantly higher than other protease and positive control cerebrolin (P＜0.05). The ABTS radical scavenging rate of the enzymolysis product was (52.54±0.89)%, the degree of hydrolysis was 14.86%, and the proportion of components with molecular weight ＜3 kDa was 96.87%. Correlation analysis showed that AChE inhibition activity was significantly positively correlated with these three indices (P＜0.05). In addition, the amino acids with the highest content in the 4 h papain hydrolysate were glutamic acid, aspartic acid, and lysine, all of which were positively or negatively charged. The proportion of hydrophobic amino acids was 34.92%, which contributed to the interaction between the hydrolysate products and AChE and free radicals, improving the inhibition rate of AChE and antioxidant activity. The binding ability of metal ions was determined. The enzymatic hydrolysis product could bind Ca²⁺ and Fe²⁺; the binding rates were (26.28±1.20)% and (14.25±0.85)%, respectively, and the AChE inhibition activity was improved after binding. Ultraviolet analysis showed that the hydrolysates interacted with calcium and iron to form new compounds. Fourier transform infrared spectroscopy showed that amino and carboxyl groups participated in the formation of the complexes. Consequently, the screened hydrolysate has good potential for treating AD. In the future, the AChE inhibition effect of AChE inhibitory peptide in vivo will be further verified, and the primary role of peptide composition will be explored, including the mechanism of action. This provides theoretical support and scientific basis for marine bioactive peptides in the improvement and treatment of AD disease.

Abstract:The rapid pace of modern society has led to fatigue becoming a prevalent health problem that affects people of different age groups. Apostichopus japonicus, the only sea cucumber species cultured on a large scale, has recently experienced notable growth in its aquaculture and processing industry. However, approximately 30% of by-products are generated during processing, leading to low utilization and resource wastage. A. japonicus gonads are rich in protein, lipids, polysaccharides, and other nutrients. Studies on its activity mainly focus on antioxidants, immune regulation, and anti-hyperlipidemic effects. However, studies on its anti-fatigue efficacy are limited. Therefore, this study aimed to verify the fatigue-relieving efficacy of A. japonicus gonads. To investigate the anti-fatigue effect of A. japonicus gonads, the basic nutritional composition of the A. japonicus gonads was determined using national standard methods. Healthy male Kunming mice were randomly divided into blank and dose groups, and they performed weight-bearing exhaustion swimming experiments after continuous gavage for 4 weeks. The fatigue model was established to calculate the swimming time and examine the relevant indices of serum and liver tissues in the mice. The results revealed that A. japonicus gonads are rich in various nutrients, with the highest protein content at (56.76±0.04) g/100 g. Lipids were the second most abundant nutrient at (14.65±0.13) g/100 g, providing the necessary energy for the fertilization of the egg. Additionally, A. japonicus gonads contain a certain amount of polysaccharides at (5.35±0.16) g/100g, significantly affecting antioxidant, anti-inflammatory and anticoagulant activity. Mice experiments showed that A. japonicus gonads did not significantly affect the weight gain when comparing blank and dose groups. However, when the liver weight and index of mice were further analyzed, an increase in the liver weight and index of the dose group was observed. However, no significant difference was observed compared with the blank group, possibly because A. japonicus gonads reduced the degree of hepatocyte damage and promoted hepatic glycogen storage. A forced weight-bearing swimming test was used to assess the effect of A. japonicus gonads on the exercise endurance of mice. Mice in the dose group swam significantly longer than those in the blank group, suggesting that A. japonicus gonads enhanced the endurance and anti-fatigue ability of mice. This finding was consistent with previous studies. Further analysis of the serum-related indices revealed that urea nitrogen (BUN) and lactic acid (LA) were significantly reduced after feeding A. japonicus gonads, which may be associated with energy metabolism and reduction of protein catabolism. The reduction of LA content in the body of the exercising mice might alleviate or eliminate the LA accumulation in the mice. Additionally, A. japonicus gonads significantly reduced serum malondialdehyde (MDA) level and creatine kinase (CK) activity and increased the activity of antioxidant-related enzymes, such as superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT). These changes indicated that A. japonicus gonads had good antioxidant capacity, which could effectively slow down or eliminate the mice's oxidative stress damage, thereby alleviating fatigue. In the liver-related index test, the MDA content in the livers of mice was significantly reduced after feeding A. japonicus gonads, whereas SOD, CAT activities and GSH content were significantly increased. These results further confirmed its antioxidant capacity and anti-fatigue effect. However, the anti-fatigue mechanism of A. japonicus gonads remains to be further verified through cellular and molecular experiments. The study results demonstrated that A. japonicus gonads had high nutritional value and were suitable for the development of functional and nutritionally enhanced products. According to the relevant provisions of procedures and test methods for functional evaluation of health food, A. japonicus demonstrated good anti-fatigue ability, effectively alleviating mice's fatigue state by reducing the content of fatigue-related metabolites. It can improve the vitality of antioxidant-related enzymes, alleviating fatigue by reducing oxidative stress damage. This study provides a scientific basis for the high-value utilization of A. japonicus gonads and a novel concept for the development of anti-fatigue-related health food.

Abstract:The National Key Research and Development Plan "Blue Granary Science and Technology Innovation" key special project (hereinafter referred to as the Blue Granary Project) has made strategic arrangements in areas such as germplasm creation, healthy aquaculture, habitat restoration, environmentally friendly fishing, and green processing. This project has been implemented to promote modern fishery science and technology innovation in China, drive the transformation and upgrading of the fishery industry and green development, the establishment of a new fishery production system and its implementation. Based on bibliometrics, this study searched the publication of the Blue Granary Project in the SCIE database, and analyzed the research progress and focus of the project, including publication and citation situation of papers, research institutions, international collaboration, research directions, and high-frequency keywords, etc. The results show that since its launch in 2018 to November 7, 2023, the Blue Granary Project as the first funded project has published a total of 5,803 SCI papers, with a total citation frequency of 55,361, a total H index of 61, and ESI highly cited papers of 55, including 1 hot paper. A total of 47 nature index journal papers have been published, distributed over 15 journals, including top journals such as Nature Cell Biology, Nature Climate Change, Nature Genetics, Nature Communications, and PNAS. Shanghai Ocean University, Ocean University of China, Ningbo University, and other institutions had the most outstanding performance in terms of publication output; 951 of the papers were published by international collaboration with partners from 57 countries, with developed countries such as the United States and Australia having the highest output. The papers cover 78 research fields, mainly focusing on fishery, marine and freshwater biology, food science and technology, environmental science and ecology, chemistry, biochemistry and molecular biology, immunology, and veterinary medicine. The information retrieved from the papers shows that the Blue Granary Project has made significant progress in basic research, key core technology development, and typical application demonstration, providing theoretical support for high-quality development in aquaculture, aquaculture breeding, disease prevention and control, and aquatic product processing. The findings provide data-driven decision support for establishing a sustainable Blue Granary system, thereby advancing the high-quality development of the marine economy.